This work was supproted by Korea Research Foundation Grant funded by
Korea Government (MOEHRD, Basic Research Promotion Fund), the Korean
Federation of Science and Technology Societies, Korean National Institute of
Health, and the Institute for Viral Disease, Korea University.
Timetable ······················································································· 2
Plenary Lectures ······································································ 17
Hantaan Lecture ······································································ 23
Symposia ···················································································· 29
Colloquia ·················································································· 145
Workshops ·············································································· 155
Poster Sessions ····································································· 159
Instrument Exhibition ··························································· 289
Author Index ··········································································· 299
Keyword Index ······································································· 323
Time Table
October 13 (Thursday)
GEOMUNGO Hall
Place
Time
Hall A
Symposium 3
Symposium 4
Hall B
08:00~09:00
09:00~11:30
Hall C
CRYSTAL
Hall
Registration
Symposium 1
Symposium 2
11:30~11:45
Opening Remarks & Ceremony of Awarding Hantaan Prize (Hall C)
11:45~12:30
Plenary Lecture 1 (Hall C)
12:30~13:30
Lunch
13:30~15:00
Workshop 1
15:00~15:50
15:50~17:30
17:30~18:30
Workshop 2
KSMi GM
Symposium 5
Symposium 7
Symposium 6
MSK GM
Symposium 8
KSMy GM
18:30~20:30
Reception (Hall B&C)
October 14 (Friday)
GEOMUNGO Hall
Place
Time
Hall A
Colloquium 1
Registration
Symposium 9
Symposium 10
11:00~11:45
Plenary Lecture 2 (Hall C)
11:45~12:30
Plenary Lecture 3 (Hall C)
12:30~13:30
Lunch
13:30~15:45
Symposium 12
Symposium 13
15:45~16:00
16:00~18:00
Symposium 15
Symposium 16
18:30~19:00
KSMi ：The Korean Society for Microbiology
KSMy：The Korean Society of Mycology
MSK ：The Microbiological Society of Korea
한국미생물학회연합
Symposium 14
Colloquium 2
Hantaan Lecture
18:00~18:30
2
Symposium 11
Hall B
08:00~09:00
09:00~11:00
Hall C
CRYSTAL
Hall
Symposium 17
Colloquium 3
KSV GM
Closing Ceremony (Hall C)
KSV ：The Korean Society of Virology
KMB：The Korean Society for Microbiology and Biotechnology
GM ：General Meeting
Program Schedule
Plenary Lectures
PL-1
October 13 (Thursday) Geomungo Hall C
Chair : Sang Ho Choi (Seoul National University)
PL-1
11:30-12:15
Yersinia enterocolitica Infection : Views from Both Sides
Virginia L. Miller (Washington University School of Medicine, USA)
PL-2,3
October 14 (Friday) Geomungo Hall C
Chair : Sunyoung Kim (Seoul National University)
PL-2
11:00-11:45
Influenza : An Emerging Disease
Peter Palese (Mount Sinai School of Medicine, USA)
PL-3
11:45-12:30
Prospects for an AIDS Vaccine
Ronald C. Desrosiers (Harvard Medical School, USA)
Hantaan Prize Awardee's Lecture
Hantaan Lecture
15:45-16:30
October 14 (Friday) Geomungo Hall C
Chair: Yong-Sun Kim (Vice-president, Korean Society of Virology)
Awardees : Pyung-Woo Lee and Luck-Ju Baek (Korea University)
The Possible Role of Genetic Reassortment and RNA Recombination in
Hantaviral Diversity and Evolution
국제학술대회
3
Symposia
S1
Microbial Diversity
October 13 (Thursday) Geomungo Hall A
Chair : Jongsik Chun (Seoul National University)
S1-1
S1-1
09:00-09:30
The Streptomyces violaceoruber Clade: A Model System for Defining a Post-Genomic
Species Concept for Streptomycetes
Wonyong Kim (Chung-Ang University)
S1-2
S1-2
09:30-10:00
Biology of Myxobacteria
Kyungyun Cho (Hoseo University)
S1-3
S1-3
10:00-10:40
Cultivation of Uncultured Microbes
Yoichi Kamagata (Advanced Industrial Science and Technology, Japan)
S1-4
S1-4
10:40-11:20
Actinomycete Diversity and Natural Product Discovery
Michael Goodfellow (University of Newcastle Upon Tyne, UK)
S2
Virulent Mechanisms of Prevalent Pathogens in Korea I
October 13 (Thursday) Geomungo Hall B
Chair : Se-Jong Kim (Yonsei Unviersity)
4
S2-1
S2-1
09:00-09:50
The Discovery of Novel Pili of Mycobacterium tuberculosis
Richard L. Friedman (University of Arizona, USA)
S2-2
S2-2
09:50-10:15
Genome Analysis of Mycobacterium tuberculosis Strain K
Taeksun Song (Yonsei University)
S2-3
S2-3
10:15-10:40
Diacyltrehalose of Mycobacterium tuberculosis Inhibits LPS- and Mycobacteria-Induced
Proinflammatory Cytokine Production in Human Monocytic Cells
Hwa-Jung Kim (Chungnam National University)
S2-4
S2-4
10:40-11:05
Characterization of Helicobacter pylori Antigens Responsible for Inflammation and Apoptosis
in the Gastric Epithelial Cell Line
Seung-Chul Baik (Gyeongsang National University)
한국미생물학회연합
S2-5
S2-5
11:05-11:30
Use of rpoB Sequence Analysis for the Study on the Pathogenesis of Helicobacter pylori Infection
Yoon-Hoh Kook (Seoul National University)
S3
Infection and Immunity
October 13 (Thursday) Geomungo Hall C
Chair : Soon-Jung Park (Yonsei University)
S3-1
S3-1
09:00-09:30
Conserved Virulence Factors of Pseudomonas aeruginosa Identified from Drosophila
melanogaster-Based in vivo Screening
You-Hee Cho (Sogang University)
S3-2
S3-2
09:30-10:00
A Vibrio vulnficus LuxR Homolog, SmcR : Its Role in Virulence Gene Regulation and a
Consensus Sequence for Its Binding
Sang Ho Choi (Seoul National University)
Chair : Kun Soo Kim (Sogang University)
S3-3
S3-3
10:00-10:30
Regulation of Biofilm Formation by NtrC in Vibrio vulnificus
Kyu-Ho Lee (Hankuk University of Foreign Studies)
S3-4
S3-4
10:30-11:00
Comparative Genomic Analysis of a Gene Cluster for Capsular Polysaccharide Synthesis in
Vibrio vulnificus
Lien-I Hor (National Cheng-Kung University, Taiwan)
S3-5
S3-5
11:00-11:30
Regulation of Biofilm Formation and Surface Interactions of the Plant Pathogen Agrobacterium
tumefaciens
Clay Fuqua (Indiana University, USA)
S4
Recent Trends in Mycological Research I
October 13 (Thursday) Crystal Hall
Chair : Hack Sung Jung (Seoul National University)
S4-1
S4-1
09:00-09:25
Key Taxa of Biodiversity of the Macro- & Higher Fungi in China
Pei-Gui Liu (The Chinese Academy of Sciences, China)
국제학술대회
5
S4-2
S4-2
09:25-09:50
Taxonomy and Geographical Distribution of the Laboulbeniales in Korea
Young-Hee Na (Chosun University)
S4-3
S4-3
09:50-10:15
Endophytic Fungi Associated with Quercus liaotungensis
Liang-Dong Guo (Chinese Academy of Sciences, China)
S4-4
S4-4
10:15-10:40
Diversity and Pigment Biology of Fungi Causing Cosmetic Damage on Wood
Seong Hwan Kim (Dankook University)
S4-5
S4-5
10:40-11:05
Molecular Identification of Armillaria spp. Symbiotic to Gastrodia ellata (BL) in China
Ji-Chuan Kang (Guizhou University, China)
S4-6
S4-6
11:05-11:30
The Sequences of Small Subunit rDNA of Physarum didermoides and Its Evolutionary
Significance
Shu-Yan Liu (Jilin Agricultural University, China)
S5
Gene Expression
October 13 (Thursday) Geomungo Hall A
Chair : Sang Ho Choi (Seoul National University)
S5-1
S5-1
15:00-15:30
Oxidative Stress Caused by Chlorin Reductase Reaction of Rhodobacter sphaeroides
Jeong K. Lee (Sogang University)
S5-2
S5-2
15:30-16:00
Interplay between SeqA Protein and Methyl-Directed Mismatch Repair Proteins at the
Replicated GATC Sequences
Deog Su Hwang (Seoul National University)
S5-3
S5-3
16:00-16:30
Biogenesis of M1 RNA, the Catalytic Component of RNase P, in Escherichia coli
Younghoon Lee (Korea Advanced Institute of Science and Technology)
Chair : Jin Mi Kim (Chungnam National University)
S5-4
S5-4
6
16:30-17:00
The Study of Stress Response in Saccharomyces cerevisiae
Joon Kim (Korea University)
한국미생물학회연합
S5-5
S5-5
17:00-17:30
Regulation of the Mitotic Exit in Budding Yeast
Kiwon Song (Yonsei University)
S6
Virulent Mechanisms of Prevalent Pathogens in Korea II
October 13 (Thursday) Geomungo Hall B
Chair : Jeong-Kyu Park (Chungnam National University)
S6-1
S6-1
15:50-16:15
Prevalence and Molecular Characterization of MRSA and VRE
Yeong Seon Lee (Korean National Institute of Health)
S6-2
S6-2
16:15-16:40
Effect of Mycobacterial Antigens on the Expression of TLRs in Dendritic Cells
Junglim Lee (Konyang University)
S6-3
S6-3
16:40-17:05
Outer Membrane Protein 38 Is a Versatile Virulence Factor of Acinetobacter baumannii
Je Chul Lee (Kyungpook National University)
S6-4
S6-4
17:05-17:30
Generation of a Neutralizing Human Antibody to Hepatitis B Virus pre-S1 with Antibody Phage
Display Technique
Sae-Gwang Park (INJE University)
S7
Recent Trends in Mycological Research II
October 13 (Thursday) Geomungo Hall C
Chair : Seung Hun Yu (Chungnam National University)
S7-1
S7-1
15:00-15:25
Pathogenic Wood-Decaying Fungi in China
Yu-Cheng Dai (Chinese Academy of Sciences, China)
S7-2
S7-2
15:25-15:50
Fungicide Resistance and Phenotypic and Genotypic Diversity of Phytophthora infestans in
Korea
Xuan-Zhe Zhang (Kangnung National University)
S7-3
S7-3
15:50-16:15
Cytology of Interaction between Wheat and Puccinia striiformis
Zhensheng Kang (Northwest A&F University, China)
국제학술대회
7
S7-4
S7-4
16:15-16:40
Characterization of an Antifungal Agent against Monosporacus cannonballus Causing
Cucurbitaceous Crops Disease
Ki-Chul Chung (Chonnam National University)
S7-5
S7-5
16:40-17:05
Pathogen Biodiversity of Sooty Blotch and Flyspeck Apple Disease Complex in Northwest
China Based on Parsimony Analysis of Ribosomal DNA
Guang-Yu Sun (Northwest A&F University, China)
S7-6
S7-6
17:05-17:30
Identification of Colletotrichum spp. from Leguminosae and Zingiberaceae in South China
Zide Jiang (South China Agricultural University, China)
S8
Recent Trends in Mycological Research III
October 13 (Thursday) Crystal Hall
Chair : Tae Soo Lee (University of Incheon)
8
S8-1
S8-1
15:00-15:25
Biodiversity of Nematophagous Hirsutella Species and Their Relationship with Nematode
Xingzhong Liu (Chinese Academy of Sciences, China)
S8-2
S8-2
15:25-15:50
A Novel RNA Virus in Mushroom
Hyun-Sook Lee (Gyeongsang National University)
S8-3
S8-3
15:50-16:15
Diversity of Nematode-trapping Fungi in Pb-Contaminated Soils and Their Tolerance to the
Heavy Metal
Ming-He Mo (Yunnan University, China)
S8-4
S8-4
16:15-16:40
Agrobacterium-Mediated Transformation of the Edible Mushroom Agaricus bisporus
Hongkyu Kim (Chungcheongnam-do Agricultural Research and Extension Services)
S8-5
S8-5
16:40-17:05
Recent Advances in the Study of Mycorrhizas in China
Runjin Liu (Laiyang Agricultural College, China)
S8-6
S8-6
17:05-17:30
Anthostomella Species from China
Bingsheng Lu (Zhongkai Agro-Technology College, China)
한국미생물학회연합
S9
Genomics and Proteomics
October 14 (Friday) Geomungo Hall A
Chair : Hyun Ah Kang (Korea Research Institute of Bioscience and Biotechnology)
S9-1
S9-1
09:00-09:30
Dynamics of Nucleosome Occupancy by Transcription
Cheol-Koo Lee (Korea University)
S9-2
S9-2
09:30-10:00
Functional Proteomic Approaches into the Elucidation of Cyanobacterial Positive Phototaxis
Jong-Soon Choi (Korea Basic Science Institute)
S9-3
S9-3
10:00-10:30
The Novel Serine/Threonine Protein Kinase of Streptomyces coelicolor A3(2) Regulates SAM
Induced-Antibiotics Production
Kwang Pyo Kim (Konkuk University)
S9-4
S9-4
10:30-11:00
Structural Studies of Site-Specific Enzymes from Thermoplasma acidophilum DSM 1728
Kwang Yeon Hwang (Korea University)
S10
Health-promoting Effect of Lactic Acid Bacteria
October 14 (Friday) Geomungo Hall B
Chair : Kun Sub Chung (Yonsei University)
S10-1
S10-1 09:00-09:30
Modulation of Immune Responses by Probiotics and Prebiotics
Akira Hosono (Nihon University, Japan)
S10-2
S10-2 09:30-10:00
Transformation of Various Functional Glycosides Using Probiotic Bacteria and Food-Grade
Fungi
Geun Eog Ji (Seoul National University)
S10-3
S10-3 10:00-10:30
Lactic Acid Bacteria and Toll-like Receptor
Dae Kyun Chung (Kyung Hee University)
S10-4
S10-4 10:30-11:00
Monitoring of Lactic Acid Bacteria in Functional Starter-Inoculated Kimchi Fermentation Using
Microarray Technology
Yong-Ha Park (Korea Research Institute of Bioscience and Biotechnology)
국제학술대회
9
S11
Emergence of New and Variant Viruses
October 14 (Friday) Geomungo Hall C
Chair : Byung-Yoon Ahn (Korea University)
09:00-09:15
Opening Remarks
Pyung-Woo Lee (President, Korean Society of Virology)
Chair : Yong-Oh Shin (Kangwon National University)
S11-1
S11-1 09:15-09:45
Overview of Viral Infectious Diseases in Korea
Hae Wol Cho (Korean National Institute of Health)
S11-2
S11-2 09:45-10:15
Does SARS Spread by the Airborne Route? A Review of Several Major SARS Outbreaks in
Hong Kong
Tze Wai Wong (The Chinese University of Hong Kong, China)
S11-3
S11-3 10:15-10:45
Hepatitis E Virus–An Emerging Virus in Industrialised Countries
Tim J. Harrison (University College London, UK)
S12
Marine & Extreme Microbiology
October 14 (Friday) Geomungo Hall A
Chair : Sang-Jin Kim (Korea Ocean Research & Development Institute)
S12-1
S12-1 13:30-14:10
Host Vector Systems of Piezophilic Bacteria, and Construction of Pressure Sensing Cells
Takako Sato (JAMSTEC, Japan)
S12-2
S12-2 14:10-14:35
Assessment and Utilization of Marine Microbial Diversity by Using Metagenomic Study
Jung-Hyun Lee (Korea Ocean Research & Development Institute)
S12-3
S12-3 14:35-15:00
Protein trans-Splicing and Characterization of a Split Family B-type DNA Polymerase from the
Hyperthermophilic Archaeal Parasite Nanoarchaeum equitans
Suk-Tae Kwon (Sungkyunkwan University)
S12-4
S12-4 15:00-15:25
Microbial Phospholipase D : Application for Functional Phospholipid Production
Jin-Cheol Yoo (Chosun University)
10
한국미생물학회연합
S12-5
S12-5 15:25-15:50
Diversity and Application of Arctic and Antarctic Marine Bacteria
Yoo Kyung Lee (Korea Ocean Research & Development Institute)
※ This session is sponsored by the Marine & Extreme Genome Research Center Program,
Ministry of Maritime Affairs & Fisheries
S13
Trends in Fungal Biosciences and Biotechnology
October 14 (Friday) Geomungo Hall B
Chair : Hee-Moon Park (Chungnam National University)
S13-1
S13-1 13:30-14:00
Aspergillus fumigatus Cell Wall and Resistance to Host Defence Reactions
Jean-Paul Latgé (Institut Pasteur, France)
Chair : Jong-Soo Lee (Paichai University)
S13-2
S13-2 14:00-14:30
Immuno-Modulating Effects and Anti-Cancer Activities of Proteoglycan Isolated from Phellinus
linteus : Phenotypic and Function Maturation of Murine Bone Marrow-Derived Dendritic Cells
Gi-Young Kim (Cheju National University)
Chair : Jin Cheol Kim (Korea Research Institute of Chemical Technology)
S13-3
S13-3 14:30-15:00
Isolation and Characterization of Fusarium proliferatum KGL0401 as a Gibberellin-Producing Fungus
Jong-Guk Kim (Kyungpook National University)
Chair : Seong Hwan Kim (DanKook University)
S13-4
S13-4 15:00-15:30
A New Metalloprotease Gene from Pleurotus ostreatus Belongs to Eucolycins Family
Chang Soo Lee (Konkuk University)
S13-5
S13-5 15:30-16:00
Gene Transfer and Fruiting Body Development in Edible Mushrooms
Young Bok Yoo (National Institute of Agricultural Science and Technology)
S14
Molecular Basis of Viral Infection I
October 14 (Friday) Geomungo Hall C
Chair : Heuiran Lee (University of Ulsan)
S14-1
S14-1 13:30-14:00
HIV, SIV, and Resistane to Antibody-Mediated Neutralization
Ronald C. Desrosiers (Harvard Medical School, USA)
국제학술대회
11
S14-2
S14-2 14:00-14:30
Redundant Strategies of Negative Sense RNA Viruses to Overcome the Innate Immune
System of the Host
Peter Palese (Mount Sinai School of Medicine, USA)
S14-3
S14-3 14:30-15:00
Induction and Functions of Viral Stress-Inducible Genes
Ganes C. Sen (The Lerner Research Institute, USA)
S14-4
S14-4 15:00-15:30
Human Tumor Inducing Kaposi's Sarcoma-Associated Herpesvirus Immune Evasion Strategy
Jae Ung Jung (Harvard Medical School, USA)
S15
Applied Microbial Physiology
October 14 (Friday) Geomungo Hall A
Chair : Hor-Gil Hur (Gwangju Institute of Science and Technology)
S15-1
S15-1 16:00-16:30 (◈ D.Sc Memorial Lecture)
Discourse on the Researches for Production of Useful Metabolites Using Various Microorganisms
Kye Joon Lee (Seoul National University)
S15-2
S15-2 16:30-17:00
Extracelluar Proteome Analysis of Streptomyces griseus
Soon-Kwang Hong (Myongji University)
S15-3
S15-3 17:00-17:30
Genome-Wide Screening for Cryptic Biosynthetic and Regulatory Genes in Stretomyces
species
Eung-Soo Kim (Inha University)
S15-4
S15-4 17:30-18:00
Recent Advances in the Degradation of High-Molecular-Weight Polycyclic Aromatic Hydrocarbons
in Mycobacterium Species : Metabolism, Proteomic and Genomic Approaches
Carl E. Cerniglia (U.S. Food and Drug Administration, USA)
S15-5
S15-5 18:00-18:30
UV-absorbing Mycosporine-like Amino Acids in Oxygenic Photosynthetic Free-Living and
Symbiotic Microorganisms
~
Maribel L. Dionisio-Sese (University of the Philippines Los Banos,
Philippines)
12
한국미생물학회연합
S16
Structural and Functional Genomics of Microbial Community
October 14 (Friday) Geomungo Hall B
Chair : Jae-Chang Cho (Hankuk University of Foreign Studies)
S16-1
S16-1 16:00-16:30
Microbial Community Dynamics during Cyanobacterial Bloom
Chi-Yong Ahn (Korea Research Institute of Bioscience and Biotechnology)
S16-2
S16-2 16:30-17:00
Biodiversity of Oligotrophic Denitrifying Bacteria in Subsurface Soil
Kyung-Sook Whang (Mokwon University)
S16-3
S16-3 17:00-17:30
Bacterial Communities Associated with Sand Dune Plants and Their Potential for Plant
Growth Promoting Activity
Seung Bum Kim (Chungnam National University)
S16-4
S16-4 17:30-18:00
The Global Climate Change and Microbial Diversity
Seung-Hoon Lee (Ewha Womans University)
S16-5
S16-5 18:00-18:30
Genome-wide and Physiological Characterization : Effect of Nitrogen-fixation on Biphenyl and
Polychlorinated Biphenyl Degradation by Burkholderia xenovorans LB400
Joonhong Park (Yonsei University)
S17
Molecular Basis of Viral Infection II
October 14 (Friday) Geomungo Hall C
Chair : Yong-Soo Bae (Sungkyunkwan University)
S17-1
S17-1 16:30-17:00
Immune Responses and Prophylactic Potential of Live Influenza Vaccine
Baik Lin Seong (Yonsei University)
S17-2
S17-2 17:00-17:30
Circularization of a RNA Template via Long-range Base Pairing Is Critical for HBV Reverse
Transcription
Wang-Shick Ryu (Yonsei University)
S17-3
S17-3 17:30-18:00
Implications of Nonstructural 5A Protein of Hepatitis C Virus in Liver Pathogenesis
Soon Bong Hwang (Hallym University)
국제학술대회
13
Colloquia
C1
09:00-10:30
October 14 (Friday) Crystal Hall
Chair : Cheol-Won Yun (Korea University)
C1-1
C1-1
09:00-09:20
Does the Innate Immune System Discriminate Non-self from Self?
Seung-yong Seong (Seoul National University)
C1-2
C1-2
09:20-09:40
Preparation and Analysis of Yeast Cell Wall Mannoproteins, Immune Enhancing Materials,
from Cell Wall Mutant Saccharomyces cerevisiae
Chang Hoon Ha (Korea University)
09:40-09:50 Break
C1-3
C1-3
09:50-10:10
Forward Genetics of Murine Gammaherpesvirus 68 Using Signature-tagged Mutagenesis
Moon Jung Song (Hallym University)
C1-4
C1-4
10:10-10:30
Genome Analyses of Streptomyces peucetius for the Identification of Cytochrome P450 and
Regulatory Elements
Niranjan Parajuli (Ewha Womans University)
C2
13:30-15:40
October 14 (Friday) Crystal Hall
Chair : Young-Bong Kim (Konkuk University)
C2-1
C2-1
13:30-13:50
Identification of 5' and 3' cis-acting Elements of the Porcine Reproductive and Respiratory
Syndrome Virus and Acquisition of Novel 5' Sequences Required for RNA Replication
Yu-Jeong Choi (Chungbuk National University)
C2-2
C2-2
13:50-14:10
Memory CTL Reaction Can Be Recruited from Chronic Hepatitis C Infection by in vitro
Culture with Pan-HLA Reactive Epitopes and Recombinant Human Interleukin-15
Nam Kyung Kim (Mogam Biotechnology Institute)
C2-3
C2-3
14:10-14:30
Antiviral Effect of Catechins in Green Tea on Influenza Virus
Jae-Min Song (Yonsei University)
14:30-14:40 Break
14
한국미생물학회연합
Chair : Nam-Hyuk Cho (Seoul National University)
C2-4
C2-4
14:40-15:00
A Small Interfering RNA Targeting Coxsackievirus B3 Protects Permissive HeLa Cells from
Viral Challenge
Jeonghyun Ahn (University of Ulsan)
C2-5
C2-5
15:00-15:20
Virus Receptor Trap, Expressing Both the Coxsackievirus and Adenovirus Receptor and the
Decay-accelerating Factor, Neutralize Coxsackievirus in Experimental Murine Viral Myocarditis
Byung-Kwan Lim (Sungkyunkwan University)
C2-6
C2-6
15:20-15:40
Time-Saving Flow Cytometry Analysis for Rapid Determination of Ganciclovir Resistant
Human Cytomegalovirus
Gyu-Cheol Lee (Korea Water Resources Corporation)
C3
16:00-17:00
October 14 (Friday) Crystal Hall
Chair : Kangseok Lee (Chung-Ang University)
C3-1
C3-1
16:00-16:20
Development of Yeast Whole-Cell Systems for the Heavy Metal Cadmium Based on Transcriptome
Profiling of Stress Response in Hansenula polymorpha
Jeong-Nam Park (Korea Research Institute of Bioscience and Biotechnology)
C3-2
C3-2
16:20-16:40
ATP-Binding Motifs Play Key Roles in Krp1p, Kinesin-Related Protein 1, Function for Bi-polar
Growth Control in Fission Yeast
Dong Keun Rhee (Korea University)
C3-3
C3-3
16:40-17:00
Specific Regression of Human Cancer Cells by Ribozyme-Mediated Targeted Replacement of
Tumor-Specific Transcript
Heung-Su Jung (Dankook University)
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Workshops
W1
13:30-14:10
October 13 (Thursday) Geomungo Hall A
W1-1 Pharmatech Inc.
13:30-14:10
Comparative Genomic Sequencing(CGS) and Mutation Discovery in Prokaryotes Using Microarrays
Daniel Clutter (NimbleGen System, USA)
W2
13:30-15:00
October 13 (Thursday) Geomungo Hall C
W2-1 KIMIDATA CORPORATION
13:30-14:10
Getting the Most out of Your Micrroarray Data with Sportfire Decision Site
Andrew Khoo (Asia Pacific Operations. Spotfire, Inc., Malaysia)
14:10-14:20 Break
W2-2 Korean Center for Disease Control
14:20-15:00
Biosafety and Biosecurity in Korea: Legislational Enforcement and Prospective
Won-Keun Seong (National Institute of Health, Korea)
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PL-1
Yersinia enterocolitica Infection: Views from Both Sides
Damon Ellison, Scott Handley, Jason Cathelyn, and Virginia L. Miller*
Department of Molecular Microbiology, Washington University School of Medicine, USA
Abstract
Yersinia enterocolitica and Y. pseudotuberculosis are enteric pathogens that invade the intestinal
epithelium and colonize the intestinal lymphoid tissue and in immunocompromised patients can cause
highly lethal systemic infections. Invasion of the intestinal epithelium can be mediated by host and/or
bacterial factors, but the primary bacterial invasion factor is invasin (encoded by the inv gene). Expression
of inv is positively regulated by RovA and negatively regulated by H-NS and YmoA. Exactly how each
of these factors interact at the inv promoter to regulate expression is not clear. RovA and H-NS bind to
a similar region of the inv promoter, and while YmoA does not directly bind to the inv promoter it
appears to form a complex with H-NS. Deletion of the predicted H-NS binding region relieves the
requirement for RovA dependent transcription of the inv promoter, consistent with RovA acting as a
de-repressor of H-NS mediated repression.
A rovA mutant was shown a number of years ago to have a more severe virulence defect in a mouse
model of infection than an inv mutant suggesting that RovA regulates additional virulence genes. The rovA
gene of Y. pestis (the causative agent of bubonic and pneumonic plague) is highly similar to that of Y.
enterocolitica and is expressed, despite the fact that inv is a pseudogene in Y. pestis. We found that a
rovA mutant of Y. pestis also has a virulence defect, supporting the idea that RovA regulates additional
virulence genes in the pathogenic Yersiniae.
Enteric pathogens such as Y. enterocolitica readily colonize and induce disease within the lymphatic
tissues of the mouse small intestine. In order to gain a comprehensive view of the host response to
pathogens within these tissues, we determined the transcriptional profiles of intestinal lymphatic tissue
infected with Y. enterocolitica. Expression analysis using Affymetrix GeneChips revealed a complex host
response in the Peyer’s patches (PP) and mesenteric lymph nodes following oral infection. Interestingly,
one of the genes demonstrating a large increase in transcript number during infection was histidine
decarboxylase (HDC), the enzyme solely responsible for the production of the biogenic amine histamine.
In this study, we provide evidence that histamine signaling through the H2 receptor is important for
controlling Y. enterocolitica infection within the PP of mice.
Acknowledgements
This work was supported by National Institutes of Health grants AI27342, AI52167, and AI53298
awarded to VLM.
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PL-2
Influenza: An Emerging Disease
Peter Palese
Mount Sinai School of Medicine New York, NY, USA
Influenza remains an important disease in humans and animals. In contrast to measles, smallpox and
poliomyelitis, influenza is caused by viruses which undergo continuous antigenic change and which
possess an animal reservoir. Thus, new variants and/or reassortants resulting from the genetic exchange
of human and animal influenza viruses will likely be responsible for new epidemics and pandemics in
the future. Although it is not clear whether an avian (H5) virus pandemic is imminent, it would be prudent
to take into account the lessons we have learned from studying different human and animal influenza
viruses. Specifically, reconstruction of the genes of the 1918 pandemic virus and studies of their
contribution to virulence will be important steps toward understanding the biological capabilities of this
lethal virus. Together with the availability of new antiviral drugs and superior vaccines this will make
us better prepared and provide us with better approaches to control influenza.
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PL-3
Prospects for an AIDS Vaccine
Ronald C. Desrosiers
Harvard Medical School, USA
Several lines of evidence indicate that development of an effective vaccine for HIV-1 is going to be,
at best, extremely difficult. The inability to solve fundamental scientific questions is the root cause for
why a successful vaccine is not currently within our grasp. A renewed, organized, focused effort is needed
to overcome these fundamental scientific obstacles.
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Hantaan Lecture
The Possible Role of Genetic Reassortment and RNA
Recombination in Hantaviral Diversity and Evolution
Pyung-Woo Lee
Department of Microbiology, The Institute for Viral Diseases, College of Medicine, Korea University,
126-1, Anam-dong 5-ga, Seongbuk-gu, Seoul 136-705, Korea
Introduction: Viruses are classified into more than 60 different families on the basis of their genome
type and gene content. Viruses vary in their use of RNA or DNA as genetic material, genome sizes, and
diverse life styles such as different transmission routes, infection of diverse tissues. These diversity of
virus reflect that virus evolve in diverse ways. Since isolation of Hantaan virus as the prototype of genus
Hantavirus more than 30 new species have been described and the number is increasing rapidly. Some
of the virus types are the etiologic agents of hemorrhagic fever with renal syndrome (HFRS), and others
cause hantavirus pulmonary syndrome (HPS). Whereas Prospect Hill, Thailand viruses are known to be
avirulent. Each of hantavirus serotypes is primarily associated with a single rodent host species.
Hantaviruses form three main groups as to be defined by phylogenetic analysis based on the complete
coding region of the S segment: (i) Hantaan(HTN)-like viruses (HTN, SEO, and DOB viruses) carried
by Murinae rodents (Old World mice and rats), (ii) Puumala(PUU)-like viruses (PUU, Tula, PH, and
Khabarovsk viruses) carried by Arvicolinae rodents (voles and lemmings), and (iii) Sin Nombre(SN)-like
viruses (SN, BAY, AND, and New York viruses) carried by Sigmodontinae rodents (New World mice
and rats). The evolution tree of the different hantavirus, based on viral genome sequences has been shown
to mirror the evolution tree of their specific rodents, based on sequences of mitochondrial DNA. This
suggests that an ancestral hantavirus infected a specific rodent at early during evolution, and was
subsequently run to the same evolutionary pressure as the rodent host. That is, hantaviruses have been
co-evolved with their natural hosts.
Molecular epidemiology indicates that hantavirus may have evolved in three ways: 1) mutations within
the genome, 2) reassortment of the segmented genome between two closely related hantavirus, and 3)
genomic recombination, a relatively rare phenomenon among negative stranded RNA viruses. Among
these possible mechanisms on hantavirus evolution, reassortment will be mostly discussed presenting the
data obtained in our lab and recombination will be also referred as well in this lecture.
The idea that mutations within the genome can raise evolution is applicable to viral field. Many RNA
viruses encode RNA dependent RNA polymerase (RdRP) and this enzyme has error-prone character. This
character will facilitate dynamic evolution of RNA virus through time flow. In addition, the reassortment
can increase divergency and it results in a new virus shows newly acquired feature. Several lines of
evidence have suggested that reassortment could play an important role in the evolution, pathogenesis, and
epidemiology of the arthropod-borne members of Bunyaviridae. Little information is available on genetic
reassortment of the rodent-borne virus, especially hantaviruses except the reports on SNV in nature.
Laboratory generation of reassortant strains by mixed infection with 2 different serotypes of
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hantavirus: To investigate possibility of genetic reassortment of the segmented genome in hantavirus, we
performed genetic analysis of progeny virus after mixed infection of hantavirus serotypes in tissue culture
cells. The serotypes inoculated were Hantaan(H) virus carried by Apodemus mice in nature, and Seoul(S)
virus carried by Rattus in nature. These serotypes can be discriminated by serologic test, such as
neutralizing antibody test. As the result, some reassortant strains were isolated from high m.o.i. infection.
Genotypes of the isolated progenies were determined by mutiplex PCR with corresponding primers to L,
M, and S segments of each serotypes. The incidence rate of reassortant hantavirus from randomly selected
plaques was up to 49.3 %. The genotypic distribution of these reassortants was mainly S/H/S (in L/M/S
order) as 71/144 isolates. The others are S/H/H as far less incidence. No other genotypes were isolated
and this finding reflects L and S segments itself or its products are so strongly associated that these
segments can not be incorporated into newly generated virus particles. By this molecular talk, viral M
segment may spread into other serotype more readily than other segments if there is no other restriction.
Because M segment encodes glycoprotein which plays a critical role in determination of host range, the
reassortant strain would select a new host in nature.
The acquisition of pathogenicity by genetic reassortment: Besides M segment reassortment,
phenotypic alteration in pathogenicity has been observed in a reassortant strain. After mixed infection with
PHV (apathogenic hantavirus) and MAA (hantavirus causes severe disease) in Vero E-6 cell, we found
S segment reassorted virus (M/M/P) in lower incidence through genetic analysis of progeny virus. When
its pathogenicity was analyzed using suckling mouse model, despite its parental virus, MAA was fatal
to mice, the reassortant strain (M/M/P) did not show any pathogenicity same as PHV. This suggests that
the locus determines pathogenesis is located in S segment in hantavirus and hantavirus can obtain or loss
its pathogenetic character through such genetic reassortment process.
RNA diploidism: The segmented RNA viruses have their encapisidated genomic RNA fragments in
virion. And also it is usual process that 1 set of RNA molecules is packed in the particle. However, the
diploid RNA molecules are observed in virion sometimes. Following co-infection or super-infection with
2 different species/strains of hantavirus, genotypes of the progeny viruses have been investigated. Progeny
virus possess additional RNA were found in plaque purified virus pool and this mixed genomes were
persist for several passages. To monitor this phenomenon, a mini replicon experiment was carried out.
HTN S-like RNA which encodes luciferase and is flanked by HTN UTR was transfected into host cell
infected with HTN virus. The progeny viruses were passaged 3 times serially and luciferase activity was
determined at each passage. The results showed that the introduced replicable RNA can be transferred
into progeny virus and the virus does not pack their genome as just one copy. This RNA diploidism could
be a useful mean to establish reassortant strain in nature/laboratory to increase chance carrying additional
RNA molecule. Thus, the virus can select more preferable segment to survive.
The recombination of RNA molecule: Recently, Finish group demonstrated mosaic-like structure of
RNA. According to this study, some recombinant viruses were rescued in Tula virus infected cell so as
to produce other type of Tula virus RNA. And this mosaic RNAs recombinant site was not restricted at
any point but distributed whole S segment even though there is highly preferable site.
Conclusion: The current data on hantavirus genome sequences indicate that the genetic diversity of
hantavirus is occurred primarily by genetic drift, such as accumulation of point mutation and
insertions/deletions of one or several nucleotides. No frame shift mutations in the coding regions of the
hantavirus genes have been observed so far. There is also evidence for a genetic shift in hantaviruses
which occurs both via reassortment of genomic RNA segments and recombination. Reassortants between
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2005 International Meeting of the Federation of Korean Microbiological Societies
hantaviruses in either inter or intra genus were observed in vitro, and reassortant SNV strains were found
in nature. Recently, a homologous recombination in hantaviruses has been also demonstrated. Detailed
knowledge of the rate and mode of genetic variation is essential for understanding how hantaviruses
induce disease in humans as well as for molecular epidemiology. And also advanced study on hantavirus
diversity will conduct the prediction of new emerging strains.
Keywords: diploidism, diversity, Hantavirus, genome reassortment, mitochondrial DNA, molecular evolution,
reassortants, recombination
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S1-1
The Streptomyces violaceoruber Clade: A Model System for
Defining a Post-Genomic Species Concept for Streptomycetes
Wonyong Kim1,2,* Alan C. Ward2, and Michael Goodfellow2
1
Department of Microbiology, Chung-Ang University College of Medicine, Seoul,156-756, Korea,
2
School of Biology, University of Newcastle, Newcastle upon Tyne, NE1 7RU, UK
"Streptomyces coelicolor" A3(2) has been the model organism for streptomycete geneticists for more than
50 years, and, as such, the basis of much of the understanding of the mechanisms of secondary metabolite
production in the pharmaceutical industry. With the publication of the whole genome sequence of this
organism "Streptomyces coelicolor" has become the reference organism for post-genomic streptomycete
systematics. "Streptomyces coelicolor" A3(2) is a member of the monomphyletic 16S rDNA clade which
includes Streptomyces violaceoruber and part of a larger clade in the 16S rDNA Streptomyces phylogenetic
tree which includes Streptomyces albidoflavus and several important antibiotic producing organisms, such as
Streptomyces ambofaciens and Streptomyces tendae. Streptomyces albidoflavus is one of the most common
streptomycetes found in the environment. This group of organisms provides a well-studied set of
streptomycetes, including two, multi-membered species-groups and a reference organism with a whole genome
sequence. These organisms provide a model set of organisms to apply the methods proposed for the
development of post-genomic systematics and the definition of a species concept for the genus Streptomyces.
In this study a comprehensive set of DNA:DNA similarities have been determined between all strains to give
a reference set of data for the ‘gold standard' for definition of prokaryotic species which has been extant for
the last 30 years. The fAFLP profiles of all the strains have been determined, and compared with
corresponding DNA:DNA similarities, giving a reproducible DNA finger-printing method which is calibrated
against DNA:DNA pairing in the streptomycetes. However, it is gene sequencing which provides the most
reliable, portable and cumulative data, and it has provided the basis for the application of multi-locus sequence
typing for the characterization of several groups of pathogens, and has become the basis for population studies
and subspecific typing schemes in medical identification. The application of gene sequencing, multilocus
sequencing, has been recommended as a method to supplement and supplant DNA:DNA pairing in defining
the bacterial species. In addition to completing the 16S rDNA gene sequencing for these organisms, six other,
protein-coding, genes have been identified as candidates for this approach, gyrA, gyrB, prcA, recA, rnpB and
rpoB. Conserved primers have been designed and the sequences of the nearly complete genes sequenced
(except for gyrA, which is ca. 2,400 bp, where a substantial fraction of the gene has been sequenced). This
data has been analysed by phylogenetic and multi-locus sequencing software programmes to provide a
comprehensive comparison of the relationships of these strains on the basis of 16S rDNA phylogeny,
DNA:DNA pairing and multi-locus sequencing to provide a consistent picture of the species boundary around
the Streptomyces albidoflavus and Streptomyces violaceoruber species.
Keywords: Bacterial species concept, Streptomyces violaceoruber, Streptomyces albidoflavus, DNA:DNA
hybridisation, fAFLP, Multilocus sequencing
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S1-2
Biology of Myxobacteria
Kyungyun Cho
Department of Biotechnology, Hoseo University, Asan 336-795, Korea
Myxobacteria. Myxobacteria are Gram-negative, rod-shaped, soil bacteria that move by gliding motility and
form multicellular fruiting bodies [1,2].
Gliding Motility. Gliding motility is defined as the movement of nonflagellated cells in the direction of
its long axis on a solid surface[3]. Studies with Myxococcus xanthus, a representative species of myxobacteria,
have indicated that myxobacteria have two gliding motility systems: social(S)-motility and
adventurous(A)-motility. S-motility appears to rely on the function of type IV pili, similar to twitching motility
of Pseudomonas aeruginosa and Neisseria gonorrhoeae. In contrast, the mechanism of A-motility has not
been identified yet.
Fruiting Body Development. When placed on a nutrient rich surface, rod-shaped myxobacterial cells grow
vegetatively and travel in large groups referred to as swarms. In the absence of nutrients, swarms of
approximately 100,000 cells move towards an aggregation center and form various shapes of raised mounds.
The shapes of the raised mounds depend on the species of myxobacteria. The individual rod-shaped cells
within the mounds are then converted into spherical resting cells called myxospores. In contrast to endospore
formation in Bacillus spp., spores of myxobacteriaare formed by morphogenesis of whole cells. The
myxospores are dormant and are resistant to various environmental stresses such as drying, heating, detergent,
and sonication. The mounds containing myxospores are called mature fruiting bodies. When exposed to a
nutrient rich environment, spores in the fruiting body germinate simultaneously and become swarms of
vegetative rod-shaped cells.
Timing of Fruiting Body Development. EspAB are implicated in the timing of fruiting body development
in M. xanthus [4]. The espA null mutation triggersearly development and aggregation-independent early
sporulation, resulting in the appearance of free spores outside of fruiting bodies. In contrast, the espB null
mutation causes delayed and reduced sporulation, resulting in the formation of translucent mounds rather than
dark fruiting bodies. It appears that EspA, a histidine protein kinase, inhibits sporulation during early
development prior to the completion of aggregation and that this inhibition is antagonized by EspB, a putative
integral membrane protein with homology to oligopeptide transporters [4]. While searching for additional
genes that might interact with the EspAB pathway, we discovered a new gene, espC, whose mutant phenotype
is very similar to that of the espA [5]. The espC null mutation caused accelerated aggregation and formation
of tiny fruiting bodies surrounded by spores which were also observed in the espA mutant and the
CsgA-overproducing cells in M. xanthus. In addition, the espC mutant appeared to produce higher amounts
of the complementary C-signal than the wild type strain. These suggest that EspC is also involved in
controlling the timing of fruiting body development in M. xanthus.
Isolation of Wild Myxobacteria. Myxobacteria are known to be a rich source of potentially useful
secondary metabolites and enzymes [6]. For instance, the German Research Centre for Biotechnology (GBF)
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2005 International Meeting of the Federation of Korean Microbiological Societies
isolated about 80 different basic compounds and 450 structural variants from about 7,000 wild myxobacterial
strains in last 30 years. They reported that many of these compounds were new and had novel action
mechanisms that were very rare with other producers. However only a fraction of the wild strains have been
isolated until now and a large portion of wild myxobacteria in nature are remained to be isolated. As an effort
to collect these valuable microbial resources, we isolated 1,000 wild myxobacterial strains in Korea and
classified them based on 16S rRNA sequences in last three years.All the isolated strains have been deposited
in MicroBank (www.microbank.re.kr) of the Microbial Genomics and Applications Center Program and are
available to the public. Culture extracts of the isolates are also available at MicroBank.
Keyword: Myxobacteria
References
1. Dworkin, M. and Kaiser, D. 1993. Myxobacteria II, ASM Press, Washington, D.C.
2. Reichenbach, H. 1999. The ecology of the myxobacteria. Environ. Microbiol. 1: 15-21.
3. Cho, K. 2002. Bacterial gliding motility. Kor. J. Microbiol. Biotechnol. 30: 199-205.
4. Cho, K. and D. R. Zusman. 1999. Sporulation timing in Myxococcus xanthus is controlled by the
espAB locus. Mol. Microbiol. 34: 714-725.
5. Lee. B., P. I. Higgs, D. R. Zusman, and K. Cho. 2005. EspC is involved in controlling the timing
of development in Myxococcus xanthus. J. Bacteriol. 187: 5029-5031.
6. Kim, Y. S., W. C. Bae, and S. J. Back. 2003. Bioactive substances from myxobacteria. Kor. J.
Microbiol. Biotechnol. 31: 1-12.
7. Park, S., B. Lee, J. Kim, C. Lee, E. Chang, and K. Cho. 2004. Isolation and characterization of
bacteriolytic wild myxobacteria. Kor. J. Microbiol. Biotechnol. 32: 218-223
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S1-3
Cultivation of Uncultured Microbes
Yoichi Kamagata
Institute for Biological Resources & Functions
National Institute of Advanced Industrial Science and Technology (AIST)
Central 6, Higashi 1-1-1, Tsukuba, Ibaraki 305-8566, Japan
Summary
A huge number of microorganisms are still unknown and uncultivated. SSU rRNA gene-based analyses of
complex microbial communities are unveiling their diversity, distribution and abundance in natural
environments. How to isolate those uncultivated microorganisms is an everlasting topic for microbiologists
even in the era of culture-independent molecular. Although conventional isolation procedures are very
laborious, we still have possibilities to significantly improve the strategies. In my talk, several important clues
to systematic isolation and cultivation of uncultured microorganisms will be discussed.
Introduction
Over the past decade, molecular approaches primarily based on SSU rRNA/DNA analyses have been
commonly used for analyzing overall structure of microbial consortia. These approaches have uncovered a vast
variety of unknown microorganisms present in all kinds of environments (Rappe and Giovannoni, 2003).
Cumulative sequence information has enabled us to know the diversity of microorganisms and what
phylotypes dominate the communities in a particular environment. Such phylogenetic information combined
with functional analyses (such as MAR-FISH, CARD-FISH and stable isotope probing) and metagenomics
could give us further insight into the functions and roles of predominating microorganisms in environment
(Pernthaler et al, 2002 Andreasen and Nielsen, 1997; Schloss and Handelsman, 2003; Tyson et al., 2004).
However, such approaches clearly have limitations in terms of better understanding what they are really doing
and how they are making a living in situ. In this context, isolation of tangible microorganisms is still the
most convincing way to know exactly what they are doing and potentially can do. This strategy is obviously
the opposite way to recent community genomics that can be accomplished by taking advantage of
well-developed DNA sequencers, cloning techniques and elaborate gene sequence analyses. However, both
strategies should coexist and be complementary and together may shed light on microorganisms that lurk in
environment.
Isolation of microorganisms is undoubtedly a time-consuming and laborious. In addition, the underlying
techniques, which have been used over a century, seem to have limitations to culture fastidious microorganisms.
Nevertheless, there are possibilities to improve the way of isolation of yet-to-be cultured microbes.
Isolation of anaerobic syntrophic microorganisms
From a number of collected data, it is strongly suggested that most of microorganisms on the earth thrive
in anaerobic environments. Apparently, oxygen is freely available on the surface environment where
O2-respiring microorganisms dominate. However, a huge number of anaerobic microorganisms are hidden
belowthe surface. Not only terrestrial and oceanic subsurfaces are the places where anaerobes exist, but
common places such as animal intestines, rice paddies, wetlands and methane fermenting processes are the
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well-known habitats for anaerobes.
Under strictly anaerobic conditions, one microorganism shares energy with another for substrate oxidation.
Under oxic conditions, simple substrates such as fatty acids could be completely oxidized by one
microorganism. In anaerobic environment, however, those substrates can not be completely oxidized by single
species of organism. For instance, an anaerobic fatty acids-oxidizing microorganism that is referred to as
syntroph could, to some extent, oxidize fatty acids to produce H2 and lower molecule intermediates, but H2
generated in this process inhibits further oxidation because of thermodynamic reasons. The organism,
therefore, requires another organism that could consume (scavenge) H2 to keep the H2 partial pressure
extremely low to make whole oxidation reaction energetically feasible. In general, H2-consuming methanogens
and sulfate reducing bacteria are in charge of this process.
To date, immense efforts to isolate syntrophic microorganisms have been made by many investigators. The
key to successfully isolating such microorganisms is either isolating them in coculture with H2-consuming
organisms or isolating them using different substrates that could allow them to grow in pure culture. The
former way needs H2-consuming organisms that can support the growth of syntrophs. Typically, H2
-consuming organisms are pregrown and mixed with inoculum so that the syntrophs would be expected to
grow and form colonies on the "lawn" of H2 consumers in solidified medium. The latter way is on a trial
and errors basis to find appropriate substrates to grow in pure culture. However, one of the important clues
is the fact that most of syntrophs are capable of fermenting some certain substrates using intermolecule
disproportionation, or they may have a respiratory system using fumarate, sulfate or several metalsas terminal
electron acceptor. We have accumulated such knowledge and have succeeded in isolating several very
fastidious syntrophic microorganisms in pure culture (Hattori et al., 2000; Imachi et al., 2000; Qui et al., 2003;
Sekiguchi et al., 2003, Kamagata and Tamaki, 2005).
Isolation of microorganisms that require growth factors produced by other microorganisms
Syntrophic association in anaerobic environment is, as mentioned above, underlain, to a large extent, by
interspecies H2 transfer between H2-producing microbes and H2-scavenging microbes. This is the common
way of life style for anaerobes. By contrast, how aerobic microorganisms interact remains unclear, although
most of microbial ecologists can imagine that cell-cell communications could routinely occur between different
species of microorganisms. There is a report on aerobic symbiotic relation between two organisms in which
one organism (Symbiobacterium thermophilum) requires a growth factor produced by a Bacillus strain (Ohno
et al., 2000). We also attempted to isolate microorganism whosegrowth is stimulated by other microorganisms.
The bacterium isolated from activated sludge and later designated Catellibacterium nectariphilumdid not show
significant growth on nutrient broth. However, the growth was significantly stimulated by addition of
supernatant from other bacterial cultures (Tanaka et al., 2004). Culture filtrate of a strain related to the genus
Sphingomonas in particular increased the cell yield and growth rate. The supernatant could not be replaced
by known cofactors or amino acids. These stories indicate that syntrophic (synergistic) interaction between
different species of microorganisms via material transfer is very likely to occur in many unknown
microorganisms.
Isolation of slow growing and oligotrophic microorganisms
Considering the concentrations of available substrates in natural environment, it would not be surprising
if a number of prokaryotic populations prefer lower concentrations of nutrient than the concentrations that we
routinely use to grow "lab-tamed microorganisms" such as E. coli and if a number of microorganisms are
slow growers. The keys to isolating those microorganisms from a complex community are how to determine
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the substrate concentrations and how to eliminate fast growing microorganisms on its way. There are not
general solutions, but we have succeeded in isolation of a variety of slow growing microorganisms by limiting
the nutrient concentrations to suppress the growth of fast growers or by using appropriate inocula in which
slow growers' populations are already predominant (Kamagata et al., 1997; Takeda et al., 2002; Hanada et
al., 2002; Lee et al., 2004).
Other factors affecting the culturability of uncultured microorganisms
There are many other factors that have to be taken into consideration when attempting to isolate uncultured
organisms. Physicochemical factors such as temperatures, pHs, redox potentials, O2 concentrations, salinity
and gelling reagents seem very important to improve or modify isolation strategy. Very recently, we have
found that the utilization of gellan gum as gelling reagent significantly improves the CFU counts and
culturability of unknown species from a lake sediment sample (Tamaki et al., 2005).
Keywords: Culturing, prokaryotes, small subunit RNA, anaerobes, syntrophy
References
Andreasen, K., and Nielsen, P. H.1997. Application of microautoradiography to study substrate uptake by
filamentous microorganisms in activated sludge. Appl. Environ. Microbiol. 63: 3662-3668.
Hanada, S., Liu, W-T., Shintani, T., Kamagata, Y., and Nakamura, K. 2002. Tetrasphaera elongata
sp. nov., a polyphosphate accumulating bacterium isolated from activated sludge. Int. J. Syst. Evol.
Microbiol. 52: 883-887.
Hattori, S., Kamagata, Y., Hanada, S., and Shoun, H. 2000. Thermoacetogenium phaeumgen. nov., sp.
nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium. Int. J. Syst. Evol.
Microbiol. 50: 1601-1609.
Imachi, H., Sekiguchi, Y., Kamagata, Y., Ohashi, A., and Harada, H. 2000. Cultivation and in situ
detection of a thermophilic bacterium capable of oxidizing propionate in syntrophic association with
hydrogenotrophic methanogens in a thermophilic methanogenic granular sludge. Appl. Environ.
Microbiol. 66: 3608-3615.
Kamagata, Y., Fulthorpe, R.R., Tamura, K., Takami, H., Forney, L.J., and Tiedje, J.M. 1997. Pristine
environments harbor a new group of oligotrophic 2,4-dichlorophenoxyacetic acid-degrading bacteria.
Appl. Environ. Microbiol. 63: 2266-2272.
Kamagata, Y. and Tamaki, H. 2005. Cultivation of uncultured fastidious microorganisms. Microb.
Environ., 20, 85-91.
Lee, T.H., Kurata, S., Nakatsu, C.H., and Kamagata, Y. 2005. Molecular analysis of bacterial
community based on 16S rDNA and functional genes in activated sludge enriched with
2,4-dichlorophenoxyacetic acid (2,4-D) under different cultural conditions. Microbial Ecol., 49: 151-162.
Ohno, M., Shiratori, H., Park, M.J., Saitoh, Y., Kumon, Y., Yamashita, N., Hirata, A., Nishida, H.,
Ueda, K., and Beppu, T. 2000. Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic
thermophile that depends on co-culture with a Bacillus strain for growth. Int. J. Syst. Evol. Microbiol.
50: 1829-1832.
Pernthaler, A., Pernthaler, J., and Amann, R. 2002. Fluorescence in situ hybridization and catalyzed
reporter deposition for the identification of marine bacteria. Appl. Environ. Microbiol., 68: 3094-3101.
Qui Y-L., Sekiguchi, Y., Imachi, H., Kamagata, Y., Tseng, I-C., Cheng, S-S., Ohashi, A., and Harada,
H. 2003. Sporotomaculum syntrophicum sp. nov., a novel anaerobic, syntrophic benzoate-degrading
bacterium isolated from methanogenic sludge treating purified-terephthalate-manufacturing wastewater.
Arch. Microbiol. 179: 242-249.
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Rappe, M.S. and Giovannoni, S.J. 2003. The uncultured microbial majority. Annu Rev Microbiol. 57:
369-94.
Sekiguchi, Y., Yamada, T., Hanada, S., Ohashi, A., Harada, H., and Kamagata, Y. 2003. Anaerolinea
thermophila gen. nov., sp. nov., and Caldilinea aerophila gen. nov.,sp. nov., two novel filamentous
thermophiles that represent a previously uncultured lineage of the domain Bacteria at the subphylum
leve l. Int. J. Syst. Evol. Microbiol. 53: 1843-1851.
Schloss, P.D. and Handelsman, J. Biotechnological prospects from metagenomics. Curr. Opin. Microbiol.
2003. 14: 303-310.
Takeda, M., Kamagata, Y., and Koizumi, J. 2002. Paenibacillus koleovorans sp. nov., able to grow on
the sheath of Sphaerotilus natans. Int. J. Syst. Evol. Microbiol. 52: 1597-1601.
Tamaki, H., Sekiguchi, Y., Hanada, S., Nakamura, K., Nomura, N., Matsumura, M., and Kamagata,
Y. 2005 Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake
as revealed by molecular and improved cultivation-based techniques. Appl. Environ. Microbiol., 71,
2162-2169.
Tanaka, Y., Hanada, S., Manome, A., Tsuchida, T., Kurane, R., Nakamura, K., and Kamagata, Y.
2004. Catellatibacterium nectariphililum gen. nov., sp. nov., which requires a diffusible compound from
a strain related to the genus Sphingomonas for vigorous growth. Int. J. Syst. Evol. Microbiol. 54:
955-959.
Tyson, G.W., Chapman, J., Hugenholtz, P., Allen, E.E., Ram, R.J., Richardson, P.M., Solovyev, V.V.,
Rubin, E.M., Rokhsar, D.S., and Banfield, J.F. 2004. Community structure and metabolism through
reconstruction of microbial genomes from the environment. Nature. 428: 37-43
국제학술대회
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October 13-14, 2005, Seoul, Korea
S1-4
Actinomycete Diversity and Natural Product Discovery
Michael Goodfellow
Division of Biology, University of Newcastle Newcastle upon Tyne, NE1 7RU, UK
Natural products, notably antibiotics have an impressive record in combating infectious diseases though
once the rediscovery of known bioactive compounds exceeded the discovery of novel products the focus in
drug discovery moved away from natural product screening towards combinatorial chemistry. While the lure
of simple molecules and rational design proved irresistible, the application of these strategies has not led to
demonstrable success. However, in the meantime it has become apparent that an impressive combinatorial
chemistry and screening experiment has taken place over billions of years in the biosphere and that this has
generated an array of complex molecules that are distinctly different from those derived from chemical
synthesis. So, pharmacological screening of microorganisms for novel metabolites is back in business!
Search and discovery for new natural product drugs is difficult as microbial diversity is immense,
biogeography and ecology means that it is distributed heterogeneously over space, time and ecosystems, while
the chemical diversity sought is dispersed within this biodiversity. However, advances in microbial systematics
and molecular ecology provide ways of addressing such issues, notably by highlighting lineages that
encompass "creative" microorganisms; enabling the classification, and thus the selective isolation and
identification of target organisms; predicting metabolite potential, and dereplicating isolates for screening.
Whole genome sequence data underline the importance of streptomycetes in this respect as the genomes of
"Streptomyces coelicolor" A3(2) and Streptomyces avermitilis MA-4680 contain more than twenty natural
product gene clusters, far more than other genomes, for example, Bacillas subtilis with three, four in
Pseudomonas aeruginosa, two in Ralstonia solanacearum, and none in representatives of most other taxa. So,
actinobacteria remain an attractive target for bioprospecting!
The selective isolation and characterisation of members of rare, uncommon and currently uncultivable
actinobacterial taxa are procedures of paramount importance in bioprospecting, the more so in light of
molecular ecological surveys which show that actinobacterial genetic diversity in natural habitats is much
greater than previously recognised. The failure to isolate representatives of actinobacterial communities in
natural habitats can be attributed to several factors, including the reliance placed on classical selective
isolation procedures, a focus on temperate and terrestrial habitats and the difficulty of recognising novel
isolates. Innovative strategies are needed for the selective isolation and characterisation of representatives of
most actinobacterialgenera, not least those known to contain isolates that produce interesting metabolites. One
such strategy will be exemplified by the selective isolation, characterisation and screening of Amycolatopsis
strains, a taxon that encompasses organismsthat produce commercially important antibiotics. So, do not forget
about the genus Amycolatopsis!
Another route to the discovery of novel actinobacteria will be considered, namely the investigation of unand under-explored habitats, as exemplified by the marine ecosystem. Until recently, actinobacteria in marine
habitats were seen as wash-in components from terrestrial ecosystems. It is now evident that some
actinobacteria are indigenous to marine habitats, notably the genus Salinispora, the source of the anticancer
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2005 International Meeting of the Federation of Korean Microbiological Societies
metabolite salinosporamide A. The potential importance of marine actinobacteria in bioprospecting will be
addressed by reference to a series of collaborative studies designed to isolate, dereplicate, characterise and
screen novel actinobacteria from deep sea muds, including sediment collected by a submersible from the
Challenger Deep (10, 898m) of the Mariana Trench. A notable development from these studies was the
discovery that a new member of the genus Verrucosispora, strain AB-18-032, produced a unique polycyclic
polyketide family, the abyssomicins, of which abyssomicin C is a potent inhibitor of para-aminobenzoic acid
biosynthesis. This compound strongly inhibits Gram-positive bacteria, including methicillin and vancomycin
resistant strains of Staphylococcus aureus. So, tiny treasures from the ocean deep can beat the "superbugs"!
The relationship between biodiversity and chemodiversity is recognised at broad taxonomic levels. However,
the use of taxonomic diversity as a surrogate for chemical diversity, that is, the concept that finding novel
actinobacterial strains should lead to the discovery of new natural habitats, has been challenged with particular
reference to streptomycetes, the most prolific source of novel bioactive compounds. Further, the extent of
actinobacterial diversity in the environment is indisputable though it has been questioned whether this applies
to the streptomycetes as they have been screened extensively. Evidence will be presented to support the
hypothesis that streptomycete taxonomy can be used as a taxonomic roadmap to biosynthetic genes and that
voids exist in the taxonomic space occupied by streptomycetes. So, do not write off streptomycetes when
bioprospecting!
Keywords: microbial systematics, selective isolation, biodiversity, chemodiversity, Verrucosispora, Salinispora,
bioprospectin
국제학술대회
39
October 13-14, 2005, Seoul, Korea
S2-1
The Discovery of Novel Pili of Mycobacterium tuberculosis
Richard L. Friedman
Department of Microbiology and Immunology, University of Arizona College of Medicine,
P.O. Box 245049, Tucson, AZ 85724, USA
Abstract
Understanding the mechanisms and bacterial factors responsible for M. tuberculosis’ ability to cause disease
in humans is critical for the development of improved treatment strategies. Using negative staining and
transmission electron microscopy it was discovered that mycobacteria, including the human pathogen M.
tuberculosis, produce fine surface structures known as pili. Mass spectroscopy analysis demonstrated that
purified pili from M. tuberculosis are comprised of protein subunits encoded by the predicted M. tuberculosis
H37Rv ORF designated Rv3312A. These pili termed M. tuberculosis pili, Mtp, are highly aggregative 2-5
nm diameter fibers and are produced during culture on plate media. Tuberculosis (TB) patient sera were found
to contain antibodies that recognized Mtp antigen in ELISA and immunofluorescent microscopy (IF)
experiments. These results indicate that Mtp are produced during human infection. Other studies found that
Mtp binds to the extracellular matrix protein laminin in vitro suggesting that Mtp are a potentially newly
identified adherence factor for M. tuberculosis.
A second pili morphotype, which appeared as rope-like bundles, were produced by M. tuberculosis when
grown in liquid cultures. It was found that the M. tuberculosis chromosome contains a type IVB pili gene
cluster. The M. tuberculosis type IV pili belong to the Flp sub-family of type IVB pili. RT-PCR analysis
reveals that flp is expressed by M. tuberculosis and the Flp protein was detected by (IF) using Flp-specific
antibodies, showing that the Flp protein is secreted from the bacteria when interacting tissue culture cell lines.
Further investigations show that the M. tuberculosis type IV pili are encoded by a novel 5-kb genomic island
that contains the flp prepilin and putative biogenesis genes. The flp genomic island is characterized by an
increased G+C content of 70%, while the mean G+C content of the M. tuberculosis chromosome is 65%,
and this region is flanked by multiple direct repeats. The identification of type IV pili in M. tuberculosis and
the genetic characteristics of the locus strongly suggest this chromosomal region was horizontally acquired
from an ancestral microorganism.
Results and Discussion
It was shown by electron microscopy that the human pathogen M. tuberculosis possessed the capacity to
elaborate at least two physically distinct pili-types. The appearance of multiple pili morphotypes produced by
M. tuberculosis seemed to coincide with different growth conditions. This suggests that the expression of pili
by the bacillus may be influenced by nutritional or other environmental signals. When the attenuated M.
tuberculosis H37Ra was grown in broth culture it had a diminished capacity to produce pili as compared to
the virulent H37Rv and clinical isolate CDC1551 strains. Pili play a key role in the pathogenesis of many
bacterial pathogens and they are expressed on the bacterial surface. Thus pili represent a very attractive target
for new vaccine and innovative therapeutic developments for the treatment and prevention of TB.
It was shown that purified Mtp is comprised of protein subunits (pilin) encoded by the M. tuberculosis
H37Rv predicted ORF designated Rv3312A. This ORF is not organized in an operon or clustered with genes
that are associated with pili biogenesis. Thus, it is possible that Mtp biogenesis genes are distantly located
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2005 International Meeting of the Federation of Korean Microbiological Societies
away from Rv3312A on the M. tuberculosis chromosome. Recently, pili have been identified in Gram-positive
Group B Streptococcus and C. diphtheriae. These pili are associated with sortase genes, implicating that they
are assembled by covalent linkage to the cell wall peptidoglycan. Interestingly, M. tuberculosis lacks any
identifiable sortase-encoding genes, and therefore it seems unlikely that Mtp are assembled in a
sortase-dependant manner and may be assembled utilizing a unique process.
Type IV pili represent a large group of bacterial adhesins produced by many bacterial pathogens. It was
found that M. tuberculosis harbours a type IVB pili locus within its chromosome. The prepilin encoded by
this locus represents a member of the Flp pili family. This low molecular weight molecule contains absolutely
conserved amino acid residues found in all type IVB pili members. Type IV pili are thought to be exclusively
produced by Gram-negative bacteria, so the question arises as to how M. tuberculosis came to possess a type
IVB pili locus. The genetic analysis presented here demonstrates that it is possible that M. tuberculosis
acquired these genes in a horizontal gene transfer event. The flp locus in M. tuberculosis has a G+C content
of 70%, significantly higher than the mean chromosomal G+C content of the chromosome (65%). It is
possible that the events leading to the acquisition of these genes by M. tuberculosis occurred some time ago.
As the bacillus evolved, the synteny with these Gram-negative flp genes was lost. The presence of multiple
direct repeats flanking the flp operon in M. tuberculosis is further evidence of the insertion of foreign DNA
into its chromosome and is consistent with the hypothesis that M. tuberculosis encodes type IV pili on a
genomic island.
The presence of Flp pili are required for the ability of the periodontal pathogen A. actinomycetmcomitans
to adhere to surfaces, a key feature of its pathogenic capability. Thus, Flp could play a similar role for M.
tuberculosis attachment to host cells. The ability of M. tuberculosis to survive within phagocytic cells is well
documented and represents the central paradigm of its pathogenesis. However, relatively little is known about
the mechanisms responsible for the actual adherence and invasion of the macrophage. Type IV pili have been
shown to play an important role for the human respiratory pathogen Legionella pneumophila capacity to
become internalized by macrophages. Like M. tuberculosis, L. pneumophila is a facultative intracellular
pathogen that survives and replicates within phagocytes during human infection. For L. pneumophila, type IV
pili were demonstrated to be required for the efficient invasion of macrophages. These studies suggest that
M. tuberculosis type IV pili may also play an important role in the bacteria’s ability to parasitize phagocytic
cells.
In summary, this is the first report that M. tuberculosis produces pili, and thus represents a significant
advance in the understanding of the basic physiology of the bacteria, and has important implications regarding
its pathogenesis. It is very likely that pili play an important role in some aspect of human TB infection. It
is well documented that M. tuberculosis adheres to and invades macrophages and epithelial cells, however
the bacterial factors involved in these processes are largely unknown. From the knowledge of pili functions
in other bacterial pathogens and the initial studies presented here, it is plausible that M. tuberculosis utilizes
pili to facilitate colonization of the human host. Future studies with Mtp and Flp pili-deficient mutants in
M. tuberculosis in various cell culture and animal models will ultimately determine their role in M.
tuberculosis pathogenesis.
Keywords: Mycobacterium tuberculosis, Mtp, pili, adherence, tuberculosis, immunofluorescence
국제학술대회
41
October 13-14, 2005, Seoul, Korea
S2-2
Genome Analysis of Mycobacterium tuberculosis Strain K
Taeksun Song1,*, Hong-Seok Park2, Hye-Eun Bang1, Sang-Haeng Choi2, Yong-Seok Lee2,
1
3
3
1,4
Hyeyoung Lee , Young-Gil Park , Gil-Han Bai , and Sang-Nae Cho
1
The Genome Research Center for Respiratory Pathogen and 4Department of Microbiology, Yonsei
University College of Medicine, Seoul; 2Korea Research Institute of Bioscience and Biotechnology, Taejeon;
3
and The Korea Institute of Tuberculosis, Seoul, Korea
Despite the availability of several drugs and the BCG vaccine, tuberculosis remains one of the most deadly
infectious diseases claiming approximately 2 million lives a year worldwide. A recent survey in Korea
indicated that there are about 40,000 new cases and more than 3,000 people are killed by the disease each
year, placing tuberculosis among the 10 leading causes of death in Korea. M. tuberculosis strain K has been
noted as a single strain with a particular restriction fragment length polymorphism (RFLP) profile in an
investigation of outbreaks among high school students, and later found to be most prevalent among clinical
isolates in Korea. The K-family consisting of strain K and K-related strains is the largest cluster comprising
18.4% of the isolates, implying that strains in K-familyare more virulent or transmissible than others. To
elucidate the genetic basis of virulent phenotype of the strains, whole genome of a representative strain, M.
tuberculosis strain K is sequenced and analyzed. A whole-genome shotgun sequencing and assembly of the
sequences combined with sequences of clones of a fosmid library followed by gap-filling between adjacent
contigs resulted in a single continuous genome of 3,879,571 bp with a G + C content of 65.1%. Among 4,089
open reading frames identified 2,528 genes were functionally annotated, which is much higher portion
compared to the annotated genomes of other M. tuberculosis strains. Global comparison of the genomic
sequence with the known sequences of M. tuberculosis H37Rv and CDC1551, a laboratory strain and a recent
clinical isolate respectively, showed no major genomic rearragement among them. Detailed analysisof the
sequences revealed the presence of 125 insertions and 153 deletions that are larger than 10 bp in the genome
of M. tuberculosis K relative to M. tuberculosis H37Rv affecting 62 and 75 open reading frames that code
for mostly PE-PPE and PE-PGRS proteins, hypothetical proteins with unknown function and transposases.
Interestingly the whole sequence of a prophage was found to be absent in M. tuberculosisK genome. A global
network of proteins was constructed based on the predicted function of them to provide clues for any
biological activity that unique to M. tuberculosis K. Thorough analysis of the genomic sequences, together
with characterization of virulence phenotype of the bacterium would provide further insight into the
pathogenesis of this particular strain, which may enable development of better vaccines and therapeutics
against tuberculosis.
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2005 International Meeting of the Federation of Korean Microbiological Societies
S2-3
Diacyltrehalose of Mycobacterium tuberculosis Inhibits LPS- and
Mycobacteria-Induced Proinflammatory Cytokine Production in
Human Monocytic Cells
Hwa-Jung Kim*, Chang-Hwa Song, Eun-Kyeong Jo, and Jeong-Kyu Park
Department of Microbiology, College of Medicine, Chungnam National University, Taejeon 301-131, Korea
M. tuberculosisis one of the most successful pathogens, as indicated by its ability to infect one-third of
the world's population and cause about three million human death per year.major challenge in the study of
the host-pathogen interaction in tuberculosis is to define the mechanisms used by M. tuberculosis to avoid
eradication by the immune response. Part of the survival strategy of M. tuberculosis may depend on bacilli
gaining entry into the host macrophage without evoking a strong antimicrobial response.
Macrophages play a central role in the first line of defense against mycobacterial infections through the production
of proinflammatory cytokines that are involved in the activation of both innate and acquired immune response.
Among the proinflammatory cytokines, IL-12 provides an important bridge between both the immune responses by
stimulating the development of type 1 T helper cells, which are critical in the eradication of intracellular pathogens.
Factors affecting the pathogenesis of tuberculosis are complex and poorly defined. The mycobacterial cell
envelope has long been thought to be related to both the pathogenicity of these bacteria and their resistance to
the hostile environments in the host. One of the unique features of mycobacterial cell wall is presence of the
lipids esterified with multiple methyl-branched fatty acids such as sulfolipid (SL), phthiocerol dimycocerosate
(DIM), di- and tri-acyltrehalose (DAT and TAT), and polyacyltrehalose (PAT). The occurrence of a large number
of such lipids in pathogenic mycobacteria has raised the possibility that they may play a significant role in
pathogenesis. Among these lipids, DIM has been implicated as a virulence factor because mutants that lack this
compound were attenuated in human monocytes and in the murine lung. Numerous biological effects of SL on
phagocytic cells have been reported. These lipids are located at or near the surface of M. tuberculosis where their
fatty acid chains have been proposed to noncovalently interact with mycolic acids of the cell wall core.
These lipids were separated on thin layer chromatography (TLC) and purified directly from TLC plate. We
examined the possible biological activities of the isolated lipids by their ability toproinflammatory cytokines
in human monocytes and THP-1 cells. None of the lipids tested could induce a significant IL-12p40 or TNF-
productions in human peripheral blood mononuclear cells. Interestingly, DAT showed a significant inhibition
of LPS induced IL-12p40 and TNF- productions by pretreatment for 30 min in human monocytes and
PMA-treated THP-1 cells while other lipids had no effect. DAT inhibited LPS-induced IL-12p40 and TNF-
productions and mRNA expression in a dose-dependent manner. However, DAT was unable to inhibit
peptidoglycan-induced IL-12p40 production in PMA-treated THP-1 cells. DAT also significantly inhibited M.
tuberculosis-induced IL-12p40, TNF- , and IL-6 productions by pretreatment for 30 min and these inhibitory
average effects were more prominent when were pretreated with DAT for 20 hrs. And also preincubation of
THP-1 cells with DAT for 20 hrs suppressed M. tuberculosis-induced IL-12p40 production in a dosedependent manner. But SL and DIM failed to inhibit M. tuberculosis induced cytokines production in
PMA-treated THP-1 cells. These results suggest that DAT is one of the many mycobacterial factors which
modulate host's immune response.
Keywords: Mycobacterium tuberculosis, Cell wall lipids, Diacyltrehalose, Proinflammatory cytokines
국제학술대회
43
October 13-14, 2005, Seoul, Korea
S2-4
Characterization of Helicobacter pylori Antigens Responsible for
Inflammation and Apoptosis in the Gastric Epithelial Cell Line
Seung-Chul Baik*, Kyung-Mi Kim, Hyung-Lyun Kang, Myung-Je Cho, and
Kwang-Ho Rhee
Department of Microbiology Gyeongsang National University College of Medicine, 92 Chilam-dong, Jinju,
Gyeong-Nam 660-280, Korea
Helicobacter pylori, a gram-negative, microaerophilic, spiral bacterium, is a human-specific gastric pathogen
that colonizes the stomachs of approximately one-half the world's population. H. pylori infection causes
accumulation of the inflammatory cells in the lamina propria of gastric mucosa, leading to development of
the active and chronic inflammation. Chronic inflammatory cells including plasma cells are prominent and
polymorphonuclear leucocytes are frequently found in the lamina propria. H. pylori has been generally known
to be noninvasive pathogens. The eradication of this infection is considered the best way to relieve mankind
of most gastric disease. Despite the clinical significance and prevalence of H. pylori, relatively little is known
about the bacterial components that induce apoptosis, and promote an inflammatory response within its host.
Chronic infection with H. pylori might result in continual stimulation of proinflammatory cytokines such as
TNF-alpha, IL-1 beta, and IL-8.
In our study, we characterized pathophysiological functions of H. pylori 26 kDa. A protein of 26 kDa
antigen which was present in large quantities in extracts of cells of H. pylori, has been reported to be a
species-specific antigen. An ORF fragment of H. pylori 26 kDa antigen gene has been cloned into the
high-expression plasmid pET15b and overexpressed in the E. coli BL21. The antigen expressed in the E. coli
was purified by the nickel-bound column. Our results demonstrate that H. pylori 26 kDa antigen is an alkyl
hydroperoxide reductase which is critical elements of the antioxidant defense system against to oxygen
radicals. Taken together, these results demonstrated that H. pylori 26 kDa antigen has physiological functions
for defense against acid and oxygen attacks in the environment as well as a pathological role by IL-8
induction in the host.
There are many reports that H. pylori infection interferes with the equilibrium between proliferation and
apoptosis of the gastric epithelium. Most of the in situstudies have shown that the number of apoptotic
epithelial cells increases during H. pylori infection. However, H. pylori-induced apoptosis has been studied
to explore the mechanism of H. pylori-mediated pathogenesis, the apoptosis-inducing factor of H. pylori is
exactly unknown. To evaluate the role ofthe apoptosis-inducing factor of H. pylori, we purifedthe recombinant
antigens including gamma-glutamyl transpeptidase (GGT)and alcohol dehydrogenase (ADH).
The aims of this study were to clarify the apoptotic signal pathway of GGT and ADH.Using the DNA
recombination techniques, GGT gene was cloned into pET17b and transformed into E. coli. The recombinant
γ-glutamyl transpeptidase was purified by a nickel-affinity column and was digested by thrombin. ADH was
isolated by anion exchange and affinity chromatography. The recombinant GGT and ADH induced apoptotic
activity in AGS cell, which was confirmed by the TUNEL staining test. AGS cells treated with the two factors
for 24 hours resulted in morphological changes and DNA fragmentation. The mechanism by which GGT and
ADH induces apoptosis in infected gastric mucosa has not yet been elucidated. To examine the signal pathway
of apoptosis leading to DNA fragmentation, we examined the activity of apoptosis-related proteins, such as
the Bcl-2 family proteins, caspase-3 and cytochrome c. In our results, the activities of caspase-3 following
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2005 International Meeting of the Federation of Korean Microbiological Societies
exposure to GGT or ADH increased in a dose-dependent manner and signal property represented upregulation
of proapoptotic Bax and a downregulation of antiapoptotic Bcl-2. Apoptotic signal also trigger changes in
mitochondria, which led to a release of cytochrome c. These results indicate that GGT and ADH genes of
H. pylori induceapoptosis mediated by the release of cytochrome c in AGS cells. Further studies are needed
to clarify the intracellular signaling machinery that controls GGT and ADH-induced apoptosis.
Keywords: H. pylori; inflammation, IL-8, apoptosis, gamma-glutamyl transpeptidase, alcohol dehydrogenase
국제학술대회
45
October 13-14, 2005, Seoul, Korea
S2-5
Use of rpoB Sequence Analysis for the Study on the
Pathogenesis of Helicobacter pylori Infection
Yoon-Hoh Kook
Department of Microbiology and Cancer Research Institute, Institute of Endemic Diseases, Seoul National
University College of Medicine, 28 Yongon-dong, Chongno-gu, Seoul 110-799, Korea
Helicobacter pylori infection typically leads to chronic inflammation of the gastric mucosa. H. pylori
carriers have a higher risk of gastro-duodenal diseases like gastric cancer which is particularly common in
Korea and Japan. However, H. pylori factors involved in gastric carcinogenesis are not well understood. The
presence of the cytotoxin-associated gene A (cagA) of H. pylori has been proposed to be an important risk
factor for the development of H. pylori-mediated gastric cancer. Recently, it was suggested that Src homology
2-containing tyrosine phosphatase (SHP-2) is an intracellular target of CagA protein and that the prevalent
CagA type in East Asian countries binds more strongly to SHP-2, and thus induces more cellular
morphological changes, than the CagA type prevalent in Western countries. Moreover, it has been suggested
that this difference may be correlated with the striking difference in the incidence of gastric cancer in these
two geographical areas. However, even though nearly 100% of Korean and Japanese isolates possess cagA
and express the East Asian type of CagA, relatively few infected individuals develop peptic ulcer or gastric
cancer. The reason for this remains unresolved. So, we hypothesized that there is an unique clonal H. pylori
strains, which are uniquely prone to cause chronic inflammation and metaplastic changes in the gastric
mucosa, in Asia and studied the population structure of H. pylori isolates from several countries by analyzing
rpoB sequences. Phylogenetic analysis based on amino acid sequences often provides more significant
information than analysis based on the nucleotide sequences of protein-coding genes. This allowed us to
analyze the H. pylori population by both nucleotide and amino acid sequence analyses which, however, have
not been performed in previous population studies with cagA, oipA, or other housekeeping genes An extremely
high level of allelic diversity among H. pylori strains was found. The rpoB sequences of Asian and non-Asian
strains were found to differ. An amino acid polymorphism (RpoBAla/Thr types) was found at the 497th residue
by deduced amino acid analysis. RpoBThr was uniquely present in East Asian countries, and two-thirds of the
H. pylori isolate population in this region was RpoBThr however, this type was rare or absent in Western
countries, where RpoBAla predominated. RpoBThr strains induced a much larger amount of IL-8, a chemokine
that plays an important role in chronic inflammation, than RpoBAla strains in cultured MKN45 cells.
Keywords: H. pylori, rpoB analysis, population study
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한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S3-1
Conserved Virulence Factors of Pseudomonas aeruginosa
Identified from Drosophila melanogaster-Based in vivo Screening
Shin-Young Park, Yun-Jeong Heo, Kelly B. Choi, and You-Hee Cho*
Department of Life Science, Sogang University, Seoul 121-742, Korea
The human opportunistic pathogen Pseudomonas aeruginosa is an interkingdom pathogen capable of
infecting diverse hosts that includes plants, insects, nematodes, fungi, and amoeba. Among those host models,
much attention has been paid to the fruit fly, Drosophila melanogaster, since its innate immune signaling
pathways exhibitstriking similarity to those in mammalian innate immunity, which involves Toll-like receptors
and NF-κB transcription factors. We have exploited the P. aeruginosa-induced killing of D. melanogaster as
an assay system to screen for virulence-attenuated mutants of P. aeruginosa strain PA14. Ten mutants were
isolated from a 2,000 random TnphoA insertion clones, and 8 of them (80%) displayed significantly reduced
virulence in murine peritonitis model. Semi-random PCR and direct cloning of the TnphoA insertion regions
from the mutant chromosomes revealed the mutation sites; two known genes (dsbA and wspF) together with
several genes such as PA2424 (pvdI), PA0253, PA0369, PA2077, PA2113, and PA2002 are identified as
virulence genes the other two insertionsare located at an intergenic region between PA1928 (rimJ) and PA1929
and at a coding region located within the 13th variable segment. Topics discussed will include the
demonstration that D. melanogaster can be used for an in vivo high throughput screen to identify novel
virulence factors and, presumably, pathogenicity islands and our recent findings on several isolated mutants
involved in biofilm-formation and biofilm-induced phenotypic variations.
Acknowledgments
This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program from
Ministry of Science and Technology to Y.-H. Cho (MG05-0104-05-0).
Selected readings
Apidianakis, Y., M.N. Mindrinos, W. Xiao, G.W. Lau, R.L. Baldini, R.W. Davis, and L.G. Rahme. 2005.
Profiling early infection responses: Pseudomonas aeruginosa eludes host defenses by suppressing
antimicrobial peptide gene expression. Proc. Natl. Acad. Sci. USA. 102: 2573-2578.
Boles, B.R., M. Thoendel, and P.K. Singh. 2004. Self-generated diversity produces "insurance effects" in
biofilm communities. Proc. Natl. Acad. Sci. USA. 101: 16630-16635.
Drenkard, E. and F.M. Ausubel. 2002. Pseudomonas biofilm formation and antibiotic resistance are linked
to phenotypic variation. Nature. 416: 740-743.
Lau, G.W., B.C. Goumnerov, C.L. Walendziewicz, J. Hewitson, W. Xiao, S. Mahajan-Miklos, R.G.
Tompkins, L.A. Perkins, and L.G. Rahme. 2003. The Drosophila melanogaster Toll pathway participates
in resistance to infection by the gram-negative human pathogen Pseudomonas aeruginosa. Infect.
Immun. 71: 4059-4066.
Lee, J.-S., S.-H. Kim, and Y.-H. Cho. 2004. Dithiothreitol attenuates the pathogenic interaction between
Pseudomonas aeruginosa and Drosophila melanogaster. J. Microbiol. Biotech. 14: 367-372.
Lee, J.-S., Y.-J. Heo, J.K. Lee, and Y.-H. Cho. 2005. KatA, the major catalase,is critical for osmoprotection
and virulence in Pseudomonas aeruginosa PA14. Infect. Immun. 73: 4399-4403.
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Rahme, L.G., Ausubel, F.M., Cao, H., Drenkard, E., Goumnerov, B.C., Lau, G.W., Mahajan-Miklos, S.,
Plotnikova, J., Tan, M.W., Tsongalis, J., Walendziewicz, C.L., and Tompkins, R.G. (2000) Plants and
animals share functionally common bacterial virulence factors. Proc. Natl. Acad. Sci. USA 97:
8815-8821.
Wolfgang, M.C., Kulasekara, B.R., Liang, X., Boyd, D., Wu, K., Yang, Q., Miyada, C.G., Lory, S. 2003.
Conservation of genome content and virulence determinants among clinical and environmental isolates
of Pseudomonas aeruginosa. Proc Natl Acad Sci USA. 100: 8484-8489
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S3-2
A Vibrio vulnficus LuxR Homolog, SmcR: Its Role in Virulence
Gene Regulation and a Consensus Sequence for Its Binding
Hye Sook Jeong2, Dong Hwan Lee1, and Sang Ho Choi1*
1
Department of Food Science and Technology, School of Agricultural Biotechnology, and Center for
2
Agricultural Biomaterials, Seoul National University, Seoul 151-742, Laboratory of Enteric Infections,
Department of Microbiology, National Institute of Health Korea, Seoul 122-701, Korea
The putative virulence factors of Vibrio vulnificus include an elastase, the gene product of vvpE. The
current authors previously demonstrated that vvpE expression is differentially directed by two different
promoters in a growth phase-dependent manner. The activity of the stationary-phase promoter, PS, is
dependent on RpoS and also under the positive control of CRP. In the present study, primer extension analyses
revealed that SmcR, the Vibrio harveyi LuxR homologue, is also involved in the regulation of vvpE
transcription by activating PS. Although the influence of CRP on PS is mediated by SmcR, the level of PS
activity observed when CRP and SmcR function together was found to be greater than the sum of the PS
activities achieved by eachactivator alone. Western blot analyses demonstrated that the cellular levels of RpoS,
CRP, and SmcR were not significantly affected by each other, indicating that CRP and SmcR function
cooperatively to activate the PS rather than sequentially in a regulatory cascade. The binding sites for CRP
and SmcR were mapped based on a deletion analysis of the vvpE promoter region and confirmed by in vitro
DNase I protection assays. The binding sites for CRP and SmcR were juxtapositioned and centered 220-bp
and 198-bpupstream of the transcription start site of PS, respectively. Accordingly, the present results revealed
that CRP and SmcR coactivate the expression of vvpE synergistically with the RpoS-dependent promoter PS,
and that the activators exert their effect by directly binding to the promoter in the stationary phase.
Keywords: Vibrio vulnificus, virulence gene regulation, SmcR
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October 13-14, 2005, Seoul, Korea
S3-3
Regulation of Biofilm Formation by NtrC in Vibrio vulnificus
Chang Beom Kim1, Soon-Jung Park2, and Kyu-Ho Lee1,*
1
Department Environmental Science Hankuk University Foreign Studies, Yongin 449-791,
Department Parasitology Yonsei University, College of Medicine, Seoul 133-791, Korea
2
Numerous bacterial species showed capabilities to form surface-associated multicellular structures called
biofilms. The biofilm formation is composed of distinct developmental stages, which include an
attachment/adhesion of a single cell, a proliferation toward monolayered coverage, a propagation to aggregated
microcolony, a maturation to three-dimensional structure, and subsequently a local degradation (Costerton et
al., 1999). The mature biofilm is equipped with extracellular polymeric matrix and internal water-filled
channels (Stoodley et al., 2002). This complex architecture can be achieved by differential expression of
several hundred genes, among which the most studied are the genes coding for exopolysaccharide
biosynthesizing/exporting proteins (Friedman and Kolter, 2004) and quorum-sensing components (Zhu and
Mekalanos, 2003).
The causative agent of septicemia, Vibrio vulnificus has been considered an important pathogen in humans
due to its rapid pathogenic progresses and its high mortality rates (Hollis et al., 1976). This pathogenhas been
shown to exhibit an ability to form a biofilm (Joseph and Wright. 2004). In the present study, we observed
the processes for biofilm formation using the light and confocal microscopies. V. vulnificus showed distinct
stages during biofilm formation, as shown in many bacterial species. The effects of growth conditions and
genetic backgrounds on biofilm formation were investigated. Among the various parameters given to growth
condition, the carbohydrate added as a sole carbon source showed great effect. The modulation of biofilm
formation in the presence of specific carbon sources was resulted from the altered production of
exopolysaccharide (EPS), when determined by SDS-PAGE analysis of EPS extracts.
Screening of about 10,000 transposon-mutants revealed that the strains showing decreased capability of
biofilm formation were mutated at the genes coding for transcriptional regulators. An NtrC-homolog is one
of the regulators obtained in this screen, which is a well-known response regulator, and thus we examined
whether this NtrC-homologplayed a role in biofilm formation by regulating the expression of several genes
involved in biofilm formation. Since the ntrC mutant showed decreased production of EPS, the genes encoding
enzymes involved in EPS biosynthesis were screened by in silico analysis. Most of all, the promoter regions
of the EPS genes were used for constructing transcriptional fusions. Assays using these fusions demonstrated
that the expression of each promoter was positively regulated by the functional NtrC-homolog. Each EPS gene
was also knockout by deletion mutagenesis, and these null mutants showed decreased abilities in both
production of EPS and formation of biofilm. These results indicate that NtrC is involved in the critical step
in biofilm development of V. vulnificus by regulating EPS biosynthesis.
Keywords: Biofilm, NtrC, Vibrio vulnificus
References
Costerton, J. W., P. S. Stewart, and E. P. Greenberg. 1999. Bacterial biofilms: A common cause of
50
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2005 International Meeting of the Federation of Korean Microbiological Societies
persistent infections. Science 284:1318-1322.
Friedman, L., and R. Kolter. 2004. Two genetic loci produce distinct carbohydrate-rich structural
components of the Pseudomonas aeruginosa biofilm matrix. J. Bacteriol. 186:4457-4465.
Hollis, D. G., R. E. Weaver, C. N. Baker, and C. Thornsberry. 1976. Halophilic Vibrio species isolated
from blood cultures. J. Clin. Microbiol. 3:425-431.
Joseph, L. A., and A. C. Wright. 2004. Expression of Vibrio vulnificus capsular polysaccharide inhibits
biofilm formation. J. Bacteriol. 186:889-893.
Stoodley, P., K. Sauer, D. G. Davies, and J. W. Costerton. 2002.Biofilms as complex differentiated
communities. Annu. Rev. Microbiol. 56:187-209.
Zhu, J., and J. J Mekalanos. 2003. Quorum sensing-dependent biofilms enhance colonization in Vibrio
cholerae. Dev. Cell. 5:647-656.
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S3-4
Comparative Genomic Analysis of a Gene Cluster for Capsular
Polysaccharide Synthesis in Vibrio vulnificus
Lien-I Hor1,2,*, Chung-Ping Shao1, Jui-Feng Wang1, Shih-Feng Tsai3,4
1
Department of Microbiology and Immunology, 2Institute of Basic Medical Sciences, College of Medicine,
3
National Cheng-Kung University, Tainan, Taiwan, R.O.C. Division of Genomic Medicine, National Health
Research Institutes, Taipei, Taiwan, Republic of China, 4Institute of Genetics, National Yang-Ming
University, Taipei, Taiwan, R.O.C.
Vibrio vulnificusis a gram-negative marine bacterium that causes wound infection and fulminant septicemia
in people with underlying diseases. The capsular polysaccharideand iron acquisition ability have been shown
to be important for virulence. A few other potential virulence factors have also been proposed however, the
pathogenesis of this organism remains unclear. The whole genome sequences of two V.vulnificus strains,
CMCP6 and YJ016, have been determined in 2003. Large-scale screening,such as comparative genomics and
transcriptome analysis, of this organism for novel virulence factors isnow possible for interested researchers.
In a pilot study, comparative genome hybridization and gene expression profile analysis with a small scale
DNA microarray were conducted. Forty-one open reading frames (ORFs)involved in the synthesis of capsular
polysaccharides or iron acquisition were chosen as the probes. Two different kinds of probes were prepared
one is the PCR products amplified from the selected overlapping clones in the shotgun library used for
determining the genome sequence. The other is PCR products amplified from each ORF with the ORF-specific
primers designed according to the genome sequence. In one experiment we compared the gene expression
profiles of strain YJ016 and its isogenic ∆fur mutant with this DNA microarray. As expected, the expression
levels of 12 ORFs involved in iron-acquisition, including the well-known Fur targets, hupA and viuA, in V.
vulnificus were shown to be approximately 3- to 10-fold higher in the ∆fur mutant. In the other experiment
we compared the gene difference in these ORFs between the encapsulated strain, YJ016, and three acapsular
strains, CG061, CG076 and CG099. We found that ORF VV0361, which was predicted to encode the
UDP-galactose phosphate transferase, was absent in all the three acapsular strains. From this study, we
concluded that satisfactory results can be obtained with the DNA microarray system for analyzing the genomic
content and gene expression in V. vulnificus. Nevertheless, compared to the probes derived from shotgun
library, the ORF-specific probes although cost much more money, are less time- and labor-consuming, and
are suitable for both comparative genome hybridization and gene expression profile analysis. We further
detected the ORFs in VV0337-0366, a region with genes involved in capsular polysaccharide and colanic acid
synthesis, in 9 encapsulated and 9 acapsular environmental V. vulnificus strains by Southern hybridization with
ORF-specific probes. Only three ORFs, wza, wzb, and wzc shown to be required for surface assembly of
capsular antigen in other bacteria, were present in all the encapsulated strains but absent from four acapsular
strains. The other ORFs were not detected in most of the strains tested, no matter they were encapsulated
or not. Introduction of an in-frame deletion in an ORF predicted to encode a nucleoside-diphosphate sugar
epimerase in strain YJ016 resulted in translucent colonies and loss of resistance to human serum killing effect.
This gene although appeared to be essential in the formation of capsule in strain YJ016, it was absent from
6 of the 9 capsulated strains tested. Our data suggest that V. vulnificus strains may use the same genes for
the surface assembly of capsule, but may synthesize the capsular polysaccharides via different pathways. Our
finding is consistent with the heterogeneity in antigenicity and carbohydrate compositions of the capsule
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2005 International Meeting of the Federation of Korean Microbiological Societies
ofvarious V. vulnificus strains demonstrated by other laboratories previously.
Keywords: Vibrio vulnificus, comparative genome hybridization, gene expression profile analysis, DNA
microarray, capsular polysaccharide synthesis
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October 13-14, 2005, Seoul, Korea
S3-5
Regulation of Biofilm Formation and Surface Interactions of the
Plant Pathogen Agrobacterium tumefaciens
Clay Fuqua*, Thomas Danhorn, Amelia D. Tomlinson, and Pei-Wei Li.
Department of Biology, Indiana University, USA
Agrobacterium tumefaciens, a rod-shaped representative of the alpha-Proteobacteria, is the causative agent
of the ubiquitous plant disease known as crown gall. Crown gall results from the transfer of a large segment
of DNA from the infecting bacterium into host plant tissues, followed by integration and expression of this
DNA within the plant genome (2). The infection process is one of the few examples of cross-kingdom gene
transfer. Virulent and avirulent derivatives of A. tumefaciens are abundant in many soils, existing as
saprophytes adhered to soil particulate matterand associated with plant tissue. A growing number of plant
pathogens are recognized to form biofilms as a component of the disease process (4). We hypothesize that
the ability of A. tumefaciens to form biofilms on both biotic and abiotic surfaces in the environment is
important for the persistence of the pathogen and the infection of plants.
A. tumefaciens forms architecturally complex biofilms on host tissues, as well as model inert surfaces. A.
tumefaciens biofilms on model surfaces are characterized by densely packed, but relatively shallow layers of
cells along the surface, punctuated by larger, globular aggregates of 20-30 cells in depth. Early stages of
biofilm formation exhibit a large proportion of cells attached to surfaces via their poles. As the biofilm grows,
more complex cellular arrangements emerge. On plant surfaces, the adherent biomass is somewhat
heterogeneous, but quite substantial and notably similar to inert surface biofilms. We have identified three
regulatory pathways that exert considerable influence on biofilm formation by A. tumefaciens. A putative
repressor called ExoR, is known to control synthesis of the A. tumefaciens exopolysaccharide succinoglycan
(SCG) and is also required for efficient adherence to surfaces (Tomlinson et al, manuscript in preparation).
Another important control circuit is the PhoB-PhoR two-component system, facilitating adaptation to limiting
phosphorous. Under limiting phosphorous PhoB-PhoR stimulates biofilm formation and modifies the eventual
structure of the mature biofilm (1).
Additionally, a regulatory cascade has been identified that is comprised of dual FNR-type transcription
factors that integrate adaptation to oxygen-limitation with the maturation of A. tumefaciens biofilms (5). FnrN
is similar to FNR from Escherichia coli and is likely to ligate an oxygen-labile 4Fe-4S cluster, via four
conserved cysteine residues in the amino-terminal region of the protein (3). Under limiting oxygen, FnrN
activates expression of a large number of A. tumefaciens genes. Among these regulatory targets, FnrN
activates expression of the gene encoding SinR (surface interaction regulator), a second FNR-type protein, but
in this case a member of the DNR subfamily. SinR also contains 4 amino-terminal cysteine residues, but in
non-conserved positions. Mutation of these cysteines to generate serine residues does not alter the activity of
the protein. SinR is required for normal biofilm maturation. Conversely, artificial elevation of sinR expression
results in far denser biofilms than ever observed for the wild type. The increased biomass of the pathogen
associated with plant tissue in strains with high levels of sinR expression can enhance infectivity, thereby
linking biofilm formation and virulence.
The three regulatory pathways thus far identified to influence A. tumefaciens biofilms are notably distinct
from one another. Mutation and limiting expression of these regulators results in strikingly different surface
interaction deficiencies. The ExoR and PhoB-PhoR regulators are required for early surface interactions, while
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2005 International Meeting of the Federation of Korean Microbiological Societies
SinR influences later stages of biofilm formation. The analysis of these regulators and the target functions
under their control, provides a glimpse of the physiological complexity of A. tumefaciens biofilms, and the
different environmental factors that influence the ultimate configuration and activity of adherent populations.
References cited:
1. Danhorn T, Hentzer M, Givskov M, Parsek M, Fuqua C. 2004. Phosphorous limitation enhances biofilm
formation of the plant pathogen Agrobacterium tumefaciens through the PhoR-PhoB regulatory system.
Journal of Bacteriology 186: 4492-501
2. Escobar MA, Dandekar AM. 2003. Agrobacterium tumefaciens as an agent of disease. Trends Plant. Sci.
8: 380-6
3. Korner H, Sofia HJ, Zumft WG. 2003. Phylogeny of the bacterial superfamily of Crp-Fnr transcription
regulators: exploiting the metabolic spectrum by controlling alternative gene programs. FEMS Microbiol
Rev 27: 559-92
4. Ramey BE, Koutsoudis M, von Bodman SB, Fuqua C. 2004. Biofilm formation in plant-microbe
associations. Current Opinion in Microbiology 7: 602-9
5. Ramey BE, Matthysse AG, Fuqua C. 2004. The FNR-type transcriptional regulator SinR controls
maturation of Agrobacterium tumefaciens biofilms. Molecular Microbiology 52: 1495-511
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October 13-14, 2005, Seoul, Korea
S4-1
Key Taxa of Biodiversity of the Macro- & Higher Fungi in China
Pei-Gui Liu
Kunming Institute of Botany, Chinese Academy of Sciences, China
China is one of the richest countries and areas of the species biodiversity of macro- and higher fungi in
the world. Different species existed in the nature show different situation, with different affection on the
ecological environment and with quite different role in the ecological system, with different signification to
the scientific research as well as different importance to a human being lives. Among of all of them, function
and effect of key taxa are of quite greater importance than others. Therefore it is great necessary and urgency
for mycologists to select and determinate the key taxa among the macro- and higher fungi recorded and
described up to now from China. Based upon many field investigations and analysis and check of reference
material as well as literature of the higher fungi, 433 species have been selected and primary determined as
the key taxa of species diversity from the macro- and higher fungi. They are divided into three grades, namely
the first grade is critically endangered species, the second is significant worthy species to scientific research
and the third is important economic taxa.
Keywords: macro-and higher fungi, endangered taxa, taxa with worth in scientific research, important
economic taxa
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S4-2
Taxonomy and Geographical Distribution
of the Laboulbeniales in Korea
Young-Hee Na
Division of Science Education, College of Education, Chosun University, GwangJu 501-759, Korea
In 1994, 46 species under 14 genera of the Laboulbeniales are known in Korean numbers of the
Laboulbeniales. Latter that time, 3 unrecorded genera, 2 new species and 20 species newly collected in korea.
As a result of this study, there are 68 species under 17 genera in Korea.
Two new species are Laboulbenia mudeungensis Y. H. Na et Y. B. Lee on Bembidion lissonotum Bates,
Laboulbenia nogodanicus Y. H. Na et Y. B. Lee on Agonum bucanani Hope and Agonum sp.. Laboulbenia
mudeungensis Y. H. Na et Y. B. Lee collected from Mt. Mudeung, Gwangju city and Muan, Jeonnam
Province. Laboulbenia nogodanicus Y. H. Na et Y. B. Lee collected from Nogodan, Mt. Jiri, Mt. Duryeun,
Jeonnam Province and Mt. Deckyou, Jeonbuk Province.
Herpomyces stylopygae on Blatta orientalis, Filariomyces forficulae on Labidura japonica, Laboulbenia
anoplogenii on Anoplogenius cyanescens, Platynus daimio and Stenolophus quinquepustulatus, L. benjaminii
on Stenolophus difficilis, L. borealis on Gyrinus japonicus, L. filifera on Harpalus sp., L. gebleri on
Haplochlaenius constiger, L. habui on Chlaenius variicornis, L. hastiana on Bembidion lissonotum, L. humilis
on Chlaenius naeviger, L. pallida on Anisodactylus signatus, L. pedicellata on Bembidion thermarum and B.
morawitzi, L. philonthi on Philonthus wuesthoffi, L. yamadae on Chlaenius variicornis, Misgomyces dyschirii
on Dyschirius ovicollis, Chitonomyces iriomotensis on Laccophilus lewisius, C. orientalis on Laccophilus
lewisius, C. zonatus on Laccophilus lewisius, Rickia pallodina on Pallodes umbratilis, Dimeromyces anisolabis
on Anisolabis maritima were newly collected in Korea.
Three unrecorded genera were Dimeromyces, Filariomyces, Misgomyces.
In 1994, 49 species under 31 genera of insects are known as the hosts of Korean numbers of the
Laboulbeniales. Latter that time, 22 species under 11 genera are add to host insects. As a result of this study,
there are 71 species under 42 genera in Korea.
33 species of Laboulbenia which were a parasite on the host of 42 species under 22 genera in total take
the dominant of them. They are Acupalpus inoratus, Agonum buchanani, A. sp., Anisodactylus punctatipennis,
A. signatus, A. tricuspidatus, Anoplogenius cyanescens, Bembidion chloreum, B. lissonotum, B. morawitzi, B.
oxyglimma, B. scopulinum, B. thermarum, Brachinus stenoderus, Chlaenius inops, C. naeviger, C. protenus,
C. variicornis, Colpodes buchani, Gyrinus japonicus, Haplochlaenius costiger, Harpalus roninus, H. sinicus,
H. sp., H. sp.(1), (2), Nebria ocohotica, Paederus fuscipes, P. parallelus, Panagaeus japonicus, Perigona
nigriceps, Philonthus longicoris, P. wuesthoffi, Platynus daimio, Pterostichus audax, P. microcephalus, P.
subovatus, Stenolophus difficilis, S. quinquepustulatus, Tachys fuscicauda, T. gradatus, T. laetifica and
Trichotichnus congruus.
In a view of distribution, 33 species of Laboulbenia were widely distributed and collected from 30 to totally
45 survey areas. 6 species of Chitonomyces were distributed and collected from 10 areas of them, 7 species
of Peyritschiella were distributed and collected from 9 areas of them and 6 species of Rickia were distributed
and collected from 8 areas of them. The other genera of Laboulbeniales were distributed and collected from
14 areas of them.
The most dominant collecting areas was Gwangju city (24 species), next one was Upo swamp (15 species),
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October 13-14, 2005, Seoul, Korea
and Mt. halla (11 species).
Keywords: Laboulbeniales, Laboulbenia mudeungensis, L. nogodanicus, Dimeromyces, Filariomyces, Misgomyces,
Korea
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S4-3
Endophytic Fungi Associated with Quercus liaotungensis
Sun Xiang and Guo Liang-Dong*
Systematic Mycology and Lichenology, Institute of Microbiology, Chinese Academy of Sciences, Beijing
100080, China
The diversity of endophytic fungi associated with Quercus liaotungensis in China was studied in August,
2004. Thirty mature plants were chosen in the Dongling Mountain, Beijing Forest Ecosystem Research Station
of the Chinese Academy of Sciences, 117 km west of Beijing (39°58'N, 115°26'E). One branch with leaves
was cut from each tree. The leaves were divided into blade and midrib, and were cut into discs with 5mm
diam. The twigs were divided into 3-age classes, i.e. 1-year-old, 2-year-old and 3-year-old segments, and were
cut into 5mm long. The surface sterilization by dipping in ethanol and sodium hypochlorite were processed
subsequently. The sterile segments (discs) were then evenly placed in each 90 mm Petri dish containing malt
extract agar supplemented with streptomycin sulphate to suppress bacterial growth, for 2 months at 25℃. A
total of 1244 isolates of endophytic fungi were obtained from 1240 tissue segments/discs, of which 1125
isolates sporulated and were identified into 20 fungal taxon based on morphological characteristics.
Fusicoccum sp. was dominant, and Alternaria alternata, Microsphaeropsis spp., Phoma spp., and Phomopsis
sp. was isolated frequently in this study. The overall colonization rate of endophytic fungi recovered was
64.1%, and the overall isolation rate was 1. The colonization and isolation rates were significantly increased
with the aging in twig tissues. The colonization and isolation rates were significantly higher in midrib than
in blade tissues. The highest endophyte species diversity occurred in blade tissues, and there was a similar
species diversity of endophytic fungi in twigs regardless of tissue ages.
Keywords: ecology, endophyte, diversity, Quercus liaotungensis
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October 13-14, 2005, Seoul, Korea
S4-4
Diversity and Pigment Biology of Fungi Causing Cosmetic
Damage on Wood
Seong Hwan Kim
Department of Microbiology, Dankook University, Korea
Physiologically weakened trees, freshly felled wood or sawn lumber, and even timber which is in service
but has been rewetted, are susceptible to attack by fungi which can cause bluestain. Bluestain fungi are
pioneer organisms which metabolize the simple carbohydrate compounds found in the tracheids and ray
parenchyma cells of the wood. The effect of the stain is dueto the production of melanin granules within their
hyphal cell walls. Although these fungi do not cause any structural degradation, their presence still results
in a devaluation of the timber. The increasingly stringent requirements of importing countries because of
quarantine issues and the demand by consumers for clean wood, in an age of great pressure to reduce
chemical use, has renewed interest in the organisms involved in sapstain. However, there has been limited
data set about the pigment biology of this group of fungi. To generate information on these organisms,
researches have been performed on their diversity and pigmentation biology.
Diversity of Ophiostoma fungi from conifers
To determine which fungi cause stain problem, a detailed survey was conducted at seven selected sawmills
across Canada from 1997 to 2001. From five important conifer species including Abies balsamea, Picea
mariana, Picea glauca, and Pinus contorta, over 2000 fungal isolates were isolated and identified based on
morphological and physiological properties and mating compatibility. Five genera and 13 species were found.
Ophiostom was the most commonly encountered genus (97%). A more diverse range of fungi was found in
logs than lumber. From the survey Ophiostoma setosum was identified as a new species. O. piceae was most
dominant species and could be differentiated from sibling species, O. quercus by PCR.
Pigment biology
C.coerulescens infected wood was considerably more pigmented than the Leptographium spp. stain area.
Leptographium isolates typically showed a non-pigmented, dead host cell zone approximately two to five cm
ahead of the stained area within the logs. The kill zone of Leptographium was not substantially darker than
the non-infected control wood. Mannose consistently yielded the densest growth and the dark color in all the
tested fungi. Leptographium and O. piliferum had identical color scoring on all the carbon sources. O. piceae
had a very similar coloring except for glucose and linoleic acid. These results suggest that carbon source is
one of factors affecting pigmentation in sapstaining fungi.
To explore the property of pigmentation in sapstain fungi, we cloned and confirmed that scytalone
dehydratases (SD), THN reductase (THNR), and pentaketide synthase (PKS) genes from O. floccosum are
melanin genes based on their high sequence similarity with other fungal SD or THNR genes and their ability
to restore melanin production in SD- or THN- deficient mutants of Colletotrichum or Magnaporthe fungi,
respectively. This work demonstrates sapstain fungi use dihydroxynaphtalene (DHN) melanin pathway for their
pigment production. To apply the use of melanin information on the control of problematic sapstaining fungi,
a potential biocontrol agent CartapipTM, an O. piliferum albino strainhas been tested. In a field trial in a
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2005 International Meeting of the Federation of Korean Microbiological Societies
sawmill in Alberta, Canada, the treatment of albino strain protected lodgepole pine logs from sapstain.With
the development of molecular probes, the presence of the albino strain could be monitored in fields.
Keywords: bluestain, pigment biology
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October 13-14, 2005, Seoul, Korea
S4-6
The Sequences of Small Subunit rDNA of Physarum didermoides
and Its Evolutionary Significance
LIU Shu-Yan* and LI Yu†
College of Agronomy, Jilin Agricultural University, Changchun130118, Jilin Province, China
Myxomycetes are an important and special group of eukaryotes. Relationships among members of the
myxomycetes, as well as the other groups of slime molds (dictyostelids and protostelids), are not well defined,
and morphological and biochemical data have not provided conclusive evidence tosupport one phylogenetic
tree (Rusk et al. 1995). Based on small-subunit rDNA (SSU rDNA) sequence phylogeny, myxomycetes are
included in the Protozoa (Cavalier-smith, 1993). However, only one myxomycetes species (Physarum
polycephalum Schwein.) was included in the analysis. Based on gene analysis of the elongation factor EF-1 ,
Baldauf and Doolittle (1999) concluded that the clade formed by Physarum, Dictyostelium and
Planoprotosteliumis the sister group of Animalia and Fungi. Furthermore, it was stressed that analyses based
on any single molecule appear to be inaccurate in reconstructing all higher order taxonomic relationships. In
general, literature on the origin and evolution of myxomycetes, based on molecular methods, is scarce. The
primary problem is the difficulty of isolating DNA from a single sporophore in sufficientquantity and quantity
to amplify and obtain sequences from the target regions. DNA extractions from myxomycetes have been done
from plasmodia in culture (Rusk et al 1995, Baldauf and Doolittle 1997). However, the plasmodium of many
species remains unknown, or the plasmodia do not grow well and form no sporophores, which makes it
impossible to establish the identity of the species. The objectives of this study were to amplify and sequence
the small subunit rDNA of Physarum didermoides and analysis the relationships of the higher taxa of
Myxomycetes.
The specimen of Physarum didermoides was collected in the field in 1998. DNA was extracted following
the methods of Liu and Li (2001). The small subunitrDNA was amplified by using primers of SMNUR101
®
and SMNUR108 (Rusk et al., 1995). The PCR product is ligated into pGEM -T Easy Vector and transferred
into Escherichia coli JM109 competent cells for sequencing. The nearly complete sequence of small subunit
rDNA of Physarum didermoides has been determined. The length is 1946bp. Similarity searches were
performed with the program BLAST in the GenBank databases. Sequences were aligned by hand using
MacLade. Then phylogenetic analysis were done using PAUP 4.0b8 (Swofford, 2001) together with the 29
sequences available from Genbank. The results showed that the Myxomycetes could be grouped into three
distinct clades ( see Fig1). The most basal group is trichiales. The second clad is Physarales. The third consist
of the Echinosteliales and Stemonitales. This result is different from Fiore_Donno's recommand on the
Echinosteliales. The further research is required to provide the powerful evidence about the relationships
among these groups.
Keywords: Physarum didermoides, Myxomycetes, Phylogeny, small subunit rDNA
※ supportedby National Natural Scientific Foundation(30300003)
corresponding author
†
62
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S5-1
Oxidative Stress Caused by Chlorin Reductase Reaction of
Rhodobacter Sphaeroides
Jeong K. Lee
Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang University,
Seoul 121-742, Korea
Rhodobacter sphaeroides has a cuprozinc-containing superoxide dismutase (CuZnSOD) in its periplasm in
addition to the cytoplasmic SOD that appears to contain iron (FeSOD). The FeSOD activity whose expression
is regulated transcriptionally is approximately twofold greater than that measured under the oxygen-limiting
(≤ partial pressure of oxygen [pO2] 2%) conditions where the light-harvesting complexes are found. The
CuZnSOD is detected only under the oxygen-limiting conditions. The CuZnSOD expression is regulated
posttranscriptionally, and the enzyme protects the photoheterotrophic cells from periplasmic superoxide that
may be generated from the photosynthetic electron flow at pO2 less than 2%. Disruption of sodB coding for
FeSOD is lethal unless the mutant SodB1 is cultured in Luria-Bertani (LB) medium, where SodB1 is
genetically stable. Component of LB medium, which is responsible for the complementation of lethality in
SodB1 growing in Sistrom's succinate-based minimal medium as well as its genetic stability, is lysine that
can be convertedinto cadaverine by lysine decarboxylase. Polyamines including cadaverine have been proposed
to scavenge superoxide. Suppressor for the lethality in SodB1 growing in the minimal medium was
predominantly localized to bchZ of chlorin reductase (BchXYZ) whose mutation is associated with reduced
pigmentation compared with wild type. BchX, BchY, and BchZ were over-expressed in R. sphaeroides,
followed by purification using histidine tag. The proteins were mixed in equimolar ratio in vitro to show
anaerobic chlorin reduction using NADH in the presence of protein Q (or PufQ), a bacteriochlorophyll-carrier
protein. An electroparamagnetic resonance (EPR)signal ascribed to superoxide was detected with
5,5-dimethyl-1-pyrroline-N-oxide (DMPO) from the reaction at pO2 less than 2%. Chlorin reductase composed
of wild-type proteins of BchX and BchY, and a mutated BchZ' from suppressor mutant, revealed less activity
not only for chlorin reduction but also for superoxide formation compared with the wild-type enzyme
BchXYZ. Consistently, a bchZ-sodB double mutant is viable in Sistrom's minimal medium. Induction of R.
sphaeroides bchXYZ in trans in Synechocystis sp PCC6803 arrested its photosynthetic growth, but the cessation
of cell growth was relieved by increasing the level of cytosolic SOD. Thus, superoxide from chlorin reductase
of R. sphaeroides, once it is expressed in Synechocystis harboring chlorin as an intermediate for chlorophyll
biosynthesis, appears to cause cellular damage to stop cell growth. The oxidative stress by chlorin reductase
in the presence of oxygen may give us an insight into a clue to possible evolutionary relations between the
biosynthetic steps forming chlororophyll and bacteriochlorophyll; the former does not employ chlorin reduction
and has shorter biosynthetic steps.
Keywords: Rhodobacter sphaeroides, SOD, sodB mutant, suppressor, chlorinreductase
Acknowledgments
This work was supported by the 21C Frontier Microbial Genomics and Applications Center Program from
Ministry of Science and Technology, Korea.
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63
October 13-14, 2005, Seoul, Korea
S5-2
Interplay between SeqA Protein and Methyl-Directed Mismatch
Repair Proteins at the Replicated GATC Sequences
Deog Su Hwang*, Sung Ho Kim, and Sukhyun Kang
Department of Biological Sciences, and Institute of Molecular Biology and Genetics, Seoul National
University, Seoul 151-742, Korea
The methyl-directed mismatch repair (MMR) system, which is composed of MutS, MutL, and MutH
proteins, repairs mismatches during DNA replication in E. coli. The system recognizes hemi-methylated
GATC sequences that are transiently produced upon replication of fully-methylated DNA, and corrects
mismatched nucleotides in the newly synthesized and unmethylated strands. Hemi-methylated regions have
been shown to be the binding sites for SeqA protein. We found that SeqA inhibits MutH from cleaving
hemi-methylated GATC sequences, and that MutS and MutL restore MutH cleavage activity in a
mismatch-dependent manner. Our results suggest that SeqA prevents the newly synthesized strands from being
cleaved by MutH. This finding is supported by the SOS response in seqAmutants. We will describe additional
aspects of the interplay of SeqA with the MMR system at the symposium.
Keywords: SeqA, methyl-directed mismatch repair, hemi-methylated DNA
64
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S5-3
Biogenesis of M1 RNA, the Catalytic Component of
RNase P, in Escherichia coli
Younghoon Lee*, Kwang-sun Kim, Yool Kim, Kook Han, Soyeong Sim,
and Jae-hyeong Ko
Department of Chemistry and Center for Molecular Design and Synthesis,
KAIST, Daejeon 305-701, Korea
Ribonuclease P (RNase P) is a processing enzyme that catalyzes the endonucleolytic removal of 5' leader
sequences from precursors of tRNAs to generate the mature 5′ ends of tRNAs. In addition to precursor tRNAs,
non-tRNA substrates of RNase P, such as the 4.5S RNA, tmRNA precursors, and some polycistronic mRNAs,
are also found in Escherichia coli. RNase P enzymes from diverse organisms have been shown to contain
both essential RNA and protein components. The E. coli holoenzyme consists of two subunits, an RNA
subunit (M1 RNA, 377 nucleotides), and a small basic protein (C5 protein, 119 amino acids). M1 RNA, as
a naturally occurring ribozyme, carries out the catalytic reaction in the absence of C5 protein in vitro, although
both components are essential for the activity of RNase P in vivo.
In vitro and in vivo studies indicate that the major primary transcript from the rnpB gene is the one
initiating from the nearest promoter, P-1, and terminating at the first terminator, T1. This primary transcript,
termed precursor M1 RNA (pM1 RNA), carries an extra stretch of about 36 nucleotides containing a
termination stem and loop at the 3′ end. These extra nucleotides are removed by a processing event in vivo.
This reaction is initiated with a cleavage by RNase E at an rne-dependent site to produce processing
intermediates with 3′ ends of +378 and +379, which are subsequently trimmed to +377 by exoribonucleases.
Although the M1 RNA processing has been a well studied process, the biological significance of this process
remains unclear. One possible function could be the generation of a functional RNA molecule, but as both
pM1 RNA and M1 RNA have comparable RNase P activity and M1 RNA retains its catalytic activity with
the additional seven nucleotides at its 3′ end, this would seem unlikely. Hence, the question of why this
processing reaction needs to occur in the cell is an intriguing one. We constructed mutant strains containing
alterations in their rne-dependent sites at the chromosomal rnpB locus and analyzed the resulting phenotypes.
The mutant cells showed growth defects, which correlated with their M1 RNA levels, and the analysis of
M1 RNA metabolism in both wild-type and mutant cells indicated that pM1 RNA undergoes not only 3′
processing but also poly(A)-dependent degradation. These results suggest that the processing of M1 RNA is
required for the protection of its primary transcript form degradation.
Besides pM1 RNA, large rnpB transcripts were observed in cells lacking RNase E. We examined how the
large rnpB transcripts are related to M1 RNA biogenesis using a model upstream transcript. Our data show
that RNase E is involved in degradation of the model transcript in vivo. An in vitro assay reveals that the
N-terminal catalytic half of RNase E cleaves the model transcript at specific sites within the structural
sequence of M1 RNA. The results suggest that RNase E can function in both the degradation and processing
modes for the expression of the rnpB gene. This dual role of degradation and processing of rnpB transcript
by RNase E may play a role in accomplishing quality control of M1 RNA biosynthesis.
The 3′ flanking region of the rnpB gene contains a repeated unit of 113 bp, which includes both the
sequence coding for the 3′ terminal 24 nucleotides of M1 RNAand the region for intrinsic transcription
termination. This unit starts at position +354 and successively reiterates almost 3.5 times. The second repeated
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65
October 13-14, 2005, Seoul, Korea
sequence has potential to code for two small proteins by the overlapping coding region. However, it is not
yet known whether the proteins are expressed and, if so, what their functions are. For these small proteins
to be translated, transcription should run through both the first and second intrinsic rnpB terminators. We
revealed that the small protein encoded by the first coding region was expressed in the cell. We also found
that an E. coli mutant strain with a deletion of the 3′ flanking region of rnpB gene had an altered phenotype
in tRNA metabolism. These data suggest that the small proteins may participate in tRNA metabolism.
Keywords: biogenesis, M1 RNA, transcription, RNA processing, RNA degradation
66
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S5-4
The Study of Stress Response in Saccharomyces cerevisiae
Joon KIM
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea
Gcn4p is a typical eukaryotic transcriptional activator which is involved in the gene expression of many
biosynthetic genes of amino acids and purine under stress conditions. Gcn4p is known as a yeast functional
homolog of a c-Jun oncoprotein and binds directly as a homodimer to a conserved regulatory sequence
'TGA(C/G)TCA' of its target genes. GCN4 expression and activity are subject to a tight series of controls
that are exerted at the transcriptional, translational and post-translational levels. In order to find the helper
protein when the DNA binding activity was defective, we performed saturation mutagenesis that introduces
point mutations in the DNA binding domain of Gcn4p. We isolated the mutants with defective DNA binding
activities which werechanged in a specific conserved residue in DNA binding domain of bZIP proteins, and
we also tried to find the unknown proteins to suppress the mutant phenotype under an amino acid depletion
condition through the multi-copy suppression test. As a result, SSB2 (Stress-Seventy subfamily B2) gene was
found to be a suppressor of mutant strains. The Ssb proteins are highly abundant nascent chain-binding
chaperones and exist as both ribosome-associated and free populations. We found that Ssb2p suppressedthe
defective phenotype of mutant gcn4 by the increase of the mRNA expression of Gcn4p target genes. We also
found that increased mRNA level was resulted from the improvement of DNA binding activity which was
induced by the increase of protein stability through the interaction between Gcn4p and Ssb2p. Among the
target genes of GCN4, some ribosomal genes are known as the repressed genes based on the transcriptional
profiling microarray data.Rps3p is a component of the ribosomal small subunit, and it also has an
extra-ribosomal function, an AP endonuclease activity. Generally, there are upstream activating sequences in
ribosomal protein genes, i.e. UASrpg. One of these isthe binding site of Rap1p, yeast transcription factor
which activates transcription of ribosomal protein genes. In this research, the UASrpg of RPS3 was identified.
Interestingly, RPS3 promoter region has several putative Gcn4p-responsive elements (UASGCRE) and Rap1
binding sites. This study revealed that Gcn4p and Rap1p bind to the promoter of RPS3, and Gcn4p and Rap1p
physically interact each other. In an amino acid starvation, a rapamycin treatment or post-confluent culture
conditions,the transcriptional level of RPS3 appears to be controlled by Gcn4p. And when Rap1 acts as
transcription activator, it recruits many transcriptional machinery proteins. This study revealed that the
physical association between Rap1 and Esa1 is changed under these RPS3 transcription repression conditions.
Therefore, the detailed roles of Rap1, Gcn4 and Esa1 in the regulation of RPS3 transcription will be discussed.
Keywords: stress-respons, transcription, GCN4, RPS3
국제학술대회
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October 13-14, 2005, Seoul, Korea
S5-5
Regulation of the Mitotic Exit in Budding Yeast
Junwon Kim and Kiwon Song*
Department of Biochemistry, College of Science, Yonsei University, Seoul 120-749, Korea
To maintain genomic stability, the exit from mitosis is tightly coupled to the segregation of one set of
chromosomes into the daughter cell. In budding yeast, mitotic exit is triggered by Tem1p-regulated signaling
cascade called Mitotic Exit Network (MEN), whose components are associated with the spindle pole body
(SPB, the functional equivalent of centrosome in yeast). The interaction of astral microtubule plus-ends with
cell cortex regulates the asymmetric association of MEN components with the SPB toward bud and thereby
couples nuclear migration and mitotic exit, although its mechanism is not well understood. In this study, we
showed several lines of evidence that propose the astral microtubule-cortex interaction acts as a sensor for
spindle position to coordinate chromosome segregation and mitotic exit, and that this interaction is transmitted
through kinesin-related microtubule motor Kar3p to polo kinase Cdc5p: (1) In cells overproducing Bfa1p that
negatively control the MEN, astral microtubule became remarkably elongated and its interaction with cell
cortex is abnormal. In addition, this phenotype is suppressed by overexpression or deletion of microtubule
associated proteins (MAPs), suggesting Bfa1p directly or indirectly regulates microtubule dynamics via MAPs.
(2) In Cdc5p kinase-dead mutant cdc5-2, Bfa1p is present on both SPBs, demonstrating Cdc5p functions to
localize Bfa1p on bud-directed SPB via phosphorylating it. (3) In cells lacking Kar3p, Bfa1p is not associated
with SPBs.
Keywords: Edible mushrooms, gene transfer, genetic recombination, protoplast fusion, synkaryons, transformation
68
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S6-1
Prevalence and Molecular Characterization of MRSA and VRE
Yeong Seon Lee
Department of Bacteriology, National Institute of Health, Korea
Serious infections caused by methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin resistant
Enterococci (VRE) are increasingly difficult to treat because of limited treatment options. MRSA has known
as one of the major pathogen of nosocomial infections and has become increasingly isolated from
community-acquired infections. And, the prevalence of VRE infection in hospitals has increased dramatically
in recent years and VRE persist in the poultry farm after the avoparcin ban. Therefore, in order to investigate
the current status and prevent the spread of MRSA and VRE, we analyzed the prevalence of MRSA strains
from hospitals and VRE strains from animals and human sourcesand their molecular characterization by the
SCCmec typing, analysis of vanA gene cluster, and Multi-locus sequencing typing (MLST).
Prevalence and Molecular typing of MRSA by hospital groups
More than 64% and 41% of S. aureus isolated in tertiary and non-tertiary hospitals respectively, were
reported to be methicillin resistant. In 8 geriatric hospitals, the nasal MRSA colonization prevalence was 228
of 632 patients (36.1%). Total 101 MRSA isolates with different PFGE patterns selected fromand tertiary care
hospitals between 1999 and 2004. An SCCmec element was classified as cassette chromosome recombinase
(ccr) gene complex and mecA gene complex. MLST analysis of MRSA isolates showed seven sequence types
(STs), ST1, ST5, ST72, ST89, ST239, ST254, ST345, ST608. The major genotypes were ST5 (44 isolates)
with SCCmec type Ⅱ and ST239 (22 isolates) with SCCmec type Ⅲ, ST1 (8 isolates) and ST254 (13 isolates)
with SCCmec type Ⅳ or Ⅰvariant. And ST72 (10 isolates) and ST89 (2 isolates) with SCCmec type Ⅱvariant
were also identified. Additionally, each one isolate for ST600, ST345 with SCCmec type invariant was
detected.
Molecualr epidemiology of VRE isolated from humans and animals
E. faecium isolates from tertiary hospitals and non-tertiary hospitals were resistant to vancomycin in
18.6-37% and 6.2%, respectively.
In analysis of vanA gene cluster of 95 strains with vanA VRE isolates from hospitals, poultry, and humans,
one strain from healthy human and 35 strains from poultry were identical with original Tn1546 (TypeⅠ),
5 strains from poultry were left-end (orf1) deletion(TypeⅡ), 14 strains were insertions of IS1542 in orf2 and
IS1216V in vanX-vanY intergenic region within original Tn1546 (TypeⅢ), 33 strains were left-end deleted
in TypeⅢ (TypeⅣ), 2 strains were right-end deleted (vanY or vanZ) in TypeⅢ (TypeⅤ), 3 strains were
left-end and right-end deleted in TypeⅢ (TypeⅥ), and 2 strains with mutation in vanX, vanR or vanS were
sensitive to vancomycin (TypeⅦ).
Of total 77 vancomycin resistant E. faecium (VREF) from hospitals (48 isolates) and animals (29 isolates),
MLST analysis were revealed 11 sequence types (STs: 17, 78, 80, 203, 204, 205, 230, 203, 204, 205) for
clinical isolates and 12 STs (8, 16, 20, 78, 231, 232, 236, 237, 238, 250, 251) for animal isolates. These
results suggest that there was low genetic relationship between clinical and animal isolates.
Keywords: MRSA, VRE, SCCmec, MLST, vanA gene cluster
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69
October 13-14, 2005, Seoul, Korea
S6-2
Effect of Mycobacterial Antigens on the Expression of TLRs in
Dendritic Cells
Junglim Lee1, Jinmin Lee2, Sang-Nae Cho2, and In-Hong Choi2
1
2
Department of Microbiology, College of Medicine, Konyang University,
Department of Microbiology, College of Medicine, Yonsei University, Korea
Toll-like receptors (TLRs) are innate immune recognition molecules that play critical roles in identification
of microbial products and the subsequent initiation of inflammatory responses. These receptors recognize
pathogen associated molecular patterns (PAMPs) that are evolutionarily conserved amongst infectious agents.
Stimulation of these receptors leads to the induction of an inflammatory response and the development of an
antigen-specific adaptive immune response. Immature dendritic cells (DCs) are the immunological sensors that
screen for pathogen entry using TLRs. Invading pathogens like Mycobacterium tuberculosis or its products
are recognized by Toll-like receptors, especially TLR2 expressed on the surface of DCs. However, the
mechanism how these TLR agonists modulate the protective immune mechanism against M. tuberculosis
infection is uncertain. In this study, immature DCs were obtained from CD14(+) monocytes of healthy human
donors by culture with GM-CSF and IL-4. Immature DCs then were stimulated with different mycobacterial
antigens and the changes of DC differentiation, the activation of mitogen-activated protein (MAP) kinase and
the production of cytokines were studied. Both AraLAM and ManLAM induced the expression of TLR2 and
TLR4 in immature DCs, while other mycobacterial antigens, CFP and CFP-10, did not. When LPS was
treated, DCs underwent maturation process and lost TLR2 and TLR4 on their surface. However, treatment
of LAM maintained the expression of TLR2 and TLR4in LPS-treated, mature DCs. Other markers for DCs
(CD80, CD86, CD83) were not changed with LAM treatment. The allostimulating effect was strong in DCs
treated with LPS or ManLAM/LPS, intermediate in DCs treated with AraLAM/LPS and low in immature
DCs. The production of TNF- and IL-6 decreased in DCs treated with ManLAM/LPS or AraLAM/LPS in
compared to DCs treated LPS. DCs stimulated with mycobacterial ManLAM, 19kDa lipoprotein, and 38 kDa
lipoprotein showed ERK activation, but AraLAM inhibited ERK activation.LPS -stimulated DCs also showed
ERK activation. The activation of p38 kinase was also observed in response to AraLAM, ManLAM, 19kDa
lipoprotein and LPS. In contrast with MAP kinase observation, AraLAM and ManLAM elicited no IL-6
production. 6kDa, 16kDa, 38kDa lipoprotein, Ag85A and LPS induced massive production of IL-6. Potent
immunosuppressive cytokine IL-10, otherwise, none of treated DCs produce this cytokine.
Keywords: TLR, mycobacterial antigen, DC, MAP kinase, cytokine
70
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S6-3
Outer Membrane Protein 38 Is a Versatile Virulence Factor of
Acinetobacter baumannii
Je Chul Lee1,*, Chul Hee Choi1, Sung Hee Hyun2, Soon Ae Kim3, Jungmin Kim1,
1
1
1
Yoo Chul Lee , Sung Yong Seol , and Dong Taek Cho
1
Department of Microbiology, Kyungpook National University School of Medicine,
2
Department of Clinical Pathology, Eulji University, College of Medicine,
3
Department of Pharmacology, Eulji University, College of Medicine, Korea
Bacteria of the genus Acinetobacter are ubiquitous microorganisms, which can be found in a variety of
ecological niches including water and soil, and in clinical specimens of human and animal origins. In recent
decades, Acinetobacter have emerged as important nosocomial pathogens mostly in severely ill patients. Of
the 32 named and unnamed species currently known, Acinetobacter baumannii is the species with the highest
prevalence in clinical specimens. Despite considerable clinical and epidemiological data regarding the role of
A. baumannii in nosocomial infection, the specific virulence factor or pathogenic mechanism of this organism
has yet to be elucidated. Outer membrane proteins (Omps) have been identified in many Gram-negative
bacteria. Some Omps have been identified as porin, Fe-receptors, and also as virulence factors, including
adhesions, invasions, and factors involved in resistance to serum opsonization and killing. Omp38 was the
most abundant in A. baumannii, but little is known about its functions. The study investigated that Omp38
of A. baumannii is a versatile virulence factor that acts as an apoptosis inducing ability to epithelial cells,
invasin, and a factor of serum resistance.
The molecular mechanism of apoptosis on the infection of human laryngeal epithelial HEp-2 cells with A.
baumannii was first investigated and the contribution of Omp38 on the ability of A. baumannii to induce
apoptosis of epithelial cells was examined. The formalin-killed A. baumannii did not induce apoptosis of
HEp-2 cells, but live A. baumannii induced apoptosis of HEp-2 cells through cell surface death receptors and
mitochondrial disintegration. To determine which components of A. baumannii are involved with apoptosis
of epithelial cells, transposon mutagenesis was performed. Of the mutant library, the Omp38-deficient mutant
KS37 was not as able to induce apoptosis as the wild-type A. baumannii strain. To confirm whether Omp38
of A. baumannii induces apoptosis of epithelial cells, Omp38 was purified from A. baumannii ATCC19606and
the recombinant Omp38 was produced. Purified Omp38 entered the cells and was localized to the
mitochondria, which led to a release of proapoptotic molecules such as cytochrome c and apoptosis-inducing
factor (AIF). The activation of caspase-3, which is activated by caspase-9, degraded DNA approximately 180
bp insize, which resulted in the appearance of a characteristic DNA ladder. AIF degraded chromosomal DNA
approximately 50 kb in size, which resulted in large-scale DNA fragmentation. The wild-type A. baumannii
has been shown to invade human laryngeal epithelial HEp-2 cells in gentamicin protection assay, while the
Omp38-deficient mutant did not invade epithelial cells. Although the aggregated Omp38-deficinet mutant
adhered to surface of the cells, those bacteria seemed to fail to invade HEp-2 cells. A. baumannii ATCC
19606 showed almost complete resistance to 60% of normal human serum, while the Omp38-deficient mutant
showed complete killing in the presence of 20% of normal human serum. Serum resistance of A. baumannii
was mediated by factor H binding to Omp38, which induced blockade of alternative pathway cascade in
complement activation. These results indicate that Omp38 may act as a potential virulence factor in A.
baumannii infection.
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Nuclear targeting of bacterial proteins is considered to be a new concept of bacterial pathogenesis. The
putative candidate nuclear localization signal (NLS) was found in the C-terminal region of Omp38. Nuclear
translocation of Omp38 was confirmed by monitoring the localization of Omp38-EGFP fusion in COS-7 cells,
HEp-2 cells, and H292 cells. When COS-7 cells were transfected with an Omp38-EGFP fusion, the cells were
highly toxic compared with the Omp38∆NLS-EGFP fusion. Now, we investigate the interaction of Omp38
with nuclear components in the nucleus of eukaryotic cells.
Keywords: Acinetobacter baumannii, Virulence factor, Nuclear localization signal, Apoptosis
72
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
S6-4
Generation of a Neutralizing Human Antibody to Hepatitis B Virus
pre-S1 with Antibody Phage Display Technique
Sae-Gwang PARK1,2,*, Yong-Joo JEONG1,2, Yong-Yi LEE1,2, Ik-Jung KIM3, Su-Kil SEO1,2,*,
4
4
4
5
1,2
Eui-Joong KIM , Heung-Chae JUNG , Jae-Gu PAN , Ik-Sang KIM , and In-Hak CHOI
1
2
Department of Microiology, College of Medicine, INJE University, Center for Viral Disease Researh, INJE
University, 3Department of Microiology, College of Medicine, Dogguk University, 4Microbial Display NRL,
Genofocus Inc, 5Deparment of Micrbioogy, College of Medicine, Seoul National University, Korea
Expressing cDNAs or peptides on filamentous phages has been a powerful tool for the identification of
functional peptides or proteins with pharmaceutical applications. Among functional proteins, antibodies are of
particular interest owing to their ability to recognize a variety of targets with high specificity and affinity.
More specifically, the use of partial or complete human antibodies, which elicit no (or a minimal) immune
response when administered to patients, is yielding a growing list of FDA protein-based drugs. Phage display
technology enables the generation of large repertoires of human antibodies, while biopanning permits the
selection of individual antibodies with a desired specificity.
Using the phage display system, various formats of antibody fragments can be displayed on the surface
of filamentous phages that contain the antibody genes. Antibody fragments such as single-chain variable region
fragment (scFv) molecules have been developed for potential clinical applications. ScFvs are the smallest type
of antibody fragment and are composed of a light and a heavy chain variable region (VL and VH,
respectively) joined by a short peptide spacer. Thus, the phage display system has been developed from
recombinant scFv techniques to clone and express cDNAs that encode the variable regions of VL and VH
chains and allows the in vitro generation of large antibody repertoires.
In this presentation, we display the generation of a neutralizing human antibody to hepatitis B virus pre-S1
by CDR3 mutagenesis of previously generated anti-pre-S1 human antibody and from large naive antibody
library.
1. Improvement of neutralizing activity against HBV binding of human scFv antibodies using CDR3 VH
mutant library
We have aimed here at improving the affinity of a human single-chain Fv (1E4) specific for preS1 of
hepatitis B virus (HBV) by random mutagenesis in CDR3 VH of the clone. By employing a BIAcore as a
panning and screening strategy, we have selected three clones with lower KD than 1E4. Afiinities of selected
-7
-8
clones were from 1.76 × 10 M to 6.3 × 10 M, which were increased factors of 1.46 to 4.08, respectively,
compared to the parental clone. Binding inhibition assay using flow cytometry and PCR revealed that B2 had
a higher neutralizing activity against preS1 or HBV virion binding to liver cell line. This anti-preS1 scFv
can be considered as a potential therapeutics for the passive immunotherapy for HBV infection.
2. Hepatitis B virus-neutralizing anti-pre-S1 human antibody fragments from large naïve antibody phage library
We report the construction of a large nonimmunized phage antibody library in single-chain variable region
fragment (scFv) format, which allowed the selection of antibodies that neutralize hepatitis B virus (HBV) in
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October 13-14, 2005, Seoul, Korea
10
vitro. We generated 1.1 × 10 independent scFv clones using the cDNA of functional variable (V) gene
segments of heavy and light chains purified from the peripheral blood mononuclear cells of 50 nonimmunized
human donors. Using BIAcore, we selected two clones that recognized pre-S1 and neutralized pre-S1- and
-7
HBV binding of Chang liver cells. Clone G10 had the highest affinity (KD = 1.69 × 10 M), which was
higher than that of clone 1E4 that was generated previously from a heavy chain-shuffled antibody library.
-3
-1
The off-rates of clones were within 10 s as determined by BIAcoreand are comparable to those of
antibodies derived from a secondary immune response. In the inhibition assays of pre-S1 and virus binding
to Chang liver cells using flow cytometry and the polymerase chain reaction, G10 had better neutralizing
activity than 1E4. The new phage library may be a valuable source of antibodies with reasonable affinities
to different targets, and the anti-pre-S1 antibody fragment G10 may be a good candidate for
immunoprophylaxis against HBV infection.
Keywords: antibody phage display technique, naive library, CDR3 mutagenesis, hepatitis B virus preS1,
neutralizing human antibody
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S7-1
Pathogenic Wood-Decaying Fungi in China
Yu-Cheng Dai
Institute of Applied Ecology, Chinese Academy of Sciences, Shenyang 110016, China
Pathogenic wood-decaying fungi in Chinawere surveyed during last 10 years, and the wood destroying
species were in particular investigated. 90 pathogenic Basidiomyceteswere found in natural forests, forest
plantations and garden forests, and among them 30 species were recorded for the first time on living trees
from China. The host(s), type of damage, type of decay and distribution of each species in China are given.
Most of these wood-destroying fungi are polypores in the Aphyllophorales, and the majority were found in
temperate and boreal forests. 80 species cause a white rot, and 10 cause a brown rot.
Keywords: Basidiomycetes, forest pathology, wood-destroying fungi, China
국제학술대회
75
October 13-14, 2005, Seoul, Korea
S7-2
Fungicide Resistance and Phenotypic and Genotypic Diversity of
Phytophthora infestans in Korea
Xuan-Zhe Zhang* and Byung-Sup Kim
Department of Applied Plant Science, Kangnung National University, Gangneung,
Gangwon-do 210-702, Korea
A total of 939 isolates of Phytophthora infestanswas isolated from the leaf samples of late blight disease
collected from five provinces in Korea over the three growing seasons of 2001-2004. To determine the mating
type of an isolate, the agar disk of unknown mating type was placed at the center of the medium, and both
standard A1 and A2 mating type isolates of P. infestans were then placed 3 ㎝apart from the center on
opposite sides. The mating type was designated by observing oospores in the contact zone between each
standard and unknown isolates. Of the 939 isolates, 875 isolates were of the A1 mating-type, and 64 isolates
were of A2 mating type, showing that the majority was A1 mating-type.
Frequencies of metalaxyl resistance isolates were gradually increased from 17% in 2001 to 84.2% in 2004,
but isolation frequencies of metalaxyl sensitive and intermediate resistant isolate were decreased. Most isolates
were grown at 0.5 ㎍/㎖ of dimethomorph and isolates grown at 1 ㎍/㎖ of dimethomorph were approximately
10.2~22.9%. However, no isolate was able to grow at 5.0 ㎍/㎖. Based on these results, minimum inhibitory
concentrations (MIC) of dimethomorph to P. infestans were determined to be 0.5~1.0 ㎍/㎖. Our results
indicated that the reason decreasing control efficacy of dimethomorph was not caused by occurrence of
resistant isolates. About 5% and 12.1% isolates among the total isolates collected in 2003 and 2004 were
grown on V-8 juice rye agar containing 1.0 ㎍/㎖ethaboxam. The 2.1 and 25.4% isolates had MICs of 0.2~0.4
㎍/㎖, and MIC values of 87.9% and 74.3% isolates were less than 0.2 ㎍/㎖ concentrations of ethaboxam.
Therefore, resistance development by P. infestans to ethaboxam is not likely to occur in the natural condition.
The strains of P. infestans obtained from five provinces in Korea were determined as 18 physiological race
clones in the experiment, and all race composed of at least five or more than multiple race. Therefore, race
differentiation were diverse in Korea, almost Korean isolates have virulence to R1, R3, R5, R6, R10 and R11
among the potato resistance gene.
To examine genetic diversity of P. infestans in Korea, this study were performed analysis of RAPD, mt
DNA, and allozyme patterns. Profiles of Gpi and Pep defined four allozyme genotypes among the total of
367 isolates tested. All four allozyme genotypes could be distinguished on the basis of Gpi profiles alone,
whereas all isolates were homozygous at the Pep locus(100/100). The mitochondrial DNA haplotype of all
isolates were the Ⅱa haplotype. Amplification of the genomic DNA's extracted from eight isolates of each
mating type by polymerase chain reaction with the selected primer (OPC-5 primer) produced a total of 20
DNA bands, of which 11 bands were polymorphic. According to the RAPD analysis by using OPC-5 primer,
106 isolates including two standard isolates were separated into 8 groups at the similarity level of 92.5%.
The RAPD groups were not correlated with the allozyme genotypes and the isolated locations. All of the eight
RAPD groups were identified in Gangwon-do, suggesting that Gangwon-do is the center of origin of the P.
infestans in Korea. A 600bp DNA band generated with OPC-5 primer was specific to A1 mating-type isolates,
but not detected with A2 mating type, showing that specific PCR primer can distinguish different mating types
in P. infestans.
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Keywords: Phytophthora infestans, Mating type, Dimethomorph, Ethaboxam, Metalaxyl, Resistance, Race
Differentiation allozyme loci (Gpi and Pep), mt DNA haplotype, RAPD
국제학술대회
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October 13-14, 2005, Seoul, Korea
S7-3
Cytology of Interaction between Wheat and Puccinia striiformis
Zhensheng Kang
College of Plant Protection, Northwest A&F University, Yangling, Shaanxi 712100, China
Wheat stripe rust, caused by Puccinia striiformis Westend f. sp. tritici, occurs worldwide and is considered
a major disease in temperate regions, particularly in China. It has been demonstrated that the reasonable use
of resistant wheat cultivars is the most effective and economical method to control wheat rust diseases.
Studying on the interaction between wheat and rust can reveal the resistant mechanism of the host plant and
the pathogenic mechanism of the rust fungi, which provide further information for selection and reasonable
use of resistant wheat cultivars, and also lay an essential foundationfor exploring the host resistant mechanism
from biochemical and molecular biologicalaspects. The studying on interaction between wheat and rust
involves in genetics, histology, cytology, physiology, biochemistry, molecular biology and so on. To reveal
the resistance mechanism of wheat to stripe rust, we recently examined the compatible and incompatible
combinations between wheat and P. striiformis by means of electron microscopic and immunogold labeling
techniques.
The infection process of Puccinia striiformis West is similar to that of other cereals rust. After penetrating
the stoma of wheat leaves, Puccinia striiformis forms substomatal vesicle, infection hypha, haustorial mother
cell and haustorium within the host tissue, respectively. The multinucleat condition, e.g. more than two nuclei,
is usually found in the intercellular hypha cells, haustorial mother cells and haustoria. In the infected wheat
leaves of the susceptible cultivar a higher hyphae number was usually detected as compared to the
corresponding tissues in the resistant cultivar, indicating that the fungal development was restricted in wheat
leaves of the resistant cultivar. The structural defense reactions such as formation of cell wall apposition,
collar or papillae, and encasement of haustorium were essentially more pronounced in the infected wheat
leaves of the resistant cultivar than in the susceptible one. Sometimes, in the wheat leaves of the incompatible
combination the typical papillae of large size, detected in the host cell subjacent to the penetration site of
the haustorial mother cell stopped the pathogen's further development. Immunogold studies demonstrated the
presence of callose in the collars or papillae, cell wall appositions and encasements formed in P.
striiformis-infected wheat leaves. Immunogold localization of lignin revealed a markedly higher labelling
density in host cell walls of the infected wheat leaves of the resistant cultivar than in the cell walls of the
infected wheat leaves of the susceptible wheat cultivar. These findings indicated that lignin accumulation in
the infected wheat leaves may play an important role in resistance to the spreading of the pathogen in the
host tissues.
Two antisera raised against acidic chitinase and acidic β-1, 3-glucanasewere used to investigate the
subcellular localization of the two enzymes in the compatible and incompatible interactions between wheat
and P. striiformis. The studies demonstrated that the labelling patterns for both enzymes were very similar
in the uninoculated healthy and infected wheat leaves. The enzymes were localized mainly in the host cell
walls, while no labeling was observed in cytoplasm and organelles of the host cells. However, the
accumulation of two enzymes in the infected wheat leaves differed markedly between resistant and susceptible
wheat cultivars. The labelling densities for the two enzymes in the infected leaves of the susceptible cultivar
increased slightly as compared to the uninoculated healthy leaves, whereas significantly higher labelling
densities of chitinase and β-1, 3-glucanase were found in the infected leaves of the resistant cultivar compared
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2005 International Meeting of the Federation of Korean Microbiological Societies
to the uninoculated healthy leaves. Furthermore, the labelling of chitinase and β-1, 3-glucanase also occurred
over the extrahaustorial matrix and the fungal cell walls in the infected wheat leaves. The extrahaustorial
matrix and the hyphal cell walls in the infected leaves of the resistant cultivar usually showed a higher density
of the labelling than those in the susceptible cultivar. These finding indicated that chitinase and β-1,
3-glucanase accumulation have potential role in the defense reactions in the incompatible interaction between
wheat and P. striiformis.
국제학술대회
79
October 13-14, 2005, Seoul, Korea
S7-4
Characterization of an Antifungal Agent against Monosporacus
cannonballus Causing Cucurbitaceous Crops Disease
Ki-Chul Chung*, Yoon-Gyo Lee1, Bang-Ung Yoon, and Jae-won Ryu2
School of Biological Sciences and Technology, 1Division of Applied Bioscience and Biotechnology,
2
Graduate School of Biotechnology, Chonnam National University, Gwangju 500-757, Korea
Watermelon is the most popular fruit in the summer time among the cucurbits in Korea, and now produced
all the year round by greenhouse cultivation. Since the greenhouse culture of watermelon as well as other
cucurbits was widespread, incidences of major diseases in the past such as Fusarium wilt, gummy stem rot
and anthracnose, have been decreased. On the other hand, a group of new borne diseases have been prevalent,
causing serious economic losses, one of which is a root rot/vine decline disease that has recently occurred
on cucurbits worldwide(Martyn and Miller, 1996; Mertely et al., 1991, 1992, 1993; Park et al., 1994;
Stanghellini et al., 1996)
The disease was first reported by Pollack and Uecker(1974), caused by Monosporacus cannonballus Pollack
and Uecker, occurring especially in hot and dry areas, Texas and Arizona, of the USA, and in other countries
such as Japan, Israel and Taiwan(Martyn and Miller, 1996; Reuveni et al., 1983; Uematsu et al., 1985). Park
et al.(1994) first reported the occurrence of the disease on bottle gourd-stocked watermelon cultivated at a
region nearby Chochiweon in Korea.
This disease is typically soil-borne and mainly occurs in desert or stress conditions, such as hot and dry
regions in other studies(Martyn and Miller, 1996). High salt soil conditions may be an important factor for
this disease development. Host range of this pathogen expands not only to cucurbits, but also to other crops
such wheat(Reuveni and Krikun, 1983) The disease has occurred mainly at the later developmental stage of
the plant growth and thereby influenced on the fruit quality. Fruits could not be harvested if plants are
severely damaged. There is no information about economic losses in Korea, but economic losses in USA
alone estimate about 10-25% losses of the crops annually(Martyn and Miller, 1996).
Control strategies for this disease have not been established well because of no information about
epidermiology of the disease, and only soil fumigation with methyl bromide has been studied(Martyn and
Miller, 1996).
This study was carried out to isolate antagonistic bacteria and fungi against M. cannonballus causal agent
of a sever root rot/vine decline disease of muskmelon and watermelon. M. cannonballus infects young
secondary and tertiary roots early in the season, colonizes in the cortical tissue, ultimately killing most of
the feeder roots. By mid to late season, most of the root system is affected and the vines begin to collapse,
typically beginning with the crown leaves and progressing distally. So we isolated potent antagonistic bacteria
and fungi against the pathogen from soil of Chonnam area and investigated antifungal activity against various
plant and animal pathogen. Among them a fungal strain MET0425 showed a powerful antifungal activity
against the M. cannonballus. Based on morphological, biochemical and 18S rDNA, ITS1, 5.8S rDNA and
ITS2 sequence analysis, the strain MET0425 was identified as Epicoccum sp. by comparing microscopic
characteristics of fungal structures with those of Epicoccum nigrum KACC40642 fungal strains. Antifungal
agent was extracted with ethyl acetate from culture filtrates and partial purified using Sephadex LH-20 column
and YMC-Pack ODS-A HPLC. The eluted fraction was exhibited broad spectrum antifungal activity. The pH
and thermal stability, and structure of this antifungal agent was determined. Therefore, we can expect that
80
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
this fungus will provide for a powerful resource in studying the biology and in developing cucurbitaceous
crops disease biocontrol agent.
References
1. Kwon, M. K., Hong, J. R. Kim, Y. H. and Kim, K. C. 2001. Soil-Environmental Factors involved in
the Development of Root Rot/vine on Cucurbits caused by Monosporacus cannonballus. Plant Pathol.
J. 17(1):45-51.
2. Larone, D. H. 1995. Medically Important Fungi: A Guide to Identification, 3rd ed. ASM Press,
Washington, D. C.
3. Martyn, R. D. and Miller, M. E. 1996. Monosporacus root rot and vine decline: An emerging disease
of melons worldwide. Plant Dis. 80:716-725.
4. Mertely, J. C., Martyn, R. D., Miller, M. E. and Bruton, B. D. 1991. Role of Monosporacus
cannonballus and other fungi in root/vine decline disease of muskmelon. Plant Dis. 75:1133-1137.
5. Mertely, J. C., Martyn, R. D., Miller, M. E. and Bruton, B. D. 1993. Quantification of Monosporacus
cannonballus ascospores in three commercial muskmelon fields in South Texas. Plant Dis. 77:766-771.
6. Mertely, J. C., Martyn, R. D., Miller, M. E. and Bruton, B. D.1992. An expanded host range for the
muskmelon pathogen Monosporacus cannonballus. Plant Dis. 77:667-673.
7. Park, K. S., Nam, S. H. and Kim, C. H. 1994. Root rot of bottle gourd stock of watermelon caused
by Monosporacus cannonballus in Korea. J. plant pathol. 10:175-180.
8. Pollack, F. G. and Uecker, F. A. 1974. Monosporacus cannonballus, an unusual ascomycete in
cantaloupe roots. Mycologia 66:346-349.
9. Pritchard, R. C. and D. B. Muir, 1987. Black Fungi: a survey of dematiaceous hyphomycetes from
clinical specimens identified over a five year period in a reference laboratory. Pathology 19:281-4
10. Reuveni, R., krikun, J., and Shani, U. 1983. The occurrence and distribution of Monosporacus
euthpoides in a collapse of melon plants in and arid area of Israel. Trans. Br. Mycol. Soc. 72:354-3556.
11. Reuveni, R., Krikun, J., and Shani, U. 1983. The role of Monosporacus euthpoides in a collapse of
melon plants in and arid area of Israel. Phytopathology 73:1223-1226.
12. Stanghellmi, M. E., Kim, D. H. and Rasmussen, S. L. 1996. Ascospores of Monosporacus cannonballus
in Arizona. Phytopathology 86:509-514.
13. Sutton, D. A., A. W. Fothergill, and M. G. Rinaldi(ed). 1998. Guide to Clinically Significant Fungi,
1st ed. Williams & Wilkius, Baltimore.
국제학술대회
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October 13-14, 2005, Seoul, Korea
S7-5
Pathogen Biodiversity of Sooty Blotch and Flyspeck Apple
Disease Complex in Northwest China Based on Parsimony
Analysis of Ribosomal DNA
G.Y. Sun1,*, J.C. Batzer2 R. Zhang1, M. Zhang1, Y.M. Zhang1, and M.L.Gleason2
1
College of Plant Protection, Northwest A&F University, Yangling, Shaanxi, 712100, China
2
Department of Plant Pathology, Iowa State University, Ames, IA, 50011, USA
The sooty blotch/flyspeck (SBFS) fungal complex poses a major threat to apple production in humid
climates worldwide, including China. These fungi colonize the apple cuticle, creating blemishes that prevent
apples from being marketable as fresh fruit, resulting in significant economic losses to growers. The fungi
are difficult to isolate, grow extremely slowly, and are not easily maintained in culture collections, in cause
of culture most strains can not product conidia. So they can not be classified and identified by traditional
method. We examined the pathogen diversity of sooty blotch and flyspeck from Shaanxi, China, by sequences
analysis of the internal transcriber spacer (ITS) and larger subunit (LSU) regions. Comparisons of sequences
obtained from isolates from China, the mid-western and southeastern U.S. were made using parsimony
analysis. The result shown: All SBFS fungi from Shaanxi orchards were ascribed to 16 species, belong to
two classes Dothidiomycetes and Chaetothyriomycetes. Among the species, 1 specie were classified as
Leptodontium sp. in the class Chaetothyriomycetes, 15 species were sorted into 11 genera in Dothideales
(Dothidiomycetes): 2 species were classified Pseudocercosporella, 1 specie in Peltaster, 2 species in
Zygophiala,2 species in Xenostigmina, 2 species in Mycovelosiella,1 specie in Pseudocercospora, 1 specie in
Dissoconium, 3 incertitude species. In all species from Shaanxi just 2 species were identical to known species
in the mid-western and southeastern U.S. These results indicated that sooty blotch and flyspeck fungi were
different in areas. Expanded investigation is needed to carried out around the world to understand the pathogen
constitution of SBFS complex.
Keywords: Sequence analysis, phylogeny, sooty blotch, flyspeck, internal transcribed spacer region (ITS),
large subunit of ribosomal DNA (LSU), biodiversity
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S7-6
Identification of Colletotrichum spp. from Leguminosae and
Zingiberaceae in South China
Peng Ren, Xi Pingge, Zeng Daxiong, and Zide Jiang*
Department of Plant Pathology, South China Agricultural University, Guangzhou 510642，China
Colletotrichum Cda. is one of the most important genera of plant pathogenic fungi worldwide, especially
in tropical and subtropical regions. The identification of Colletotrichum spp. is rather difficult due to their
similar and unstable characteristics in morphology. In order tomake further confirmation on species from the
plants of Leguminosae and Zingiberaceae, 80 isolates of the fungi collected from South China were compared
in morphology, cultural characters, and host ranges, and the intra-specific and inter-specific phylogenic
relationships of them were studied by ITS sequence analysis.
The results indicated that 80 isolates of Colletotrichum were belonged to 4 species (3 with straight conidia
and 1 with falcate conidia): C. gloeosporioides (Penz.) Sacc., C. boninense J. Moriwaki, Toy. Sato & T.
Tsukiboshi, C. acutatum Simmonds and C. capsici（Syd.）Butler & Bisby. Among them, C. gloeosporioides,
C. boninense and C. capsici could be isolated from Leguminosae or Zingiberaceae, but C. acutatum only from
Zingiberaceae. C. boninense was a newly recorded species for China. The results also showed that the main
pathogen of anthracnose was C. gloeosporioides on Leguminosae, but C. gloeosporioides or C. capsici on
Zingiberaceae according to the host plant.
Internal transcribed spacers (ITS) of the ribosomal RNA gene (rDNA) were sequenced for the 80 isolates.
A neighbor-joining phylogenetic tree was constructed by representative ITS sequences from the isolates and
some referenced sequences as well as sequences of 2 out-groups [Gibberella sacchar (AF455450) and
Pestalotia thujae (AF377295)] from GenBank. The results showed that all of the isolates of Colletotrichum
were clustered into one group in 100 percent bootstrap, while the Gibberella sacchar and Pestalotia thujae
were not included in this group.
The phylogenetic tree supported that C. gloeosporioides was a complex species and should involve different
independent taxa. The primary reason for its heterogeneity was likely not the selective pressure of the host
plants but the long independent evolution in respective environment owing to the far geographical segregation.
Some isolates of C. boninense showed some difference of pathogenicity to different host plants. Correspondingly,
the ITS sequence analyses indicated there was some variation in genetic background within the species.
The ITS sequence data revealed that C. acutatum, first reported on the plants of Leguminosae in China,
was 100% identical to the reference C. acutatum isolate from GenBank. Moreover, it could produce some
atypical conidia, which further confirmed its complexity in morphology and universality in host range.
The ITS1 sequence data suggested that 20 isolates with falcate conidia from Leguminosae and
Zingiberaceae and 2 isolates with same conidia from Capsicum fructescens were clustered into one group in
99 percent bootstrap, and theintra-specific sequence identity were between 95.1 to 100%. The result
manifested that all of the isolates with falcate conidia from Leguminosae and Zingiberaceae should be
identified as C. capsici, and C. zingiberis was its synonym. In addition, the result of pathogenicity test
indicated that there were some differentiations in pathogenicity of the isolates of C. capsici from
Zingiberaceae. The phylogenetic tree also revealed that they were dispersed in 3 sub-groups and had some
genetic diversity, which suggested there should be new forms within the species.
Keywords: C. gloeosporioides, C. boninense, C. capsici, C. acutatum, ITS, Sequence analysis
국제학술대회
83
October 13-14, 2005, Seoul, Korea
S8-1
Biodiversity of Nematophagous Hirsutella Species and Their
Relationship with Nematode
Xingzhong Liu
Key Laboratory of Systematic Mycology & Lichenology, Institute of Microbiology, Chinese Academy of
Sciences, Beijing 100080, China
Soybean cyst nematode (SCN), Heterodera glycines, is widely distributed in soybean-producing areas, and
causes great yield loss in China. Hirsutella rhossiliensis and H. minnesotensis are two prominent parasites
of SCN juveniles. Their occurrence and frequency were investigated in the soybean plant area in China. H.
minnesotensis was detected in 20.5% of the soil samples and H. rhossiliensis in 1.4%. Variability of
morphology, parasitism of nematodes, and ITS-5.8S rDNA sequences among Hirsutella isolates were
determined. There were differences among species and among isolates within species. Most of the isolates
of Hirsutella species had host specificity in the agar plate tests. All isolates of H. rhossiliensis were clustered
into one clade, in which two sub-clades were formed corresponding to host specificity. The isolates of H.
minnesotensis and H. thompsonii were clustered into one clade, but separated into two sub-clades. A real-time
PCR for quantitative detecting and monitoring H. rhossiliensis in soil was developed and compare with the
parasitism assay. The effect of environmental factors such as temperature and moisture was determined. The
DNA amount yield by real-time PCR assay was higher at lower temperature and moisture of soil. The
population dynamics of H. rhossiliensis in soil and its biological control effectiveness on SCN was also
evaluated in greenhouse. The density-dependent parasitism between percentage of J2 parasitized and the
nematode density (number of H. glycines J2) was examined in vial soil. A novel subtilisin, designated as Hrp1
(Hirsutella rhossiliensis protease 1), was purified from H. rhossiliensis OWVT-1 by ammonium sulphate
precipitation, ion exchange chromatography on Q Sepharose Fast Flow and gel filtration chromatography on
SuperdexTM 75. Molecular weight of the subtilisin Hrp1 was estimated to be around 32 kDa by SDS-PAGE.
N-terminal of the protease Hrp1 was AVIDTGVEASHPEF. DNA sequence consists of 1170 bps including
3 introns and 4 extrons coding 389 amino acids which resulted in a mature peptide of 246 amino acids. The
sequences of protease and DNA were similar to that of other nematophagous fungi.
Keywords: Soybean cyst nematode, Juvenile parasites, Hirsutella rhossiliensis, Hirsutella minnesotensis,
Variability, Serine protease
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S8-2
A Novel RNA Virus in Mushroom
Hyun Jae Yu1, So Yeon Kim1, Eun Joo Kang1, Sung-Soon Kim1, Jun-Oh Choi1, Hyeon-Su Ro1,
2
3
4
1,
Jae San Ryu , Jong Kyu Lee , Moo Ung Chang , and Hyun-Sook Lee *
1
Department of Microbiology and Research Institute of Life Science, Gyeongsang National University, Jinju
660-701, 2Division of Plant Environment, Gyeongnam Agricultural Research and Extension Services, Jinju
660-360, 3Department of Forest Resources Protection, Kangwon National University, Chunchon 200-701,
4
Department of Biology, Yeungnam University, Kyongsan 712-740, Korea
Natural viral epidemic was observed in commercial oyster mushroom farms in Korea. The disease was
always accompanied by the presence of a ssRNA spherical virus. All isolates obtained from different
geographic farms characterized by a dramatic reduction in yield and the formation of abnormal fruiting bodies
contained the ssRNA virus. The virus, named oyster mushroom spherical virus (OMSV), encapsidated a
single-stranded RNA (ssRNA) of approximately 5.8 kb. To examine a correlation between the presence of
OMSV and the disease, we treated the diseased isolates with Adenosine 3',5'-cyclic monophosphate (cAMP)
in order to eliminate OMSV. Curing of the virus converted abnormal phenotypes into normal phenotypes
involving normal mycelial growth, the formation of normal fruiting bodies, and increased yield. To determine
that conversion of malformed phenotypes into normal phenotypes is due to elimination of the ssRNA virus,
we carried out reverse transcription-polymerase chain reaction (RT-PCR) using total RNA from the virus-cured
strain. RT-PCR analysis of primers targeted to the coat protein gene of 5.8 kb viral genome of OMSV showed
that the 5.8 kb RNA of the virus was eliminated from the virus-cured strains. This suggests that OMSV could
be a causative agent for the disease symptoms.
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85
October 13-14, 2005, Seoul, Korea
S8-3
Diversity of Nematode-trapping Fungi in Pb-Contaminated Soils and
Their Tolerance to the Heavy Metal
Wei-Min Chen, Ke-Qin Zhang, and Ming-He Mo*
Laboratory for Conservation and Utilization of Bio-resources, Yunnan University, Kunming, 650091, Yunnan
Province, China
Nematode-trapping fungi (NTF) are a group of hyphomycetes able to capture, kill and digest nematodes
by special modified hyphae including sticky nets, sticky knobs, sticky branches, constricting rings,
non-constricting rings. For their potential of controlling nematode diseases of plants and animals, these fungi
have attracted more and more attention of researchers all around the world. Numerous ecological surveys have
shown these fungi are abundant in various habitats examined only with a few species restricted geographically.
Though numerous previous reports have given us a clear insight on the general ecological characteristics of
NTF in soil, there are still some ecological questions are uncertain. Up to now, only one study has been
performed to examine the heavy metals associated with the distribution of the major types of NTF or
individual species, and one in vitro to investigate the influence of several heavy metals on growth, trap
formation and collagenase activity of NTF. In this study, we investigated the NTF in Pb contaminated soils
and gave the particular attention to the following three questions: 1. Does the diversity of NTF in the area
contaminated by Pb increases or decreases with the increase of degrees of Pb contamination? 2. Do the fungi
from Pb-polluted soils exhibit higher tolerance to Pb toxicity than those from lead-unpolluted soils? 3. Are
the trap formation and predacious activity of NTF affected by Pb?
An investigation was conducted to establish whether the heavy metal Pb exerted adverse effect on species
diversity, mycelial growth, trap formation and predacious ability of nematode-trapping fungi. Totally, 21
nematode-trapping fungi, belonging to Arthrobotrys Corda, Monacrosporium Oudem., Dactylella Grove, were
recorded from 500 samples collected in Lanping Mine and Huize Mine where the Pb concentration was
216-7150 mg kg-1 and 132-13380 mg kg-1, respectively. These fungi fell into five groups according to their
trapping mechanisms. The net former was the most frequent group in which 9 species were isolated and its
occurrence frequency (OF=41.43%) was higher than those of the others. Two predators M. ellipsosporum
(OF=32.85%), A. oligospora(OF=15.41%), which were found in all sites, were frequent species. Liner
regression analysis indicated, in Pb-polluted soils, the numbers of microorganisms (including fungi,
actinomycetes and bacteria) and nematodes were negatively correlated with Pb concentration. However, the
distribution of nematode-trapping fungi was not restricted by the Pb contamination. The diversity indexes of
these fungi were positively correlated with the Pb pollution levels in Lanping Mine (r=0.66) and Huize Mines
(r=0.72). The mycelial growth of nematode-trapping fungi derived from either Pb-polluted or unpolluted soils
was completely inhibited by 1.8 mmol of PbCl2. At the Pb concentration of 0.35 mmol, the inhibition growth
rates varied between 7.03-18.27%. For the strains of a given species, there was no significant difference
(P>0.01) in the Pb tolerance regardless the strains were derived from habitats with or without Pb pollution
except three strains from Pb-polluted soils showing more tolerance against Pb toxicity than the control strains.
The Pb exerted similar adverse effect on trap formation and predacious capability of nematode-trapping fungi.
With the presence of PbCl2, except those strains easily forming more traps, nematode-trapping fungi obviously
decreased their abilities to form traps and capture nematodes. In most cases, the distinct differences of trap
formation and predacious ability of these fungi were observed among species but not occurred among strains
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2005 International Meeting of the Federation of Korean Microbiological Societies
of the same species and this characteristic was steady in spite of the strains'origin and the conditions with
or without Pb addition.
Keywords: nematode-trapping fungi, Pb-polluted soil, diversity, heavy metal tolerance
국제학술대회
87
October 13-14, 2005, Seoul, Korea
S8-4
Agrobacterium-Mediated Transformation of the Edible Mushroom
Agaricus bisporus
Hongkyu Kim1,*, Inka Borchers2, and Mike Challen2
1
Chungcheongnam-do Agricultural Research and Extension Services, Korea,
2
Horticulture Research International, UK
In order to improve the Agrobacterium tumefaciens mediated transformation efficiency of the edible
mushroom Agaricus bisporus, the use of sonication and biolistics has been tested alongside the standardised
vacuum infiltration method that presently yields the highest numbers of transformants. A. bisporus fruiting
body gill tissue was sonicated between 10 sec and 10 min in the presence of induced A. tumefaciens
harbouring a binary vector containing the Echerichia coli hygromycin phosphotransferase gene (hygromycin
resistance).
All putative hygromycin resistant transformants were obtained from transformation experiments using A.
tumefaciens strain AGL-1 and the binary vector pBGgHG. No transformants were recovered using AGL-1
with pBIN7-1. This suggests thatthe structure of the binary vector has great impact on the efficiency of the
transformation. The binary vector pBGgHG has a homologous promotor sequence whereas pBIN7-1 contains
a heterologous promotor (gpd) from A. nidulans.
Isolation of putative transformants on fresh Selection Medium confirmed the stability of hygromycin
resistance. The difference in growth between putatively transformed and non-transformed pieces of gill tissue
on Selection Medium is clearly visible. Non-transformed tissue can make very limited growth, but this is
restricted to the periphery of tissue, and does not proceed further onto the Selection Medium. To fully
determine transgene stability, cultivation of the isolates under non-selective conditions combined with repeated
transfer back to Selection Medium would be necessary, but this procedure could not be accomplished within
the timespan of this project.
The PCR Screening of 25 putative transformants confirmed the presence of the hph gene in at least 19
of the tested isolates. The negative results for the other 6 isolates tested do not prove the absence of the
gene, but only means that it could not be detected. The fact that all the isolates grow on Selection Medium
strongly suggests that the transgene is present. Another factor supporting presence of the hph gene is the
observation that PCR produce yield from different transformants was variable. It is possible that the extraction
of the genomic DNA was differentially successful leading to different quantities of the amplified gene after
the PCR depending on the number of the copies in the template DNA. Further work would be required to
optimise PCR screening of these samples. The PCR Screening is only sufficient to prove the presence of the
gene, but does not confirm integration into the genome of A. bisporus. To confirm integration of the transgene,
Southern blot analysis is required.
The transformation experiments VIAT I-III and SAT I-III have been carried out parallel and the recovery
of 41 putative transformants from VIAT I and 56 from SAT I shows that, if all the other conditions are right,
the SAT method seems to be at least as effective for the recovery of transformants as the VIAT method.
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2005 International Meeting of the Federation of Korean Microbiological Societies
Further experiments are needed to fully determine whether one method is better than the other. It is difficult
to say, why no transformants could be recovered from VIAT and SAT I and II. One factor that might have
influenced the results is, that the Agrobacterium were cultured from 80°C storage shortly before VIAT and
SAT I and again before SAT IV, which was also successful. It is possible that the age of the cell line has
an impact on the Agrobacterium infection process. It might also influence the subsequent growth of the
Agrobacterium as the plates of VIAT and SAT II were particularly heavily contaminated with Agrobacterium,
which hinders the recovery of any transformed tissue. The possible connection between the age of the
Agrobacterium culture and the transformation efficiency merits further investigation. Another factor that might
have influenced the recovery of transformants is the competence of Agaricus cells. Even though only stage
2 and 3 mushrooms are used for the transformation experiments, the physiological state of the cells and their
ability to up take DNA could differ.
Thesonication of pure gill tissue demonstrated that the duration of treatment had no impact on the
regeneration of the gill tissue. The recovery of transformants from SAT I also shows, that long periods of
sonication (10 min) do not significantly reduce the efficiency of Agrobacterium to infect gill tissue. The
recovery of 362 putative transformants from SAT IV with durations of treatment between 10 sec and 5 min
indicates that shorter periods of sonication seem to be more effective than longer periods. Considering the
number of putative transformants obtained from SAT IV, the optimal duration of sonication seems to lie
within the range of 10 sec until 1 min. The maximal average number of putative transformants (18) was
obtained with 45 sec of sonication. The result of SAT I shows that long durations of sonication do not hinder
the recovery of putative transformants, but shorter times of treatment might maintain a higher number of
viable cells within the gill tissue, which would facilitate the recovery of the tissue. Electron microscopy could
be used to investigate if the duration of treatment affects the number of viable cells. And even though gill
tissue regenerates readily after long periods of sonication in the absence of Agrobacterium, the additional
stress factor of the Agrobacterium infection might reduce its ability to regenerate. Additionally, shorter times
of treatment might also do less damage to the Agrobacterium cells.
To draw conclusions from only two successful experiments about the effectiveness of the SAT method
compared to the VIAT method is hardly possible, but the results of the project show that the SAT method
does successfully yield transformants. Using gill tissue, the transformation efficiency observed with 45 sec
of sonication (90%) suggests that the SAT method can enhance the recovery of transformants above that
achievable with the VIAT method. The SAT method therefore merits further investigation and future work
would be needed to determine the optimal duration of sonication. Future experiments should include
non-sonicated and sonicated controls to determine the actual effect of the sonication compared to a normal
Agrobacterium infection. Within this project only one set of sonication parameters has been used, but
optimisation of the method could also include a variation of the wavelength.
Keywords: Agaricus bisporus, transformation, Agrobacterium tumefaciens
국제학술대회
89
October 13-14, 2005, Seoul, Korea
S8-5
Recent Advances in the Study of Mycorrhizas in China
Runjin Liu
Mycorrhiza Laboratry, Laiyang Agricultural College, Laiyang, Shandong 265200, China
Mycorrhizal fungi are ubiqutous soil inhabitants, and form a symbiotic relationship with roots of most
terrestrial plants. There are 7 types of mycorrhizas: ectomycorrhizas(ECM), ectoendomycorrhizas(EEM),
arbuscular mycorrhizas(AM), arbutoid mycorrhizas (ARM), monotropoid mycorrhizas(MM), ericoid
mycorrhizas(ERM) and orchid mycorrhizas(OM). The largest group, which is predominantly associated with
agricultural crops, is the AM fungi. Another important one is ECM, which is essential in forest development.
Recently, knowledge of mycorrhizas and mycorrhizal fungi has developed at a rapid pace. In fact, it has been
a subject of scientific interest since 1950's and studies are concentrated on AM, ECM, ERM and OM in
China.
1. The resources and biodiversity of mycorrhizal fungi in China
China has a vast territory with a diverse flora. So the resources and biodiversity of mycorrhizal fungi are
rich in China. Total 12 new species and 91 new record species of China in 7 genera of AM fungi and
143species in 33 genera of ectomycorrhizal fungi were reported.
2. The anatomy and morphology of mycorrhizas
The rain tropical tree in Dipterocarpaceae used to beconsidered as ECM. However, typical arbuscular
mycorrhizal structures were observed for the first time in roots of Parashorea chinesis, Vatica astrotricha,
Dipterocarpus turbinatus, Hopea exalata, Shorea assamica, and S. robustagrown in Yunnan Province and
Hainan Island, China.
3. The physiology of mycorrhizas
The mineral, water, and endogenous hormone metabolism of mycorrhizal fungi, and the pure culture of AM
fungi were investigated.
4. The ecology of mycorrhizas
The influences of environmental factors on species distribution, diversity, development and function of
mycorrhizal fungi were paid more attention to.
5. The effect and mechanismof mycorrhizal fungi on plants and soils
Effects of mycorrhizal fungi on mineral nutrition, water status, plant disease, stress with salt, and heavy
metals, growth, and yield of plants, soil fertility and health, and their functioning mechanisms were widely
investigated.
6. Research methods
Several experimental techniques were developed.
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2005 International Meeting of the Federation of Korean Microbiological Societies
7. The application of mycorrhizal fungi
The development and research center of mycorrhizas was set up in the Chinese Academy of Forestry
Science in 1993. The development of application of ectomycorrhizal fungi was faster than that of AM fungi.
8. Research trends and prospects
More and more attention has been paid to the study, development and application of mycorrhizas. However,
the study of mycorrhizas need to strengthed in China. And there are wide prospects in the study and
application in low-input sustainable agricultural production.
Keywords: Mycorrhizas, Mycorrhizal fungi, Taxonomy, Morphology, Anatomy, Ecology, Physiology, Application,
Research methods
국제학술대회
91
October 13-14, 2005, Seoul, Korea
S8-6
Anthostomella Species from China
Bingsheng Lu1,* and Kevin D. Hyde2
1
Department of Plant Protection, Zhongkai Agro-Technology College, #24 Dongsha St., Textile Rd.,
2
Guangzhou 510225, China, Center for Research in Fungal Diversity, Department of Ecology and
Biodiversity, The University of Hong Kong, Pokfulam Rd., Hong Kong
During the study of genus Anthostomella, the most type specimens and new collections from China were
examined. In this paper, all twenty-two species of Anthostomella from China are tabulated. Three species are
regarded as doubtful and two are excluded. These doubtful and excluded species are also tabulated with notes.
Considering China is a big country, it is obviously arbitrary to make a conclusion about the genus in China
based only on these limited collections. Therefore, further collections from various provinces and different
sites are urgently needed so that an intensive study on the genus can be carried out.
Keywords: Anthostomella, Xylariaceae, Systematics, China
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2005 International Meeting of the Federation of Korean Microbiological Societies
S9-2
Functional Proteomic Approaches into the Elucidation of
Cyanobacterial Positive Phototaxis
Jong-Soon Choi
Proteomics Team, Korea Basic Science Institute, Daejeon 305-333, Korea
The cyanobacterium Synechocystis sp. PCC 6803 displays phototaxis through gliding motility. All
disruptants of histidine kinase (HK)-response regulator (RR) domains-harboring gene cluster I (sll0038-sll0043)
showed negative phototaxis whereas those of dispersed two-component gene cluster III genes (sll0073,
slr0322) did non-motile. In order to understand the regulation of cyanobacterial positive phototaxis, the
proteomic profiles of sll0043 (hybrid sensory kinase)-deleted mutant were compared with wild type. In the
phototaxis sign-switched mutant, sll0043, eight proteins were identified to be up-regulated but two proteins
down-regulated by 2-D gel-based MALDI-MS analysis. Out of proteins identified, one twitching motility
protein encoded by slr0161 (pilT1, motility-involved gene) was over-expressed in sll0043 null mutant,
indicating that hybrid sensory kinase Sll0043 suppresses the gene expression of motility motor protein PilT1).
The quantitative real-time PCR analyses confirmed that the pili biosynthesis and regulatory genes (pilA1,
pilT1, pilT2) were negatively inhibited by positive phototaxis mutant. In addition, we examined the
protein-protein interaction and interplay between two-component domains of gene cluster I and expanded to
test the possible interaction between RR domains of cluster I and twitching motility protein using a pairwise
yeast two-hybrid analysis. Sll0043-HK can recognize specifically the response regulator moiety from Sll0043,
Sll0038 and Sll0039 while Sll0073 alone recognizes Slr0322. Interestingly, Hpt1-deleted Sll0043-HK can
interact with either RR domain of Sll0038, Sll0039 and Sll0043, indicating that Hpt2of Sll0043-HK binds only
response regulator domain of locus I. Interestingly, Sll0043-RR and Sll0038-RR interact specifically with PilT2
(motility motor protein) regulating phototaxis sign but did not interact with PilT1 (twitching motility protein)
facilitating gliding motility. In conclusion, the positive phototaxis of Synechocystis sp. is governed by hybrid
sensory kinase Sll0043, in which the phototaxis signals from Sll0043-HK flow predominantly through
Sll0043-RR and Sll0038-RR via Hpt2 domain of Sll0043 to a putative signal switch molecule and Sll0043
constituently suppresses the gene expression of pilin protein, pili motor and switch protein. These results
presented here give the insight of understanding the positive phototaxis of cyanobacteria by the communication
between two-components and the direct connection to pili biosynthesis and regulatory apparatus.
Keywords: cyanobacteria, positive phototaxis, two-component, proteomics, real-time PCR, yeast two-hybrid
국제학술대회
93
October 13-14, 2005, Seoul, Korea
S9-3
The Novel Serine/Threonine Protein Kinase of Streptomyces
coelicolor A3(2) Regulates SAM Induced-Antibiotics Production
Soo Jae Lee, Choonshik Shin, Yoongho Lim, Joong-Hoon Ahn, and Kwang Pyo Kim*
Department of Molecular Biotechnology, Konkuk University, Seoul 143-701, Korea
Abstract
Streptomyces coelicolor A3(2) is a good model system to investigate the regulation of antibiotics
production.S-adenosylmethionine(SAM) is known to regulate antibiotics production from Streptomyces
coelicolor A3(2). Using proteomics approach, we studied the molecular mechanism of the antibiotics
production induced by 1 mM SAM from the plasma membrane of Streptomyces coelicolor A3(2). Interestingly,
the putative serine/threonine kinase(gi:32141327) was induced when the cell was treated with SAM and the
putative kinase has very high sequence homology with AfsK family.It suggested that the antibiotics induction
by SAM was regulated by a kinase of AfsK family.To further characterize the role of AfsK family in
SAM-induced antibiotics production, we cloned and expressed 12 putative AfsK family kinases from
Streptomyces coelicolor A3(2) based on the their expected sequence homology with AfsK.
Materials and Methods
Treatment of SAM Streptomyces coelicolor A3(2) was obtained from ATCC.The spore of S. coelicolor was
precultured for 1 day at 28℃ in LB media (0.1% beef extract, 0.1% yeast extract, 0.2% tryptone, and 1%
glycerol).After preculture 1ml of preculture solution was inoculated into 50ml of LB media and cultured for
7 days with or without 1mM SAM.
Preparation of membrane protein The cultured cells was lysed in lysis buffer (0.1M Tris-HCl pH 7.5,
14mM Mercaptoethanol, 5mM EDTA, and 10% Glycerol) and sonicated for 2 min. at 4℃. The supernatant
was ultracenrifuged 100,000 × g for 90 min. at 4℃. pellet was taken as membrane fraction.
Mass Analysis The amount of 20ug of protein was loaded in a lane of SDS-PAGE. The gel percentage
was 12.5.in-gel trypsin digestion, the sample was analyzed by LTQ (Thermo Finnigan) using a reverse phase
capillary column.
Cloning and Expression Forward and reverse primers of a family of AfsK were designed and PCR was
carried out using total genomic DNA as a template.IsolatedPCR productswere inserted into pET15b vector
and the inserted plasmid was transferred into BL21strain in order to overexpress target proteins
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2005 International Meeting of the Federation of Korean Microbiological Societies
Results
CA-3: SAM (-)
ecrE2
ecrF
hypothetical protein (21234326)
putative secreted hydrolase
(32141249)
hypothetical protein (21234196)
transcription termination factor Rho
(32141261)
putative TetR-family transcriptional
regulator (32141296)
phosphoenolpyruvate-utilizing
enzyme (32141277)
putative acetolactate synthase
(32141318)
putative ATP/GTP-binding protein
(21234165)
hypothetical protein (21234117)
putative peptide transport system
ATP-binding (32141254)
hypothetical protein (21234319)
putative 4-hydroxy-2-oxovalerate
aldolase (21234299)
putative flavin-binding protein
(32141333)
hypothetical protein (21234199)
histidine kinase (32141257)
hypothetical protein (21234154)
putative oxidoreductase (32141335)
CA-3 & CB-3
CB-3: SAM (+)
ecrE1
putative phospholipase C
(32141312)
putative oxidoreductase
(21234083)
guanosine pentaphosphate
synthetase (32141272)
putative ATP/GTP-binding protein
(21234142)
putative acyl-CoA
putative TetR-family regulator
carboxylase complex A subunit (21234309)
(32141294)
spore associated protein
(21234301)
putative oxidoreductase
hypothetical protein (21234193)
(32141253)
insertion element IS466S
transposase (21234275)
hypothetical protein (21234099)
hypothetical protein (21234277)
putative serine/threonine protein
kinase (32141327)
putative integral membrane
protein (32141299)
Table 1. List of Identified proteins
Conclusion
• We found that a family of AfsK, a serine/threonine kinase, was involved in the induction of secondary
metabolism by 1mM SAM.
• Above 30 serine threonine protein kinase family proteins have a possibility of regulation of antibiotics
induction in S. coelicolor A3(2)
• These proteins have high homology of kinase domain compared with AfsK.
• Most of Afsk family genes were obtained by PCR.
• It is needed that the proteins are overexpressed and are analyzed by LC-MS/MS in order to find
phosphorylated serine/threonine resides because a family of AfsK is to be autophosphorylated.
Keywords: streptomyces, proteomics, SAM, AFSK
국제학술대회
95
October 13-14, 2005, Seoul, Korea
S9-4
Structural Studies of Site-Specific Enzymes from Thermoplasma
acidophilum DSM 1728
Ki Hyun Nam1,†, Young Kwan Kim1,†, Kyung Hee Rhee1, Won-ho Lee1, Sam-Yong Park2,
1
1,3,
Eunice Eyungkyung Kim , and Kwang Yeon Hwang *
1
2
Biomedical Research Center, Life Science Division, KIST, Korea, Protein Design Laboratory, Yokohama
City University, Japan, 3College of Life & Environmental Sciences, Division of Biotechnology and Genetic
Engineering, Korea University, Seoul 136-701, Korea
Thermoplasma acidophilum is a thermoacidophilic archaeon that inhabits a hot (59°C) and highly acidic
environment (pH 2) in which few organisms are viable. The genome of T. acidophilum is one of the smallest
among free-living organisms (Ruepp et al., 2000). Species of the genus Thermoplasma do not possess a rigid
cell wall, but are only delimited by a plasma membrane. Many macromolecular assemblies from
Thermoplasma, primarily proteases and chaperones, have been pivotal in elucidating the structure and function
of their more complex eukaryotic homologues. Because of its biological importance, the complete genome
sequence of strain DSM 1728 has been reported (Ruepp et al., 2000). In order to elucidate the mechanism
of the site specific character of proteins from T. acidophilium, X-ray crystal structures of site-specific
recombinase (XerCD) and t-RNA splicing enzyme endonuclease (EndA) were determined at 2.2 Å and 2.5
Å resolution, respectively. Structural information of XerCD and EndA from T. acidophilium will provide the
basis for a better understanding of its properties and mechanism of site-specific enzymes. Here, I will present
the detailed mutant and structural studies of two enzymes from T. acidophilium.
Keywords: site-specific enzyme, XerCD, t-RNA splicing endonuclease, EndA, T. acidophilium, thermostability,
x-ray structure
96
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2005 International Meeting of the Federation of Korean Microbiological Societies
S10-1
Modulation of Immune Responses by Probiotics and Prebiotics
Akira Hosono* and Shuichi Kaminogawa
Department of Food Science and Technology, College of Bioresource Sciences, Nihon University, Japan
Recently, "probiotic" bacteria and "prebiotics" have attracted considerable attention regarding their benefits
to our health as physiologically functional foods. In particular, their immunomodulatory effects are expected
to apply to the prevention of infection or allergy. The present studies demonstrated that the
immunomodulatory components derived from Bifidobacterium acting as probiotics induced IgA production by
Peyer's patch (PP) cells, and that oligosaccharides acting as prebiotics modulated immune responses following
oral feeding.
The mitogenic activity of 27 strains of Bifidobacterium and 22 strains of other food microobacteria were
examined, and Bifidobacterium pseudocatenulatum 7041 has shown the strongest mitogenic activity in murine
splenocytes or PP cells in in vitro assay. A soluble high molecular weight fraction with mitogenic activity
was successfully isolated from the Bifidobacterium. The isolated soluble immunopotentiating fraction was
found to consist of polysaccharides containing -4Galp1- (and/or -5Galf1-) and -6Glcp1- as the major residues
and Galf1- and -6Galf1- as characteristic galactofuranosyl residues. In animal experiments, BALB/c mice were
administered sonicated B. pseudocatenulatum 7041 as a Bifidobacterium immunomodulator (BIM) for 7
consecutive days. We demonstrated that BIM administration augmented total IgA production by PP cells.
+
Moreover, the secretion of cytokines (IFN-γ, IL-6) by CD4 PP T cells was upregulated by BIM feeding.
It is suggested that BIM feeding immunomodulated IgA production on Peyer's patches, the inductive sites
of mucosal immunity in the gut. Furthermore, we demonstrated that live Bifidobacterium reached the PP and
lamina propria (LP) in the small intestine within hours of its oral administration. As such, it suggested that
orally administered Bifidobacterium might directly stimulate immune cells in the PP and LP in the small
intestine.
Dietary fructooligosaccharides (FOS) as prebiotics stimulate the growth of selected intestinal microflora. In
this study we could observe the immunological modulation by orally administered FOS. BALB/c mice were
administered 0-7.5% FOS for 4-6 weeks. In the FOS-fed group, total IgA in the intestinal contents was
significantly increased compared to controls. IgA secretion by PP cells was upregulated in a dose-dependent
+
manner in response to FOS administration, and CD4 T cells from PP showed a dose-dependent increase in
production of IFN-γ, IL-5 and IL-6. In contrast, FOS-feeding suppressed serum IgG1 concentration. The
genomic variation of Bacteroides in feces was also found to be augmented following FOS-feeding. Our
findings suggest that FOS supplementation changes the intestinal microflora, upregulates IgA secretion in
+
CD4 PP cells in the intestinal mucosa, and suppresses the systemic Th2 dominant immune response.
Therefore, dietary supplementation with bacterial components from probiotics or prebiotics will modulate
both innate and adaptive immunity to prevent infectious disease and allergy.
Keywords: immunomodulation, Peyer's patch, probiotics, prebiotics
국제학술대회
97
October 13-14, 2005, Seoul, Korea
S10-2
Transformation of Various Functional Glycosides Using Probiotic
Bacteria and Food-Grade Fungi
Geun Eog Ji
Department of Food and Nutriotin, Seoul National University & Research Institute, Bifido Inc. Korea
We explored the possibility of transforming various functional glycosides using probiotic bacteria and edible
food-grade fungi. We suggest that the proper combination of food microorganisms and transformation conditions
improve the functionality and the sensory value and reduce the cytotoxicity of the functional glycosides present in
various functional food raw materials.
1) Platycodon grandiflorum A. DC (Campanulaceae) is known to contain abundant glycoside conjugates of
platycodin saponins. Among the experimental microorganisms such as bifidobacteria, lactobacilli, leuconostocs,
yeasts and aspergilli, Aspergillus niger KCTC 6906 showed the greatest transformation of platycodin glycosides
during fermentation especially when the organism was incubated in the presence of rhamnose and platycodins. On
the other hand, among the cell extracts of various microorganisms the cell extracts of Bifidobacterium sp. Int57
showed the greatest transformation of the platycodin glycosides. The main site of partial-hydrolysis of 28-sugar
chains in platycodin D lied between rhamnose and xylose. The cytotoxicity on LLC (Lewis Lung Carcinoma), the
hemolytic toxicity, the sensory scores responsible for the pungency, bitterness and after-tastes were significantly
reduced by the hydrolysis of platycodin glycosides.
2) Ginseng saponins (ginsenosides) have been regarded as the principal components responsible for the
pharmacological and biological activities of ginseng. Among the various bacteria, Bifidobacterium sp. Int57
exhibited the most potent transforming activity of ginsenosides to 20-O-D-glucopyranosyl-20(S)-protopanaxadiol
(compound K), followed by Bifidobacterium sp. SJ32. In fungi, Aspergillus niger KCTC 6906 was more potent in
transforming ginsenosides to compound K than A. usamii var. shiro-usamii KCTC 6956. Ginsenoside Rb1 was
transformed into compound K via Rd and F2 by Bifidobacterium sp. Int57, Bifidobacterium sp. SJ32, Aspergillus
niger, and Aspergillus usamii. Lactobacillus delbrueckii, and Leuconostoc paramesenteroides transformed Rb1 into
Rh2 via Rd and F2. Bifidobacterium sp. SH5 transformed Rb1 into F2 via Rd. Re was transformed into Rh1 via Rg2
by Bifidobacterium sp. Int57 and Bifidobacterium sp. SJ32. A. niger transformed Re into Rh1 via Rg1. A. usamii
transformed Re into Rg2. Transformation of Rb1 proceeded at a higher rate and needed less amount of enzymes than
that of Re.
3) Soybeans and Puerariae radix (PR) are known to contain abundant glycoside conjugates of isoflavones. Among
the strains tested, Bifidobacterium sp. Int-57 showed the greatest daidzin hydrolysis activity. When yeast extract
(2.5%) was added to the PR medium during fermentation with Bifidobacterium sp. Int-57, all of the daidzin was
hydrolyzed to near completion. The addition of skim milk and whole milk also improved the conversion of daidzin
into daidzein. Puerarin, a C-glucoside of daidzin in PR, was not hydrolyzed into daidzein by any of the experimental
strains during fermentation. The isoflavone glycosides, daidzin and genistin, in soymilks were completely
hydrolyzed by Bifidobacterium sp. Int-57 in 18 hr. This study demonstrates that daidzin and genistin can be
efficiently converted into daidzein and genistein, respectively, by proper combination of probiotic strains and
fermentation conditions.
4) Trignoella foenum-graecum (TFG), a widely used medicinal and dietary herb, is abundant in saponins, such as
diosgenin. Its seeds are known for antidiabetic and hypocholesterolemic agents and also employed as a condiment
and improvement of appetite. TFG saponins were not fully transformed by live microorganisms in culture media but
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2005 International Meeting of the Federation of Korean Microbiological Societies
transformed by extracted crude microbial enzymes. Among those microbes tested, Bifidobacterium sp. Int57 showed
the greatest enzyme activities and effectively transformed TFG saponins. We examined the antidiabetic and
anti-inflammatory activity of TFG and its transformed saponins by Int57. Antidiabetic activity was measured by
assessing the inhibition of  -glucosidase and  -amylase. The Griess assay was used for the measurement of nitric
oxide scavenging. Deglycosylated fenugreek saponins inhibited  -glucosidase and pancreatic  -amylase activity
higher than glycoside saponins at the concentration of 0.1 mg/ml. The Griess assay indicated that TFG saponins are
more potent in nitrite scavenging activity than its metabolites, especially, at pH 1.2. Overall, these finding suggested
that transformed TFG saponins using microbe enzymes may contribute to hypoglycemic effect and to the
improvement of various bioavailability.
국제학술대회
99
October 13-14, 2005, Seoul, Korea
S10-3
Lactic Acid Bacteria and Toll-like Receptor
Han Geun Kim and Dae Kyun Chung*
School of Biotechnology and Institute of Life Science and Resources, Kyung Hee University, Suwon, Korea
Lactic acid bacteria (LAB) are known for their health-promoting effects such as nonspecific enhancement
of the immune system, protection against intestinal infection, decreasing cholesterol levels in serum, and
antioxidative activity. Especially it is well known that LAB contributes to the health of the host by immune
modulation. The mechanism of such immune modulations are not clearly known, but it has been demonstrated
that the cell components of these bacteria contain immuno-modulatory components such as cell surface
components and peptidoglycan that may play important role in activating immune-competent cells in the
intestine. Several LAB strains were reported to exert in vitro stimulatory properties on cells of the innate
immune system, including macrophages and natural killer cells. They induced cytokine production such as
IL-12, IL-10, TNF- , TGF-β, IL-8, and RANTES, and cell proliferation of human intestinal epithelial cells.
Interestingly substantial differences were found among LAB in their capacity to induce a specific cytokine
profile. Cell surface molecules such as Toll-like receptor (TLR) and CD14 interact with peptidoglycan or LPS
to control expression of several specific, inducible immune responses. Signaling pathway initialized from the
recognition of conserved unique products from microorganism by TLR maturates antigen-presenting cell
(APC), and then matured APC generates pro-inflammatory cytokines such as IL-12, IL-23, and IL-27, which
promote Th1 cell proliferation. Because LAB can be efficient immune modulators, certain strains could be
particularly useful for delivery of biotherapeutics and vaccines.
Keywords: lactic acid bacteria, toll like receptor
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S10-4
Monitoring of Lactic Acid Bacteria in Functional Starter-Inoculated
Kimchi Fermentation Using Microarray Technology
Yong-Ha Park
Biological Resource Center, KRIBB, Yousung, Daejon, Korea
Kimchi, a kind of pickled vegetable, was first created as a representative food in Korea around the 7th
6
century. At the moment, more than 1.5×10 tons of Kimchi is consumed per year in South Korea, a
phenomenon that is now rapidly growing and spreading to other Asian countries. Despite its great impact on
Asian health, the microbiology of Kimchi has only been explored by non-quantitative PCR-based pattern
analyses. In order to find correlations between Kimchi types and their bacterial compositions, genome probing
microarrays (GPM) comprising 150 distinct lactic acid bacterial (LAB) genomes deposited on a glass
slidehave been developed to meet the pressing need for a more sensitive, quantitative and high-throughput
analysis tool. Compared to current oligonucleotide microarrays, GPM offers a specificity approaching that of
the species-specific level with a sensitivity of 0.25 ng of genomic DNA(100 times higher sensitivity). In an
assessment of the applicability of GPM in monitoring the community dynamics of lactic acid bacteria, about
100 species were quantitatively analyzed and shown to be actively involved in Kimchi fermentation. Several
species from the genera Lactobacillus, Leuconostoc and Weissella were also found to be the most dominant
microflora in Kimchi fermented at 4°C. GPM profiles of Kimchi samples evolved significantly after 7-9 days
of fermentation, showing that some Streptococcus and Lactobacillus species disappear after a decrease in pH.
Recently, we were able to design Kimchi with special features, such as anti-Corona virus or anti-Helicobacter
activity, by using probiotic Lactobacilli as starter cultures. In order to monitor whether starter LAB can
proliferate in the fermented Kimchi or not, we are fabricating starter strain-specific microarrays by using genes
amplified after subtractive suppression hybridization as microarray probes.
Keywords: Lactic Acid Bacteria, Kimchi, Fermentation, Microarray Technology
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October 13-14, 2005, Seoul, Korea
S11-1
Overview of Viral Infectious Diseases in Korea
Hae Wol Cho
National Institute of Health, Seoul, Korea
Today, various kinds of infectious diseases are emerging everyday and we are facing an unprecedented era
of emerging and re-emerging infectious diseases. The World Health Organization predicts that the next
pandemic of Influenza may kill tens of millions of people and the ADIS epidemic continues to add more
people carrying HIV today. The research on infectious diseases is one of the most urgent and important area
of biomedical sciences. We must control communicable diseases in order to make the world better place to
live. Therefore, we will explore the occurrence of main viral infectious diseases present in Korea and defense
methods against these diseases.
Back in the 1970s and before, smllpox, polio, measles, mumps, Japanese encephalitis were major acute
infectious diseases in Korea. Since 1970s rapid economic growth in Korea improved living conditions in this
country. Accordingly, in mid 1970s vaccine were introduced to Korea which dramatically decreased the
occurrence of these diseases.
One of the aims of the World Health Organization is elimination or eradication of infectious diseases. In
1980, smallpox was completely eradicated in nature for the first time. Afterwards, WHO initiated the global
eradication of poliomyelitis in 1988. There has been no report of poliomyelitis in Korea since the last five
cases of polio in 1983. However, the Republic of Korea introduced Acute Flaccid Paralysis (AFP) surveillance
in 1998 with 70 reporting hospitals, and this surveillance system has been operated under the coordination
of the National Institute of Health (KNIH) to meet requirements for certification of poliomyelitis eradication.
Recent importation cases of wild poliovirus from Nigeria to Yemen and Indonesia, which have been polio-free
for about 10years also demonstrated that sustaining high polio immunization status is critically important to
maintain polio-free status. Therefore, sustaining high polio immunization coverage rate and maintaining the
AFP surveillance system, even in countries that have already achieved polio eradication, are essential for
global eradication of poliomyelitis.
During the 1970s - 1980s Measles were occurred 4,000 - 6000 cases annually.
As a result of mass immunization in 1985, the report of measles has been decreased under 1,000 case per
year. However, in 1994 explosive measles outbreak occurred in which the reported cases were 7,883. The
recent nationwide measles outbreak during 2000-2001 caused approximate 50,000 cases of suspected measle
patients. National Immunization Programme in Korea decided to conduct a nationwide measles catch-up
supplementary immunization activity campaign, targeting all children aged 8-16 years old. The Republic of
Korea started a routine two-dose measles-mumps-rubella vaccination with 95% coverage, added a school-entry
requirement certification in 2001, held a catch-up campaign for children 8-16 years and increased surveillance
to monitor elimination. The comprehensive surveillance programme was clearly and precisely delineated, and
a strict case definition of measles was developed. Republic of Korea has made significant progress towards
achieving elimination of measles and plans to undertake a final evaluation in 2005. The emphasized area
during this fourth year is enhancing and evaluating measles surveillance after the national measles
immunization campaign of 2001.
The number of Japanese encephalitis (JE) cases and deaths reported during the past 30 years, 1949 through
1983, in the republic of Korea has shown certain fluctuations. Initial period, from 1949 to 1958, epidemic
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JE outbreaks were noticed every three years with over 1,000 cases for nine consecutive years. The reduced
case level, which is considered to be a recent JE trend, has persisted until 1973 and 1982 when the JE cases
increased with a total of 769 and 1,197 cases reported, respectively. In the incidence of JE in 1982, it was
noticed that outbreak occurred in an unusually large scale and the age pattern of patients had changed. JE
usually occurred dominantly in children under 14 years old, but the age group shifted towards an older age
group. In this regards, we have to look carefully at the age patterns of JE patients in the future in connection
with mass vaccination programs. Since the efficacy of JE vaccine has been clearly proven in field trials and
serological studies in Korea, there is hope for effective vaccination controling outbreaks of JE among people.
AIDS is another important viral disease which needs continuous monitoring, prevention and publicity. In
1985, the first infected person of Human Immunodeficiency Virus (HIV) was reported. Since 1987, blood
centers performed screening tests from blood sample of more than two million people for HIV. By tests before
surgery in clinics and hospitals, health checkups, tests on pregnant women and volunteer-based tests in public
health centers, nearly two million tests were performed every year. Based on the HIV infection rate in these
epidemiological groups, the prevalence rate of HIV in Korea is estimated to be 0.01-0.1%. The number of
HIV-infected persons was increased by 30 to 40% than that of previous year, but there was 15% increase
in 2004 and 5% in 2005 increase compared to the same period of previous year, respectively. The male to
female ratio is 10 to 1, and most of males infected inside Korea are characterized to have been infected by
HIV subtype B.
Influenza is one of the several diseases causing fever and respiratory symptoms that might raise suspicions
of SARS. However, influenza is of particular concern because of its potential for institutional and community
outbreaks and regional epidemics. Influenza typically infects 10% t0 20% of the total population during
seasonal epidemics, resulting in three to five million cases of severe illness and at least 250,000 to 500,000
deaths each year worldwide. The Korean government has extended the nationwide influenza surveillance by
launching Korean Influenza Surveillance Scheme (KISS) to monitor the trends of influenza activities and to
detect influenza epidemics as early as possible in Korea. KISS consists of two components, clinical and
laboratory surveillance, two input lines are required.
The presence of avian influenza in humans is of concern worldwide because humans have no immunity
against the H5N1 strain of avian flu, which is more virulent than the 1997 version that killed six people in
Hong Kong. There are several unknowns, however, including when or whether a pandemic will occur and,
if so, the level of reassortment that may take place as the virus crosses interspecies barriers. Although we
do not know the evolving rate of the virus, it has been known to be moderate. Since 1997 there have been
only 106 cases and 54 deaths worldwide, which indicates that the virus has not evolved to the point that it
would readily infect humans. However, this infectious virus continues to circulate among chickens and ducks,
thereby posing the threat of a pandemic.
Recently, the emerging-reemerging infectious diseases have been threatening human beings globally. Most
of these diseases are caused by viral agents such as SARS, Avian influenza, Niphavirus, Dengue and etc.
The appearance of emerging-reemerging viruses has been resulted by their survival mechanisms through
endless mutations which will further invade human beings in the future.
These diseases have resulted in a great number of deaths and economical destruction in the world.
Therefore, stronger national preparedness of health care against the attacks by a new viral agent and
development of vaccines and antivirals are more than necessary.
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S11-2
Does SARS Spread by the Airborne Route? A Review of Several
Major SARS Outbreaks in Hong Kong
Tze Wai Wong
Department of Community and Family Medicine, The Chinese University of Hong Kong, 4/F, School of
Public Health Prince of Wales Hospital Shatin, BT Hong Kong
The epidemic of Severe Acute Respiratory Syndrome (SARS) in 2003 left many questions unanswered.
Although scientists worldwide have been quick in identifying the causative virus the SARS coronavirus
(SARS-CoV), the epidemiological features surrounding the mode of transmission remain unclear in several
well-known outbreaks: (i) the Metropole Hotel outbreak, which kick-started the spread of the disease from
Guangzhou in Southern China to Hong Kong and from there to many countries of the world, (ii) the Prince
of Wales Hospital (PWH) outbreak, one of the earliest hospital outbreak, and (iii) the Amoy Gardens outbreak,
the largest community outbreak of SARS and most mysterious of all in terms of mode of transmission.
Descriptive epidemiological features (temporal and spatial distribution) of this outbreak led to several
hypotheses regarding the mode of transmission, and an airborne route have been suggested. I shall describe
the key epidemiological features of these outbreaks, and examine the various hypotheses regarding the mode
of transmission of SARS in Amoy Garden and in Ward 8A of the Prince of Wales Hospital outbreak. The
objective of this study is to examine the evidence for and against these hypotheses, so that we can get a
better understanding of the mode of transmission of SARS under different environmental conditions.There is
strong evidence that aerosols played an important role in disease transmission in the PWH outbreak. Fro the
Amoy Gardens outbreak, epidemiological data collected thus far are in support of an aerosol hypothesis of
spread, although other modes of disease transmission cannot be definitely eliminated.
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S11-3
Hepatitis E Virus-An Emerging Virus in Industrialised Countries
Tim J. Harrison
Windeyer Institute of Medical Sciences, University College London, London, UK
Hepatitis E originally was thought to be a disease limited to developing countries. The hepatitis E virus
(HEV) genome was cloned in 1990 from two closely related viruses implicated in epidemics in Burma
(Myanmar) and Mexico and referred to originally as Old World and New World isolates. Other virus isolates
from epidemics and sporadic cases in Asia and Africa resembled the Burmese prototype closely and these
are now recognised as Genotype 1 viruses. Partial HEV sequences described more recently from Nigeria
resemble the Mexican prototype (Genotype 2). Later, rare cases of hepatitis E in the USA in patients who
had not travelled abroad were found to be attributable to Genotype 3 viruses which are endemic in domestic
swine. Genotype 3 viruses been found in pigs from five continents and rarely may be transmitted to humans.
A fourth HEV genotype has been described in mainland China, Taiwan, Vietnam and Japan. Again, this virus
is found in domestic swine but may be transmitted to humans. Rare cases of hepatitis E in the UK are in
travellers returning from endemic areas, and attributable to genotype 1 HEV, but also occur sporadically in
individuals who have not travelled. HEV sequences derived from non-travellers are closely related to those
found indomestic swine in the UK. Many such cases may be asymptomatic and, therefore, undetected.
Epidemics of severe hepatitis E in humans are caused by Genotype 1 viruses (and Genotype 2 in Mexico)
and Genotypes 3 and 4 have not been implicated in epidemics. It is not clear whether Genotype 1 (and 2)
HEV are truly human viruses or may also be zoonotic, withreservoirs in other species of mammals.
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S12-1
Host Vector Systems of Piezophilic Bacteria, and Construction of
Pressure Sensing Cells
Takako Sato
Extremobiosphere Res. Ctr., JAMSTEC, Yokosuka, Japan
Deep-sea bacteria are adapted to extreme environments, such as high pressures and cold temperatures. We
have isolated many piezophiles which grow well even under high pressures from deep-sea sediment.
Shewanella violacea DSS12 and Moritella japonica DSK1 have the ability to grow at up to 70 MPa, and
1)
those bacteria have unique mechanisms of gene expression in response to high pressure conditions . Recently,
green fluorescent protein (GFP) from Aequorea victoria has been developed as a reporter protein, and it is
an attractive candidate to monitor "real time" gene expression in living bacteria. Thecombination of gene
expression systems in piezophiles, like the high pressure-dependent promoters and GFP reporter gene, may
reveal highly fluorescent cells when exposed to high hydrostatic pressure conditions. It is predicted that a
novel bio-sensing system can be made to probe high pressure environments using living bacteria.
First, gene transformation into our piezophiles, strains DSS12 and DSK1, were examined. Eschericha coli
S17-1, which has the ability to transfer bearing plasmid DNAs to other microorganisms, was used for bacterial
conjugation with those piezophiles. Since the optimum growth temperatures of both E. coli and deep-sea
piezophiles are different, the transformation efficiency was fairly low compared with other bacterial
conjugations. As a result, the broad host range vector, pKT231, and the shuttle vector, pTH10, were
2)
successfully introduced to DSS12 and DSK1, respectively .
Next, the fluorescent tolerance of GFP tohigh pressure was confirmed as a reporter gene in the living
organisms. The fluorescent spectrum profile of intensity ratio and wavelength of GFP up to 100MPa was
almost the same as the under atmospheric pressure. Therefore, we consider that GFP is suitable as a high
pressure reporter protein. The pressure regulated promoters from DSS12 and DSK1 were cloned into
propervectors and combined with GFP as a reporter gene downstream of each promoter. The transformants
of DSK1 and DSS12 with the recombinant pTH10 and pKT231 plasmid, which has cadA and glnA
promoters(each of them is a pressure regulated promoter from DSK1 and DSS12, respectively) and GFP, were
grown under high pressure and gene expression of GFP promoted by 50 MPa pressure was confirmed. This
is a critical point to create a pressure-sensing bacteria which will indicate the level of environmental pressure
3)
using fluorescence of GFP as a reporter gene .
1) Nakasone K, Ikegami A et al: "Transcriptional regulation under pressure conditions by the RNA
54
polymerase σ factor with a two components regulatory system in Shewanella violacea" Extremophiles
6 (2002) 89-95
2) Fukuchi J, Sato T et al: "The host-vector system for deep-sea piezophilic bacteria, Shewanella violacea
DSS12 and Moritella japonica DSK1" JAMSTECR 46 (2002) 157-161
3) Sato T, Fukuchi J: "High pressure activated glow cells" Kaiyo Monthly 36 (2004) 665-672
Keywords: Deep-sea bacteria Shewanella violacea, Moritella japonica, pressure-sensing bacteria
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S12-2
Assessment and Utilization of Marine Microbial Diversity by Using
Metagenomic Study
Jung-Hyun Lee*, Jeong Ho Jeon, Hyun Sook Lee, Seung Seob Bae, Jae Kyu Lim,
Sung Gyun Kang, and Sang-Jin Kim
Marine Biotechnology Center, KORDI, Ansan, Korea
Recent studies onmarine environments have led to significant discoveries of unusual microbial diversity,
genetic potential and metabolic activity in a variety of marine habitats cold waters, sinking particles,
sediments, and animal guts or other biological surfaces (at temperatures from -1.7℃ in polar oceans to ~4℃
at temperate and tropical latitudes); hydrothermal vent environments that encompass a wide range of
temperatures (2-350℃) and nutritional conditions (organic compounds, reduced chemicals, heavy metals); and
seafloor habitats in deeply buried sediments (>500m below the seafloor). Among the various microbial
habitats, the marine sediment of coastal area and deep sea are considered to be eutrophic habitats rich in
nutrients which may contain higher level of microbial diversity. The access of microbial diversity by culture
techniques has been limited due to low cultivability less than 1%. An alternative procedure for the obtaining
of the collective microbial genome in a given habitat (termed as metagenome) has been extensively studied;
i.e. extracting total DNA and cloning it into proper vector systems.
We have collected samples from varioussediments of deep-sea clam beds community (depth, 1,400 m) in
low-temperature venting area located in western Pacific area, intertidal zone of Ganghwa-island, and coasts
of Korean Arctic Station, Dasan in Svalbard, Norway. The environmental DNAs of high quality and long size
(>30 kb) werecloned into a fosmid vector system. The library contained 220,000 clones with average size
of 31 kb, which equivalent to 6,820 Mb in total. The individual clone in the metagenomic library from a
special microbial community of clam beds area has been analyzed by end-sequencing and useful enzymatic
activity was screened. The end-sequences (~500 bp in length) of 223 clones were obtained and sequence
analysis was accomplished. The sequences of 101 clones (45%) have showed similarity to proteins from γ
-Proteobacteria (32%),  -Proteobacteria (10%), β-Proteobacteria (9%),  -Proteobacteria (5%), Firmicutes
(4%), CFB group (15%), cyanobacteria (6%), actinobacteria (3%), euryrarchaeta (6%), and other groups (10%)
with a value less than e-20. Two clones were selected for showing hydrolyzing activity; one lipase and one
protease, and the lipase clone has been further analysed. Full sequence of insert DNA of the lipase clone
has been determined to predict the origin of lipase activity. This study has shown that metagenomic approach
could be used as new genetic resources for obtaining metabolic activity.
Keywords: Marine metagenomics, Deep-sea sediment
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October 13-14, 2005, Seoul, Korea
S12-3
Protein trans-Splicing and Characterization of a Split Family B-type
DNA Polymerase from the Hyperthermophilic Archaeal Parasite
Nanoarchaeum equitans
Jeong Jin Choi1, Ki Hoon Nam1, Bokkee Min2, Sang-Jin Kim3, and Suk-Tae Kwon1,*
1
Department of Genetic Engineering, Sungkyunkwan University, Suwon 440-746, 2Department of Clinical
3
Laboratory Science, Eulji University, Daejeon 301-832, Microbiology Laboratory, Korea Ocean Research
and Development Institute, Ansan 425-600, Korea
The domain Archaea is recognized as constituting a third major branch of living organisms, together with
Bacteria and Eukarya. From a phylogenetic perspective on the basis of rRNA sequences, the Archaea has been
classified into three phyla, the Crenarchaeota, Euryarchaeota and Korarchaeota. Recently, a novel phylum of
the Archaea was identified, namely the Nanoarchaeota (1). Nanoarchaeum equitans Kin4-M, representative of
the new phylum, was isolated from a submarine hot vent at the Kolbeinsey ridge, north of Iceland. N. equitans
is a nano-sized, hyperthermophilic anaerobe. This organism is an extremely tiny cell, only about 400 nm in
diameter, growing on the surface of a specific crenarchaeal host, Ignicoccus sp. strain KIN4/I. N. equitans
can only be cultivated in coculture with Ignicoccus sp. strain KIN4/I, and the growth of these cells occurs
under strictly anaerobic conditions between 70 and 98°C (optimally 90°C). The genome (490,885 bp), one
of the smallest microbial genomes, of N. equitans has completely been sequenced (2). N. equitans possesses
components for information processing and repair, but lacks genes for the de novo biosyntheses of amino
acids, nucleotides, lipids and cofactors. It has been deduced by the analyses of the genome sequence and
membrane lipid composition that N. equitans is a parasite for Ignicoccus sp. strain KIN4/I. N. equitans is
regarded as the most ancient archaeon because of its parasitic nature and its deepest location in the archaeal
phylogenetic tree.
Inteins are protein insertion sequences that are embedded in-frame within precursor protein sequences and
must be removed during splicing process of the precursor protein. Protein splicing is a post-translational
processing event in which the intein is precisely self-excised from a precursor protein with concomitant
ligation of the flanking protein sequences, exteins, by a normal peptide bond. Naturally occurring inteins can
be grouped into three types according to the structural organization (3): inteins that have both self-splicing
and homing endonuclease domains, mini-inteins that lack the endonuclease domain, and split mini-inteins in
which the splicing domain exists as a split form on two separate genes, and are therefore spliced in trans.
N. equitans family B-type DNA polymerase (Neq DNA polymerase) is encoded by two open reading frames
(ORFs), separated by 83,295 bp on the chromosome, including a deduced split mini-intein sequence (2).
Excepting the Neq DNA polymerase, the sequences of naturally occurring split mini-inteins, among about 180
known inteins, have been only found in several cyanobacterial family C-type DNA polymerase III α subunits
(DnaE proteins), including experimentally shown in Synechocystis sp. PCC6803 (Ssp) DnaE protein. The
protein trans-splicing was first identified in the split mini-intein of Ssp DnaE protein, and its splicing
mechanism has been reported (4). In addition, Methanobacterium thermoautotrophicum (Mth) family B-type
DNA polymerase is splited, but two constituting polypeptides are active as a dimer because of the absence
of a self-splicing intein (5).
In this study, we report the protein trans-splicing and characterization of Neq DNA polymerase from the
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hyperthermophilic Archaeal parasite Nanoarchaeum equitans. Neq DNA polymerase is encoded by two
separate genes, the large gene coding for the N-terminal part (Neq L) of Neq DNA polymerase and the small
gene coding for the C-terminal part (Neq S), including a split mini-intein sequence. The two Neq DNA
polymerase genes were cloned and expressed individually, together (for the Neq C) and as a genetically
protein splicing-processed form (Neq P) in Escherichia coli. The protein trans-spliced Neq C was obtained
using the heating step at 80°C after the co-expression of the two genes. Neq S has no catalytic activity and
Neq L has the lower 3′→5′ exonuclease activity; whereas Neq C and Neq P have polymerase and 3′→5′
exonuclease activities, indicating that both Neq L and Neq S are needed to form the active DNA polymerase
possessing the higher proofreading activity. The genetically protein splicing-processed Neq P showed the same
properties with the protein trans-spliced Neq C. The protein trans-splicing of the N-terminal and C-terminal
parts of Neq DNA polymerase was examined in vitro using the purified Neq L and Neq S. The trans-splicing
was mainly influenced by temperature, and occurred only at temperatures above 50°C. The trans-splicing
reaction was inhibited in the presence of zinc ion. These results are the first evidence showing experimentally
that the protein trans-splicing occurs in archaeal protein, thermostable protein and family B-type DNA
polymerase.
Acknowledgments
This work was supported by the Marine and Extreme Genome Research Center Program, Ministry of
Maritime Affairs & Fisheries, Republic of Korea.
Keywords: B-type DNA polymerase,Nanoarchaeum equitans,Neq DNA polymerase,protein trans-splicing,
hyperthermophilic archaeal parasite
References
1. Huber, H., Hohn, M. J., Rachel, R., Fuchs, T., Wimmer, V. C., and Stetter, K. O. (2002) A new phylum
of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63-67
2. Waters, E., Hohn, M. J., Ahel, I., Graham, D. E., Adams, M. D., Barnstead, M., Beeson, K. Y., Bibbs,
L., Bolanos, R., Keller, M., Kretz, K., Lin, X., Mathur, E., Ni, J., Podar, M., Richardson, T., Sutton,
G. G., Simon, M., Söll, D., Stetter, K. O., Short, J. M., and Noordewier, M. (2003) The genome of
Nanoarchaeum equitans: insight into early archaeal evolution and derived parasitism. Proc. Natl. Acad.
Sci. U.S.A. 100, 12984-12988
3. Martin, D. D., M.-Q. Xu, and T. C. Evans, Jr. 2001. Characterization ofa naturally occurring
trans-splicing intein from Synechocystis sp. PCC6803. Biochemistry 40:1393-1402.
4. Evans, T. C., Jr., D. Martin, R. Kolly, D. Panne, L. Sun, I. Ghosh, L. Chen, J. Benner, X.-Q. Liu, and
M.-Q. Xu. 2000. Protein trans-splicing and cyclization by a naturally split intein from the dnaE gene
of Synechocystis species PCC6803. J. Biol. Chem. 275:9091-9094.
5. Kelman, Z., S. Pietrokovski, and J. Hurwitz. 1999. Isolation and characterization of a split B-type DNA
polymerase from the archaeon Methanobacterium thermoautotrophicum  H. J. Biol. Chem.
274:28751-28761.
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S12-4
Microbial Phospholipase D : Application for Functional
Phospholipid Production
Jin-Cheol Yoo
Department of Pharmacy, College of Pharmacy, Chosun University, Kwangju 501-759, Korea
Phospholipase D (PLD; EC 3.1.4.4) is an enzyme that occurs ubiquitously in various organisms including
mammals, plants, yeast and bacteria. The enzyme catalyzes two types of reactions (Fig.1): hydrolysis of
phosphatidylcholine (PC), a major substrate of PLD, to phosphatidic acid (PA) and choline (hydrolytic
reaction), and transfer of polar head groups to others (transphosphatidylation reaction).
Fig. 1. Schematic representation of PLD reactions.
Phospholipids, as an abundant lipid component in nature, possess glycerol structure with a polar head
moiety and two fatty acid chains.This amphiphilic property facilitates the application of phospholipids in
various industrial fields such as pharmaceuticals, cosmetics, and foods. Phosphatidylserine (PS) is used as a
liposome for drug delivery systems (DDS), a surfactant, an emulsifier for cosmetics, and a dietary supplement
for foods. Additionally, PS is known to improve memory performance in patients suffering from
age-associated memory impairment or Alzheimer's disease. The enzymes from Actinomycetes show higher
transphosphatidylation activity than those from other sources and exhibit broad substrate specificity to
phospholipids. It is therefore expected that the Actinomycetes PLD is applicable for synthesizing various
phospholipids from inexpensive PC in industrial fields. The genes coding for PLD were cloned from bacteria,
plants, yeast, and mammals, and their nucleotide sequences were determined. The bacterial genes encoding
PLD were cloned from the chromosomal DNA of various Actinomycetes strains. The polypeptide length of
bacterial enzymes (500600 amino acid residues) are usually shorter than those of the eukaryotic enzymes
(>1000 amino acid residues). The amino acid sequence similarity among bacterial enzymes is about 70%. The
similarity between the Streptomyces PLD and eukaryotic enzymes is less than 30%. The primary structures
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of eukaryotic PLD contain four highly conserved regions (regions I-VI). Bacterial PLD enzymes lack region
III, which is postulated to possess a regulatory function for eukaryotic enzymes. Within regions I and IV,
a highly conserved sequence, HxKxxxxD.
Recently, we have screened PLD-producing Actinomycetes species, and evaluated several PLDs in culture
supernatants. Among them, two Actinomycetes strain, Streptomyces Cs57 and Streptomyces Cs 528, release
high activity of PLD in the medium. Subsequently, two enzymes werepurified from the culture medium and
one PLD gene was cloned from Streptomyces CS 57. We are developing, for the first time, an efficient
expression system for the active PLD and industrial phospholipid synthesis system. The use of PLD gene from
extreme microorganisms will further improve the phospholipid synthesis.
Keywords: phospholipase D, phospholipids, streptomyces, purification, gene cloning, protein engineering
References
1. T. Hatanaka, T. Negishi, M. Kubota-Akizawa and T. Hagishita1, Purification, characterization, cloning
and sequencing of phospholipase D from Streptomyces septatus TH-2, Enzyme Microb Techno 31 (2002),
pp. 233241.
2. Chiaki Ogino, Shun'ichi Kuroda, Shinji Tokuyama, Akihiko Kondo, Nobuaki Shimizu, Katsuyuki
Tanizawa and Hideki Fukuda, Phospholipase D from Streptoverticillium cinnamoneum: protein
engineering and application for phospholipid production Journal of Molecular Catalysis B: Enzymatic,
23(2003), pp. 107-115
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S12-5
Diversity and Application of Arctic and Antarctic Marine Bacteria
Yoo Kyung Lee*, Joung Han Yim, and Hong Kum Lee
Korea Polar Research Institute, KORDI, Ansan, Korea
The Arctic and the Antarctic are representative cold habitats that offer good sources of useful enzymes with
activity at low temperature. At low temperature, rate of enzymatic reactions, fluidity of cellular membrane,
and affinity of uptake and transport systems decrease. Therefore, biomolecules of organisms living in cold
habitat may show distinctive physical properties. Study on bacterial diversity of the Arctic and the Antarctic
provides potential benefits by storing new gene poolas well as by finding new bacterial taxa. The
understanding bacterial diversity also gives new insights into the biological mechanisms of adaptation to and
tolerance of cold environments. In this study, we report Arctic and Antarctic bacteria that inhabit around the
Korean Arctic Research Station Dasan and the Korean Antarctic Research Station King Sejong. We also give
examples of useful bacteria such as protease-producing Arctic bacteria and a chitinase-producing Antarctic
bacterium.
Arctic vs Antarctic Bacteria
Various bacterial strains were isolated from marine environment around the Korean Arctic Research Station
Dasan located at Ny-Alesund, Svalbard, Norway (79°N, 12°E), and the King Sejong Station in on King
George Island, Antarctica (62°S, 58°W). The collected samples were diluted in distilled seawater, and spread
on marine agar plates. They were cultured at 10℃ and 25℃, and bacterial isolates were preserved in glycerol
media (15%, v/v) at-80℃. Phylogenetic analysis of 16S rDNA sequences indicated that the marine bacteria
belonged to alpha-, beta-, and gamma-Proteobacteria, CFB group, high GC Gram-positive bacteria, and low
GC Gram-positive bacteria. Although specific species names of the cultivable Arctic bacteria were quite
different from Antarctic species, general composition of Arctic bacteria were similar to that of Antarctic
bacteria (Fig. 1). Among the marine bacteria, we found several candidates for new species or genus. The
closest cultured bacteria with validly published names belonged to Cellulophaga, Colwellia, Devosia,
Janibacter, Polaribacter, Plantibacter, Rheinheimera, Roseobacter, Sanguibacter, Subtercola, and Thiobacillus.
Useful bacteria
We screened protease- or chitinase-producing bacteria. Protease-producing Arctic bacteria shared high 16S
rDNA sequence similarities with Pseudoalteromonas elyakovii, Exiguobacterium oxidotolerum and
Pseudomonas jessenii (Fig. 2). We also isolated a cold-adapted chitinase-producing bacterium, Sanguibacter
sp. from the Antarctic. The chitinase was active at broad temperature range from 0℃ to 37℃. It contained
about 71% of relative activity at 0℃ compared with 100% at 37℃. This bacterium could grow faster at higher
temperature; it produced much more chitinase enzyme at low temperature.
Further studies are needed to elucidate specific activity and optimal conditions for these Arctic and
Antarctic bacterial enzymes. We expect that they can be used to develop new industrial enzymes that are
active at low temperatures.
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Arctic
Antarctic
Fig. 1. Composition of Arctic and Antarctic bacteria cultivated from marine sediments.
Fig. 2. Arctic marine bacteria showing protease activity.
Keywords: Arctic, Antarctic, Marine bacteria, Diversity, protease, chitinase
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S13-1
Aspergillus fumigatus Cell Wall and Resistance to Host Defence
Reactions
Jean-Paul Latgé
Institut Pasteur, Paris, France
Aspergillus fumigatusis a world wide thermophyllic saprophytic species playing an essential role in
recycling the earth carbon and nitrogen. This fungal species has a very simple biological cycle ; one of its
characteristics is its high sporulating capacity which results in the ubiquitous presence of high concentrations
3
of conidia (1-100 co/m ) in the atmosphere, indoors and outdoors. Conidia of A. fumigatus are continuously
inhaled by humansand are eliminated efficiently by innate immune mechanisms. Human infections have been
however regularly reported since mid 1800's. Until 1980-1990, aspergillosis was considered as a rare disease,
occuring among people with prexisting cavities resulting from previous lung infection such as tuberculosis.
The exotic status of A. fumigatus has however changed over the last years owing to the increase in the number
of immunosuppressed patients and the degree of severity of immunosuppressive therapies. A. fumigatusis now
the most prevalent airborne fungal pathogen causing numerous, severe and usually fatal invasive aspergillosis
infections in bone marrow and solid organ transplant recipients and leukemic patients.
To date, the study of the virulence of A. fumigatusis based on the analysis of mutants constructed by gene
disruption, and the detection of attenuated virulence of the mutant through an animal screen No true virulence
factors viz a gene or a protein essential for growth in vivo whose deletion does not affect mycelial growth
in vitro, have been identified to date in A. fumigatus. Avirulence has been associated to genes such as PABA
or PyrG which deletion lead to auxotrophs unable to germinate inthe lung environment because uridine/uracle
and p-aminobenzoic acid are not freely available in the lung. Strains with reduced growth rate are less
pathogenic than wild type parental strains. All virulence studies suggested that virulence of A. fumigatus seems
polygenic. Further analysis of A. fumigatus virulence would require the identification of global regulators or
multiple genes expressed concomittantly in the same metabolic pathway. To date, the most efficient way to
reach this objective is a comparative transcriptome analysis of the fungus grown in vivo and in vivo. Such
approach is now feasible and has been undertaken since the sequence of the entire genome of A. fumigatus
is now available.
The pathobiology of A. fumigatus can be analysed also fromthe host side. In our laboratory we focused
on the response of the major phagocytic cell of the lung, the alveolar macrophage, since survival and
germination of A. fumigatus in the phagocyte are essential for fungal virulence. Conidial engulfment by the
alveolar macrophage (AM) is very quick (1-2h) and not affected by the immune status of the host. Killing
of conidia, mainly due to Reactive Oxidant intermediates (ROIs) starts 6-8 hours after phagocytosis. The
killing rate is surprisingly slow with only a 10% or less reduction of conidia viability after 6 hours of
phagocytosis. Immunosuppression by corticoids is associated to a reduction in ROI production that is
correlated to the intramacrophagic germination seen in these patients. Our working hypothesis was that
molecules produced by the fungus and fungal strutures can inhibit the host defence reactions. Attenuated
virulence of this fungus has been indeed associated to the disruption of genes involved in the resistance of
the conidium to its killing by the alveolar macrophage (AM) that is the most efficient resident phagocyte of
the lung. Two cell wall molecules promote resistance to phagocyte reactions: (1) hydrophobins that are
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responsible for the formation of the rodlet layer on the surface of the conidia and (2) melanin that is
responsible for the green colour of the A. fumigatus conidia. The melanin layer of the conidial cell wall
efficiently scavenges reactive oxygen species and plays a role in protecting the fungus against the phagocytic
reactions. Accordingly, mutants with white conidia resulting from the inactivation of ALB1, a polyketide
synthase essential for the synthesis of dihydroxynaphtalene melanin present on the surface of the A. fumigatus
conidia, were more efficiently damaged by phagocytesthan wild-type.
These finding have lead us to study in more details the structure and biosynthesis of the cell wall of A.
fumigatus. Indeed, the hyphal cell wall is essential for A. fumigatus to penetrate solid nutrient substrates and
to resist host cell defence reactions. The polymer organization of the A. fumigatus cell wall is very specific,
a specificity reflected also at the genomic level. The cell wall of Aspergillus fumigatus is mainly composed
of  1-3, glucan, branched β 1-3 glucan, linear. β 1-3, 1-4 glucan, chitin, and galactomannan. All these
polysaccharides are interconnected to make a three dimensional network responsible for the cell wall integrity.
Anecdotically, the galactomannan is one of the antigens that circulate in the biologicalfluids of the patients
and that has been at the basis of the development of an enzyme immunoassay developed in collaboration with
our laboratory, that is the best commercial diagnostic kit for early diagnosis of IA.
Membrane polysaccharide synthases and remodelases involved in the biosynthesis of the A. fumigatus cell
wall have been investigated in our laboratory. A special emphasis is directed towards proteins anchored to
the plasma membrane through a glycosylphosphatidylinositol (GPI) moiety since the first β 1-3
glucanosyltransferase described in the fungal kingdom and discovered in our laboratory has been shown to
be GPI-anchored. Comparative proteomic and genomic approaches have identified 6 GPI-protein families
common to both A. fumigatus and yeast. The function of the SPS2, CRH and GEL families putatively involved
in organizing the cell wall structure of A. fumigatus has been specially analysed. Mutations in the genes
coding for some members of these GPI-anchored proteins result in a reduced mycelial growth, an abnormal
conidiation with altered conidial survival, that can be responsible for a reduction in fungal virulence.
Among the 82 putative GPI-anchored proteins identified in A. fumigatus, no orthologues of the yeast
GPI-anchored proteins that covalently bind to cell wall polysaccharides were found. A. fumigatus also lacked
homologues of the yeast PIR proteins that are putatively bound to the β 1,3 glucans through an alkali-labile
bond. It had been hypothesized previously, based on work in yeast, that the linkage of proteins to cell wall
polysaccharides was important in establishing the three-dimensional polysaccharide network that constitutes the
skeleton of all fungal cell walls. Based on these comparative genomic analyses, it is more likely that binding
to polysaccharides is merely a way for certain yeast proteins to remain at the surface of the cell wall to fulfill
their biological function in adhesion, flocculation, and mating -- all events absent in mold biology.
Hydrophobins, proteins not found in yeast, are the only cell wall linked GPI-proteins detected in the A.
fumigatus genome sequence, Hydrophobins play a major role in mold biology since they are required for air
transport and conidial survival. Allthe other GPI-proteins unique to A. fumigatus or common between A.
fumigatus and yeasts have putative glycosylhydrolase and transferase activities and thus are likely to play an
enzymatic role in cell wall construction.
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S13-2
Immuno-Modulating Effects and Anti-Cancer Activities of
Proteoglycan Isolated from Phellinus linteus: Phenotypic and
Function Maturation of Murine Bone Marrow-Derived Dendritic Cells
Gi-Young Kim1,*, Kyeong-Jun Lee1, Kwang-Sik Choi1, Ki-Wan Lee1, Moon-Soo Heo1,
2
2
Tae-Ho Lee , and Jae-Dong Lee
1
2
School of Applied Marine Science, College of Ocean Science, Cheju National University, Department of
Microbiology, College of Natural Sciences, Pusan National University, Korea
Proteoglycan (PG) isolated from Phellinus linteusare known to stimulate the proliferation of T lymphocytes
and humoral immune functions to act as a polyclonal activator of B cells, and to inhibit tumor growth and
metastasis. However, little is known about their immunomodulating effects or the effects of its mechanisms
on murine bone marrow (BM)-derived dendritic cells (DC). In this study, it profoundly increased CD80,
CD86, MHC I, and MHC II expression in murine, GM-CSF and IL-4 stimulated, BM-derived myeloid DC.
The ability of unstimulated DC to uptake dextran was higher than that of PG- or LPS-stimulated DC. We
analyzed the concentration of IL-12 secreted by DC using flow cytometry and ELISA. Untreated DC secreted
a low concentration of IL-12, while PG- or LPS-stimulated DC secreted higher levels of IL-12 than untreated
DC. There were no remarkable differences in the concentrations of IL-12 produced by PG- or LPS-stimulated
DC. However, polymyxin B (PB; an LPS inhibitor) effectively inhibited the surface molecules and IL-12
production induced by LPS, but had no effect on the PG in DC. PG-treated DC were much more potent
antigen-presenting cells in allogeneic immune response than untreated DC. PGtreatment not only formed
morphologically mature DC but also induced predominant migration to lymphoid tissues. Moreover, the
inhibitors of protein tyrosine kinase (PTK) or protein kinase C (PKC) significantly blocked the expression
of surface molecules and IL-12 production in PG-stimulated DC. Treatment of DC with PG directly induced
PKC activity and phosphorylated PTK.
DC are known to not only induce the activation of T cells, but are also associated with the polarization
of T cells. This study investigated whether or not proteoglycan (PG) isolated from Phellinus linteus induces
+
the phenotypic and functional maturation of CD11c DC in vitro and in vivo. PG was found to induce the
phenotypic and functional maturation of bone marrow-derived DC via Toll-like receptors (TLR) 2 and 4 in
vitro. Administration of PG in vivo strongly inhibited the MCA-102 tumor growth and increase in vivo. The
+
ratio of CD8 DC to CD8 DC increased, and PG enhanced IL-12 and IFN-γ production, and expression of
surface molecules including major histocompatibility complexes (MHC) classes I, MHC II, CD80, and CD86
in MCA-102-challenged mice. PG also caused a marked increase in the production of Th (helper T cells)-1
cytokine (IFN-γ) and a decrease in the production of Th-2 cytokine (IL-4) by splenic cells and inguinal lymph
+
+
node cells in MCA-102 tumor-bearing mice. Furthermore, PG stimulated the proliferation of CD4 and CD8
T cells. In addition, a combination of PG and tumor lysate-pulsed DC inhibited completely the growth of
MCA-102 cells in tumor-bearing mice. These results indicate that the administration of PG inhibited the
tumorgrowth through a mechanism leading to a Th-1 dominant immune state and the activation of
+
+
CD11c CD8 DC.
In summary, this study found that the administration of PG induced anti-tumor and immunomodulating
+
+
activities via maturation of CD11c CD8 DC in tumor-bearing mice. The inhibitory effect of PG on the
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growth of MCA-102 tumor cells is associated with its immunoregulatory properties, including the induction
of IL-12 and IFN-γ production leading to a Th-1 dominant state. These effects were made more pronounced
by the substantial expression of surface molecules. Therefore, PG would be useful in preventing tumor growth,
and it also has the advantage of having no side effects. Attempts to clarify the mechanism responsible for
inhibitory effects of PG on tumor growth are now under study.
Keywords: Phellinus linteus, Proteoglycan, Dendritic cells
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October 13-14, 2005, Seoul, Korea
S13-3
Isolation and Characterization of Fusarium proliferatum KGL0401
as a Gibberellin-Producing Fungus
Soon-Ok Rim, Jin-Hyung Lee, In-Jung Lee1, In-Koo Rhee2 and Jong-Guk Kim*
Department of Microbiology, 1Department of Agronomy, 2Department of Agricultural Chemistry, Kyungpook
National University, Daegu 702-701, Korea
GAs are included in a large family of diterpenoid compounds, some of which are bioactive growth
regulator, that control such diverse developmental processes as seed germination, stem elongation, leaf
expansion, trichome development, flower and fruit development. But the most major trait is the enhancement
of stem growth.
As well as being phytohormones, GAs are present in some fungi and bacteria. To date a total of 130 GAs
have been identified from natural sources. It was first report on GAs in 1828 that disease of rice plants could
affect on the elongation of stem excessively. The cause of rice disease was identified as fungus G. fujikuroi
by Hori in 1898. It was shown that the substance produced by G. fujikuroi was responsible for the disease
effect by Kurosawa in 1926. And crystallization of GA was succeeded in 1938 by Yubuta and Sumiki. GAs
are secondary metabolites of the fungus G. fujikuroi. The commercial source of the bioactive GAs was known
as GA1, GA3, GA4 and GA7 produced by fermentation of the fungus G. fujikuroi. GAs show commercially
precious applications. Most seedless grapes are grown with treatment of GA3. The rind of citrus fruit
tenderizes at maturity. By inhibiting senescence, GAs maintain the rind in better condition. A variety of
ornamental plants can be induced to flower either earlier than usual, or in off-seasons. In the brewing of beer,
malt production is time consuming step; 2-3 days may be saved by the addition of 25-500 ㎍ of GA3 for
each ㎏ of barley.
We isolated GAs-producing fungi from the root of various plants. 715 kinds of fungi were isolated from
the root of 73 kinds of plants. Fifteen strains of fungi were isolated from the roots of P. alkekengi var.
francheti. Four isolate (PA03, PA05, PA08 and PA15) were identified to have GAs-production activity by
Holbrock method. Bioassay was carried out with culture fluid of the four isolated fungi. The strain PA08
showed to have highest activity in bioassay and it had two times greater plant growth promoting activity than
G. fujikuroi. The fungal strain PA08 showed the highest productivity of GAs by GC-MS analysis and GAs
productivity of the strain PA08 was higher than G. fujikuroi. The fungal strain PA08 was identified as F.
proliferatum KGL0401 from the sequence analysis of ITS region.
The waito-c rices were treated with 10 ㎕ of various concentration 1 ng to 1 ㎎ GA3 and they were grown
at 30℃ for 7 days, and the height was measured with scale. When bioassay with culture broth of F.
proliferatum KGL0401 was carried out on waito-c rice, the height of waito-c rice became 28.5 ㎝ in 7 days.
But the amount of GA3 in the culture media of F. proliferatum KGL0401 was estimated to 143.7 ng/㎖ by
GC-MS analysis. Therefore it can be thought that the growth of waito-c rice was promoted by not only GA3
but also other kinds of GA and metabolic intermediates of GAs contained in culture fluid of F. proliferatum
KGL0401. It could be inferred that the quantity of GAs produced by F. proliferatum KGL0401 was gradually
increased as the cultivation was continued for 7 days.
The amount of GA1, GA3, GA4, GA7, GA9, GA20 and GA24 in the medium was measured by Gas
chromatography-mass spectrometer(GC-MS). The quantity of GAs produced by the isolate PA08 were 4.85
ng/㎖, 4.79 ng/㎖, 17.30 ng/㎖, 6.01 ng/㎖, 16.61 ng/㎖, 0.08 ng/㎖ and 17.30 ng/㎖, respectively.
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Proteomic and genetic analysis of Fusarium proliferatum KGL0401 was also carried out.
References
1. Neil Olszewki, Tai-ping Sun, and Frank Gubler. 2002. Gibberellin Signal: Biosynthesis, Catabolism, and
Response Pathways. The Plant Cell, S61-S80.
2. Peter Hedden and Andrew L. Phillips. 2000. Gibberellin metabolism: new insights revealed by the genes.
Trands in Plant Sci. 5: 523-530.
3. Peter Hedden, Andrew L. Phillips, Maria Cecilia Rojas, Esther Carrera, and Bettina Tudzynski. 2002.
Gibberellin Biosynthesis in Plants and fungi: A Case of convergent Evolution? J. Plant Growth Regul.
20: 319-331.
4. Rademacher W. 1997. Gibberellins. In: Anke T (ed) Fungal biotechnology. pp 193-205 Champman and
Hall, New York USA.
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October 13-14, 2005, Seoul, Korea
S13-4
A New Metalloprotease Gene from Pleurotus ostreatus Belongs to
Eucolycins Family
Chang Soo Lee
Department of Applied Biochemistry, College of Biomedical and Health Science,
Konkuk University, Chung-Ju, Korea
Pleurotus ostreatus known as oyster mushroom is one of the most widely cultivated edible mushrooms.
Because almost interested substances of P. ostreatusare specific to the fruiting body and human used the
fruiting body as food, the study of fruiting mechanism is important, but the molecular mechanism of fruit
body initiation and maturation is still poorly understood. Metalloproteases are involved in numerous
physiological functions, dependingon their substrate specificity and the tissues and cellular compartments in
which they are expressed. Although various metalloproteases are widely distributed from bacteria to mammals,
metalloproteases of higher basidiomycetes have received relatively little attention. Only a few metalloproteases
have been purified or cloned from mushroom. Some of them were characterized to belong to gluzinsins
(HExxH + E motif), aspzincins (HExxH + D motif), pitrilysins (HxxEH motif) or thimet oligopeptidase
(HExxH motif) family, but not to metzincins (HExxHxxGxxH + M motif) family. Since metalloprotease
members have a critical role in a broad spectrum of developmental processes (Stocker et al., 1995), we
reported the metalloprotease gene (PoMTP) belonging to the new eucolysins subfamily and the
characterization by quantitative analysis of mRNA (Joh et al. 2004). A metzincin family metalloprotease
cDNA (PoMTP) was cloned from Pleurotus ostreatus. Full-length cDNA sequence (1,140bp) of PoMTP
contained a 870bp open reading frame encoding a protein product of 290 amino acids. The deduced
amino-acid sequences of PoMTP containedan extensive zinc-binding consensus sequence and a so-called
Met-turn sequence which are typical for the metzincin family of metalloproteases. Four cysteine residues were
also observed in the zinc-binding region of PoMTP amino-acid sequence, which are known to be important
for the structure and the function of some subfamilies of the metzincins. Searches of the GenBank protein
database showed that the amino-acid sequence of PoMTP over the whole amino-acid sequence has a high
identity with metalloproteases or hypothetical proteins from one basidiomycete of Ustilago maydis, and four
ascomycetes of Magnaporthe, Metarhizium anisopliae var. anisopliae, Aspergillus nidulans and Neurospora.
Although no information about the function of these proteins was known at present, all these proteins have
an extended zinc-binding sequence and the Met turn sequence. Furthermore, the putative metzincin PoMTP
metalloprtease indicated no significant homology with any other prereported mushroom metalloproteases,
which are fibrinolytic metalloprotease, mitochondrial intermediate peptidase and mitochondrial processing
peptidase. Real-time PCR, qunatitative RT-PCR and Northern blot analysises indicated the PoMTP mRNA to
be abundant at primordial and fruit body stages, but scarce at the mycelial stage, suggesting that the PoMTP
metalloprotease plays an important role in mushroom fruiting.
Keywords: Pleurotus ostreatus, metalloprotease, mushroom development
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S13-5
Gene Transfer and Fruiting Body Development in Edible
Mushrooms
Young Bok Yoo1,*, Won Sik Kong1, Se Jong Oh1, Jong Chun Cheong1, Chang Sung Jhune1,
2
3
Beom Gi Kim , and Gyu Hyun Kim
1
Applied Microbiology Division, National Institute of Agricultural Science and Technology, RDA, Suwon
441-707, 2National Institute of Agricultural Biotechnology, RDA, Suwon 441-707, 3Department of
Biotechnology, Yonam College of Agriculture, Sungwan, Choongnam 330-802, Korea
Distinct methods of gene transfer in fungi areprtoplast fusion, uptake of foreign genetic materials and
transformation. Somatic hybrids of inter-compatible and inter-incompatible strains were obtained by protoplast
fusion. The fusion products between compatible strains, Pleurotus ostreatus and P. florida, formed
heterokaryons, while fusants between incompatible strains such as P. cornucopiae + P. florida, P. ostreatus
+ Ganoderma applanatum, P. florida + Ganoderma lucidum, and P. ostreatus + Flammulina velutipes formed
synkaryons that retained genes from both parents. The heterokaryons between compatible species showed the
same level of variability and contained both parental RAPD bands. In contrast, most of thesynkaryons between
incompatible species showed similarity to those of either parental bands and non-parental RAPD bands. There
are several factors related to the mechanism of clamp connection formation and fruiting body development
of synkaryons. Major one of them may be associated with self-fertility and mating type switching as like
homokaryotic fruiting of wild type P. ostreatus.
Transfer of the isolated nuclei from Lentinula edodes into protoplasts of Pleurotus florida was induced with
polyethlene glycol (PEG) and CaCl2. The intergeneric nuclear transfer products were classified into nuclear
hybrid, synkaryon, and reconstituted cell. Two synkaryon were analysed with distribution of progenies and
segregation of genetic markers by random spore analyses. The genetic markers were segregated into wild type
and riboflavine requiring auxotrophs.
Restriction enzyme-mediated integration (REMI) was used to transform uracil auxotrophs of Pleurotus
ostreatus to prototrophy. When protoplasts of P. ostreatus were treated by the reaction mixture containing
10 units of BamH1, efficiency was increased by 14.2 times compared with that of the conventional PEG
method. Uracil auxotrophs of P. ostreatus were isolated using the selectable marker, resistant to
5'-fluoro-orotic acid (5'-FOA). Two of the nine uracil auxotrophs obtained were transformed to prototrophy
using plasmid pTRura 3-2 that contains the orotidine monophosphate decarboxylase (ura3) gene from
Trichoderma reesei. Normal fruiting bodies were induced in the transformants by crossing them with wild-type
monokaryons, and the basidiospores collected from these fruiting bodies showed a biased segregation rate to
prototrophy.
The major species of Pleurotus are all bifactorial heterothallism. Single basidiospore isolates from fruiting
bodies are homokaryotic and self-sterile. However, we found that homokaryons derived from one strain could
develop fruiting bodies of three different types.. This review will discuss these aspects.
Keywords: Edible mushrooms, gene transfer, genetic recombination, protoplast fusion, synkaryons, transformation
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S14-1
HIV, SIV, and Resistance to Antibody-Mediated Neutralization
Ronald C. Desrosiers
Harvard Medical School. New England Primate Research Center, USA
HIV replication is continuous during all stages of infection. Although some individual cells in an infected
individual may be latently infected, persistent viral replication is generally believed to be the predominant
force responsible forthe prolonged disease course. It is now clear that HIV has evolved specific strategies to
evade ongoing immune responses and it is these immune evasion strategies that allow the continuous,
unrelenting viral replication. There are two facets to the evasion of humoral immunity. First is an inherent
resistance of natural isolates of HIV-1 to antibody-mediated neutralization. Primary isolates of HIV-1 and its
close monkey relative SIV are inherently difficult to neutralize even using antisera from individuals infected
by the same strain being used as the target of neutralization assays. This inherent resistance likely results from
the way the virus has constructed its outer envelope and from the stepwise two receptor entry system used
by the virus to gain access to the cell. The outer envelope gp120 proteins of HIV and SIV are among the
most heavily glycosylated proteins known to science. More than 50% of the mass of gp120 is carbohydrate.
This carbohydrate is believed to form a "glycan shield" that is difficult for antibodies to permeate.
Furthermore, the tightly-packed conserved domains are largely shielded by highly variable loops on the
exterior that are displaced during the stepwise conformational changes that occur in the process of viral entry.
The second facet to evasion of humoral immunity is escape. Naturally-infected humans and
experimentally-infected monkeys generally do make antibodies with some level of neutralizing activity, but
unfortunately this neutralizing activity is highly strain specific, i.e. specific for the particular envelope
sequence present on the virus with which an individual has become infected . Sequence variants appear during
the course of infection that result in escape from this neutralization. It is likely that the strain-specific
neutralizing activity is directed to the variable loops. Both facets of this resistance to antibody-mediated
neutralization pose formidable obstacles to antibody-based approaches to vaccine development.
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S14-2
Redundant Strategies of Negative Sense RNA Viruses to
Overcome the Innate Immune System of the Host
Peter Palese
Mount Sinai School of Medicine, New York, NY, USA
The innate immune or interferon (IFN) response is the primary host defense mechanism againstviral
infection. The induction of IFN synthesis in response to virus infection leads to the establishment of an
antiviral state through a highly coordinated signaling cascade, which serves to inhibit virus replication. Not
surprisingly, many (perhaps all) viruses have devised ways of preventing the induction of the IFN response
through the action of viral IFN antagonist proteins. Our research has focused on the IFN evasion strategies
of negative sense RNA viruses, specifically influenza and Nipah viruses. The demonstration that an influenza
virus lacking the NS1 was compromised for growth in IFN competent systems but grew well in IFN deficient
systems was the first indication of anti-IFN activity encoded by an RNA virus (Garcia-Sastre et al., Virology
252, 1324, 1998). When expressed alone, NS1 has been shown to prevent the transcriptional induction of IFN
by viruses, probably through its dsRNA binding domain. In addition it has been found that the influenza virus
NS1 protein inhibits IFN-signaling. Nipah virus has an even higher redundancy in countering the host's
antiviral system; it encodes four different IFN antagonist proteins. The P, V and W proteins act by binding
to STAT1 and thereby inhibit the action of IFN-signaling. The unique nuclear localization of the W protein
also gives it the ability to block IFN synthesis, while the Nipah virus C protein appears to be a general
transcriptional repressor. These multiple viral IFN antagonists likely act in concert to efficiently suppress the
host innate immune response and allow viral replication to proceed. Different viruses have evolved different
and redundant mechanisms by which to counteract the antiviral response of the infected host.
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S14-3
Induction and Functions of Viral Stress-Inducible Genes
Ganes C. Sen
Department of Molecular Biology/NC20, The Lerner Research Institute, The Cleveland Clinic Foundation,
9500 Euclid Avenue, Cleveland, OH 44195, USA
Innate immune response plays an important role in determining the outcome of virus infection in mammals.
An integral part of this response is mediated by proteins encoded by the viral stress-inducible genes (VSIG),
whose transcription is induced immediately after virus infection. Many of the same genes can also be
independently induced by double-stranded (ds) RNA and type I interferons. The different inducers use distinct
signaling pathways to activate specific members of the IRF-family of transcription factors that use the ISRE
elements present in the promoters of VSIG to induce their transcription. Several proteins encoded by these
genes lead to inhibition of protein synthesis using distinct mechanisms. P56 and its family members bind to
specific subunits of the translation initiator factor eIF-3 and block its functions. In contrast, 2-5 OAS enzymes
synthesize 2'-5' oligoadenylates, which activate RNase L and cause degradation of mRNAs. Another enzyme,
PKR, is a latent protein kinase that can be activated by dsRNA or the PACT protein. The inactive
conformation of PKR is maintained by an intramolecular interaction between its N-terminal domain and
C-terminal domain. Binding of dsRNA to the N-terminal domain or PACT to the C-terminal domain disrupts
the intramolecular interaction and activates the enzyme. Activated PKR phospharylates another translation
initiator factor eIF-2 and blocks protein synthesis. Many viruses encode proteins or RNAs that block PKR
activation and thus evade its antiviral actions.
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S14-4
Human Tumor Inducing Kaposi's Sarcoma-Associated Herpesvirus
Immune Evasion Strategy
Jae Ung Jung
Department of Microbiology and Molecular Genetics, Harvard Medical School and Tumor Virology
Division, New England Primate Research Center, USA
Kaposi's sarcoma-associated herpesvirus (KSHV) has been consistently identified in Kaposi's sarcoma
tumor and primary effusion lymphoma that are major AIDS associated malignancy. While worldwide, classical
KS has a low prevalence rate, the more aggressive endemic KS, seen primarily in Africa, can account for
nearly half of the reported cancer within some regions and is the leading cause of cancer death.
Host immune responses including cytotoxic T lymphocytes (CTLs) and Natural killer (NK) cells play a role
in the elimination of virus-infected cells. To avoid these immune responses, herpesviruses have evolved
elaborate mechanisms that target and modulate different aspects of the host's immune system. KSHV encodes
two genes, K3 and K5, whose products are able to down regulate MHC class I from the surface. The K3
and K5 proteins are both type III integral membrane proteins containing a Ring Finger-like ubiquitin ligase
domain at the N-termini, two hydrophobic transmembrane regions and a series of protein motifs important
in cellular trafficking in the C-termini. Both K3 and K5 membrane proteins efficiently ubiquitinate MHC class
I molecules and deliver to the lysosome for degradation. Without MHC class I on the cell surface, no peptides
are presented to induce CTL activation and K3/K5 expressing cells are able to escape CTL killing.
Furthermore, K5 protein additionally targets ICAM-1 and B7.2 for destruction by the ubiquitin:proteasome
system. By removing these two molecules from the surface of infected cells, K5 reduces the average time
that the NK cell stays in contact with the K5-expressing target cell and thus, the NK cell releases the
K5-expressing cell unharmed. This demonstrates a novel immune evasion strategy of KSHV to survive
destruction by host immune effector cells and to achieve persistent infection.
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S15-1
Discourse on the Researches for Production of Useful Metabolites
Using Various Microorganisms
Kye Joon Lee
Department of Biological Sciences, Seoul National University, Seoul 151-742, Korea
This summary is based on over 120 articles published mainly in refereed international academic journals
over the past 20 years which are evidence of a significant scientific achievement and substantial application
to industries. The articles are summarized into 4 parts according to the disciplines and their application as
follows:
Part 1: Ethanol production using Zymomonas mobilis.
This part covers research continued after completion of my PhD and includes studies which are currently
in progress. We have been involved in pioneering work to sequence the full genome of Z. mobilis ZM4. The
achiements of the ethanol fermentation using Z. mobilis to produce fuel ethanol from renewable resources have
been considered as the most promising processes that are in situ operated in few countries.
In addition, it is thought that the new principles and techniques based on the genomics, transcriptomics,
proteomics, and metabolomics would give us great benefits in the molecular construction of industrial strains.
And the researches currently carried out in our group are very highly advanced topics opening a new paradigm
for the molecular construction of industrial strains of using Z. mobilis that produce ethanol more economically
and also other primary metabolites rather than ethanol.
Part 2: Morphological Differentiation of Streptomyces spp.
Streptomycetes are important of their unique metabolism to produce secondary metabolites (physiological
differentiation)and also an unusual morphological differentiationforming substrate mycelia, aerial mycelia, and
arthrospores on solid media. The physiological and morphological differentiations in Streptomycetes are
closely related to the shift-down of essential nutrients such as carbon and nitrogen sources in culture media.
Although production of various proteases coincides with the cell differentiation, the molecular roles of the
various proteases on the mycelium formation and the morphogenesis are not well determined.
In this sense, our studies have focused on the elucidation the role of proteases in association with mycelium
growth and morphological differentiation in. As the conclusion, it is postulated that chymotrysin-like protease
participate primarily in utilization of a proteinaceous nitrogen source for cell growth and trypsin-like protease
function as essential enzymes involving in the metabolism of cell proteins during the morphological
differentiation. One remarkable finding is that autogenous protease inhibitors, named as leupeptin, inhibits the
activity of trypsin-like protease when mycelium growth is active. However the leupeptin is inactivated by a
metallo-protease named leupeptin inactivating enzyme. The cascade roles of the proteases and the inhibitor
may provide selective advantagess to the Streptomycetes over the limitation of various nutrients in nature
where the fluctuations of the nutrients are so profound.
The findings from our researches were the first elucidation to show the roles of thevarious proteases and
the protease inhibitor in the morphological differentiation of Streptomycetes. Since leupeptin analogues are
extremely important in the therapeutic applications against various human diseases caused by irregularly
expressed proteases, leupeptin fermentation process developed the researches arecurrently operated in
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anindustrial companies and recognized as being the most advanced in achieving the highest yields and
productivities for the products.
Part 3: Regulation of production of antibiotics in Actinomycetes.
It has been well understood that secondary metabolites are only produced in the idiophase but not in the
trophophase. And the intermediates originally derived from the primary metabolism are participated in the
formation of the secondary metabolites. However, the repression of the secondary metabolism in the
trophophase and the de-repression of the secondary metabolisms in the idiophase are not fully understood.
In this background, we have attempted to elucidate the role of various nutrients, particularly nitrogen
sources, in the regulation of antibiotic production. As the results, we found that aspartate aminotransferase,
methylmalonyl-CoA carboxyl-transferase were the key enzymes toregulate the formation of intermediates for
mycelium growth or the biosynthesis of tylosin in S. fradiae. And we found that valinedehydrogenase and
threonine dehydratase were the key enzymes for the deamination of the correspond amino acids under the
starvation of nitrogen sources and that the deaminated residues were used in biosynthesis of tylosin.
From the studies, the identification of the key enzymes regulating cell growth and formation of tylosin in
Streptomyces fradiae was a significant for the construction of strain through metabolic engineering techniques
to produce tylosin with the faster production rate and higher production yield.
Since the accumulationof highly phosphorylated guanine nucleotides, (p)ppGpp, is the one of the first
responses of bacteria to a nutritional shift down, we have identified the involvement of (p)ppGpp in the
initiation of antibiotics biosynthesis and morphological differentiation of S. clavuligerus. Two genes, relA and
rsh, were found regulate the synthesis and degradation of (p)ppGpp in S. clavuligerus where the relA gene
rather than rsh is essential for morphological and physiological differentiation in S. clavuligerus and that RelA
primarily governs the stringent response of S. clavuligerus to starvation for amino acids. And it is thought
that the stringent factor is a powerful factor regulating the physiological and morphological differentiation in
Streptomyces spp. The global expression of genes involved in the antibiotics production or genes specifically
regulated by the stringent factor are currently investigated. From the approaches we are expecting to construct
strains producing antibiotics of commercially important.
Part 4: Microbial β-lactamases and the inhibitors.
β-lactam antibiotics have been used with great importance in clinical treatment against various infections.
However the occurrence of β-lactam resistance in a wide range of bacterial pathogens via the production of
β-lactamases is the most important in nosocomial infections. In order to develop small molecule β-lactamase
inhibitors possessing relatively stable inhibitory activity against β-lactamases, we have characterized
β-lactamases from clinical isolates with the identification the active sites of action of β-lactamases and
structural determination of the β-lactamases are being progressed for the development β-lactamase inhibitors
based on the rational drug design approaches.
We have screened strains of Streptomyces spp. producing β-lactamase inhibitors found that most of species
of Streptomyces produced extra-cellular β-lactamases in association of mycelium growth. Two proteineous
β-lactamase inhibitors, BLIP-I and BLIP-II from S. exfoliatus were characterized in relation to their possible
roles. Although the biological roles of BLIP-1 and BLIP-II are not well understood, it can be hypothesized
that they may function as a regulator of cell wall synthesis by interacting with penicillin-binding proteins
(PBPs). It is thought also that BLIPs may play a role as possible protectors of autogenous β-lactam antibiotics
against β-lactamases produced by other organisms in the soil micro-environment as well as being regulators
of cell growth and morphogenesis.
It is thought that β-lactam antibiotics, β-lactamases, and the autogeous β-lactamase inhibitors may play roles
in the selective advantages of the producers over the competitors in the environmentswhere various
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microorganisms are interacting. And it is suggested that β-lactamase participates as a protector against
β-lactam antibiotics produced by neighbor organisms in the environment and that β-lactamase inhibitory
proteins may involve in the morphological differentiation in relation to the cell wall synthesis by interacting
with penicillin-binding proteins (PBPs). Comparative genomics of the genes encoding DD-caboxypetidase,
β-lactamase, and β-lactamase inhibitory protein are now in progressed and elucidation the structure of
β-lactamases isolated from pathogenic bacteria resisting to β-lactam antibiotics is the first step to design novel
β-lactam antibiotics.
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S15-2
Extracelluar Proteome Analysis of Streptomyces griseus
Mi-Soon Kim, Won-Jae Chi, Yoon-Hee Kim, Dae-Kyung Kang1, and Soon-Kwang Hong*
Department of Biological Science, Myongji University, Kyunggi-do 449-728, 1Bio-Resources Institute, Easy
Bio System Inc., Chonan, Chungnam 330-820, Korea
Streptomycetes can produce a variety of biologically active compounds and the biosynthesis of secondary
metabolites is closely correlated with cultural and physiological conditions of cells. The purpose and regulation
of synthesizing secondary metabolites has always been the main question for the scientist who works on
Streptomyces. Many scientists insist that secondary metabolites are produced as a mean of signals to
discriminate the species in the spatial distribution in environmental condition.
Streptomyces griseus is one of the best-studied strains in the genus Streptomyces, because it can produce
many kinds of secondary metabolites and various proteases. Streptomycin and Pronase are the representatives
of the metabolites produced by S. griseus. The regulatory cascade concerning streptomycin production and
morphological differentiation has extensively been studied in this strain, and the A-factor (2-isocapryloyl-3-Rhydroxy-methyl-γ-butyrolactone) has been known to play a central role in those regulatory cascade through
switching on the transcription of adpA encoding a transcriptional activator by binding to ArpA, the A-factor
receptor protein, and dissociating the DNA-bound ArpA from the target DNA. AdpA activates a number of
genes required for morphological and physiological differentiation, composing an AdpA regulon. The strR(a
pathway-specific transcriptional activator for streptomycin biosynthetic genes), adsA (ECF sigma factor of
RNA polymerase essential for aerial mycelium formation), sgmA (a metalloendopeptidase involved in
degradation of substrate hyphae), ssgA (a small acidic protein for spore septum formation), and amfR (essential
for aerial hyphae formation) genes have been identified as a member of the AdpA regulon.
It has been reported that more than 30 kinds of intracellular proteins are inducibly produced by the presence
of A-factor. However there have been few reports about extracellular proteins that are deeply involved in
morphogenesis in Streptomyces. From the importance of the A-factor in the differentiation process of
Streptomyces we expected that some extracellular proteins concerned with cell differentiation in S. griseus
should be produced in an A-factor dependent manner. Therefore, the total extracellular proteins of S. griseus
IFO13350 and HH1, an A-factor deficient strain, were analyzed by the 2-D electrophoresis, which made us
find several up- or down-regulated extracelluar proteins from the culture broth. Therefore the expression of
various extracellular proteins was supposed to be dependent on the presence of the A-factor.
Five extracellular proteins were identified only from the culture broth of wild-type strain and their
N-terminal sequencing revealed that protein-B was a S. griseus trypsin (SGT). Thus several trypsin- and
chymotrypsin-coding genes such as, sprA, sprB, sprC, sprD, sprT, and sprU that encode for S. griseus protease
A (SGPA), S. griseus protease B (SGPB), S. griseus protease C (SGPC), S. griseus protease D (SGPD), S.
griseus trypsin (SGT), and S. griseus trypsin (SGU) were isolated from S. griseus and their expressions
depending on A-factor were studied. In addition the chromosomal gene disruption of individual gene was
carried out to elucidate the in vivo function of those protease-encoding genes.
One protein named as SghC was also obtained from the A-factor deficient mutant strain, S. griseus HHI.
The cloned gene for SghC was found to encode a 214 amino acid protein with an alkaline phosphatase motif
in the internal portion. The functional anaysis of sghC were also performed by the analysis of gene dosage
effects, chromosomal gene disruption, and protein activity.
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Thus, the regulatory cascade for the expression of several extracellular proteins and their in vivo function
will be suggested in this presentation. [supported by the Driving Force Project for the Next Generation of
Gyeonggi Provincial Government in Republic of Korea]
Keywords: S. griseus, Extracellular proteins, A-factor
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S15-3
Genome-Wide Screening for Cryptic Biosynthetic and Regulatory
Genes in Stretomyces species
Eung-Soo Kim
Department of Biological Engineering, Inha University, Korea
The polyene antibiotics, a category which includes nystatin, pimaricin, amphotericin and candicidin,
comprise a family of most promising antifungal polyketide compounds, and are typically produced by soil
actinomycetes species. The biosynthetic gene clusters for these polyenes have been previously investigated,
revealing the presence of highly homologous cytochrome P450 hydroxylase (CYP) genes. Using polyene
CYP-specific PCR screening with several actinomycetes genomic DNAs, the Pseudonorcardia autotrophica
strain was determined to contain a unique polyene-specific CYP gene. Genomic DNA library screening using
the polyene-specific CYP gene probe revealed a positive cosmid clone which contained a DNA fragment of
approximately 34.5 kb. The complete sequencing of this DNA fragment revealed a total of seven complete
and two incomplete open reading frames (ORFs), which were found to be highly homologous, but also unique
to the previously-known polyene biosynthetic genes. These results suggest that the polyene-specific screening
approach may constitute an efficient method for the isolation of potentially-valuable cryptic polyene
biosynthetic gene clusters from various rare actinomycetes species.
Doxorubicin is a highly-valuable anthracycline-family anticancer drug, produced by a high G+C
Gram-positive soil bacterium, Streptomyces peucetius. Although traditional strain improvement via recursive
random mutagenesis has been practiced to improve doxorubicin productivity over the past several years, the
molecular genetic basis underlying the overproducing nature has remained largely unknown. To identify
cryptic regulatory systems responsible for doxorubicin overproduction, genome-wide comparative transcriptome
analyses were performed using the sequenced S. coelicolor genome microarrays and doxorubicin pathway
chips. The pathway chip analyses revealed that early-and-steady expressions of most pathway genes seem to
be the necessary requirement for the overproducing nature, which could be regulated by higher-level cryptic
regulatory systems. Accordingly, several previously-unidentified regulatory genes whose perturbation
significantly affected doxorubicin overproductionwere identified in the S. coelicolor microarray analyses.
Identification and manipulation of cryptic regulatory genes described here could maximize the genetic
potentials of productivity in S. peucetiusas well as in other valuable pharmaceutical-producing Streptomyces
strains, especially whose complete genome sequence information is not available.
Keywords: Antifungal polyene polyketide, Pseudonorcardia, doxorubicin, cryptic regulatory gene, Streptomyces
DNA Microarray
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S15-4
Recent Advances in the Degradation of High-Molecular-Weight
Polycyclic Aromatic Hydrocarbons in Mycobacterium Species:
Metabolism, Proteomic and Genomic Approaches
Carl E. Cerniglia
Division of Microbiology, National Center for Toxicological Research, U.S. Food and Drug Administration,
Jefferson, AR, 72079, USA
Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environnemental polluants. Chemically, they
constitute a class of organic compounds containing two or more fused benzene rings in a linear, angular or
cluster arrangement. Because of their human and eco-toxicity, there is a considerable interest to determine
the fate of these compounds in the environment and to consider possible use of microorganisms for
remediation of polluted sites. Whereas low molecular PAHs (two or three rings) are usually degraded high
molecular weight PAHs such as benz[a]anthracene and benzo[a]pyrene and chrysene resist extensive
degradation in soils and sediments. A variety of microorganisms including algae, fungi, cyanobacteria and
heterotrophic bacteria play a role in PAH degradation. Interestingly, research has shown that mycobacteria
have the ability to degrade low- and high molecular weight PAHs. Mycobacterium vanbaalenii PYR-1 was
isolated from oil-contaminatedsediment based on its ability to mineralize pyrene. This organism is capable of
degrading low and high molecular weight PAHs including carcinogenic benz[a]anthracene and benzo[a]pyrene.
The biodegradation pathways for anthracene, phenanthrene, fluoranthene, benzo[a]pyrene and benz[a]
anthracene have been elucidated. M. vanbaalenii PYR-1 was the first organism known to produce both
cis-dihydrodiol and trans-dihydrodiol metabolites of high molecular weight PAHs, indicating that both
dioxygenase(s) and cytochrome P-450 monogygenase(s) can initiate PAH degradation in this bacterium.
Proteomic and genomic approaches indicated that M. vanbaalenii PYR-1 uses multiple dioxygenases and
cytochrome-P-450 monooxygenases to degrade PAHs. PAH degrading genes that encode for the aromatic
ring-hydroxylating dioxygenase genes have been cloned and sequenced from M. vanbaalenii PYR-1. In
addition to the dioxygenases, three cytochrome P-450 genes were detected and the complete sequences
determined. The genes for phenanthrene and the lower pathway degradation enzymes were also cloned and
sequenced. We found that soil mycobacteria have both PAH-hydroxylating dioxygenases and cytochrome
P-450 which enhance their ability to degrade the highly recalcitrant PAHs. The overall organization of the
PAH degradation genes in M. vanbaalenii differs significantly from that of similar genes in other bacterial
genera. Protein expression profiles of M. vanbaalenii PYR-1 grown in the presence of high molecular weight
PAHs showed the over expression of several proteins for the PAH treatment over uninduced control samples.
Some proteins were found to be expressed uniquely upon exposure to specific PAHs, whereas others were
common for more than one PAH. M. vanbaalenii PYR-1 has been used infield trial; bioreactor and microcosm
studies to successfully biotreat PAH contaminated sediments. These studies demonstrate the bioremediation
potential of Mycobacterium vanbaalenii PYR-1 species to degrade PAHs.
Keywords: mycobacteria, polycyclic aromatic hydrocarbons, bioremediation, dioxygenase, cytochrome-P-450
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S15-5
UV-absorbing Mycosporine-like Amino Acids in Oxygenic
Photosynthetic Free-Living and Symbiotic Microorganisms
Maribel L. Dionisio-Sese
Institute of Biological Sciences, College of Arts and Sciences, University of the Philippines Los Baños,
College, Laguna 4031, Philippines
The diminishing ozone concentration in the stratosphere due to anthropogenic activities has the potential
to increase solar ultraviolet radiation at the ground level. Especially in the tropical aquatic environment, high
levels of UV radiation are expected which can cause detrimental effects on survival, physiology and growth
of many photosynthetic organisms. Besides repair of UV-induced DNA damage by excision repair or
photoreactivation and accumulation of enzymic defenses against oxygen toxicity, an important mechanism to
prevent UV-induced photodamage is the synthesis of UV-absorbing compounds. For higher plants, which are
the foremost primary producers in the terrestrial environment, several studies provide evidence that harmful
UV radiation is absorbed by epidermally located flavonoid derivatives. In the aquatic environment where
oxygenic free-living or symbiotic microorganisms play prominent roles, studies revealed that UV-absorbing
mycosporine-like amino acids (MAA) predominate.
The MAA is a family of water-soluble compounds characterized by a cyclohexenone or cyclohexenimine
chromophore conjugated with the nitrogen substituent of an amino acid. By intercepting UV wavelengths
between 310 and 360 nm and dissipating the energy as heat without the formation of radical intermediates,
MAA afford protection from UV and photooxidative stress.
This paper presents a review of the specific MAA identified and reported in several free-living and
symbiotic populations of photosynthetic microorganisms found in the tropical environment. Using as model
the Prochloron-ascidian symbiosis, an association exclusively found in tropical marine waters, the distribution
and importance of MAA in this symbiosis will be discussed.
Keywords: Mycosporine-like amino acids, Prochloron, Photoprotection, Photosynthetic microorganisms,
UV-absorbing compounds
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S16-1
Microbial Community Dynamics during Cyanobacterial Bloom
Chi-Yong Ahn*, Seung-Hyun Joung, So-Ra Ko, and Hee-Mock Oh
Environmental Biotechnology Laboratory, KRIBB, Daejeon 305-333, Korea
Algal bloom is a worldwide environmental problem. Developed countries spend enormous costs for its
management and treatment in eutrophic lakes as well as in the oceans. Because most of Korean lakes and
reservoirs entered into eutrophic state, algal bloom has become a yearly event in summer. Freshwater bloom
is usually caused by cyanobacteria and its occurrence sometimes accompanies toxin production, threatening
human health. Microcystin, a representative cyanobacterial hepatotoxin, was frequently detected in a large
number of Korean lakes, whereas anatoxin-a, a cyanobacterial neurotoxin, rarely detected (7). Decades of
phytoplankton researches, to cope with such problems, revealed various aspects of physiological characteristics
and environmental conditions that enable them to proliferate in eutrophic waters (3). However, much about
their molecular network systems and interactions with other aquatic microorganisms are still unknown. The
advent of useful molecular techniques and high throughput analysis tools began to make it possible to
investigate detailed mechanisms underlying complex phenomena of cyanobacterial bloom (2). Systems biology
is emerging recently and it integrates whole results of genomics, proteomics, metabolomics, and informaticsto
produce new findings. In a larger scale, ecosystems biology tries to conceptualizeecosystem as biochemical
network, unifying genomics and microscale biochemistry (1).
1. Community structure of freshwater microorganisms
Vertical profiles of microbial community structure in temperate lakes show a variety of spectrum,
particularly when water column is stratified in summer. Even when the focus is confined only to surface
water, phytoplankton community composition varies continuously with seasonal succession. Furthermore,
DGGE analysis showed that prokaryotic community rapidly changed before and after cyanobacterial bloomin
a relatively short time. The microbial community during cyanobacterial bloom was distinguished from those
ofother periods by cluster analysis (5). For the present, one major barrier to community analysis is that
bacterial identification is often hindered by poor sequence databases for freshwater microorganisms (6).
2. Interactions between phytoplankton and other microorganisms
There is no doubt that high concentrations of nutrients and favorable environmental conditions, such as high
temperature and irradiance, directly stimulate algal growth, finally leading to algal blooms. However, dynamic
community structure in aquatic microorganismsassociated with cyanobacterial bloom forced researchers to
conceive that cyanobacterial interactions with other bacteria and viruses play a crucial role in bloom
occurrence and extinction. Compared with such researcheson marine cyanobacteria, relatively little has been
revealed about freshwater cyanobacteria and their related microbial partners, waiting for further studies to be
done (4).
Keywords: bacteria, bloom, cyanobacteria, freshwater, microbial community
References
1. Azam, F., and A. Z. Worden. 2004. Microbes, molecules, and marine ecosystems. Science
303:1622-1624.
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2. Castiglioni, B., E. Rizzi, A. Frosini, K. Sivonen, P. Rajaniemi, A. Rantala, M. A. Mugnai, S.
Ventura, A. Wilmotte, C. Boutte, S. Grubisic, P. Balthasart, C. Consolandi, R. Bordoni, A.
Mezzelani, C. Battaglia, and G. De Bellis. 2004. Development of a universal microarray based on the
ligation detection reaction and 16S rRNA gene polymorphism to target diversity of cyanobacteria. Appl.
Environ. Microbiol. 70:7161-7172.
3. de Figueiredo, D. R., U. M. Azeiteiro, S. M. Esteves, J. M. G. Fernando, and M. J. Pereira. 2004.
Microcystin-producing blooms - a serious global public health issue. Ecotox. Environ. Saf. 59:151-163.
4. Eiler, A., and S. Bertilsson. 2004. Composition of freshwater bacterial communities associated with
cyanobacterial blooms in four Swedish lakes. Environ. Microbiol. 6:1228-1243.
5. Ko, S.-R., S.-J. Park, C.-Y. Ahn, A. Choi, J.-S. Lee, H.-S. Kim, B.-D. Yoon, and H.-M. Oh. 2004.
Analysis of microbial communities during cyanobacterial bloom in Daechung Reservoir by DGGE. Kor.
J. Microbiol. 40:205-210.
6. Page, K. A., S. A. Connon, and S. J. Giovannoni. 2004. Representative freshwater bacterioplankton
isolated from Crater Lake, Oregon. Appl. Environ. Microbiol. 70:6542-6550.
7. Park, H.-D., B. Kim, E. Kim, and T. Okino. 1998. Hepatotoxic microcystins and neurotoxic anatoxin-a
in cyanobacterial blooms from Korean lakes. Environ. Toxicol. Wat. Qual. 13:225-234.
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S16-2
Biodiversity of Oligotrophic Denitrifying Bacteria in Subsurface Soil
Kyung-Sook Whang1,* and Tomoyoshi Hashimoto2
1
Department of Biotechnology, Mokwon University, Korea, 2National Agricultural Research Center for
Kyushu Okinawa Region, Japan
Previous paper (Hashimoto & Niimi 2001) identified the vertical and seasonal changes in copiotrophic
denitrifying bacterial populations in upland soil. We found no clear correlation between the denitrifying
bacterial populations and potential denitrification activity in the subsurface soil, though there was a positive
correlation in the surface soil. This denotes that improved methods are necessary to identify and enumerate
the denitrifying bacterial population in subsurface soils.
The subsurface soil is limited in nitrogen sources (Hashimoto & Niimi 2001), so it might be more enriched
by oligotrophic denitrifying bacteria rather than common copiotrophic bacteria using the diluted medium. We
examined the vertical distribution of denitrifying bacterial populations in upland soil by using two kinds of
enumeration media. The number of denitrifying bacteria enumerated by the 100 fold diluted nutrient broth
(DNB) medium with nitrate was two to three orders of magnitude greater than those enumerated by the
conventional nutrient broth medium with nitrate in subsurface layers suggesting the dominance of oligotrophic
denitrifying bacteria. Sixty-one isolates were obtained by the isolating procedure from the different depth of
upland soil. Isolates were tested for their capability of growing on the full strength NB medium and divided
into two categories: those capable of growing on NB were referred to as 'NB organisms' and those incapable
of growing on NB were referred to as 'DNB organisms'. Among 61 denitrifying isolates, 45 isolates (74%
of the total denitrifying isolates) were DNB organisms and considered to be oligotrophs type.
Figure 1 presents the phylogenetic positions of 61 denitrifying bacterial isolates. The strains, categorized
in three major groups, belong to the alpha (35 strains) and beta (19 strains) subdivisions of proteobacteria
and high G+C gram-positive bacteria (7 strains). The alpha-proteobacterial isolates, most of which were
oligotrophic type, distributedwidely from surface to subsurface soil layers. The phylogenetic analysis clarified
that some isolates belonged to groups of few or no cultivated representatives and an isolate indicated its
possibility as a member of new genera. This isolation procedure of the diluted media is valuable in detecting
diverse and novel denitrifying bacteria in the subsurface soil. On the phylogenetic tree, we confirmed two
major clusters in the proteobacteria alpha-subdivision, the Rhizobium-related genera cluster and the BANA
(Bradyrhizobium, Agromonas, Nitrobacter, and Afipia) cluster, a name which was proposed for a cluster of
the bacteria that are very close phylogenetically to Bradyrhizobium japonicum. A domain in the alpha
subdivision of proteobacteria included large clusters of oligotrophic bacteria. Denitrifying bacteria are already
identified in widespread phylogenetic groups from Proteobacteria Pseudomonas, Alcaligenes, Agrobacterium
to gram-positive Bacillus groups. However, there have been few references to high G+C gram-positive
denitrifying bacterial isolates. Six of seven isolates were NB organisms isolated from subsurface layers.
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β-Proteobacteria
High G+C
Gram Positive
Bacteria
Fig. 1. Phylogenetic tree including strains of denitrifying bacteria found in subsurface upland soil. The strains
marked with grey boxes were oligotrophic denitrifying isolates.
Keywords: denitrification, oligotrophic denitrifying bacteria, soil biodiversity, subsurface
References
1. Hashimoto, T., and H. Niimi. Seasonal and vertical changes of denitrification activity and denitrifying
bacterial populations in surface and subsurface upland soils with slurry application. Soil Sci. Plant Nut.
(2001) 47:503-510.
2. Hashimoto, T., and K. Whang, and K. Nagaoka. A quantitative evaluation and phylogenetic
characterization of oligotrophic denitrifying bacteria harbored in subsurface upland soil using improved
culturability. Biol. Fertil Soils (2005) in press.
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S16-3
Bacterial Communities Associated with Sand Dune Plants and
Their Potential for Plant Growth Promoting Activity
Seung Bum Kim
Department of Microbiology, School of Bioscience and Biotechnology, Chungnam National University, 220
Gung-dong, Yuseong, Daejeon 305-764, Korea
Little is known about the bacterial communities associated with the plants inhabiting sand dune ecosystems
and their roles in the plant growth. In this study, the bacterial populations associated with two major sand
dune plant species, Calystegia soldanella (beach morning glory) and Elymus mollis (wild rye), growing along
the costal areas in Tae-An, Chungnam Province, were analyzed using both culture-based and
culture-independent approaches, and the potential of the isolated bacteria for the facilitation of plant growth
was examined.
The first part of the study was the isolation and characterization of rhizospheric and root endophytic
bacteria. A total of 212 bacteria were isolated from the root and rhizosphere samples of the two plants, and
subjected to further taxonomic analysis. Based on the analysis of the 16S rDNA sequences, all the bacterial
isolates were classified into six major phyla of the domain Bacteria. Significant differences were observed
between the two plant species, and also between the rhizospheric and root endophytic communities. The
isolates from the rhizosphere of the two plant species were assigned to 27 different established genera, and
the root endophytic bacteria were assigned to 21. Members of the phylum Gammaproteobacteria, notably the
Pseudomonas species, comprised the majority of both the rhizospheric and endophytic bacteria, followed by
members of Bacteroidetes and Firmicutes in the rhizosphere and Alphaproteobacteria and Bacteroidetes in the
root. A number of isolates were recognized as potentially novel bacterial taxa. Fifteen out of 27 bacterial
genera were commonly found in the rhizosphere of both plants, which was comparable to 3 out of 21 common
genera in the root, implying the host specificity for endophytic populations. This study of the diversity of
culturable rhizospheric and endophytic bacteria has provided the basis for further investigation aimed at the
selection of microbes for the facilitation of plant growth.
The second part of the study was to examine various plant growth promoting effects by testing the
antagonistic activities against major plant pathogenic bacteria, and production of plant growth promoting
substances such as indoleacetic acid (IAA) and siderophores. A total of 120 bacteria were shown to exhibit
in vitro antagonistic activities against plant pathogenic fungi, namely Rhizoctonia solani, Pythium ultimum,
Fusarium oxysporum and Botrytis cinerea. In particular, Pseudomonas sp. (PHA04-11, PHA 4-16, PHA04-17)
and Erwinia sp. (PSB01-13) were shown to possess broad-spectrum anti-fungal activity. Some strains showed
narrow-spectrum antagonistic activity to Fusarium oxysprum (Rhizobium sp. PSB01-13 and Chryseobacterium
sp. PSB01-20), Rhizotonia solani (unidentified bacterium PSB07-07), and Pythium ultimum (Agrobacterium sp.
PSB07-11). One hundred twenty isolates were also screened for in vitro production of degradative enzymes,
siderophores, and the plant growth hormone indole-1.3 acetic acid (IAA). A number of strains showed protease
(55.5%) and chitinase (76.9%) activities, and secreted siderophores (84.6%).
The final part of the study was to investigate the diversity using the culture independent analyses including
16S rDNA clone library construction and denaturing gradient gel electrophoresis (DGGE). The amplified
rDNA restriction analysis (ARDRA) of the clones using HaeIII exhibited significant differences in the
community composition between the two plant species as well as regional differences, but also identified a
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specific ARDRA patternthat was most common among the clones regardless of plants species. Subsequent
sequence analysis indicated that the pattern was that of Lysobacter enzymogenes, which is a member of the
family Xanthomonadaceae, class Gammaproteobacteria. The Lysobacter clones comprised 50.6 % of the
clones derived from C. soldanella and 62.5 % of those from E. mollis. Other minor patterns included those
of Pseudomonas sp., species of Rhizobium, Chryseobacterium sp. and Pantoea sp. among C. soldanella clones,
and Pseudomonas sp. and Aeromonas hydrophila among E. mollis clones.
The dominance of Lysobacter sp. was further confirmed by the DGGE analysis, in which a single major
band was observed in all of the samples in the rhizosphere or root of both plants. The sequences of the major
bands were found to be identical to the ones obtained from the clones. It is not yet clear what kind of roles
Lysobacter plays in association with sand dune plants, but its universal presence in rhizosphere, together with
the potential of this taxon for the antagonistic activity against plant pathogens, suggests that Lysobacter might
form a symbiotic relationship with its host plants.
Keywords: sand dune plant, DGGE, 16S rDNA clone, Lysobacter, bacterial diversity
국제학술대회
139
October 13-14, 2005, Seoul, Korea
S16-4
The Global Climate Change and Microbial Diversity
Seung-Hoon Lee*, Seon-Young Kim, and Hojeong Kang
Department of Environmental Science & Engineering, Ewha University, Korea
The global warming is due to increased concentrations of greenhouse gases such as CO2, CH4 and N2O
in atmosphere. Amongst, atmospheric CO2 concentrations have increased annually due to anthropogenic
activities and increased CO2 cause the enhanced greenhouse effect. Wetland ecosystems play a pivotal role
in global biogeochemical cycles including the trace gas flux such as CH4 and N2O, which is produced by
specific functional bacterial community. This indicate that the response of wetland on the climate change such
like increasing atmospheric CO2 is not only the biogeochemical change in wetland ecosystem, but feedback
action by biological processes on the climate change. As a result, the study about the effect of climate change
on biological processes in the wetland ecosystem may give significant information for understanding the
impact ofglobal change. In particular, as the role of bacterial community is essential to the recycle of material
in natural ecosystem and it is suggested that change in microbial community structure can influence the
function of ecosystem including trace gas dynamics at a large scale, it is important to study the impact of
climate change on the wetland ecosystem in terms of microbes, especially involved in trace gas flux.
In this study, we aimed to analyze the effect of elevated CO2 on dynamics of the community structure
of denitrifiers and methanogens in wetland soils, the genetic heterogeneity of the nitrite reductase gene (nirS)
and methyl-coenzyme M reductase gene (mcrA) was analyzed by using terminal restriction fragment length
polymorphism (T-RFLP) analysis. To investigate the effect of elevated CO2 on seven species of wetland plants
have been continuously exposed to ambient and elevated to CO2 at twice-ambient concentration (740ppm) for
110 days in growth chambers. The result showed that different genetic structures were observed between each
samples treated with various conditions. In case of richness & diversity, significant change was observed in
methanogen while not in denitrifier by elevated CO2. Changes in structure of community was observed in
both of denitrifiers and methanogens. It was supposed that the difference of CO2 concentration and N supply
was significant factor causing the change in bacterial community structure. In particular, dynamics of
methanogen community structure was related to level of atmospheric CO2 and N supply. Overall, the data
in this study showed that elevated CO2 is expected to cause the change of bacterial community structure in
wetland soil, while the magnitude and pattern of change might show some differences between
specificfunctional groups and be affected by other environmental factors such as N supply and vegetation
types.
Keywords: climate change, CO2, denitrifier, methanogen, microbial diversity, wetland
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S16-5
Genome-wide and Physiological Characterization: Effect of
Nitrogen-fixation on Biphenyl and Polychlorinated Biphenyl
Degradation by Burkholderia xenovorans LB400
Joonhong Park1,*, Myoung-Su Park2, and James M. Tiedje3
1
Department of Civil and Environmental Engineering, Yonsei University, Korea, 2School of Agriculture,
3
Chung-Buk National University, Korea, Center for Microbial Ecology, Michigan State University, USA
Fixed nitrogen is often very limited in soil. Because nitrogen-fixing bacteria provide soluble nitrogen by
transforming N2 into ammonia, use of nitrogen-fixing biodegradative bacteria would be advantageous for the
in-situ bioremediation of organic pollutants in soil. However, little is known about microbial degradation and
physiology under fixed-nitrogen limitation, mainly because most biodegradation studies are evaluated in
ammonia-rich conditions. In this study, we evaluated the effect of nitrogen-fixation on the degradation of
biphenyl and polychlorinated biphenyl (PCB) by a nitrogen-fixing and biphenyl-degrading bacterial strain,
Burkholderia xenovorans LB400. For this evaluation, we characterized the effect of nitrogen fixation on (i)
cellular growth rate, (ii) PCB degradation and toxicity response, and (iii) the transcriptome of biphenyl-grown
cells. When N2 is the only available nitrogen source, cells grew on biphenyl in a mineral medium. The rate
of growth on biphenyl was slower for the nitrogen-fixing condition than for the ammonia-rich condition. The
slower biphenyl growth was attributable tothe nature of nitrogen fixation and not due to the xenobiotic nature
of biphenyl, because cellular growth on more favorable carbon sources (succinate and sucrose) was similarly
slower in the nitrogen-fixing condition than in the ammonia-rich condition. Resting cell assays showed that
the nitrogen-fixing and biphenyl-growing cells can also degrade PCBs with a similar degradation specificity.
However, biphenyl-growing cells under the nitrogen fixing condition showed that cellular growth was inhibited
by PCB exposure. This response of nitrogen-fixing cells to PCB toxicity may be energy-dependent, because
nitrogen-fixing cells were PCB-tolerant under resting conditions while PCB-sensitive under growing
conditions. To further understand the effect of nitrogen-fixation on cell physiology, the transcriptome of
nitrogen-fixing and biphenyl-growing cells was characterized using microarray hybridization.
Keywords: transcriptomics, PCB degradation, bioremediation, nitrogen fixation, bacterial stress response
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October 13-14, 2005, Seoul, Korea
S17-1
Immune Responses and Prophylactic Potential of Live Influenza
Vaccine
Baik Lin Seong
Department of Biotechnology, College of Engineering, Yonsei University, 134 Shinchon-Dong,
Seodaemun-Ku, Seoul 120-749, Korea
Influenza virus remains an essentially uncontrolled infectious agent causing frequent outbreaks of epidemics
and pandemics. Influenza pandemics are caused by sudden emergence of a new influenza subtype in humans.
New subtypes most probably derive from the vast animal influenza reservoir, where 15 different influenza
A subtypes freely circulate including aquatic migratory birds. Only two subtypes (H3 and H1) are presently
circulating in humans. However, if a subtype extends its host range to include humans, it would probably
ignite the next pandemic. With the unprecedented outbreak of avian influenza in Asia (caused by H5N1), the
world has come closer than ever to the first pandemic since 1968. Our previous experience suggests that
vaccines would serve the best line of defense against the high morbidity and mortality invariably associated
with influenza pandemic.
Although trivalent subunit vaccine has been available, the influenza vaccine has been under-utilized because
of cumbersome route of vaccination and low level of protection. Therefore, there has always been a great
need to develop live attenuated influenza vaccine which can be administered through nasal route and elicit
better immunogenicity. Introduction of attenuation phenotype into the viral genome through conventional
'cold-adaptation' or reverse genetic methodology has been proven useful for the generation of live attenuated
influenza vaccine.
In part of our efforts for developing live influenza virus vaccine, a live vaccine carrier was established
through conventional repeated passage at low temperature. The virus was evaluated for several properties:
ca, ts, att phenotype and protective immune responses. By reassortant formation between the 'cold-adapted'
vaccine carrier and virulent strains, a prototype of trivalent live influenza vaccine is developed. This strain
was evaluated for their ability to protect mice from challenge with same subtype and different subtype of influenza
A virus. The vaccination of mice with live attenuated influenza virus provided complete protection against
homologous and heterologous virus challenge.
Besides well known prophylactic effect, a desirable trait for live attenuated vaccine may include potential
therapeutic effect by interfering with wild-type viruses. On the basis of the ability of the ca virus to
suppress wt viral replication, we examined in mouse infection model if the X-31 cavirus could interfere
with the virulent virus. Prior vaccination with the ca virus about 1-4 days before challenge with virulent
virus or even simultaneous infection of vaccine virus and virulent virus resulted in marked improvement
in clinical signs associated with influenza infection. The administration of this ca virus may confer
immediate protection and block further spread of the virulent virus, and could be considered useful for
minimizing morbidity and mortality associated with natural outbreak or intentional use of influenza virus.
Keywords: influenza virus, live vaccine, immune response, prophylactic, therapeutic effect
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S17-2
Circularization of a RNA Template via Long-range Base Pairing Is
Critical for HBV Reverse Transcription
Wang-Shick Ryu
Department of Biochemistry, Yonsei University, Seoul 120-749, Korea
Human hepatitis B virus (HBV) is a major cause of liver disease worldwide, ranging from acute and
chronic hepatitis to liver cirrhosisand hepatocellular carcinoma. HBV is unique among the animal DNA
viruses in that it replicates through reverse transcription of an RNA intermediate. The reverse transcription
reactions require multiple template switching events that are mediated primarily through complementary
sequences in the terminal redundancy regions of their RNA genomes.
Hepadnaviral reverse transcription occurs inside nucleocapsids that follow selective encapsidation of the
pregenomic RNA (pgRNA). Encapsidation proceeds by recognition of a stem-loop structure (ε) near the 5′
end of the pgRNA by the viral polymerase, P. Initiation of minus-strand DNA synthesis occurs througha novel
mechanism in which P itself acts as a primer by forming a covalent linkage with the first nucleotide of the
DNA. In other words, P directs the packaging of the pgRNA as well as the priming, and 5′ ε serves not
only as an encapsidation signal but also as the initiation sitefor minus-strand DNA synthesis.
Following synthesis of the first 3 or 4 nucleotides via protein priming, the P-linked oligonucleotide
translocates to the acceptor site at the opposite end, where minus-strand DNA synthesis resumes. This process
is termed minus-strand transfer or template switching. An immediate question arises as to how accurate
transfer to the acceptor site can be ensured. Recently, we and others independently identified a novel
cis-acting sequence―a 28-nucleotide sequence located upstream of the acceptor―that is required for
minus-strand DNA synthesis, termed β or  , respectively (1, 2).
To account for the role of the β /  element, Tang et al. hypothesized a novel circular structure of the
pgRNA formed via significant base pairing between the 5′ε and the β /  sequences (1). In support of the
hypothesis, we now present evidence for the 5′-3′, long-range interaction that occurs during minus-strand DNA
synthesis.
Keywords: hepatitis B virus replication, RNA template, long-range base-pair
References:
1. H. Tang, A. McLachlan, Virology 303, 199-210 (2002).
2. M.-K. Shin, J. Lee, W.-S. Ryu, J. Virol. 78, 6252-6262 (2004).
국제학술대회
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October 13-14, 2005, Seoul, Korea
S17-3
Implications of Nonstructural 5A Protein of Hepatitis C Virus in
Liver Pathogenesis
Soon Bong Hwang
Ilsong Institute of Life Science, Hallym University, Chuncheon 200-702, Korea
Hepatitis C virus (HCV) is the major etiologic agent of chronic hepatitis, which often leads to liver
cirrhosis and hepatocellular carcinoma (HCC). The most prominentfeature of hepatitis C virus is its ability
to persist in virus-infected patients (> 80% as compared with 5% in hepatitis B virus). However, the
mechanisms of liver pathogenesis by HCV infection are not well understood. Transforming growth factor-β
(TGF-β) has been implicated in the pathogenesis of liver disease. TGF-β is involved in liver regeneration and
in the fibrotic and cirrhotic transformation with hepatitis viral infection. To investigate the molecular
mechanism underlying HCV pathogenesis, we have explored the potential involvement of HCV nonstructural
5A (NS5A) protein in TGF-β-induced signaling pathways. The HCV NS5A is a multifunctional
phosphoprotein that leads to pleiotropic responses, in part by regulating cell growth and cellular signaling
pathways. We show that NS5A protein inhibits TGF-β-mediated signaling pathway in hepatoma cell lines as
determined by reporter gene assay. To further investigate the role of NS5A, we examined the protein-protein
interaction between NS5A and TGF-β signaling transducers. Both in vitro and in vivo binding data show that
NS5A protein directly interacts with type I TGF-β receptor I (TβR-I) in hepatoma cell lines. This interaction
was mapped to the amino acids 148-238 of NS5A. We also found that NS5A protein was colocalized with
TβR-I in the cytoplasm of Huh7 cells and inhibited TGF-β-mediated nuclear translocation of Smad2.
Furthermore, we demonstrated that NS5A protein abrogated phosphorylation of Smad2 and heterodimerization
of Smad3 and Smad4. To further explore the relevance to the viral infection, we examined the effect of HCV
subgenomic replicon on TGF-β signaling pathway. Indeed, we show that HCV subgenomic replicon also
inhibits TGF-β-induced signaling cascades. HCV NS5A protein is also involved in modulation of
transcriptional activation of cellular genes, cell proliferation and cell death. To investigate whether NS5A
protein has an effect on pathological phenotypesin the liver, we established transgenic mice lines carrying
either HCV core or NS5A gene. Transgenic animals harboring NS5A gene but not HCV core gene developed
HCC. Three independent lines of transgenic mice harboring NS5A gene developed HCC at 15 months of age.
These mice developed progressive histopathological changes in the liver at 10 months of age with multifocal
areas of altered hepatocytes and developed hepatic tumors containing prominent steatosis thereafter. This work
provides a good animal modelto study molecular and pathological events during the development of HCC.
Studies on molecular mechanisms for HCC development in transgenic mice are in progress at this time. Taken
together, our findings suggest that HCV NS5A protein modulates TGF-β signaling of the host cells and NS5A
may play a critical role in HCV-associated liver pathogenesis.
Keywords: Hepatitis C virus, Nonstructural 5A protein, Liver pathogenesis, Signal transduction pathways,
TGF-beta
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백지
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C1-1
Does the Innate Immune System Discriminate Non-self from Self?
Seung-yong Seong
Hyppo Lab., Department of Microbiology and Immunology, Seoul National University College of Medicine, Korea
It is currently thought that innate immune responses are initiated by pathogen-associated molecular pattern (PAMP)
or by damage-associated molecular pattern (DAMP). However, these two groups of molecules might not be mutually
exclusive. Many of them might be part of an evolutionarily ancient alert system in which the hydrophobic portions
(Hyppos) of biological molecules act, when exposed, as universal DMAP to initiate repair and innate immunity.
In this study, we showed that Toll like receptor 4 and CD14 is crucial in initiating innate immunity in response to
DAMP as much as PAMP. DCs or peritoneal cells from TLR4 KO mice or CD14 KO mice were less responsive to
NC than wild type counterpart in terms of costimulatory molecule expression, IL12p40 secretion and naïve T cell
activation. On microarray analysis, we found that various genes were regulated by TLR4 or CD14 pathways in response
to necrotic cells and endogenous lipids molecules. These findings suggest that innate immune system has been evolved
to recognize DAMP as like as PAMP.
Keywords: innate immunity, dendritic cell, toll like receptor, CD14, pathogen-associated molecular pattern, damage-associated
molecular pattern
C1-2
Preparation and Analysis of Yeast Cell Wall Mannoproteins, Immune
Enhancing Materials, from Cell Wall Mutant Saccharomyces cerevisiae
Chang Hoon Ha1, Hye Young Kim1, Cheol Won Youn1, Hyun Dong Paik3, Seung Wook Kim2, Chang
3
1
Won Kang , and Hyo-Ihl Chang *
1
School of Life Sciences and Biotechnology, Korea University, 5-1, Anam-dong, Sungbuk-ku, Seoul 136-701, Korea,
Department of Chemical and Biological Engineering, Korea University, 5-1, Anam-dong, Sungbuk-ku, Seoul 136-701,
3
Korea, Division of Animal Life Science, Konkuk University, Hwayang-Dong, Kwangjin-Gu, Seoul 143-701, Korea
2
Yeast cell wall matrix particles are composed entirely of mannoprotein and β-glucan. The mannoproteins of yeast cell
wall can systemically enhance the immune system. We previously purified and analyzed alkali-soluble β-glucans (β
-(1,3)-and β-(1,6)-glucans). First, In the present study, a wild type strain was first mutagenized with ultraviolet light, and
the cell wall mutants were then selected by treatment with 1.0 mg/ml laminarinase (Endo-β-(1,3)-d-glucanase).
Mannoproteins of Saccharomyces cerevisiae were released by laminarinase, purified by concanavalin-A affinity and ion
exchange chromatography. The results indicated that the mutants yielded 3-fold more mannoprotein than the wild type.
The mass of mannoprotein of the mutant, K48L3 was 2.25 mg / 100 mg of yeast cell dry mass. Carbohydrate analysis
revealed that they contained mannose, glucose and N-acetylglucosamine. Saccharomyces cerevisiae cell wall components,
mannoproteins, are known to interact with macrophages through receptors, thereby inducing release of tumor necrosis
factor alpha (TNF-α) and nitric oxide. Mannoprotein fractions in the present study had higher macrophage activity of
secretion of tumor necrosis factor alpha (TNF-α) and nitric oxide and direct phagocytosis than positive control (one ㎍
of lipopolysaccharide). Especially, F1 and F3 fractions in mannoproteins of K48L3 enhanced and upregulated activity
of nitric oxide secretion and macrophage phagocytosis approximately two and four-fold, respectively.
Keywords: mannoprotein, yeast cell wall, random mutation, immune activity
국제학술대회
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October 13-14, 2005, Seoul, Korea
C1-3
Forward Genetics of Murine Gammaherpesvirus 68 Using
Signature-tagged Mutagenesis
Moon Jung Song1,*, Seungmin Hwang2, Wendy H. Wong2, Ting-Ting Wu2, and Ren Sun2
1
Department of Microbiology, College of Medicine, Hallym University, Chuncheon 200-702, Korea, 2Department of
Molecular and Medical Pharmacology, the UCLA AIDS Institute, the Jonsson Comprehensive Cancer Center, and the
Molecular Biology Institute, University of California at Los Angeles, Los Angeles, CA 90095, USA
Gammaherpesviruses, Epstein-Barr virus and Kaposi’s sarcoma-associated herpesvirus, are important human pathogens,
as they are involved in tumor development. Murine gherpesvirus-68 (MHV-68 or gHV-68) has emerged as a small animal
model system for the study of gherpesvirus pathogenesis and host-virus interactions. To identify the genes required for
viral replication in vitro and in vivo, we generated 1152 mutants using signature-tagged transposon mutagenesis (STM)
on an infectious bacterial artificial chromosome (BAC) of MHV-68. Almost every ORF was mutated by random insertion.
For each ORF, a mutant with aproximal insertion to the N-terminus of each ORF was examined for the ability to grow
in fibroblasts. Our results indicate that 41 genes are essential for in vitro growth, while 26 are nonessential and 6
attenuated. Replication-competent mutants were pooled to infect mice, which led to the discovery of ORF54, being
important for MHV-68 to replicate in the lung. This genetic analysis of a tumor-associated herpesvirus at the whole
genome level validates STM screening as an effective genetic system to identify important virulent genes in vivo and
define interactions with the host immune system.
Keywords: functional mapping, herpesvirus, transposition, bacterial artificial chromosome, deoxyuridine-triphosphate
C1-4
Genome Analyses of Streptomyces peucetius for the Identification of
Cytochrome P450 and Regulatory Elements
Niranjan Parajuli1,*, Yeo Joon Yoon1, Hei Chan Lee2, Kwangkyoung Liou2, Jin Suk Woo3, Byung Gee
4
2
Kim , and Jae Kyung Sohng
1
Division of Nano Science and Department of Chemistry, Ewha Womans University, Seoul, Korea, 2Institute of
Biomolecule Reconstruction, Sun Moon University, Asan, Korea, 3GeneChem Inc. 59-5 Jang-dong, Yoosung-ku, Daejeon,
4
Korea, School of Chemical and Biological Engineering, Seoul National University, Seoul, Korea
We have determined the genome sequence of 8.7 Mb chromosome of Streptomyces peucetius ATCC 27952.The
cytochrome P450 (CYP) superfamily is represented by 19 sequences in the S. peucetius. CYP252A1 is the new family
found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2). CYP105P2 (cytochrome
P450) from S. peucetius was cloned and expressed in E. coli. In our best knowledge, cis-5,6,7,8,9-pentahydroxycinnamic
acid is a novel compound, which was isolated by enzymatic reaction by CYP105P2 with 7-ethoxycoumarin.
Several regulatory genes are analysed from S. peucetius. These include afsR, afsK, abaA, abaB, adsA, vbrA, pknG
and rarA. AfsR-p from S. peucetius shares 60% sequence identity with AfsR from Streptomyces coelicolor A3 (2). afsR-p
was expressed under the control of the ermE* promoter in its hosts S. peucetius, Streptomyces lividans TK 24,
Streptomyces clavuligerus and Streptomyces griseus. We observed overproduction of doxorubicin (4-fold) in S. peucetius,
γ-actinorhodin (2.6-fold) in S. lividans, clavulanic acid (1.5-fold) in S. clavuligerus and streptomycin (slight) in S. griseus.
Keywords: Streptomyces peucetius, CYP105P2, cis-5,6,7,8,9-pentahydroxycinnamic acid and global regulatory gene
(afsR-p)
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C2-1
Identification of 5' and 3' cis-acting Elements of the Porcine
Reproductive and Respiratory Syndrome Virus and Acquisition of
Novel 5' Sequences Required for RNA Replication
Yu-Jeong Choi1,*, Sang-Im Yun1, Shien-Young Kang2, and Young-Min Lee1
1
Department of Microbiology, College of Medicine and Medical Research Institute, Chungbuk National University,
Cheongju, Korea, 2Research Institute of Veterinary Medicine, College of Veterinary Medicine, Chungbuk National
University, Cheongju, Korea
We here demonstrated the successful engineering of the RNA genome of porcine reproductive and respiratory
syndrome virus (PRRSV) as an infectious bacterial artificial chromosome. Run-off transcription from this cDNA by SP6
polymerase resulted in capped synthetic RNAs bearing authentic 5' and 3' ends of the viral genome that had a specific
infectivity of >5x105 PFU/㎍ of RNA. The synthetic viruses recovered from the transfected cells were genotypically and
phenotypically indistinguishable from the parental virus. Using our system, a series of genomic RNAs with nucleotide
deletions in their 5' end produced viruses with decreased or no infectivity. Various pseudorevertants were isolated and
acquisition of novel 5' sequences of various sizes, composed predominantly of A and T bases, restored their infectivity,
providing a novel insight into functional elements of the 5' end of the PRRSV genome. In addition, our system was
further engineered to generate a panel of self-replicating, self-limited, luciferase-expressing PRRSV viral replicons bearing
various deletions. Analysis of these revealed the presence and location of a 3' cis-acting element in the genome that
was required for replication. Moreover, we produced EGFP-expressinginfectious viruses, which indicate that the
PRRSVcDNA can be developed as a vector that expresses heterologous genes in a wide variety of cell types. Thus, our
PRRSV reverse genetics system not only offersa means of directly investigating the molecular mechanisms of PRRSV
replication and pathogenesis, it can also be used to generate new heterologous gene expression vectors and genetically
defined antiviral vaccines.
Keywords: reverse genetics system, porcine reproductive and respiratory syndrome virus, Infectious cDNA molecular
clone, Viral replicons, cis-acting element
C2-2
Memory CTL Reaction Can Be Recruited from Chronic Hepatitis C
Infection by in vitro Culture with Pan-HLA Reactive Epitopes and
Recombinant Human Interleukin-15
Nam Kyung Kim*, Yu Kyeong Hwang, OkJae Lim, Jung Min Park, Mahnhoon Park
Division of Immunotherapy, Mogam Biotechnology Institute 341, Pojung-Ri, Guseong-Eup,
Yongin-City, Kyonggi-Do 449-913, Korea
It is well known that the immune responses are impaired in chronic patients who had infected with hepatitis C virus
(HCV). Especially, the patients have less number of NK cells and higher expression of inhibitory molecules on cell
surface than normal subjects. Furthermore, dendritic cells infected with HCV can not be matured even if they are treated
with type I IFN (IFN-α, -β) stimuli. Therefore, to induce CTL responses from chronic HCV patient's peripheral blood
mononuclear cells (PBMCs), we provided recombinant IL-15 with pan-HLA reactive epitope peptides which do not
restricted to specific HLA molecule. Pan-HLA reactive epitopes from consensus sequences of HCV antigen were
predicted by computer algorithm according to binding score, and selected with high or intermediated affinity to various
HLA molecules. Epitope-specific CTL responses were measured by IFN-γ secreting cells among CD8+ T cells after in
vitro culture for 5 days. Most of patients who had chronic hepatitis having various HLA phenotypes recruited strong
CTL responses after in vitro culture with these epitope peptides and IL-15 in this study. These results indicate that
pan-HLA reactive epitopes can recruit memory CTL reactions from immune suppressed chronic hepatitis C patientswho
have various HLA polymorphisms. Furthermore, IL-15 might be used as a candidate of new therapeutics to overcome
immune suppression for chronic hepatitis caused by HCV.
Keywords: pan-HLA restricted epitope, CTL, hepatitis C virus, chronic hepatitis, IL-15
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October 13-14, 2005, Seoul, Korea
C2-3
Antiviral Effect of Catechins in Green Tea on Influenza Virus
Jae-Min Song*, Kwang-Hee Lee, and Baik-Lin Seong
Department of Biotechnology, College of Engineering, Yonsei University, 134, Shinchon-dong, Seodaemun-gu,
Seoul 120-749, Korea
Polyphenolic compound catechins (EGCG, ECG and EGC) from green tea were evaluated for their ability to inhibit
influenza virus replication in cell culture and for potentially direct virucidal effect. Among the test compounds, the EGCG
and ECG were found to be potent inhibitors of influenza virus replication in MDCK cell culture and this effect was
observed in all influenza virus subtypes tested, including A/H1N1, A/H3N2 and B virus. The 50% effective inhibition
concentration (EC50) of EGCG, ECG, and EGC for influenza A virus were 22-28, 22-40, and 309-318  M, respectively.
EGCG and ECG exhibited hemagglutination inhibition activity, EGCG being more effective. However, the sensitivity in
hemagglutination inhibition was widely different among three different subtypes of influenza viruses tested. Quantitative
RT-PCR analysis revealed that, at high concentration, EGCG and ECG also suppressed viral RNA synthesis in MDCK
cells whereas EGC failed to show similar effect. Similarly, EGCG and ECG inhibited the neuraminidase activity more
effectively than the EGC. The results show that the 3-galloyl group of catechin skeleton plays an important role on the
observed antiviral activity, whereas the 5'-OH at the trihydroxy benzyl moiety at 2-position plays a minor role. The
results, along with the HA type-specific effect, suggest that the antiviral effect of catechins on influenza virus is mediated
not only by specific interaction with HA, but altering the physical properties of viral membrane.
Keywords: Influenza virus, catechins, green tea, EGCG, ECG
C2-4
A Small Interfering RNA Targeting Coxsackievirus B3 Protects
Permissive HeLa Cells from Viral Challenge
Jeonghyun Ahn1,*, Eun-Seok Jeon2,3, Hui Sun Lee1, Seung Yong Yoon4, Dong Hou Kim4,5, Chul Hyun
1,5
1,5
1,5
Joo , Yoo Kyum Kim , and Heuiran Lee
Departments of Microbiology1, Anatomy & Cell Biology4, and Research Institute for Biomacromolecules5, University of
2
Ulsan College of Medicine, Department of Internal Medicine , Sungkyunkwan University School of Medicine, Cardiac
3
and Vascular Center , Samsung Medical Center, Seoul, Korea
To investigate the antiviral potential of RNA interference (RNAi) for coxsackievirus 3 (CVB3)infection, we examined
the ability of small interfering RNAs (siRNAs) to disrupt infection by CVB3. The incorporation of siRNAs decreased
cell death in HeLa cells in parallel with a reduction in viral replication. Thus, the results demonstrate that CVB3-specific
antiviral effect can be readily acquired by siRNA pretreatment via the down-regulation of viral replication. In addition,
the CVB3-specific siRNA inducedsimilar antiviral effects in other related enteroviruses possessing sequence homology
in the targeted region. To investigatethe potential as a universal anti-enteroviral strategy of RNAi, we further developed
siRNA design software, providing siRNA candidates targeting highly conserved virus genome regions. We are currently
examining the antiviral effects of multi-enteroviral targeting siRNA in various enteroviruses.
Keywords: small interfering RNA, coxsackievirus B3, enterovirus, antiviral effect, CAPSID program
150
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
C2-5
Virus Receptor Trap, Expressing Both the Coxsackievirus and
Adenovirus Receptor and the Decay-accelerating Factor, Neutralize
Coxsackievirus in Experimental Murine Viral Myocarditis
Byung-Kwan Lim* and Eun-Seok Jeon
Department of Medicine, Sungkyunkwan University School of Medicine, Samsung Medical Center, Seoul, Korea
The coxsackie and adenovirus receptor (CAR) and the decay-accelerating factor (DAF) are receptors for coxsackievirus
B3 (CVB3), which is known as the major cause of human viral myocarditis. We designed a novel virus receptor trap
(hCAR-hDAF:Fc) consisting of both CVB3 receptors and the Fc portion of human IgG1, and evaluated its antiviral
effects in experimental CVB3 myocarditis. Soluble virus receptor fusion proteins, hCAR:Fc and hCAR-hDAF:Fc in the
supernatant of transfected cells neutralized echovirus, adenovirus, and various serotypes of CVB in a dose-dependent
manner. Both soluble viral receptor proteins bound to the VP0 and VP1 capsid proteins of CVB3. The in vivo efficacy
of viral receptor proteins was evaluated by intramuscular injection of plasmid (hCAR:Fc or hCAR-hDAF:Fc) followed
by electroporation in a murine model of CVB3 myocarditis. Serum levels of the virus receptor proteins increased relative
to baseline values from day 3 and peaked on day 14 at 12.9-fold for hCAR:Fc and 7.1-fold for hCAR-hDAF:Fc. The
three-week survival rate was significantly higher in hCAR-hDAF:Fc-treated mice (61%) than in hCAR:Fc-treated mice
(29%) and in controls (15%; p<0.05). Myocardial inflammation, fibrosis, and myocardial virus titers were all significantly
reduced in the hCAR:Fc and hCAR-DAF:Fc groups compared to the controls. Our soluble virus receptor trap,
hCAR-hDAF:Fc, attenuated viral infection, myocardial inflammation and fibrosis, resulting in higher survival rates in
mice with coxsackieviral myocarditis. This virus receptor trap may be a novel therapeutic and/or preventive strategy for
the treatment of coxsackieviral diseases.
Keywords: coxsackie and adenovirus receptor; coxsackievirus B3 decay-accelerating factor; virus receptor trap
C2-6
Time-Saving Flow Cytometry Analysis for Rapid Determination of
Ganciclovir Resistant Human Cytomegalovirus
Gyu-Cheol Lee
1
1,2,
*, Dong-Gun Lee1, Su-Mi Choi1, Jin-Hong Yoo1, Sun-Hee Park1, Jung-Hyun Choi1,
Woo-Sung Min1, Chan-Hee Lee3 and Wan-Shik Shin1
The Clinical Research Institute, St. Mary's Hospital, The Catholic Hemopoietic Stem Cell Transplantation Center, The
Catholic University of Korea, College of Medicine, Seoul 150-713, Korea, 2Water Quality Analysis Team I,
International Water Analysis Center, Korea Water Resources Corporation, Daejeon 306-711, Korea, 3Department of
Microbiology, Natural Sciences College, Chungbuk National University, Cheongju 361-763, Korea
There are two categories of methods assessing the susceptibility of HCMV to ganciclovir (GCV): one is genotypic
test that detects the resistant-related mutated gene and the other is phenotypic test that actually assesses the phenotypic
expression of resistance. The advantages of genotypic tests analyzing UL97 gene are its rapidity and accuracy. However,
for the detection of novel mutations or a mutation in UL54 DNA polymerase, the phenotype test such as plaque reduction
assay (PRA) is also required. To overcome the shortcomings of PRA such as time consuming and labor intensiveness,
we developed the time saving (TS)-FACS analysis. We obtained GCV inhibitory concentration of 50% (IC50) from 5
clinical isolates and a HCMV laboratory strain (AD169), and compared the results with those of PRA. The laboratory
strain and three clinical isolates were sensitive to GCV. Although one out of three isolates showed a minor discrepancy,
GCV IC50 obtained by TS-FACS analysis was well correlated to that of PRA. The remaining two isolates were resistant
to GCV, one contained the GCV resistance related mutation M460V, and the GCV IC50 result by TS-FACS analysis
and by PRA was also comparable. In the GCV susceptibility assay, the advantages of TS-FACS analysis are shorter time
to take, the possibility of automation, and its comparable result to that of PRA regarded as the golden standard. In
conclusion, TS-FACS analysis might be applicable as an alternative method to PRA in clinical practice.
Keywords: human cytomegalovirus, ganciclovir, resistance, flow cytometry
국제학술대회
151
October 13-14, 2005, Seoul, Korea
C3-1
Development of Yeast Whole-Cell Systems for the Heavy Metal
Cadmium Based on Transcriptome Profiling of Stress Response in
Hansenula polymorpha
Jeong-Nam Park*, Min Jeong Sohn, Doo-Byoung Oh, Joo-Hyung Heo,
Sang Ki Rhee, and Hyun Ah Kang
Metabolic Engineering Lab., KRIBB, Daejeon 305-333, Korea
The methylotrophic yeast Hansenula polymorpha is characterized with high tolerance to various environmental stresses
induced by heavy metals, xenobiotics (drugs) and environmental pollutants. The transcriptome profiling using the H.
polymorpha whole-genome cDNA microarray allowed us to identify novel genes responded to cadmium treatment.
Among a number of H. polymorpha genes induced more than 4-fold upon cadmium treatment for 2 hours, three genes
(HpHSP12, HpFEN2, HpSEO1) were chosen for further analysis of promoter activities. About 1,000-bp upstream
fragments of these genes were fused with the reporter green fluorescence protein (GFP) gene. The resultant reporter
cassettes were introduced into H. polymorpha NCYC495 strain and analyzed for their response to cadmium. The
recombinant yeasts showed the increased intensity of GFP in a cadmium concentration-dependent manner. Among them,
the recombinant strain carrying HpSEO1promoter-GFP reporter cassette showed the highest increase of GFP intensity
upon exposure to cadmium, but negligible signals to the treatment of other metals. Deletion up to 500 bp upstream of
the HpSEO1 promoter retained the whole Cd-inducible activity, but the 250-bp short fragment showed a decreased
Cd-inducible activity. Furthermore, the recombinant strains immobilized onto 96-well microplates displayed Cd-dependent
expression. The results strongly demonstrate the potential of the recombinant yeasts genetically engineered with the
HpSEO1 promoter-GFP reporter cassette as whole-cell biosensor to monitor cadmium contamination.
Keywords: Hansenula polymorpha, Transcriptome, Stress Response, Cadmium, Whole-cell biosensor
C3-2
ATP-Binding Motifs Play Key Roles in Krp1p, Kinesin-Related Protein
1, Function for Bi-polar Growth Control in Fission Yeast
1
Dong Keun Rhee * and Hyong Bai Kim
2
1
School of Life Sciences and Biotechnology, Korea University, Seoul 136-701, Korea, 2Department of Bioinformatics,
Korea University, Yeongigun, Chungnam 339-700, Korea
Kinesin is a microtubule-based motor protein with variousfunctions related to the cell growth and division. It has been
reported that Krp1p, kinesin-related protein 1, which belongsto the kinesin heavy chain superfamily, localizes on
microtubules and may play an important role in cytokinesis. However, the function of Krp1p has not been fully
elucidated. In this study, we overexpressed an intact form and three different mutant forms of Krp1p in fission yeast
constructed by site-directed mutagenesis in two ATP-binding motifs or by truncation of the leucine zipper-like motif
(LZiP). We observed hyper-extended microtubules and the aberrant nuclear shape in Krp1p-overexpressed fission yeast.
As a functional consequence, a point mutation of ATP-binding domain 1 (G89E) in Krp1p reversed the effect of Krp1p
overexpression in fission yeast, whereas the specific mutation in ATP-binding domain 2 (G238E) resulted in the altered
cell polarity. Additionally, truncation of the leucine zipper-like domain(LZiP) at the C-terminal of Krp1p showed a
normal nuclear division. Taken together, we suggest that krp1p is involved in regulation of cell-polarized growth through
ATP-binding motifs in fission yeast.
Keywords: Fission yeast, Microtubule, Polymerization, Kinesin, Krp1, Krp1p, ATP-binding motif, Polarity
152
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
C3-3
Specific Regression of Human Cancer Cells by Ribozyme-Mediated
Targeted Replacement of Tumor-Specific Transcript
Heung-Su Jung* and Seong-Wook Lee
Department of Molecular Biology, Institute of Nanosensor and Biotechnology,
Dankook University, Seoul 140-714, Korea
In this study, we describe a novel approach to human cancer therapy that is based upon trans-splicing
ribozyme-mediated replacement of cancer-specific RNAs with new transcripts that exert therapeutic activities. We have
developed a specific ribozyme that can reprogram human telomerase reverse transcriptase (hTERT) RNA to induce
transgene activity selectively in cancer cells that express the RNA. The ribozyme-mediated triggering of the transgene
expression was accomplished via a high-fidelity trans-splicing reaction with the targeted residue in the hTERT-expressing
cells. The ribozyme also induced cytotoxic activity in various hTERT-expressing cancer cells, hence selectively retarding
the growth of those cells. Efficient and specific cell regression was also detected with ganciclovir (GCV) treatment only
in hTERT-positive cancer cells, which were established to express stably the specific ribozyme that contains the herpes
simplex virus thymidine kinase (HSV-tk) gene. Tissue-specific expression of the ribozyme could further augment the
target specificity of the ribozyme. Importantly, we observed efficient regression of tumors with GCV treatment in mice
that had been inoculated subcutaneously with hTERT-positive cancer cells that stably expressed the specific ribozyme
that contains HSV-tk. These results suggest that the hTERT RNA-targeting trans-splicing ribozyme could be a powerful
agent for tumor-targeted specific gene therapy.
Keywords: Gene therapy, Human telomerase reverse transcriptase (hTERT), Trans-splicing ribozyme
국제학술대회
153
백지
154
한국미생물학회연합
국제학술대회
155
백지
156
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
Pharmatech Inc.
W1-1
Comparative Genomic Sequencing (CGS) and Mutation Discovery
in Prokaryotes Using Microarrays
Daniel Clutter
NimbleGen System, USA
NimbleGen has created a technique to rapidly determine the identity and location of mutations (SNPs,
deletions and insertions) in prokaryotes. This technique has been used to investigate mechanisms of antibiotic
resistance, identify specific strains of SARs, E. coli, plasmodium, etc. The CGS process involves 2 steps:
mutation mapping & sequencing. The first step uses high density microarrays (>385,000 features) to locate
all of the nucleotide differences between two strains and the second step uses array based sequencing to
determine the identity of those mutations. This approach yields a very low false positive rate (almost zero).
The results are comparable to capillary sequencing for about one tenth the cost and half the time.
Keywords: microarray, sequencing, mutation mapping, prokaryote
국제학술대회
157
October 13-14, 2005, Seoul, Korea
KIMIDATA CORPORATION
W2-1
Getting the Most out of Your Microarray Data with Spotfire
DecisionSite
Andrew Khoo
Asia Pacific Operations. Spotfire, Inc., Malaysia
If you are a researcher, biologist, statistician or bioinformatics professional, and involved in gene expression
data analysis, this is an important workshop for you. Learn how to get the most out of your microarray data
with the Spotfire technologywhich is used by leading research institutions to accelerate genomics research.
This workshop is designed to give an overview of the basics of microarray analysis and the critical features
and benefits of Spotfire DecisionSite for Functional Genomics. Learn how to access your microarraydata and
integrate gene expression data with known biological annotations from public databases; quality control
analysis, basic filtering to find interesting and reliable genes and various statistics like cluster analysis and
pattern detections to mine results sets. Lastly, you will learn how to export interesting micorarray results that
can be used for presentations.
158
한국미생물학회연합
A. Systematics and Evolution
B. Environment and Ecology
C. Differentiation and Morphology
D. Physiology and Biochemistry
E. Fermentation and Metabolites
F. Genetics and Genome
G. Infection and Pathogenesis
H. Immunology and Signal Transduction
I. Biotechnology
J. Food Microbiology
K. Others
면지
2005 International Meeting of the Federation of Korean Microbiological Societies
A001
A003
Conservative Gene Analyses of Thermophilic
and Hyperthermophilic Bacteria
Taxonomic Discussions on Fomitopsis, Rhodofomes
and Fomitopsis incarnatus sp. nov.
Dong-Geun LEE, Nam Young KIM, Eo-Jin LEE, Jae-Hwa LEE,
Jong-Myung HA, Bae Jin HA, and Sang-Hyeon LEE*
Kyung Mo KIM*, Jin Sung LEE, and Hack Sung JUNG
Department of Bioscience and Biotechnology, Silla University
*Corresponding author: slee@silla.ac.kr
Department of Biological Sciences, College of Natural Sciences, Seoul
National University
*Corresponding author: minervas@snu.ac.kr
For investigation of conservative genes among 13 thermophilic and
hyperthermophilic bacteria, COG(clusters of orthologous groups of
proteins) algorithm was applied. Totally 16,299 conservative genes
were common and all genes were belong to 167 COGs. Eight COGs
were related to protein metabolism among 167 COGs. However
conservative gene was not limited only thermophiles and hyperthermophiles,
meaning thermal stability is independent of specific protein. Reverse
gyrase was only found in all hyperthermophilic archaebacteria and
eubacteria, indicating DNA stability is important in hyperthermophiles.
Phylogenetic tree revealed that hyperthermophilic eubacteria and
thermophilic archaebacteria had different position between gene
content and 16S rRNA. Thermophilic archaebacteria, hyperthermophilic
eubacteria and archaebacteria had similar values by the statistical
analysis of distance values with 167 COGs in each organism.
Keywords: thermophile, hyperthermophile, COG(clusters of orthologous
groups of proteins), conservative gene
For many years, the split of Rhodofomes from Fomitopsis has been
based on the relatively thin wall of basidiospores, but our sequence
analyses of nuclear ITS, mitochondrial SSU, and the second largest
subunit of RNA polymerase II showed that the wall thickness of the
spores was not a synapomorphic character, indicating that the
segregation of Rhodofomes from Fomitopsis increases the artificial
attribute of the genus Fomitopsis. A new polypore was discovered
from Kangwon Province of Korea and has been recognized as
Fomitopsis rosea or F. cajanderi due to morphological similarities
of basidiocarps. However, the lighter red color and the number of
pores made it possible to discriminate the fungus from other species.
Microscopically, the basidiospores of the new fungus were more
globular than those of F. cajanderi and tended to be more curved
than those of F. rosea. Based on sequence analyses, we again
determined that the fungus was distinct as an independent lineage
from both F. rosea and F. cajanderi. In comparison with the generic
concepts of Rhodofomes and Fomitopsis, we propose this new
polypore as Fomitopsis incarnatus sp. nov. Supported by grants
from RDA and BK21.
Keywords: Fomitopsis, Rhodofomes, Fomitopsis incarnatus sp.
nov.
A002
A004
Clostridium dongmakense sp. nov., Isolated
from Tidal Flat
Epiphytic Fungi on Needles of Pine Trees
Seil KIM
1,2,3
, Hyunyoung JEONG
1,2,3
*, and Jongsik CHUN
1,2,3
*
1
Laboratory of Prokaryotic Biology and Bioinformatics, 2School of Biological
3
Science, Seoul National University
*Corresponding author: jchun@snu.ac.kr
A Gram-negative, strictly anaerobic, halophilic, motile, sporulating
T
and rod-shaped bacterium, designated strain HY-45-18 , was
isolated from tidal flat sediment from Ganghwa Island in South
Korea. The isolate produced butyric acid, propionic acid, glycerol
and H2 as fermentation end products from glucose. Strain
T
HY-45-18 grew optimally at 30℃, pH 7.0 and 4 (w/v)% artificial
sea salts. No growth was observed in the absence of sea salts. In
phylogenetic analyses based on 16S rDNA sequence, strain
T
HY-45-18 showed a distinct phyletic line within the members of
clusterⅠof the order Clostridiales. The closest phylogenetic
T
neighbor to strain HY-45-18 was Clostridium ganghwense KCTC
T
5146 (96.53% 16S rDNA sequence similarity). Several phenotypic
characters readily differentiate the tidal flat isolate from phylogenetically
T
related clostridia. On the basis of polyphasic evidence, strain HY-45-18
should be classified as a novel species, for which the name Clostridium
dongmakense sp. nov. is proposed; the type strain is HY-45-18T
T
T
T
(=IMSNU 40129 = KCTC 5147 = JCM 13194 ). This work was
supported by the 21C Frontier Microbial Genomics and Applications
Center Program (grant MG02-0101-001-2-1-0) and the Strategic
National R&D Program through the Genetic Resources and Information
Network (grant M1-0219-00-0018).
Keywords: Clostridium dongmakense sp. nov., getbol, tidal flat,
16S rDNA sequencing
Jae Young PARK* and Hack Sung JUNG
Department of Biological Sciences, College of Natural Sciences, Seoul
National University
*Corresponding author: minervas@snu.ac.kr
The existence of diverse and abundant microbial populations on
external aerial plant surfaces has been well documented. However,
the majority of studies on epiphytic fungi used to be focused on
filamentous fungi. In this study, filamentous fungi, yeasts, and
yeast-like fungi were collected and investigated from healthy
needles of pine trees (mostly Pinus densiflora) at various sites from
January 2004 to June 2005. Filamentous fungi and yeasts were
isolated from needles using the washing isolation method and a
total of 235 strains of fungi were obtained during this period.
Among them, yeast-like fungi predominated in spring and summer
seasons. The most frequently occurring fungi were Aureobasidium
pullulans, Hormonema dematioides, and Peucimoniella zeimoides.
Hormonema dematioides was confused with A. pullulans as they
have very similar cultural morphology but was discriminated from
each other by molecular analysis. The remaining 71 strains were
mostly filamentous Deuteromycetes. Supported by grants from
KOSEF and BK21.
Key words: Epiphytic Fungi, Pine Needles, Pinus densiflora
국제학술대회
161
October 13-14, 2005, Seoul, Korea
A005
A007
Taxonomic and Systematic Studies on Korean
Aphyllophorales
Identification of Genomic Differences between
Tsukamurella spumae and Pathogenic Tsukamurella
paurometabola Using Suppression Subtractive
Hybridisation Analysis
Jin Sung LEE* and Hack Sung JUNG
Department of Biological Sciences, College of Natural Sciences, Seoul
National University
*Corresponding author: minervas@snu.ac.kr
The Aphyllophorales is one of important orders classified in the
Hymenomycetes (Basidiomycota) whose fruitbodies possess pores,
ridges, teeth, or smooth surfaces but usually lack gills. Recently,
molecular works showed that traditional classifications were
artificial and the Aphyllophorales were phylogenetically subdivided
into some independent lineages. The total Korean Aphyllophorales
consisted of 411 species which belonged to 144 genera and 23
families. Through the specimen examination of Seoul National
University Fungus Collection (SFC), 180 species and 240 strains of
Korean Aphyllophorales were analyzed. To infer their higher level
relationships, nuclear large subunit (nLSU) rDNAs were sequenced
and compared with those of 600 taxa of the Homobasidiomycetes. In
results, several new phylogenetic groups were identified and some
new species of the Aphyllophorales were determined, which
indicated the uniqueness of the Korean fungal flora. Supported by
grants from KOSEF, RDA and BK21.
Keywords: Taxonomy, Systematics, Korean Aphyllophorales
2
1
1
1
Department of Microbiology, Chung-Ang University College of Medicine,
KHIDI
*Corresponding author: kimwy@cau.ac.kr
2
The genus Tsukamurella is well defined and currently encompasses
seven species with validly published names, namely T.
paurometabola (Collins et al. 1988), T. inchonensis (Yassin et al.
1995), T. pulmonis (Yassin et al. 1996), T. strandjordii (Kattar et al.
2001), T. tyrosinosolvens (Yassin et al. 1997), T. spumae (Nam et al.
2003) and T. pseudospumae (Nam et al. 2004). Members of these
taxa share very high 16S rDNA similarity values but can be
distinguished by DNA.DNA relatedness. Tsukamurellae have been
described in association with clinical disease, but T. spumae and T.
pseudospumae were isolated from foaming in activated sludge
plants and have not so far been associated with pathogenicity.
Suppression subtractive hybridization strategy was applied to
uncovering genomic differences between T. spumae (tester) and T.
paurometabola (driver), the well characterised pathogenic strain.
We determined the sequence of 172 tester strain-specific DNA
fragments and further determined novel DNA fragments using each
DNA fragment as a probe in Southern hybridisations of reference
strains. Our results support the idea of a considerable genetic
variability among tsukamurellae species.
Keywords: Tsukamurella spumae, Tsukamurella paurometabola,
Suppression subtractive hybridis
A006
A008
Cryseobacterium buyeonense sp. nov., from
Puddle
Phylogenetic Analysis of Antarctic Marine
Bacteria Jung-Sook LEE*, Keun Chul LEE, Yong-Ha PARK, and Hee
Mock OH
Hyun Hee CHO *, Kyeung Hee CHO , Yoo Kyung LEE ,
1
1
Joung Han YIM , and Hong Kum LEE *
Korean Collection for Type Cultures, KRIBB
*Corresponding author: jslee@kribb.re.kr
1
T
Chryseobacterium strains, 10-59 and 10-60, were isolated from
puddle, Buyeo, Korea and examined by phenotypic,
chemotaxonomic, and genetic characterization. They were
Gram-negative, non-motile, non-spore-forming rod with
yellow-pigmented colonies on nutrient agar. These strains
contained MK-6 as the main respiratory quinone. The major cellular
fatty acids of the isolates were 15:0 iso and 17:0 iso 3OH. The DNA
base composition of them was 34.6-35.2 mol%. Phylogenetic
analysis based on 16S rDNA sequence revealed that isolate formed
an evolutionary lineage distinct from other Chryseobacterium
species. Based on the evaluation of morphological, physiological
and chemotaxonomic characteristics, DNA-DNA hybridization
values and 16S ribosomal DNA sequence comparison, we propose
the new species Chryseobacterium buyeonense sp. nov., type strain
is 10-59T (= KCTC 12485T).
Keywords: Cryseobacterium buyeonense, taxonomy
162
1
Ji Yeon KIM , Sun-Woo NAM , Sung Lim CHO , Kijeong KIM ,
Sang-In CHUNG1, and Wonyong KIM1*
한국미생물학회연합
1,2
1
1
2
Polar BioCenter, KOPRI, KORDI
*Corresponding author: hee2138@nate.com
Antarctic bacteria were isolated from various marine environments,
and identified using phylogenetic analysis on the basis of 16S rDNA
sequences. Sediments, macro algae, marine animal and sea water
samples were collected from around the Korean Antarctic Research
Station King Sejong on King George Island, Antarctica (62°13′ S,
58°47′ W). The collected samples were transported to KOPRI under
frozen conditions, diluted in sterilized seawater, and cultured on
zobell agar plates at 10℃ and 25℃. Total 230 bacterial isolates were
cultured and preserved in glycerol media (15%, v/v) at –80℃.
Phylogenetic analysis of the bacterial isolates indicated that they
belonged to alpha-Proteobacteria (1.3%), beta-Proteobacteria
(0.4%), gamma-Proteobacteria (42.2%), CFB group (23.5%), High
GC Gram-positive bacteria (22.2%), and Low GC Gram-positive
bacteria (10.4%). Among them, eleven isolates were candidates for
new species; the closest cultured bacteria with validly published
names were Cellulophaga baltica, Colwellia piezophila, Devosia
neptuniae, Janibacter marinus, Polaribacter irgensii, Plantibacter
flavus, Rheinheimera baltica, Roseobacter gallaeciensis,
Sanguibacter keddieii, Subtercola boreus, and Thiobacillus
prosperus.
2005 International Meeting of the Federation of Korean Microbiological Societies
A009
A011
Phylogenetic Relationship of Species and Strains
related to Pleurotus eryngii var. ferulae by
Molecular Genetic Analysis
1
1
1
Sun-Gyu CHOI , Min-Goo KIM , Hyun-Min KANG , Won-Sik
KONG2, Young-Bok YOO2, Kab-Yeul JANG2, and Gyu-Hyun
3
KIM *
1
Institute of Mushroom Science, Cheonan Yonam College, 2Division of Applied
Microbiology, NIAST, RDA, 3Department of Horticultural Bio-Industry,
Cheonan Yonam College
*Corresponding author: loger12@nate.com
Pleurotus eryngii var. ferulae is classified to Basidiomycota,
Basidiomycetes, Agaricales, Pleurotaceae, Pleurotus in taxonomy.
P. eryngii var. ferulae has not only high nutritional quality but also
potential of functional food, and showed improvement possibility as
new genetic resource. P. eryngii var. ferulae is known by variety
species of Pleurotus eryngii. But it is classified several species of P.
eryngii var. ferulae, P. ferulae, P. eryngii var. fossulatus, P. eryngii
var. nebrodensis and then happens to be controversial in their
taxonomic location. This study was carried out to classify P. eryngii
var. ferulae using of molecular genetic method with several species
related to P. eryngii var. ferulae, collected and preserved strains from
abroad and the country. It was identified the phylogenetic relationship
of P. eryngii var. ferulae by comparing the DNA sequences of the
internal transcribed spacer regions (ITS Ⅰ, 5.8S rDNA and ITS Ⅱ)
in ribosomal DNA repeat unit and analyzed polymorphism through
randomly amplified polymorphic DNAs (RAPD).
Keywords: Pleurotus eryngii var. ferulae, phylogenetic relationship,
the internal transcribed spacer regions (ITS), randomly amplified
polymorphic DNA (RAPD)
A010
Diversity of Marine Bacterial Communities in
Arctic Biofilms Using Cultivation and CultivationIndependent Methods
Kyeung Hee CHO, Hyun Hee CHO, Yoo Kyung LEE, and Hong
Kum LEE*
PolarBioCenter, KOPRI, KORDI
*Corresponding author: cell93@empal.com
Bacterial diversity in Arctic marine biofilms was analyzed through
cultivation and cultivation – independent methods. We collected
biofilms formed on a floating-pier and a plastic cable under seawater
which were located around the Korea Arctic Research Station Dasan
at Ny-Alesund, Svalbard, Norway (79ON, 12OE). A total of 95
strains were cultured, and 22 species were finally identified by
phylogenetic analysis on the basis of 16S rDNA sequences. They
belonged to γ-proteobacteria, Gram high GC+, CytophagaFlavobacterium group. The phylogenetic analysis of 110 clones
which were randomly selected from 16S rDNA clone libraries,
showed that about 54% of the clones belonged to CytophagaFlavobacterium group. The most frequent clone was an uncultured
Cytophaga sp. DGGE of the partially amplified 16S rDNA was
performed and DNA fragments in the most intense bands were
sequenced. The majority of retrieved sequences were associated
with an uncultured bacterium and an uncultured Cytophaga sp.
Diversity of cultured bacteria was quite different from bacterial
diversity obtained from cultivation-independent methods.
Keywords: Arctic biofilm, DGGE, culture-independent
A012
Antarctic Lichen Flora around Korean Antarctic
Station (King Sejong Station)
1
2
3
Soon-Ok OH , Kwang-Mi LIM , Young-Jin KOH , Jae-Seoun
1
4
4
HUR *, Hosung CHUNG , and Jee Hee KIM
1
Department of Environmental Education, Sunchon National University,
2
3
Department of Biology, Sunchon National University, Department of Applied
Biology, Sunchon National University, 4Korean Polar Research Institute
*Corresponding author: jshur1@sunchon.ac.kr
Twenty six Lichen specimens were collected around Korean
Antarctic Station (King Sejong Station) which is located on the
o
o
Barton Peninsula, King George Island (62 13’S, 58 47’W) on
January of 2005. Thirteen lichen species were identified by
morphological characteristics, TLC analysis and ITS nucleotide
sequence analysis. The species were Cladonia borealis, Cladonia
gracilis, Himantormia lugubris, Lepraria straminea, Pertusaria
erubescens, Placopsis contortuplicata, Pseudephebe pubescens,
Psoroma fruticulosu, Sphaeroglobus globosus, Stereocaulon
alpinum, Usnea aff. igniaria, Usnea antarctica and Usnea
aurantiaco-atra. All the collected specimens are deposited in
Korean Lichen Research Institute (KoLRI) at Sunchon National
University in Korea, and are duplicated to Korean Polar Research
Institute, Korea. This is the first report on the lichen flora in this area.
Keywords: lichen flora, Antarctic, King Sejong Station
Differentiation of Korean Bacillus anthracis
Isolates and Related Bacillus Species by
Cellular Fatty Acid Composition Profiles
Kyunghee LEE
Research Center for Pathogen Control, NIH
*Corresponding author: wksung@nih.go.kr
Capillary gas chromatography (GC) with flame ionization detection
was used to determine the cellular fatty acid profiles of various
microbial pathogens. To evaluate the availability of cellular fatty
acid analysis for the differentiation of Bacillus species, we analyzed
the extracted fatty acid methyl esters (FAMEs) of thirty-five
Bacillus strains including 18 B. anthracis strains and 11 B. cereus
strains. Thirty-five Bacillus strains produced 2 major branched fatty
acids of 15:0 iso and 15:0 anteiso. Major fatty acids of B. anthracis
ATCC 14578T appeared 15:0 iso, 15:0 anteiso, 16:0 iso, 17:0 iso and
sum in feateue3 (15:0 iso 2OH/16:1w7c), especially fatty acid
content of 15:0 anteiso (6.78±0.42 %) was higher than that of B.
cereus and B. thuringiensis. Eighteen B. anthracis strains were
divided into cluster A and B by fatty acid composition profiles.
Among them fifteen Bacillus anthracis strains belonged to Cluster
A and only 3 strains were separated to Cluster B containing 12:0 iso,
16:1 ω 7 alcohol, 16:1 ω 11c, iso 17:1 ω 10c. Moreover this result
corresponds to previous MLVA genotyping data that Korean B.
anthracis isolates classified into 2 clusters, A and B. Therefore we
suggest fatty acid composition analysis is rapid and useful method
for the determination of Bacillus anthracis strains from other
Bacillus species, considering the cellular fatty acids in a sample can
be analyzed within 2 hrs.
Keywords: Gas chromatography, fatty acids, Bacillus anthracis,
Bacillus
국제학술대회
163
October 13-14, 2005, Seoul, Korea
A013
A015
Taxonomic Revision of Peltigera (Lichenized
Ascomycota) in South Korea
1
2
3
Soon-Ok OH , Kwang-Mi LIM , Young Jin KOH , and Jae-Seoun
HUR1*
1
Department of Environmental Education, Sunchon National University,
Department of Biology, Sunchon National University, 3Department of Applied
Biology, Sunchon National University
*Corresponding author: jshur1@sunchon.ac.kr
2
Based on extensive field studies and herbarium research, the
taxonomy and distribution of 18 species of Peltigera in South Korea
are discussed. A key to the species is provided and their Korean
names are suggested. Our specimens of P. degenii, P. horizontalis,
P. leucophlebia, P. neopolydactyla, P. polydactylon, P. ponojensis,
P. praetextata are deposited in Korean Lichen Research Institute
(KoLRI) at Sunchon National University. P. polydactylon was the
common species of Peltigera in South Korea. Eleven more species
previously reported in South Korea were also discussed in this
communication. Japanese collections were used to describe the
eleven species. This is the first report on taxonomic revision of
Peltigera in South Korea.
Keywords: taxonomy, Peltigera, lichen, South Korea
Identification of Colletotrichum spp. Associated
with Anthracnose of Chinese Matrimony (Lycium
chinense Mill.) Based on Morphological
Characteristics and Sequence Analysis of ITS
Regions of rDNA
1
1
2
Anthracnose of Chinese matrimony is one of the severe diseases of the plant. It occurred
mostly on green fruits from late July to October. On the basis of morphological
characteristics and sequence analysis of ITS regions of rDNA, anthracnose fungi were
divied into four groups, namely Colletotrichum acutatum, C. dematium, Glomerella
cingulata (C. gloeosporioides) and C. sp. The predominant species was C. acutatum and
the isolation frequency of the species was 86.7%. The species showed strong pathogenicity
on non-wounded immatured fruits of Chinese matrimony. Conidia of the species were
fusifrom in shape and had pointed ends. It produced orange colored colonies when cultures
on PDA at 30 ºC in darkness. The species which formed falcate conidia setae was identified
as C. dematium. Isolation frequency of the species was 10%. It showed severe symptoms
on wounded, immatured fruits but showed slight symptoms on non-wounded, matured
fruits. It formed dark gray to black colonies when cultured on PDA at 25∼30 ºC in darkness.
G. cingulata, perfect stage of C. gloeosporioides, was isolated and identified for the first
time from the anthracnose of Chinese matrimony. The isolation frequency of G. cingulata
and C. gloeosporioides was 2%. They produced symptoms on matured and wounded fruits
but showed slight symptoms on non-wounded fruits. A unrecorded species of
Colletotrichum was isolated from the anthracnose of Chinese matrimony. More work is
needed for identification of the species.
Keywords: Anthracnose, Chinese matrimony, Colletotrichum, ITS
regions of rDNA
A016
Characterization of Trichoderma spp. Associated
with Green Mold of Oyster Mushroom by
PCR-RFLP and Sequence Analysis of ITS
Regions of rDNA
1
2
3
Myung Soo PARK *, Geon Sik SEO , Kyung Sook BAE , and
1
Seung Hun YU *
1
Department of Applied Biology, College of Agriculture and Life Sciences,
2
Chungnam National University, Korea National Agricultural College,
3
KRIBB
*Corresponding author: shunyu@cnu.ac.kr
Molecular profiles of PCR-RFLP and sequence analysis of internal
transcribed spacer (ITS) regions of rDNA were compared between
morphologically distinguishable species of Trichoderma isolated
from substrates of oyster mushroom in Korea, T. atroviride, T.
citrinoviride, T. harzianum, T. longibrachiatum, T. virens, and two
unidentified species, Trichoderma sp. 1 and 2. PCR-RFLP analysis
divided the Trichoderma spp. into six RFLP groups, A, B, C, D, E,
and F. The RFLP groups were generally agreed with described
morphological species, except that the RFLP group A containing the
two unidentified species. A neighbor-joining tree based on ITS
sequences well supported RFLP groups observed by RFLP analysis
of ITS regions of rDNA. Additionally, the two unidentified species,
Trichoderma sp. 1 and 2, which could not be distinguished by
PCR-RFLP analysis, were separated in sequence analysis of ITS
regions of rDNA.
Keywords: ITS regions of rDNA, oyster mushroom, PCR- RFLP,
Trichoderma
한국미생물학회연합
2
2
Chongyang Boxthorn Experimental Station, Department of Applied Biology,
College of Agriculture and Life Sciences, Chungnam National University
*Corresponding author: shunyu@cnu.ac.kr
A014
164
1
Boo Hee LEE , Joo Chan LEE , Myung Soo PARK , and Seung Hun YU *
Tsukamurella pseudospumae sp. nov., a Novel
Actinomycete Isolated from Activated Sludge
Foam
1,2
1,3
4
Sun-Woo NAM *, Wonyong KIM , Jongsik CHUN , and
1
Michael GOODFELLOW
1
2
3
School of Biology, University of Newcastle, UK, KHIDI, Department of
4
Microbiology, Chung-Ang University College of Medicine, School of
Biological Sciences, Seoul National University
*Corresponding author: swnam@khidi.or.kr
The taxonomic position of two Tsukamurella strains isolated from
activated sludge foam was clarified. The organisms, isolates JC85
T
and N1176 , were found to have chemical and morphological
properties typical of members of the genus Tsukamurella.
DNA:DNA relatedness studies showed that the strains formed a
distinct genomic species which was most closely related to
Tsukamurella spumae. The two isolates also share a range of
phenotypic properties that distinguishes them from representatives
of all of the validly described species of Tsukamurella. It is evident
from the data that the two organisms should be classified as a new
Tsukamurella species. The name proposed for this new taxon is
T
Tsukamurella pseudospumae sp. nov., the type strain is N1176 (=
T
T
DSM 44118 = NCIMB 13963 ).
Keywords: Tsukamurella pseudospumae sp. nov., polyphasic
taxonomy; activated sludge isolates
2005 International Meeting of the Federation of Korean Microbiological Societies
A017
A019
Genotype Variation of Virulence-Associated
Factors among Korean Isolates of Bordetella
pertussis
1
1
1
Kyung-Tae JUNG , Jeong-Hoon CHUN , Gi-Eun RHIE ,
Ki-Joong KIM2, and Won-Keun SEONG1
1
Research Center for Pathogen Control, Department of Microbiology, NIH,
2
School of Life Sciences and Biotechnology, Korea University
*Corresponding author: jfoxdr@lycos.co.kr
Antigenic divergences have been reported between the Bordetella pertussis
vaccine strain and circulating strains in each country. In Korea, the vaccination
program against pertussis using acellular pertussis vaccine produced from
Tohama strain was started in 1982. In this study, we investigated the antigenic
divergences of B. pertussis 26 isolates in Korea by analyzing genes of three
virulence-associated proteins, pertussis toxin (ptx), adhesion pertactin (prn) and
fimbria (fim3). Sequence analyses of the ptx gene revealed three types of
variants, ptxS1A, ptxS1B and ptxS1D, while prn gene revealed one type, prn1.
The fim3 gene revealed two type, fim3A and fim3A*. 5 strains isolated in 1972
before vaccination revealed the combination of ptxS1B and prn1 alleles,
ptxS1B/prn1, which is the same as the vaccine strain used in Korea, Tohama. On
the other hand, 21 strains isolated from 1999 to 2005 contained two combination
alleles, ptxS1A/prn1 and ptxS1D/prn1. The ptxS1D/prn1 allele was found in the
only KNT39 strain. Combinations of ptxS1 and prn alleles well corresponded
with those of pulsed-field gel electrophoresis (PFGE). In accordance with
previous reports, antigenic variation were observed in strains isolated after
pertussis vaccine program. Further study will be needed to systemically analyze
the diversity of the B. pertussis strains in Korea, and these results will be useful
as a baseline for selecting appropriate B. pertussis strain for future vaccine
development in Korea.
Keywords: B.pertussis, ptx, prn, fim3 gene
Genomic Species Identification of Environmental
Isolates of Tsukamurella by rep-PCR Genomic
Fingerprinting Techniques
1,2
1,3
1
Sun-Woo NAM *, Wonyong KIM , and Michael GOODFELLOW
1
2
3
School of Biology, University of Newcastle, UK, KHIDI, Department of
Microbiology, Chung-Ang University College of Medicine
*Corresponding author: swnam@khidi.or.kr
In the present investigation the rep-PCR fingerprinting techniques
based on five different primers (REP, ERIC, BOX, SERE, LR REP)
were used to evaluate for the rapid delineation of Tsukamurella
reference strains and isolates and to determine the extent to which
the resultant groups corresponded to those based on chemical and
numerical phenetic taxonomy. The combination of all five different
rep-PCR methods showed the highest discriminatory power, while
LR REP analysis had the lowest discrimination of the techniques
used. The fact that fewer banding patterns were generated by LR
REP-PCR analysis than by the other four techniques and
conservation of these patterns from year to year suggest that the LR
REP sequences within this locus are relatively conserved. The
choice of more variable genetic loci for rep-PCR analysis may
provide more detailed information about genetic relationships
among isolates. The classification derived from the combined
analyses of the five different rep-PCR fingerprint analyses showed
the highest congruence with individual rep-PCR analysis, namely
REP-, ERIC-, BOX-, SERE- and LR REP-PCR.
Keywords: Genus Tsukamurella, rep-PCR fingerprinting, REPPCR, ERIC-PCR, BOX-PCR, SERE-PCR, LR REP-PCR
A018
A020
Analysis of Genetic Variation by Mating in
Pleurotus eryngii var. ferulae
1
1
1
Phylogenetic Implications of Glyceraldehydes
-3-Phosphate Dehydrogenase Gene Sequences
in Cordyceps
Sun-Gyu CHOI , Dong-Soon BAE , Jung-Hwan CHA , Won-Sik
2
2
2
KONG , Young-Bok YOO , Kab-Yeul JANG , and Gyu-Hyun
3
KIM *
Hyuk Gu PARK , Han Gyu KO , Seong Hwan KIM , Jae Mo
3
1
SUNG , and Won Mok PARK *
1
Institute of Mushroom Science, Cheonan Yonam College, Division of Applied
Microbiology, NIAST, RDA, 3Department of Horticultural Bio-Industry,
Cheonan Yonam College
*Corresponding author: loger12@nate.com
1
Pleurotus eryngii var. ferulae is mushroom of the genus Pleurotus in
Agaricales and this species is improved with possibility of new
commercial cultivar. Breeding of Pleurotus is performed by mating
and easy to be compared to some other cultivated mushrooms. This
study carried out analyzing the genetic variation between original
strain and new strain produced by their mating in Pleurotus eryngii
var. ferulae. Original strain was collected and its fruiting body was
produced through bottle cultivation. Spores were germinated and
monokaryons were isolated through plate incubation in the
laboratory. And then it was determination of mating types in
monosporous isolates, A1B1, A1B2, A2B1, and A2B2. It was mated
and induced production of fruiting bodies. In new strains, growth of
mycelia and characters of fruiting body were measured. Their
genetic variation was analyzed polymorphism through randomly
amplified polymorphic DNAs (RAPD). It is to offer basic data and
effective method for breeding of new excellent cultivar in Pleurotus
eryngii var. ferulae.
Keywords: Pleurotus eryngii var. ferulae, Genetic Variation,
RAPD, Mating
The present study was carried out to determine the phylogenetic
relationships of eight Cordyceps species using glyceraldehydes3-phosphate dehydrogenase gene sequences data. For this work, the
target gpd gene was amplified by PCR using gpd1-gpd2 primers
from genomic DNA of 28 isolates of Cordyceps and their nucleotide
sequences determined. Analysis of sequence data showed that the
size of gpd gene of all isolates of Cordyceps militaris is about 730
base pairs, while that of C. pruinosa, C. longissima, C. pentatom,C.
yongmoonensis, Paecilomyces tenuipes, Shimizuomyces poradoxa
and C. scarabaeicola is 693bp, 526bp, 729bp, 951bp, 867bp, 938bp
and 719bp, respectively, indicating there is size variation in gpd
gene among Cordyces spp. Phylogram based on the gpd gene
sequence analysis revealed that the 28 isolates could be divided into
three groups. The first group included one species, C. militaris. The
second group included four species, C. yongmoonensis, C. pruinosa,
Shimozuomyces poradoxa. The third group was one species, C.
scarabaeicola.With the comparison of sixteen isolates of C.
militaris, we found that there was not exists intraspecific variation in
the gpd gene of C. militaris.
Keywords: Cordyceps, gpd gene, phylogenetic relationship
2
1
1
2
College of Life Sciences and Graduate School of Biotechnology, Korea
2
3
University, Department of Microbiology, Dankook University, Department
of Applied Biology, Kangwon National University
*Corresponding author: hyukgu@korea.ac.kr
국제학술대회
165
October 13-14, 2005, Seoul, Korea
A021
A023
Notes on Two Species of the Laboulbeniales
from Tibet
1
2
3
Yong-Bo LEE *, Chae-Kyu LIM , Dong-Kyoung JANG , In-Ha
JUNG3, Sang-Hee PARK3, In-Hwa JANG3, Sung-Eun YUN3,
4
and Han-Su PARK
1
Division of Science Education, College of Education, Chosun University,
Department of Herbal Medicine Resources Development, Naju College,
3
Major in Biology Education, Graduated School of Education, Chosun
University, 4Major in Biology Education, Graduated School, Chosun
University
*Corresponding author: ybalee@chosun.ac.kr
2
Two species of the Laboubeniales based on the preparations of Tibet
are described. They are new to the mycological flora of Tibet. Laboulbenia polypaga were found on several parts of Amara
majuscula, this species have the outer appendages not ramified,
simple. Peyritschiella protea were found on the lower abdomen of
Philonthus wuesthoffi, this species have two perithecia and two
antheridia produced on the third layer of receptacle. Keywords: Laboulbenia, Peyritschiella, Laboulbeniales. Tibet
Lactobacillus koreensis sp. nov., Isolated from
Wheat Flour in South Korea
Zubair ASLAM, Kyoug-Ho KIM, Ju Hyoung LIM, Wan-Taek IM,
and Sung-Taik LEE*
Department of Biological Sciences, KAIST
*Corresponding author: e_stlee@kaist.ac.kr
The taxonomic position of two lactic acid producing bacteria, isolated
from wheat flour in South Korea, was studied using polyphasic
approach. Biochemical and physiological characteristics indicated
these to be the members of the genus Lactobacillus, showing more
similarity (98.8 to 98.9%) to Lactobacillus rossii than to any other
species. These strains (M1-212T and M2-236) were facultative
anaerobic, gram-positive, non-spore forming and non-motile, short
rod-shaped bacteria. The optimum temperature for these strains was
30 ºC and did not grow on 15 and 45 ºC and tolerated 5% (w/v) NaCl.
The G+C content of the type strain M1-212T was 45.4 mol% which
is in the range of genus Lactobacillus (32-53 mol%). Physiological,
biochemical and genotypic data, as well as results of DNA-DNA
hybridization of genomic DNA with one of the closest phylogenetic
relatives, L. rossii DSM 15814T, indicated that strains (M1-212T and
M2-236) represent a novel species of the genus Lactobacillus for
which the name Lactobacillus koreensis sp. nov. is proposed. The
type strain of this species is M1-212T (KCTC 3985T). This work was
supported by the 21C Frontier Microbial Genomics and Application
Center Program, Ministry of Science &Technology (Grant
MG05-0101-4-0), Republic of Korea.
Keywords: Lactobacillus koreensis sp. nov., Wheat flour
A022
A024
Determination of the Most Closely Related
Bacillus Isolates to Bacillus anthracis by
Multilocus Sequence Typing
1,2
1
2
Kijeong KIM *, Eunhee CHEON , Katherine WHEELER ,
3
2
2
Youngchul YOUN , Chulmin PARK , Terrance LEIGHTON ,
1
1
Wonyong KIM , and Sang-In CHUNG
1
Department of Microbiology, College of Medicine, Chung-Ang University,
Children's Hospital Oakland Research Institute, USA, 3Institute of Medical
Science, Chung-Ang University
*Corresponding author: kimkj@cau.ac.kr
2
There have been many efforts to develop Bacillus anthracis
detection assays, but the problem of false-positive results has often
been encountered. Therefore, to validate an assay for B. anthracis
detection, it is critical to examine its specificity with the most closely
related Bacillus isolates that are available. To define the most closely
related Bacillus isolates to B. anthracis in our Bacillus collections,
we analyzed by multilocus sequence typing(MLST) the phylogeny
of 77 closely related Bacillus isolates selected from 264 Bacillus
isolates. The selection includes all the Bacillus isolates that have
been shown in our previous studies to produce false-positive results
by some anthrax-detection assays. The MLST phylogenetic analyses
revealed that 27 of the non-B. anthracis isolates clustered within the
B. anthracis clade, and four of them (three sequence types, STs) had
the highest degree of genetic relatedness with B. anthracis, 18 (11
STs) had the second highest, and five (fiveSTs) had the third highest.
We anticipate that the inclusion of the 19 ST isolates when analyzing
B. anthracis detection assays will prove to be useful for screening for
their specificity to detect B. anthracis.
Keywords: Bacillus anthracis, Bacillus cereus group, MLST,
phylogeny, detection of Bacillus anthracis
166
한국미생물학회연합
Erwinia Suwonensis, a Novel Pathogen that
Affects Pleurotus eryngii
1
1
1
Mi-Soon KIM *, Gyong-Yel KWON , Han-Kyung KIM ,
2
1
1
Soon-Wo KWON , Kang-Hyo LEE , Wan-Gyu KIM , and
1
Hang-Yeon WEON
1
2
Applied Microbiology Division, NIAST, Korean Agricultural Culture
Collection, Genetic Resources Division, NIAST
*Corresponding author: hyweon@rda.go.kr
Gram-negative bacteria were isolated from soft rot of Pleurotus
eryngii. A collection of 38 strains belonging to the four genus of
Enterobacteriaceae (genus Erwinia, Ewingella, Pantoea and
Pectobacterium) were identified by 16S rDNA sequences and REP
(repetitive extragenic palindromic element)-PCR fingerprinting
patterns. Among them, strain PED 3-4 showed strong pathogenesis
against Pleurotus eryngii. The strain was studied by phenotypic,
genotypic and chemotaxonomical methods. The major fatty acids of
the strain were C18:1 ω7c (36.1 %), C16:0 (23.0 %) and the highest 16S
rRNA gene sequence similarities were found with Pectobacterium
cypripedii (97.4 %), Pantoea ananatis (97.1 %) and Pantoea
stewartii (97.1 %). On the basis of polyphasic approaches, strain
PED 3-4 should be placed in the genus Erwinia and designated a
novel species, for which the name Erwinia Suwonensis is proposed.
Keywords: Erwinia suwonensis, Polyphasic taxonomy, Novel
bacterium, Pleurotus eryngii, Mushroom pathogen
2005 International Meeting of the Federation of Korean Microbiological Societies
A025
A027
Evolution of HIV-1 Subtype B env Gene in Men
who Sex with Men (MSM) of Korea
1
1
1
Jeong-Gu NAM *, Gab Jung KIM , Bo-Gyeong SHIN , Han Na
YANG1, Mee Kyung KEE1, Joo-Shil LEE2, and Sung Soon KIM1
1
2
Center for AIDS Research, Department Virology, NIH, KCDC, Department
Virology, NIH, KCDC
*Corresponding author: jeonggu@nih.go.kr
HIV-1 infections in Korea are mainly by sexual contacts and quite a
portion of the infections is due to men who sex with men (MSM). To
investigate whether the evolution of HIV-1 env gene is different by
transmission mode, we have analyzed the evolution of HIV-1
subtype B env gene in group of MSM the transmission mode of
which were identified. We selected 197 HIV-1 subtype B infected
MSM and they were infected in Korea. The nucleotide sequences of
HIV-1 env gene were analyzed through phylogenetic tree using the
Neighbor-Joining method with PAUP* program. The evolution of HIV-1 env gene was also analyzed through its diversity and
divergency by year of virus isolation. In phylogenetic tree analysis
of HIV-1 subtype B env gene, 189 (96%) of 197 MSM were formed
monoclade (Bootstrap value = 86%). The diversity by year of virus
isolation (1996-2005) was 11.5-17.6%. The divergency from
persons infected in early 1990s to those infected in later periods was
9.3-12.3% by year of virus isolation. We could not find statistically
significant increase in diversity and divergency of HIV-1 env gene
by year of virus isolation (p=0.89 and p=0.65, respectively). The
prevalent HIV in MSM of Korea are very closely related and the
evolution is not well progressed. This study suggests that further
analysis of female infected individuals by heterosexual contacts are
necessary to identify the evolution of HIV by transmission mode.
Keywords: HIV-1 env gene, MSM, Diversity, Divergency,
Evolution
A026
SIMPLOT Analysis of the Coronavirus Genomes
Ok-Ju KIM*, Chan-Seung PARK, and Chan-Hee LEE
Division of Life Sciences, Chungbuk National University
*Corresponding author: chlee@chungbuk.ac.kr
Severe acute respiratory syndrome (SARS) is a newly emerged
disease caused by a novel coronavirus (SARS-CoV), which spread
globally in early 2003, affecting over 30 countries. The origins and
evolutionary history of the SARS-CoV remain an issue of
uncertainty and debate. Previous phylogenetic studies suggest that
SARS-CoV appears to be related to Group 2 coronavirus (G2-CoV).
SIMPLOT was used to provide further evidence for the close
relationship between SARS-CoV and G2-CoV. Side-by-side
sequence similarity between SARS-CoV and other groups of
coronaviruses was speculated by the SIMPLOT analysis of the
coronavirus genes including polymerase (orf1a&b), spike (S),
envelope (E), membrane (M) and nucleocapsid (N). SIMPLOT
curve is a comparison between the query sequence (SARS-CoV) and
other coronavirus (G1-CoV, G2-CoV and G3-CoV) sequences. The
result of the SIMPLOT analysis suggested that G2-CoV is the most
similar than other coronavirus groups. Especially orf1b region
showed a similarity over 50% both in nucleotide and amino acid
sequence. Further and in detailed analysis of the SIMPLOT may
provide information on the origin and evolution of coronaviruses
including SARS-CoV.
Keywords: SIMPLOT, SARS-CoV
A028
Phylogenetic Analysis of Nuclear Ribosomal
DNA Intergenic Spacer (IGS)ⅠRegion of Genus
Agaricus
1
1
1
Aspromonas composta gen. nov., sp. nov. and
Aspromonas terrae gen. nov., sp. nov., Isolated
from the Compost
YoungHyun RYU *, WooSik JO *, SungGuk CHOI *, JongGuk
2
3
4
KIM , JaeTak YOON , and JungSik PARK
Long JIN, Kwang-Kyu KIM, Hee-Chan YANG, Wan-Taek IM,
and Sung-Taik LEE*
1
Department of Biological Sciences, KAIST
*Corresponding author: e_stlee@kaist.ac.kr
2
Department Plant Environment, GyungBuk ATA, Department Microbiology,
3
4
Kyungpook National University, GyungBuk ATA, NIAST, RDA
*Corresponding author: molgene@gba.go.kr
Phylogenetic relationship of genus Agaricus was studied by
comparing the nuclear ribosomal intergenic spacer(IGS)Ⅰ region
and structural characteristics was analyzed. IGSⅠ region of 5
species(A. bisporus, A. edulis, A. bitorquis, A. campestris, A.
potobella) of Agaricus was from 1,323bp(A. bisporus ASI 1056) to
1,491bp(A. potobella ASI 1210) long and GC contents were 47%(A.
bisporus) to 49%(A. potobella). Size difference of IGS 1 region
among Agaricus species was caused by sequence insertion in 3' end
region, especially A. potobella. and inserted sequence was higher
GC content than other IGS sequence and have repeated GG/GC
patterns. ITS region was widely used in phylogenetic relation of
basidiomycetes, but fungal ribosomal IGS region was not well
understood and applied yet. Our study indicated that IGS region can
be a good tool in phylogenetic study of basidiomycetes.
Keywords: IGS, Agaricus, ribisomal
A polyphasic taxonomic study was carried out on TR7-09T and
T7-07T, two strains isolated from the compost in Republic of Korea.
Comparative 16S rRNA gene sequence studies showed a clear
affiliation of these two bacteria into the ‘Gammaproteobacteria’,
which were most closely related to Silanimonas lenta
KCTC12236T(90.1% 16S rRNA gene sequence similarity),
showing 95% 16S rRNA gene sequence similarity between them.
These strains were Gram-negative, aerobic, motile, and rod-shaped
bacteria. The predominant ubiquinone was Q-8 and major fatty acids
were C15:0iso, C16:0 iso, C17:0 iso and C11:0 iso 3-OH for both stains.
The G+C content of the genomic DNA of the strains TR7-09T and
T7-07T were 71.4% and 64.9%, On the basis of polyphasic
evidences, it is proposed that strains TR7-09T and T7-07T should be
placed in a new genus Aspromonas gen. nov., as two distinct new
species, for which the names Aspromonas composta sp. nov. and
Aspromonas terrae sp. nov. are proposed with the type strains
TR7-09T and T7-07T, respectively. (This work was supported by the
Eco-Technopia-21, Ministry of Environment)
Keywords: Aspromonas composta, Aspromonas terrae, compost
국제학술대회
167
October 13-14, 2005, Seoul, Korea
A029
A031
Kineococcus marinus sp. nov., Isolated from a
Marine Sediment of Jeju Coast in Korea
Nocardia caverna sp. nov., Isolated from a
Natural Cave in Jeju Island
Soon Dong LEE
Jae Pyo SEO and Soon Dong LEE
Department of Science Education, Cheju National University
*Corresponding author: sdlee@cheju.ac.kr
Department of Science Education, Cheju National University
*Corresponding author: sdlee@cheju.ac.kr
A new marine actinomycete, strain KST3-3T, which was isolated
from a sediment sample of Jeju coast, was subjected to polyphasic
taxonomic characterization. The organism was morphologically
characterized by the formation of motile, coccoid cells. A
phylogenetic analysis based on 16S rDNA sequence studies
showed that the organism was related to the genera Kineosporia,
Kineococcus and related genera of the suborder Frankineae and
occupied the deepest branch outside a taxon encompassing members
of the genus Kineococcus. The morphological and chemotaxonomic
characteristics, albeit with a higher level of sequence similarity to
members of the genus Kineosporia, supported the classification to
the genus Kineococcus. It was evident, on the basis of phylogenetic,
phenetic and genetic data, that the organism should be assigned as a
new species of the genus Kineococcus, for which Kineococcus
marinus sp. nov. is proposed. The type strain is strain KST3-3T (=
KCCM 42250T = NRRL B-24439T).
Keywords: Kineococcus marinus sp. nov., marine actinomycete,
16S rDNA sequence
A new actinomycete, strain N2-1T, were isolated from a natural cave
in Jeju, Republic of Korea, using a dilution method and was
subjected to polyphasic taxonomy. The almost complete 16S DNA
sequence was determined by direct sequencing of the purified PCR
product and was compared with those of representatives of the genus
Nocardia. It was revealed from the phylogenetic analysis that the
organism forms a distinct clade between Nocardia salmonicida
cluster and Nocardia alba branch within the evolutionary radiation
occupied by the genus Nocardia of family Nocardiaceae. The
organism showed 16S rDNA sequence similarity of 98.9% with its
nearest phylogenetic neighbors, Nocardia vaccinii. The
chemotaxonomic properties, such as the principal amino acid of
peptidoglycan, major menaquinone, phospholipid composition and
the presence of mycolic acid, supported the classification in the
genus Nocardia. The organism was readily differentiated from the
validly described Nocardia species by a broad set of phenotypic
properties and its unique phylogenetic position, for which the name
Nocardia caverna sp. nov. is proposed. The type strain is strain
N2-1T (= KCCM 42249T = NRRL B-24438T). Keywords: Nocardia caverna sp. nov., actinomycete, natural cave,
polyphasic taxonomy
A030
A032
The Identification and Characterization of
Halophilic Fungi, Eurotium spp., Isolated from
Marine Anchovies
1
1,2
1
Su Young KIM , Hee-Gon CHOI , Mira JIN , and Kwang-Yeop
1,3
JAHNG *
1
2
Division of Biological Sciences, Chonbuk National University, Research
3
Institute of Water Environment, CNU, Basic Science Research Institute, CNU
*Corresponding author: goodear@chonbuk.ac.kr
We have isolated halophilic fungi from Korean traditional food,
jeotkal, which is made of salted anchovies. These filamentous fungi
that formed the sexual structure cleistothecium could not grow
without salt in growth medium. Spores were lysed by osmostress
under no salt condition so that they could not germinate. All strains
from anchovy jeotkal were also isolated from the surface tissue such
as scales and gills of raw anchovy, but not from liver and digestive
organs, implying that these isolates originated from sea water. The
phylogenetic analyses based on the sequence comparison of 18S,
28S and ITS region of rDNA suggested that they classified into the
genus Eurotium. Species of Eurotium typically produce bright
yellow cleistothecia and dull green spinose conidia which are
formed from phialides only. The nucleotide sequence of 28S region
of isolates showed 100 - 99% identity to that of E. herbariorum and
E. rubrum, suggesting that they should be highly related to these
species. Morphological, physiological and molecular characteristics
further supported that these fungal isolates belong to halophilic
species of the genus Eurotium.
Keywords: Eurotium, halophilic fungi, phylogeny, rDNA
168
한국미생물학회연합
Polyphasic Taxonomic Research about Newly
Isolated 4 Strains in the Genus Deinococcus
1
1
1
Hae-Min JUNG , Wan-Taek IM , Myung Kyum KIM , Nagamani
2
2
1
BORA , Michael GOODFELLOW , and Sung-Taik LEE *
1
Environmental and Molecular Microbiology Laboratory, Department of
2
Biological Sciences, KAIST, School of Biology, King George VIth Building,
University of Newcastle, UK
*Corresponding author: e_stlee@kaist.ac.kr
Gram-negative, non-spore-forming, non-motile, rod or coccusshaped bacteria, designated PB314, DaeR-4, Ho-08, BE4-4 were
isolated from freshwater with sediment of GapCheon and stream
under Daechung-Dam, activated sludge, and root of an oak tree,
respectively. They were characterized in order to determine their
taxonomic positions. 16S rRNA gene sequence analyses revealed that
the strains belong to the genus Deinococcus, and they were most
T
closely related to Deinococcus grandis DSM 3963 (96.3%),
T
Deinococcus radiophilus DSM 20551 (91.4), Deinococcus grandis
T
DSM 3963 (96.7%), and Deinococcus radiopugnans (97.9%),
respectively. Chemotaxonomic data revealed that these 4 strains
possess quinone system MK-8 as the predominant compound, C16:1 ω
7c and C16:0 as predominant fatty acids, and ornithine as a diamino acid
in a peptidoglycan structure, corroborating our assignment of the
strains to the genus Deinococcus. The results of phylogenetic analyses
based on 16S rRNA gene sequences and physiological and
biochemical tests clearly demonstrated that the strains represent a
distinct species. This work was supported by the 21C Frontier
Microbial Genomics and Application Center Program, Ministry of
Science &Technology (Grant MG05-0101-4-0), Republic of Korea.
Keywords: Deinococcus, Polyphasic taxonomy, 16S rRNA gene
sequence
2005 International Meeting of the Federation of Korean Microbiological Societies
A033
A035
Polyphasic Characterization of Paenibacillus
spp. Isolated from Various Habitats of Korea
1
2
2
Hang-Yeon WEON , Soon-Wo KWON , Byung-Yong KIM ,
Hyung-Jun PARK1, Soon-Ja SEOK1, and Wan-Gyu KIM1
1
Applied Microbiology Division, National Institute of Agricultural Science and
Technology, RDA, 2KACC, Genetic Resources Division, National Institute of
Agricultural Biotechnology, RDA
*Corresponding author: hyweon@rda.go.kr
The genus Paenibacillus comprise at least 62 validly published
species and have been isolated from a wide variety of sources
including soil, water, the plant rhizosphere, plant materials, food,
fodder, faeces and diseased insect larvae. Fifty-eight Paenibacillus
strains isolated from air samples, soils and composts, were
phenotypically and genotypically characterized. This study
illustrated the diversity of Paenibacillus and demonstrates a need for
further clarification and definition of additional species within this
genus.
Keywords: Polyphasic taxonomy, Paenibacillus, Novel bacteria,
Diversity
Cultivation of the Three Hundreds of Bacterial
Species from the Soil of the Ginseng Field and
Mining the Novel Lineage Bacteria
Wan-Taek IM, Hae-Min JUNG, Ying-Shun CUI, Qing-Mei LIU,
Xiu-Li ZHANG, and Sung-Taik LEE*
Environmental and Molecular Microbiology Laboratory, Department of
Biological Sciences, KAIST
*Corresponding author: e_stlee@kaist.ac.kr
The culturability of bacteria in the soil of the ginseng field (Pocheon,
Korea) was investigated by using diluted R2A agar as the growth
medium. About 1,000 isolates obtained from plate counting
experiments were identified by comparative analysis of partial 16S
rRNA gene sequences. A large proportion of these isolates can be
genomic novel species or genus of globally distributed group of soil
bacteria within the divisions Actinobacteria, Proteobacteria, and
Bacteroidetes. Among these 1,000 strains, a few bacterial strains
were related with phylum Acidobacteria and Verrucomicrobia.
Some bacterial strains are found to show the deep distant branch
(more than 15% 16S rRNA gene sequences dissimilarity) compared
with the previous reported strains. Among these novel lineage
strains, strain GS348 which was lineage in the candidate division
OP10 was cultivated first and appeared to us. This work was
supported by the 21C Frontier Microbial Genomics and Application
Center Program, Ministry of Science &Technology (Grant
MG05-0101-4-0), Republic of Korea.
Keywords: Ginseng soil, OP10, 16S rRNA sequence, Cutivation of
bacteria
A034
A Polyphasic Taxonomic Study on Two New
Halomonas Species Isolated from a Solar
Saltern
Kwang Kyu KIM, Long JIN, Hee Chan YANG, and Sung-Taik
LEE*
Department of Biological Sciences, KAIST
*Corresponding author: e_stlee@kaist.ac.kr
Totally 34 Halomonas strains were isolated from a solar saltern in
Anmyeondo, Korea. Five strains of them, considered to belong to
new species, were subjected to a polyphasic taxonomic
investigation. The strains were Gram-negative, non-motile and
non-spore-forming rods, and could grow at salinities of 1-25% (w/v)
NaCl and at temperatures of 10-42°C. Colonies were white,
translucent and shiny with entire edges. They contained Q-9 as the
predominant ubiquinone, C18:1 ω7c, C16:0 and C16:1 ω7c/15:0 iso
2-OH as the major fatty acids. Phylogenetic analysis, based on 16S
rRNA gene sequencing, showed three strains including M-29T,
M-69 and M-70 were most similar to Halomonas ventosae CECT
T
T
5797 (97.7-98.0%), and two strains including M-27 and M-67
were most similar to Halomonas desiderata DSM 9502T (95.4 and
95.7%, respectively). However, DNA-DNA hybridization data and
their phenotypic properties showed that five strains could be
distinguished from all known Halomonas species, representing two
distinct new species. Consequently Halomonas salinaria sp. nov. is
proposed for three strains and Halomonas anmyeonensis sp. nov. is
proposed for two strains; the type strains are M-29T and M-27T,
respectively. (This work was supported by the Eco-Technopia-21,
Ministry of Environment.)
Keyword: Halomonas
국제학술대회
169
October 13-14, 2005, Seoul, Korea
B001
B003
Comparative Analysis of Genes for Initial
Catabolism of Aromatic Hydrocarbons from a
Microbial Consortium and Soils in Reed
Rhizosphere of Sunchon Bay
1
2
Sung-Mi KANG , Kye-Heon OH , and Hyung-Yeel KAHNG
1
1
Department of Environmental Education, Sunchon National University,
2
Department of Life Science, Sunchoonhyang University
*Corresponding author: kahng@sunchon.ac.kr
In this study catabolic genes from a polycyclic aromatic hydrocarbon
(PAH) or benzene, toluene, ethylbenzene, and xylene (BTEX)
consortium was compared with indigenous ones from soils in reed
rhizosphere of Sunchon Bay, Korea. Our initial efforts were
attempted to obtain a PAH or BTEX consortium from the rhizosphere
as well as to elucidate indigenous dioxygenases for initial catabolism
of aromatic hydrocarbons. Two bacterial consortia capable of
degrading PAH or BTEX were obtained by enrichment culture using
PAH containing anthracene, naphthalene, phenanthrene, and pyrene
or BTEX mixtures as sources of carbon and energy. A comparative
analysis of the catabolic genes for aromatic hydrocarbons obtained
through PCR process using both consortia and soil genomic DNAs
revealed that there were of significant differences between the
diversity of catabolic genes from both consortia and indigenous soils
of reed rhizosphere. Structural analysis of bacterial community in
reed rhizosphere showed that bacterial diversity greatly decreased in
PAH- or BTEX-consortium as well as some bacteria in PAH or BTEX
consortium were a few or not detected. It suggested that most
members of PAH or BTEX consortium were minor ones in reed
rhizosphere ecosystem. (This work was supported by Korea Research
Foundation Grant (KRF-2004-041-D00375).
Keywords: Reed rhizosphere, BTEX, PAH, Catabolic genes
Vertical and Spatial Variation of Bacterial
Community Structure in Tidal Flat Sediment
Using Denaturing Gradient Gel Electrophoresis
Bong Soo KIM and Jongsik CHUN
School of Biological Sciences, Seoul National University
*Corresponding author: spore1@empal.com
We investigated vertical distributions and spatial variations of
bacterial community structure in tidal flat sediment samples of
Dongmak in Ganghwa Island. Bacterial community composition
was characterized with denaturing gradient gel electrophoresis
(DGGE) of PCR-amplified 16S ribosomal DNA products. Vertical
DGGE profiles and principal components analysis (PCA) were
shown four separated groups illustrating structural shifts along
depths. The differences of bacterial community structure were not
related to distances in two groups, but related to distances in other
two groups. Phylotypes identified through sequencing of DNA from
DGGE bands were related to organisms within the α-, γ-, δ- and ε
-Proteobacteria, CFB group and Chroloflexi phyla. Most sequences
were closely related to clones and isolates from other marine
sediments. Our findings demonstrate that a sample may not be
representative of bacterial community in a sampling site.
Keywords: Tidal Flat, DGGE, Bacterial community, PCA
B002
B004
Molecular Characterization of Bacterial Community
Structure in Forest Soils Contaminated with
Fuel Hydrocarbon
1
1
1
Jae-Hyung AHN , Mi-Soon KIM , Min-Cheol KIM , Hye-Chul
1
2
2
3
SHIN , Jong-Sung LIM , Goon-Taek LEE , Jun-Ki YUN , and
1
Jong-Ok KA *
1
2
School of Agricultural Biotechnology, Seoul National University, National
Instrument Center for Environmental Management, Seoul National University,
3
Research Institute of Technology, Samsung Corporation
*Corresponding author: joka@snu.ac.kr
Oil spill occurred since 1998 from the diesel fuel storage facility of US army
which is located at the top of Baekun Mountain in Uiwang city. For efficient
bioremediation, bacterial communities of the contaminated site and its
uncontaminated control site were comparatively analyzed using both molecular
and cultivation techniques. Soil bacterial populations were observed to be
stimulated to grow on diesel fuel contamination, while fungal and actinomycetes
populations are negatively affected by diesel fuel contamination. Most of the
diesel-degrading bacteria isolated from contaminated forest soils were strains of
Pseudomonas, Ralstonia, and Rhodococcus species. DGGE analysis revealed
that the DGGE profiles were different among the three contaminated sites,
whereas those of the uncontaminated sites were identical to each other. Analysis
of 16S rDNA sequences of dominant isolates and clones showed that bacterial
community was less diverse in the oil-contaminated site than its uncontaminated
control site. Sequence analysis of the alkane hydroxylase genes cloned from soil
microbial DNAs indicated that the diversity and distribution of the alkB genes
was different between the contaminated site and control site. The results indicated
that contamination of diesel fuel exerts a strong selection on the indigenous
microbial community in the contaminated site, leading to predominance of
well-adapted microorganisms in concurrence with decrease of microbial
diversity.
Keywords: Bioremediation, Fuel, 16S rDNA, Soil DNA, DGGE, Bacterial
community
170
한국미생물학회연합
Isolation of the Pseudomonas sp. HK-6 Mutant
that Degrades TNT More Slowly than the
Wild-type Strain
1
2
3
Joo-O KANG , Jae Woo CHUN , Hyung-Yeel KAHNG ,
2
1
Kye-Heon OH , and Bheong-Uk LEE *
1
2
Kosin University, Division Biological Sciences, Soonchunhyang University,
3
Department Genetic Engineering, Sunchon National University, Department
Environmental Education
*Corresponding author: bulee@kosin.ac.kr
A TNT-degrading bacterium, Pseudomonas sp. HK-6 was mutagenized
with nitrosoguanidine (NTG) in order to isolate mutants which show
higher sensitivities to TNT. Among 12,000 NTG-treated colonies
inoculated on replica plates of LB and LB supplemented with 0.8 mM
TNT, ten mutants that grew much more slowly on TNT-containing LB
agar were identified and designated N1 - N10, respectively. To examine
rates of TNT degradation, five mutants (N1, N2, N3 N4 and N5) were
cultured in LB liquid broth supplemented with 0.5 mM TNT. Then
concentrations of remaining TNT in liquid culture were measured using
HPLC every 6 or 12 hrs. The wild-type strain were able to completely
degrade 0.5 mM TNT within 18 hrs, meanwhile, N5 took 48 hrs, that is,
about 2.7-fold slower degradation. In addition to this, the lag phase of N5
was extended longer than that of the original strain. As far as rate of
degradation is concerned, N1, N2, N3 and N4 mutants were almost
equivalent to the wild-type strain, indicating that different types of
mutations may render N1 - N4 TNT-sensitivities. Now a genomic library
is under construction. Each of TNT-sensitive mutants will be transformed
with this genomic library, selected on LB agar with TNT, and the
complemented survivors will be further studied. [This work was
supported by grant No. R01-2005-000-106080 from the Basic Research
Program of the Korea Science &Engineering Foundation.] Keywords: TNT degradation, Pseudomonas sp. HK-6, TNTsensitive mutants
2005 International Meeting of the Federation of Korean Microbiological Societies
B005
B007
Effects of Endospore Germinations on Nutrient
Removal Efficiency
1
2
1
Ji-Hyun NAM , Woo-Keun BAE , and Dong-Hun LEE *
1
2
Department of Microbiology, Chungbuk National University, Department of
Environmental Engineering, HanYang University
*Corresponding author: donghun@chungbuk.ac.kr
Bio Best Bacillus (B3) and Rotating Activated Bacillus Contactor
(RABC) processes have been known as an advanced wastewater
treatment system in which Bacillus strains have predominated.
Bacillus strains have been known to be involved in the wastewater
treatment system, but nutrient removal characteristics of the Bacillus
strains have not been studied in detail so far. In order to study
nutrient removal characteristics depending on Bacillus life cycle,
CODcr, T-N, and T-P were measured during the incubation of
endospore and vegetative cells of Bacillus subtilis in 0.2% nutrient
broth. Although the degradation rate was affected by the population
size, nutrient removal efficiency of endospore was 2-3 times more
than that of vegetative cells. Further work is needed to determine the
role of Bacillus strains predominantly occurring in a wastewater
treatment system by isolating and characterizing pure cultures.
[Supported by grants from Eco-STAR Project]
B006
Application of Membrane-Electrode Assembly
(MEA) for Microbial Fuel Cell Type BOD Sensor
1
2
2
Mia KIM , Chang-Ho CHOI , Seok-Min YOON , Dong-Hee
LEE2, Moon-Sik HYUN1, and Hyung-Joo KIM2*
1
2
KORBI Co. Ltd., Department of Microbial Engineering , Konkuk University
*Corresponding author: csquareh@konkuk.ac.kr
Membrane-Electrode Assembly (MEA) was applied to the
microbial fuel cell (MFC) type BOD sensor to improve its
sensitivity. MEAs fabricated using a hot pressing method were
installed as a separator of the fuel cell type sensor. The optimal hot
pressing condition for MEA fabrication was 120°C and 150 kgf/㎠
for 30 seconds. Using MEA with Pt catalyzed carbon cloth
electrode, the current density increased 8.6 folds compared with the
conventional MFC type sensor without MEA. The application of
MEA could improve accuracy of the sensor especially under
oligotrophic environment.
Keywords: membrane-electrode assembly, MEA, BOD sensor,
MFC type, oligotrophic
B008
Isolation and Characterization of Cryptococcus
neoformans from Environmental Sources in
Busan
1
2
Kwang Seok OH , and Soo Myung HWANG *
1
Maritime Safety Team, Korea Institue of Maritime and Fisheries Technology,
2
Department of Clinical Laboratory Science, Catholic University of Pusan
*Corresponding author: smhwang@cup.ac.kr
Twenty nine samples of pigeon droppings (n=12) and soil
contaminated with avian guano(n=19), collected from different sites
in Busan area, were examined for isolation and characterization of
Cryptococcus neoformans. Of these samples, 5 strains of C.
neoformans were recovered from pigeon droppings (5/12 : 41.7%).
All isolates were belonged to C. neoformans serotype A. The extracellular enzyme activities of the strains by using the API-ZYM
system showed two different enzymatic patterns. The genetic
variability among C. neoformans isolates was analyzed by random
amplified polymorphic DNA (RAPD) using three 10-mer primers.
Two different RAPD patterns (I,II), which clearly distinguished
among isolates, were identified. Analysis of RAPD patterns
provided a good characterization of environmnetal strains of C.
neoformans serotype A as a heterogeneous group and in good
agreement with enzymatic profiles. This is the first report on the
phenotypic and genotypic variability of C. neoformans isolates from
environmental sources in Korea. Keywords: Cryptococcus neoformans,, Serotyping, Enzymatic
activity, API-ZYM system, RAPD
Fluoroquinolone Resistance in Environmental
Isolates of Escherichia coli
Da Hye JUNG and Yeonhee LEE*
Department of Biology and Culture Collection of Antimicrobial Resistant
Microbes, Seoul Women’s University
*Corresponding author: daimonj@empal.com
Between 2004 and 2005, 310 isolates of norfloxacin resisitant (MIC
16 µg/ml) Escherichia coli were obtained from environment. Their
resistance to fluoroquinolones was tested with nalidixic acid,
norfloxacin, ciprofloxacin, levofloxacin, moxifloxacin, gemifloxaxin
and gatifloxacin. MICs were determined by the agar dilution method
according to the guideline suggested by Clinical and Laboratory
Standards Institute. Twenty isolates resistant to both nalidixic acid
(MIC 128 µg/ml) and norfloxacin (MIC 128 µg/ml) were further
studied. When the quinolone resistance-determining regions of gyrA
and parC were sequenced, every strain had the same mutationsSer83Leu, Asp87Asn of the gyrA and Ser80Ile of par C. Six isolates
(30%) showed the decreased amount of Opm F which is known to be
responsible for quinolone uptake. Pulsed-field gel electrophoresis
using XbaI showed six distinct profiles of these twenty isolates
suggesting various origins of these resistant isolates. [supported by
grant from MHW]
Keywords: Fluoroquinolone, Resistance, Escherichia coli
국제학술대회
171
October 13-14, 2005, Seoul, Korea
B009
Characterization of Aminoglycoside–resistance
in Environmental Isolates of Escherichia coli
Min-Young LEE and Yeonhee LEE*
B011
Anaerobic Degradation of Di-(2-Ethylhexyl
Phthalate) through Multiple Pathways by Mixed
Microbial Consortium
Department of Biology and Culture Collection of Antibiotics Resistant
Microbes, Seoul Women’s University
*Corresponding author: banymin@empal.com
Javed NIAZI*, Sun-Mi LEE, and Byoung-In SANG
Aminoglycosides are commonly used antimicrobial agents in the
treatment of infections by Gram-negative bacteria such as
Escherichia coli. This study is focused on characterization of
aminoglycoside-resistance in environmental isolates of E. coli. A
total of 310 antimicrobial resistant E. coli strains were isolated from
six rivers during the last 2 years from 2004~2005. Thirty-five
percent of the isolates were resistant to aminoglycosides such as
gentamicin, streptomycin, and kanamycin. Eighteen isolates of
high-level aminoglycoside-resistant E.coli (GEN, MIC ≥ 32 µg/ml;
KAN, MIC ≥ 128 µg/ml) were analyzed for the presence of
aminoglycoside modifying enzymes and integron. Integrons were
detected in 14 (78%) isolates and all of integrons was located on
plasmid. Filter mating method was performed to check whether
aminoglycoside-resistance and integron gene are transferable to E.
coli J53 (sodium-azide resistant). On pulsed field gel electrophoresis
(PFGE), eighteen isolates of aminoglycoside-resistant E. coli
showed four patterns suggesting various origins of these resistant
isolates. [supported by grant from MHW]
Keywords: E. coli, aminoglycoside-resistance, integron
Phthalate esters (PE’s), in nature, are environmental pollutants owing
to their inferred recalcitrance to microbial degradation and potential
toxicity to higher organisms. These PE’s enter into the environment
through effluents and aerosol. Di-(2-ethylhexyl phthalate) (DEHP) is
one among various PE’s that is most toxic as evaluated by US-EPA.
This study describes the degradation of DEHP by mixed microbial
consortium under anaerobic conditions. Soil samples from sea-side
were used for the anaerobic degradation of DEHP in minimal medium
with SO4 or NO3 as electron acceptor. Biodegradation of DEHP was
measured by GC every five days per cycle followed by DEHPreloading. The intermediates of DEHP-breakdown were noticed after
five-cycles (25-days) and were extracted and characterized by
GC-MS. We found that the DEHP is degraded through multiple
pathways when SO4 was used as electron acceptor leading to phthalic
acid or 2,6-bis(1,1-dimethylethyl)-2,5- Cyclohexadiene-1,4-dione as
common intermediate through five routes of bioconversion of DEHP
to 1,2-benzenedicarboxylic acid esters of (a) mono-2-ethylhexyl-, (b)
butyl-octyl-, (c) dihexyl-, (d) butyl-2-ethylhexyl- and (e) 2-hexyl-1,
2-benzenedicarboxylic acid ester as intermediates. These intermediates
were then give rise to phthalic acid through the formation of dibutylor diethyl phthalate. However, with NO3 as electron acceptor, the
degradation of DEHP followed a single pathway through dibutyl
phthalate to phthalic acid.
Keywords: Multiple pathways, DEHP, Anaerobic metabolism
Water Environment and Remediation Center, KIST
*Corresponding author: javedkolkar@gmail.com
B010
Isolation and Characterization of Soil Microorganisms
Degrading Chlorpyrifos-Methyl
Jun-Ran KIM* and Young-Joon AHN
School of Agricultural Biotechnology, Seoul National University
*Corresponding author: uncovered@paran.com
The purpose of this study was to isolate bacteria from soil and to
determine their ability to degrade chlorpyrifos-methyl and identify
the intermediates in culture broth using GC-MS. Aerobic bacteria
capable of degrading chlorpyrifos-methyl was isolated by
enrichment culture. Ten chlorpyrifos-methyl degrading bacteria
were isolated from Korean soil. The 16S ribosomal DNA sequence
of the isolates had identity to the sequence from Burkholderia
cepacia, Chryseobacterium prolyticum, Enterobacter pyrinus,
Bacillus pseudofirmus, Paenibacillus kribbensis, Pseudomonas
putida, Achromobacter xylosoxidans, Lysobacter gummous,
Pseudomonas aeuginosa, and Fiexibacterium oleovorans. Of these
bacteria, B. cepacia degraded chlorpyrifos-methyl up to 94.17% in 7
days. Degradation of the insecticide occurred concomitant with B.
cepacia growth reaching an optical density(OD550) of 0.238. Studies
with chlorpyrifos-methyl in liquid culture of B. cepacia
demonstrated that the isolate hydrolyzed chlorpyrifos-methyl to
3,5,6-trichloro-2-pyridine, and utilized this compound for growth
and energy. We performed SDS-PAGE and two-dimensional gel
electrophoresis and identified proteins whose expression pattern is
affected by chlorpyrifos-methyl using mass spectrometry. The
results revealed various proteins that can be grouped according to
their respective cellular function. These results highlight the
potential of this bacterium to be used in the cleanup of contaminated
pesticide waste in the environment. Keywords: Insecticide, Insecticide degrading bacteria, Chlrorpyrifosmethyl, Burkholderia cepacia
172
한국미생물학회연합
B012
Effect of Electron Acceptors on the Anaerobic
Biodegradation of BTEX and MTBE at Contaminated
Sites 1
2
Ji Eun KIM , Ji Hye BAEK , and Byoung In SANG
1
1
2
KIST, College of Pharmacy, Sungkyunkwan University
*Corresponding author: hohomiin@hotmail.com
Methyl tert-butyl ether (MTBE) contamination in groundwater often
coexists with benzene, toluene, ethylbenzene, and xylene (BTEX) near
the source of the plume. Then, groundwater contamination problems have
been developed in areas where the chemical is used. Common sources
of water contamination by BTEX and MTBE include leaking
underground gasoline storate tanks and leaks and spills from above
ground fuel storage tanks, etc. In oil-contaminated environments,
anaerobic biodegradation of BTEX and MTBE depended on the
concentration and distribution of terminal electron acceptor. In this study,
effect of electron acceptor on the anaerobic biodegradation for BTEX and
MTBE -contaminated soil was investigated. This study showed the
anaerobic biodegradation of BTEX and MTBE in two different soils by
using nitrate reduction, ferric iron reduction and sulfate reduction. The
soil samples from the two fields were enriched for 220 days by providing
BTEX and MTBE as a sole carbon source and nitrate, sulfate or iron as
a terminal electron acceptor. This study clearly shows that degradation
rate of BTEX and MTBE with electron acceptors is higher than that
without electron acceptors. Degradation rate of Ethylbenzene and Xylene
is higher than that of Benzene, Toluene, and MTBE. In case of Benzene,
Ethylbenzene, and MTBE, nitrate has more activation. In case of Toluene and Xylene, sulfate has more actrivation.
Keywords: Methyl tert-butyl ether (MTBE), Benzene, toluene,
ethylbenzene, and xylene (BTEX), Anaerobic biodegradation,
Electron acceptor, Contaminated soil
2005 International Meeting of the Federation of Korean Microbiological Societies
B013
B015
Effect on Decomposition of Food Garbage by
Application of Some Cellulolytic Bacteria and
Photosynthetic Bacteria
Biocontrol Efficacy of Lyophilized Albino
Strain of Ophiostoma quercus on Blue Stain of
Coniferous Woods
Rae-Hyun KO and Hong-Gyu SONG*
Byung Ju CHO
Jong Kyu LEE1
Division of Biological Sciences, Kangwon National University
*Corresponding author: hgsong@kangwon.ac.kr
Disposal of food garbage in most large cities is very troublesome
task, and moreover, food garbage has been one of the important
contaminants in soil and groundwater. To date, microbiological
treatment has been received an attention as a new garbage
decomposition accelerator. Recently anaerobic photosynthetic
bacteria and cellulolytic bacteria are being used in food garbage
treatment although the precise effects of inoculation have not been
elucidated. In this study, some cellulolytic bacteria and
photosynthetic bacteria were added into food garbage and incubated
at 30℃ for 30 days, and influence of their addition was analyzed.
The complete cellulase complex activity was measured by filter
paper assay. Maximal filter paper activity of isolated cellulolytic
bacteria was 53 unit/ml after 48 hours incubation with filter paper
strip. Inoculated sample showed a higher decomposition rate of
cellulose than uninoculated sample. Their accelerating effects on
food garbage decomposition were elevated according to the increase
of the inoculum size. Dried residual matter of food garbage was
lower in the inoculated sample than those in the uninoculated
sample. This decomposing capability of food garbage by cellulolytic
bacteria and photosynthetic bacteria may be used for the rapid
treatment of food garbage.
Keywords: Food garbage, decomposition, cellulolytic bacteria,
photosynthetic bacteria
B014
1
1,2,3
1
1
*, Keum Chul SHIN , Nam Kyu KIM , and
2
Tree Pathology and Mycology Laboratory, Department of Forest Resources
Protection, 3Kangwon National University
*Corresponding author: jongklee@kangwon.ac.kr
In order to find proper culture and preservation conditions of
biocontrol agent for the blue stain of coniferous woods, albino strain
of Ophiostoma quercus, which had been selected as a effective
biocontrol agent in the previous work, was cultured in 8 liter
fermenters with liquid culture media, freeze-dried, and stored at
room temperature, refrigerator, and liquid nitrogen. Mycelial growth
of albino strain cultured in various culture media was compared, and
dry weight of mycelium from the culture medium containing brown
sugar(30%) and yeast extract(3%) was 3.8 times higher as that of
mycelium harvested from Potato dextrose broth. Pre-treatment of
-3
mycelial suspension of lyophilized powder (10 dilution in distilled
water) inoculated onto sterilized wood chips of P. densiflora, P.
rigida by spraying showed satisfactory stain-free results. In the field
experiment, water suspension of the lyophilized powder also
showed excellent biological activity until at least 1 year after storage
against blue stain on the wood logs of P. rigida. Keywords: Biocontrol, Blue stain, albino strain, Ophiostoma
quercus, coniferous woods, lyophilized powder
B016
Sawdust Cultivation of Lentinus lepideus
Using Liquid-Cultured Spawn
PCR Primers that Detect Arbuscular Mycorrhizal
Fungi in Roots
Keum Chul SHIN*, Nam Kyu KIM, Byung Ju CHO, and Jong
Kyu LEE
Jaikoo LEE, Sang-Hyeon PARK, and Ahn-Heum EOM
Kangwon National University
*Corresponding author: jongklee@kangwon.ac.kr
To obtain the basic data for large scale cultivation of Lentinus
lepideus, mushroom cultivation using Korean pine (Pinus
koraiensis) sawdust and liquid-cultured spawn were carried out. L.
lepideus showed the most favorable growth in culture medium, BPYM (Brown sugar, Peptone, Yeast extract, and Malt extract). The
optimal culture period and aeration rate for liquid spawn were 7
days and 1.0 vvm(vol. of air / vol. of medium / min), respectively.
The effects of spawn type (liquid-cultured spawn and sawdust
spawn) on the total periods required for the mushroom cultivation
and fruiting body yields were compared. Artificial cultivation of L.
lepideus using liquid-cultured spawn took 42 days less than the
normal period required for the cultivation using sawdust spawn. In
addition, dry weight of fruiting bodies from the cultivation using
liquid-cultured spawn was much higher than that from sawdust
spawn cultivation. Thus, mushroom cultivation using liquid-c
ultured spawn seems to be effective for the large scale cultivation of
L. lepideus.
Keywords: Lentinus lepideus, liquid-cultured spawn, sawdust
spawn, mushroom cultivation, Korean pine sawdust
Department of Biology, Korea National University of Education
*Corresponding author: eomah@knue.ac.kr
A PCR primer pair was designed to amplify rDNA from arbuscular
mycorrhizal fungi (order Glomales). PCR with the primers showed
that the pair amplified DNA from Glomalean fungi but not from
non-AM sources. To test the ability of the primers to amplify
Glomalean fungal rDNA from root samples, clones from two root
samples were analyzed. In this analysis, no non-AM fungal rDNA
sequences were identified, illustrating the selectivity of these PCR
primers. This work demonstrates the ability of the newly developed
PCR primer set to amplify and detects AM fungal rDNA from plant
roots, thereby providing tools to examine this community of
organisms in roots.
Keywords: arbuscular mycorrhiza, 18s rDNA, PCR primers
국제학술대회
173
October 13-14, 2005, Seoul, Korea
B017
B019
Biodiversity and Population of Higher Fungi by
Forest Types in Odaesan National Park
1,2,3
Nam Kyu KIM
Jong Kyu LEE1
1
1
1
*, Keum Chul SHIN , Byung Ju CHO , and
2
Mutation of the rpoS Gene Results in Enhanced
Biofilm Formation in Pseudomonas sp. KL28
1
2
1
2
Tree Pathology and Mycology Laboratory, Department of Forest Resources
Protection, 3Kangwon National University
*Corresponding author: jongklee@kangwon.ac.kr
Department of Microbiology, Changwon National University, Division of
Natural Sciences, Hoseo University
*Corresponding author: klee@sarim.changwon.ac.kr
The flora of higher fungi in Odaesan National Park was surveyed to
investigate the biodiversity and population of higher fungi in various
forest types from October, 2004 to August, 2005. Survey sites were
categorized into 3 forest types; narrow-leaved tree forest, broadleaved tree forest, and mixed tree forest. Each forest type is composed
of three forest stands with 20 x 20 m size each: Manchurian fir(Abies
holophylla), Japanese red pine(Pinus densiflora) I, II for narrowleaved tree forest; Mongolian oak(Quercus mongolica), Amur
linden(Tilia amurensis), and Japanese eurya(Eurya japonica) for
broad-leaved tree forest; Manchurian fir and broad-leaved trees I, II,
and Japanese red pine and Oriental oak(Quercus variabilis) for mixed
tree forest. A total of 1,988 specimens were collected, and classified
into 167 species, 130 genera, 104 families, 54 orders. Among
collected specimens, saprophytic, symbiotic, and parasitic fungi were
68%, 30%, 2%, respectively. The most common genus was identified
as Mycena spp. The most diverse flora was observed in the mixed
forest of Manchurian fir and broad-leaved tree, while Japanese eurya
had relatively simple flora. Diversity and population of fungal flora
within forest type was various and might be dependant on forest
environmental factors, i.e., tree species, understory vegetations, and
physical or chemical soil properties, and so on.
Keywords: Flora of higher fungi, Odaesan National Park,
Biodiversity, forest type, forest environmental factors
The stress and stationary phase sigma factor RpoS induces genes
during the transition from exponential to stationary phase,
producing proteins related to resistance to stress in a variety of
Gram-negative bacteria. In this study, the effect of rpoS mutation on
the production of biofilm formation was investigated from an
alkylphenol degrader, Pseudomonas sp. KL28. Nucleotide sequence
analysis of the genes flanking the transposon inserted rpoS gene
showed an order of nlpD (a lipoprotein), rpoS, rsmZ (a small RNA)
and fdxA (ferredoxin), which is typically found in P. putida and
related strains. The rpoS strain named S23 showed wrinkles on agar
plate and also showed faster spreading on soft agar. In addition, the
formation of pellicle (biofilm at the interface between liquid and air)
was enhanced in the knockout strain. However, the effects were
opposite in the isogenic strain with rpoS complementation. This
result indicates that rpoS in strain KL28 negatively affects biofilm
formation.
Keywords: Pseudomonas, rpoS, biofilm
B018
B020
Genetic Organization and Gene Induction of the
ctg Genes Encoding a Cresol Efflux Pump from
Pseudomonas sp. KL28
Isolation, Identification and Characterization of
MTBE Degradaing Bacteria
Ji In YUN, Soo O LEE, and Kyoung LEE*
1
Department of Microbiology, Changwon National University
*Corresponding author: klee@sarim.changwon.ac.kr
The detailed mechanism for resistance to cresol has not been known
in Pseudomonas. In a previous study we have reported a
Pseudomonas sp. KL28 that has a novel genetic organization and a
new degradation pathway for alkylphenols. The strain also showed
resistance to alkylphenols with an ability to grow in basal salts
medium containing up to 0.5 g/l m-cresol. In this study, by using a
transposon mutagenesis we identified a gene locus conferring cresol
resistance to Pseudomonas. Nucleotide sequence analysis of the
genes flanking the insertion site showed that the periplasmic
component of the RND efflux pumps was inactivated. RND efflux
pumps generally consist of three components and have been known
to afford resistance to incoming solvents in Pseudomonas. The
newly identified ctgR2R1ABC gene cluster (named ctg for cresol
tolerance genes) was most homologous to those encoding toluene
efflux pumps in Pseudomonas. The transcriptional activity of the
ctgA promoter was measured with a gfp reporter vector and the
results showed that the genes are constitutively expressed at a basal
level and further induced by o-cresol and naphthalene.
Keywords: Pseudomonas, cresol, RND efflux pump
174
1
Ji In YUN , Kyung Soon CHOI , Kyungyun CHO , and Kyoung
LEE1*
한국미생물학회연합
1
2
Sang-Woo AN *, Sang-Sup LEE , and Soon-Woong CHANG
1
2
Department of Environmental Engineering, Kyonggi University, Department
of Microbiology, Kyonggi University
*Corresponding author: asw03@naver.com
Due to the widespread use of fuels, fuel components such as
petroleum hydrocarbons and methyl tert-butyl ether(MTBE)among
the most frequent groundwater contaminants. MTBE has been
added to gasoline as an octane enhancer and fuel oxygenate to
provide a cleaner burning fuel with reduced vehicle exhaust
emissions. Studies on MTBE removal by bioremediation techniques
is still at its infancy and requires both basic and applied research to
optimize the technology. In this study, we characterized MTBE
degradation by various bacteria and its isolates. A total 14 strains of
aerobic soil bacteria were isolated form gasoline contaminated soil
in Korea, respectively. In a mineral medium(BSM) containing
MTBE as the sole source of carbon and energy, the highest rate of
MTBE elimination was achieved with MTBE degrading bacterium
which degraded over 82.7 % in 3days, respectively. They were
identified to Alcaligenes, Chryseobacterium, Methylobacterium,
and Stenotrophomonas according to the classification keys of
Bergey's Manual of Systematic Bacteriology.
Keywords: MTBE, bioremediation, MTBE degradaing bacteria
2005 International Meeting of the Federation of Korean Microbiological Societies
B021
B023
Soft Rot of Tomato Caused by Mucor racemosus
in Korea
1
Jin-Hyeuk KWON * and Seung-Beom HONG
2
1
Gyeongsangnam-do Agricultural Research and Extension Services, 2Korean
Agricultural Culture Collection, Genetic Resources Division, National
Institute of Agricultural Biotechnology
*Corresponding author: Kwon825@mail.knrda.go.kr
A soft rot of fruits caused by Mucor racemosus occurred on cherry
tomato in Jinju city Agricultural Products Wholesale Market in
Korea. The disease infection usually started from wounding after
cracking of fruits. At first, the lesions started with water soaked and
rapidly softened and diseased lesion gradually expanded. Colonies
were white to brownish to gray in color. Sporangia were 70~85 ㎛ in
size and globose in shape. Sporangiophores were 8~14 ㎛ in width.
Sporangiospores were 5~12×4~8 ㎛ in size, ellipsoidal to subglobose
in shape. Columella was 27~42 ㎛ in size, obovoid, ellipsoidal,
cylindrical-ellipsoidal, slightly pyriform in shape. Chlamydospores
were numerous in sporangiophores and barrel-shaped when young,
subglobose in old cultures. Optimum growth temperature was about
25℃. The fungus was identified as M. racemosus Fres.. This is the
first report of soft rot on cherry tomato caused by M. racemosus in
Korea.
Keywords: Cherry tomato, Mucor racemos, Soft rot
B022
Bactericidal Effects of TiO2/UV Photocatalytic
Air Sterilizer on Airborne Bacteria
Yoonjung CHOI, Mihee CHO, Hyojin PARK, and Jiyong PARK*
Department of Biotechnology, Yonsei University
*Corresponding author: love171321@paran.com
Antibacterial effects of titanium dioxide (TiO2) photocatalytic
reaction under irradiation of ultraviolet (UV) against airborne
bacteria were studied. Bacillus subtilis, Escherichia coli, Salmonella
typhimurium and Staphylococcus aureus were sprayed
5
3
(approximately 10 CFU/m ) into an aseptic chamber (total volume
3
= 1.53 m ). The chamber was sterilized with UV before each
experiment. Airborne bacteria were collected by air sampler at
5-min intervals for up to 90 min and incubated at 37°C (B. subtilis at
30°C) for 24 hr. Bactericidal effects of air circulation fan alone were
measured as control. B. subtilis, E. coli, S. typhimurium and S.
aureus were completely inactivated by TiO2/UV photocatalytic air
sterilizer within 60 min, 40 min, 30 min and 25 min, respectively,
while bacteria of the control group were not sterilized after 90 min.
The TiO2/UV photocatalytic air sterilizer appeared to control
airborne microorganisms effectively.
Keywords: Airborne bacteria, Titanium dioxide (TiO2), Photocatalytic
reaction, Air sterilizer, Antibacterial effect
Specific Detection of Phytophthora capsici in
Nutrient Solution and Soil by PCR
Y. S. LEE, J. Y. SONG, N. J. JUN, and H. G. KIM
Department of Agricultural Biology, Chungnam National University
*Corresponding author: leeyunsoo7@msn.com
One of destructive soil-borne pathogen is Phytophthora capsici that
causes a serious disease of pepper cultivation in Korea as well as
worldwide. Species-specific primers, CSP21A and CPS23B,
developed in this study newly were analysed in detection ability for
P. capsici in nutrient solution and soil. The primer set amplified
specific PCR product of 970 bp with P. capsici only. In sensitivity
test using the dilution series of genomic DNAs and zoospores, the
species-specific primer set could detect to the limit of 100 fg and
showed minimal detectable quantity of one zoospore/ml in nutrient
solution and one zoospore/g in sterilized soil. The results in this
study indicate that new species- specific primer set can demonstrate
existence of low level of P. capsici in nutrient solution and soil and
can be applied for diagnosis of Phytophthora pepper blight in pepper
cultivation field.
Keywords: species-specific primer, soil-borne pathogen
B024
Effects Cultivation Condition on Growth of the
Hydrogen Sulfide-Degradating in Bacterium
Isolated from Barnyard Manure in High
Temperature.
Chi Un JOO*, Jin Wook KIM, Chun Mea DONG, Jin Young
PARK, Jea Woong HWANG, and Jea Hwa LEE
Department of Bioscience and Biotechnology, Silla University
*Corresponding author: goodguy17@nate.com
A hydrogen sulfide-degradation bacterium isolated from barnyard
manure in high temperature. After the strain that cell growth and
sulfide oxidation was rapid was selected in agar plate media (30oC ~
60oC. 120rpm). The isolate was identified on the basis of its
morphological, physiological and chemotaxonomical characteries.
The growth conditions of the hydrogen sulfide-degradation
bacterium were investigated and the isolate characterized as
Thiobacillus sp. The thiosulfate oxidation was made of sulfate ion
by the isolated bacterium. Cell growth and pH were reduced by the
sulfate ion. The concentrations of sulfate ion (thiosulfate and sulfate)
were measured by 5% BaCl2․2H2O adsorption method using
spectrophotometer. As hydrogen sulfide-degradation bacteria was
rapid growth, this strain has the potential to be an effective
bacterium in biological deodorizing system if a suitable proper
combination with other bacteria was elaborated. The results
described above indicated that this strain was a candidated for
improving the removal efficiency in biological deodorizing system.
Keywords: sulfide-degradation, barnyard manure, Thiobacillus sp.,
thiosulfate oxidation, biological deodorizing system
국제학술대회
175
October 13-14, 2005, Seoul, Korea
B025
B027
New Method Development for Detection and
Identification of Clostridium botulinum types A,
B, E, and F and Its Application in Sediment
Samples
So-Yeon YOON*, Na-Ri SHIN, Ji-Hun SHIN, Gi-Eun RHIE, and
Won Keun SEONG
Research Center for Pathogen Control, Department of Microbiology, NIH
*Corresponding author: yunsy7@hotmail.com
A protocol for a series of the detection, isolation and identification of
Clostridium botulinum in sediment samples with specific
identification of neurotoxin types A, B, E and F was developed and
applied in 44 soil samples collected from Yeong-am lake located in
Dangdu-ri, Haenam-gun, Jeonranam-do, Korea. The soil samples
were treated with distilled water and 70% ethanol in order, and were
cultured anaerobically in TYG broth at 37℃. The each culture was
analyzed by nested PCR method constructed in previous study
(published in International meeting of the microbiological society of
Korea, 13 May 2005). 11.36% (5/44) of soil samples showed
positive results, and all positive products were identified as C.
botulinum type B neurotoxin genes. Two isolates from 5 positive
samples were found out, and identified as C. botulinum type B by
nested PCR and mouse bioassay. A newly developed series of
protocol including treatment of samples, nested PCR, isolation and
identification of organism is the effective method for specific
detection of C. botulinum types A, B, E and F from soil, and makes
practicable environmental survey.
Keywords: Clostridium botulinum, nested PCR, soil samples
Analysis of the Bacterial Communities Associated
with Two Sand Dune Plant Species, Calystegia
soldanella and Elymus mollis Over Two Seasons 1
1
3
1
Department of Microbiology, School of Bioscience and Biotechnology,
Chungnam National University, 2Department of Applied Microbiology,
College of Agriculture and Life Sciences, Chungnam National University,
3
Department of Microbiology, College of Natural Sciences, Daejeon
University, 4KRIBB
*Corresponding author: sbk01@cnu.ac.kr
Little is known about the seasonal changes of the bacterial communities
associated with the plants inhabiting coastal sand dune ecosystem. In this
study, the seasonal changes of the bacterial populations associated with two
major sand dune plant species, Calystegia soldanella (beach morning glory)
and Elymus mollis (wild rye), growing along the coastal areas in Tae-An,
Chungnam Province, were analyzed using a culture based approach. A total
of 119 bacteria were isolated in August 2003, and 217 were isolated in
February 2004. Through the amplified 16S rDNA restriction analysis
(ARDRA), the isolates were grouped and the representative ARDRA
patterns were sequenced. The isolates from the rhizosphere of the two plant
species were assigned to about 25 different established genera. Member of
Pseudomonas species comprised majority at both seasons. However,
differences were observed in the overall composition, as the enteric
bacteria such as Entrobacter sp. (Calystegia soldanella) and Klebsiella sp.
(Elymus mollis) were the second most abundant groups in August samples
but various taxa such as Chryseobacterium sp. (Calystegia soldanella and
Elymus mollis) and Arthrobacter sp. (Calystegia soldanella ) or Bacillus sp.
(Elymus mollis) were recovered in February samples. Also, obvious
seasonal differences were observed, where Pseudomonas spp. were
predominant in most cases.
B026
B028
Analysis of Bacterial Community Diversity
Associated with Sand Dune Plants by Denaturing
Gradient Gel Electrophoresis And Clone
Library.
1
2
3
Jin Ok DO *, Sera JUNG , Myong Soo PARK , Seung Bum
4
1
5
KIM , Seong Joo PARK , and Kyung Sook BAE
Population and Transfer Factor of Basidiomycota
Collected in the Heavy Metal-Contaminated and
Healthy Soils
1
2
1
Kab-Yeul JANG *, Sun-Gyu CHOI , Kang-Hyo LEE , Soon-Ja
1
3
4
5
SEOK , Gu-Bok JUNG , Gyu-Hyun KIM , and Jae-Mo SUNG
1
Department of Microbiology, Daejeon University, Department of
Microbiology, School of Bioscience and Biotechnology, Chungnam National
University, 3Department of Applied Microbiology, College of Agriculture and
4
Life Sciences, Chungnam National University, Department of Microbiology,
5
College of Natural Sciences, Daejeon University, KRIBB
*Corresponding author: candle1457@nate.com
Division of Applied Microbiology, National Institute of Agricultural Science
2
and Technology, RDA, Department of Horticultural Bio-Industry, Cheonan
Yonam College, 3Division of Environment and Ecology Management, National
4
Institute of Agricultural Science and Technology, RDA, Department of
5
Horticultural Bio-Industry, Cheonan Yonam College, Department of Applied
Biology, Kangwon National University
*Corresponding author: gabriel@rda.go.kr
The bacterial community structure of sand dune soil and plant was investigated
using the rDNA based approach. Denaturing gradient gel electrophoresis
(DGGE) analysis with eubacterial primers GC341F and 518R and analysis of
community 16S ribosomal DNA clones amplified with universal bacterial
primer sets were carried out to describe the geographical and seasonal variation
in the bacterial community structure within the rhizosphere and root of the
coastal sand dune plants inhabiting Tae-an in 2003 and 2004. The analysis of
DGGE band profiles identified a single band that occurred as the major band in
all of the samples regardless of plant species or sampling locations. The band was
identified as that of Lysobacter sp., a member of the family Xanthomonadaceae,
Gammaproteobacteria. The dominance of Lysobacter sp. was also consistent
with the results obtained from the 16S rDNA clone analysis, as the specific taxon
comprised majority of the total clones in all of the samples taken from both
rhizosphere and root. Bacterial community structure and diversity were
investigated by amplified 16S ribosomal DNA restriction analysis (ARDRA)
and diversity indices were calculated. It is not yet clear what kind of roles
Lysobacter plays in association with the sand dune plant, but the universal
presence of them in the rhizosphere and root suggests that they might form a
symbiotic relationship with their hosts.
Keywords: DGGE, clone library, Sand Dune
Two hundred seventy-four microorganisms were isolated from the soil
of abandoned mines around for selecting the heavy metal-degrading
strains and their microbial diversity was analyzed. The specimens of
macrofungi were isolated from the soil of abandoned mines around for
selecting the heavy metal-degrading strains. Eunseong and Dogok in
Gyeongsangbuk-do, for selecting the heavy metal-degrading strains.
Soil was also collected from same location. Contents of heavy metals
(Cd, Cu, Pb, Zn, Ni, Cr, and As) were determined spectrometically in
fruiting bodies of forty eight wild macrofungi specimens with soils.
When the analysed their transfer factor from soil to fruiting body,
Amanita volvata have the highest transfer factor of cadmium and
arsenic. And Mycena pura showed the highest in mercury, Marasmius
pulcherripes in zinc, Laccaria laccata in nickel, and Collybia confluens
in chrome. When compare the population of mushrooms between the
contaminated mines and Mt. Chiak as the healthy area, Genus Russula
and Collybia were collected both area, but Leucocoprinus, Coprinus,
Suillus, Lepiost, Gyroporus, Lepista, Microstoma, Stropharia, and
Agrocybe were only in the contaminated mine area.
Keywords: Heavy metal-contaminated Soils, Basidiomycota,
Transfer Factor, Population
1
176
2
Dong Sung SHIN , Myung Soo PARK , Sera JUNG , Jin Ok DO ,
Kang Hyun LEE4, Seung Bum KIM1*, and Kyung Sook BAE4
2
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
B029
B031
Degradation of Polycyclic Aromatic Hydrocarbons
by Pleurotus ostreatus and Coriolus versicolor
1
2
1
Molecular Identification of Ectomycorrhizal Fungi
from Pinus densiflora in Abandoned Coal Spoils
Kab-Yeul JANG *, Sun-Gyu CHOI , Jong-Cheon CHEONG ,
Soo-Muk CHO3, Gyu-Hyun KIM2, and Jae-Mo SUNG4
Hyeon-Suk JEONG, Bong-Hyung LEE, Yoo-Mi LEE, and
Ahn-Heum EOM
1
Department of Biology, Korea National University of Education
*Corresponding author: eomah@knue.ac.kr
Division of Applied Microbiology, National Institute of Agricultural Science
and Technology, RDA, 2Department of Horticultural Bio-Industry, Cheonan
Yonam College, 3Division of Agriproduct Science, National Institute of
4
Agricultural Science and Technology, RDA, Department of Applied Biology,
Kangwon National University
*Corresponding author: gabriel@rda.go.kr
Mycelial growth of fungi, Pleurotus ostreatus and Coriolus versicolor,
was examined on PDA medium contain the different PAHs(Polycyclic
Aromatic Hydrocarbons). When the mycelial growth of P.ostreatus was
examined, the control (PDA media not contain the PAHs), F-10 (contain
the fluorene of 10ppm), and A-10(contain the anthracene of 10ppm)
showed good mycelial growth and A-10 was the best growth in all
treatment. When the concentrations of PAHs in the PDA media were
increased, most of mycelial growth was decreased exceedingly. Mycelial
growth of C. versicolor was higher than P.ostreatus collectively except
media contain the fluorine of 50ppm (F-50). Like a C. versicolor, when
the concentrations of PAHs in the PDA media were increased, most of
mycelial growth was decreased exceedingly except A-10. Anlalsis of
fungal degradation of PAHs were evaluated PDB in the presence of 50
ppm anthracene and phenanthrene using the HPLC. The maximum
laccase activity of C. versicolor was 129.4 U/1 at 25 days incubation at
which 99.3% of added phenanthrene was degraded, and also the
degradation of phenenthrene reached maximum level. And also
P.ostreatus was reached maxmum at 25 days.
Keywords: Pleurotus ostreatus, Coriolus versicolor, Polycyclic
Aromatic Hydrocarbons, Degradation
B030
B032
Bacterial Diversity in Subgingival Plaque of
Healthy Subjects and Periodontitis Subjects
of Jeju Individuals
1
1
2
Dong-Heon LEE , Byoung-Jun YOON , Youn-Hwa KIM , and
1
Duck-Chul OH *
1
This study was conducted to select native ectomycorrhizal (ECM)
fungi colonizing Pinus densiflora for revegetation of abandoned
coal mine spoils. Seedlings of ECM pine roots growing on mine
spoils in Taebaek, Kangwon, Korea were collected. Mycorrhizal
root tips were classified according to morphological characteristics
and DNA was then extracted from each group of morphotyped tips.
PCR primer pairs specific to fungi, ITS1F and ITS4, were used to
amplify fungal DNA and restriction enzymes, AluI and HifI were
used for RFLP. ECM fungal species including Thelophoraceae,
Pezizales, Lacaria, Pisolithus were identified from the sequence
analysis. Results indicate that the most frequently found ECM fungi
in this sites was a species in Thelophoraceae and this species and
others could be useful for revegetation of abandoned coal mine
spoils with P. densiflora.
Keywords: ectomycorrhizal fungi, 18s rDNA, RFLP
2
Department of Life Science, Cheju National University, Department of
Dental Hygiene, Ulsan College
*Corresponding author: duckoh@cheju.ac.kr
This study provides the basic data on bacterial flora in subgingival plaque
of healthy subjects and periodontitis subjects of Jeju individuals. 16S
rDNA clonal library from subgingival plaque was constructed using the
16S rDNA PCR products. A total of 437 clones were examined by
amplified rDNA restriction analysis(ARDRA) using HaeIII. In healthy
subjects, 38 RFLP types were detected from 204 clones. In periodontitis
subjects, 40 RFLP types were detected from 233 clones. The 92 clones
were selected and sequenced according to different RFLP types.
Sequenced clones revealed that bacterial community belonged to six
phyla and 21 genera in subgingival plaque of healthy subjects, while
bacterial community belonged to seven phyla and 18 genera in
subgingival plaque of periodontitis subjects. In subgingival plaque of
periodontitis subjects, the most abundant bacterial species in the genus
level belonged to Firmicutes which occupied 38% of total clones. The
other bacterial clones were Fusobacteria (16%), Bacteroidetes (15%),
Proteobacteria (7%). Spirochetes (2%), Actinobacteria (2%) and phylum
TM7(2%). Whereas, the bacterial clones in subgingival plaque of healthy
subjects, belonged to Firmicutes(52%), Proteobacteria (23%),
Fusobacteria (9%), Bacteriodetes (5%), Spirochetes (3%), and phylum
TM7(2%).
Keywords: ARDRA, subgingival plaque, bacterial community,
Jeju individuals
Ericoid Mycorrhizal Fungi Found in Roots of
Rhododendron micranthum
In-Yong KIM, Jin-A KIM, and Ahn-Heum EOM
Department of Biology, Korea National University of Education
*Corresponding author: eomah@knue.ac.kr
This study was conducted to describe ericoid mycorrhizal fungi
colonized in Rhododendron micranthum and their effect on the
host plant. The roots of R. micranthum collected in Mt. Joryeong,
Chungbuk were stained and obserbed under light microscope and
electronmicroscopes(SEM). Ericoid mycorrhizal fungi were
isolated from surface sterilized roots of R. micranthum. DNA of
fungal Isolates were extracted to identify using sequence analysis
as well as morphological characteristics. Inoculation experiment
of one of fungal isolate, Hymenocyphus ericae to the seedling of
R. micranthum confirmed that these fungi gave beneficial effect
on host plant growth.
Keyword: ericoid mycorrhizal fungi
국제학술대회
177
October 13-14, 2005, Seoul, Korea
B033
B035
Ecological Analysis of Pseudomonads Associated
with the Sand Dune Plants
1
2
3
1
Sera JUNG *, Myung Soo PARK , Jin Ok DO , Dong Sung SIN ,
Kang Hyun LEE4, Kyung Sook BAE4, and Seung Bum KIM1
1
Department of Microbiology, School of Bioscience and Biotechnology,
Chungnam National University, 2Department of Applied Microbiology,
College of Agriculture and Life Sciences, Chungnam National University,
3
Department of Microbiology, College of Natural Sciences, Daejeon
University, 4KRIBB
*Corresponding author: sbk01@cnu.ac.kr
Pseudomonads are intimately associated with plant. Diverse populations
of Pseudomonas occur in several sand dune plants, and directly and
indirectly contribute to plant health. In this study, strains of Pseudomonas
were isolated from the rhizosphere and root of various sand dune plants,
and taxonomically characterized. A number of isolated strains were found
to suppress plant pathogenic microbes and pests by producing antibiotic
metabolites, or to promote plant growth in a variety of ways. All of the
Pseudomonas isolates produced indoleacetic acid (IAA), and 97.4 %
produced siderophores, both plant growth promoting substances. All of
the isolates again produced pectinases and chitinases, while 81.6 %
produced proteases. The enhanced germination and growth rates on
Calystegia soldanella (beach morning glory) were observed in 26.3 % of
the isolates. Enhanced germination rates on Oryza sativa (rice) were
observed in 39.5 % of the isolates, while growth rates were in 15.8 %. The
combined germination rates and growth promoting activity was observed
in 13.2 % of the isolates. Strains 2PBSB1-1 and 2PBSD8-3 exhibited
good germination rates in both tested plants. For the antagonistic activity
against plant pathogens, 76.3% of Pseudomonas were antagonistic
against Phytium ultimum, 52.6 % of the strains were against Fusarium
oxysporum, and 57.9 % were against Botrytis cinerea, respectively.
Keywords: Pseudomonas, sand dune plants, antagonistic activity,
plant growth promoting
Characteristics of Microbial Populations and
Enzyme Activity in Composting of Turfgrass
Clippings
Sang-Dong BAE*, Jung-A YOON, Keun-Whan LIM, and
Kyung-Sook WHANG
Department of Biotechnology, Mokwon University
*Corresponding author: kswhang@mokwon.ac.kr
The composting was performed in a 6 reactor system. The material
used was three kinds of turfgrass clippings with chicken slurry added
as nitrogen source. Microbial and enzyme activity were investigated
intensively, at four degrees intervals until the end of the fermentation.
In zoysiagrass clipping composting system, the temperature was
rapidly increased about 60~70℃ in the early stage. The
characteristics of composting bacteria isolated from samples taken
at the different composting stages were characterized based on a
phylogenetic analysis using 16S rDNA sequences. Species
diversity decreased markedly at thermophillic phase, enterobacteria
group was low after the thermophillic phase, and at the end of the
composting process were showed Cellulomonas, Streptomyces and
Bacillus. The major activity of enzyme as a protease occurred
gradually after the thermophilic phase as a result of degradation of
turfgrass. A total of 677 isolates, 455 isolates were as protease
producing bacteria. High number of themophilic bacteria growing at
temperatures above 60℃ were from the thermogenic phase of the
composting process, Bacillus subtilis are dominated all samples.
Keywords: composting, phylogeny, protease, turfgrass
B034
B036
Protection of Polaromonas naphthalenivorans
CJ2 from Naphthalene Toxicity by Extracellular
Polysaccharide Capsules
1
1
2
Minjeong PARK *, Shipeng LU , Eugene L. MADSEN , and
1
Che Ok JEON *
1
1,2
3
Ki Young CHOI , Dockyu KIM , Jong-Chan CHAE , Gerben J.
3
1
ZYLSTRA , and Eungbin KIM *
1
2
Division of Environmental Biotechnology and EBNCRC, Gyeongsang
2
National University, Department of Microbiology, Cornell University, USA
*Corresponding author: cojeon@gsnu.ac.kr
Department of Biology, Yonsei University, Microbial Resources Bank,
3
Microbial Genomics and Applications Center, KRIBB, Biotechnology Center
for Agriculture and the Environment, Rutgers University, USA
*Corresponding author: eungbin@yonsei.ac.kr
An extracellular polysaccharide (EPS) producing bacterium, Polaromonas
naphthalenivorans CJ2, responsible for naphthalene degradation at a coal
tar contaminated site, was isolated on MSB agar with naphthalene vapor
o
as the sole carbon source at 10 C. The strain is not isolated under the same
o
isolation condition using the same soil sediment at 20 C although its
o
optimum temperature is 20 C. Dispersed CJ2 cells in PBS buffer formed
o
colonies on MSB agar with naphthalene vapor at 10 C of low naphthalene
o
vapor pressure, but not at 20 C of high naphthalene vapor pressure.
However, streaked cells without resuspension formed colonies on MSB
o
o
o
agar with naphthalene at 10 C, 20 C, and even 25 C. Investigation of
scanning electron microscopy and light microscopy after EPS staining
showed that CJ2 cells formed EPS capsules and the capsules were released
easily from CJ2 cells by just dispersion. Insertional inactivation of one of
EPS producing genes using Campbell-type single-crossover homologous
recombination was carried out to verify the protection of strain CJ2 from
naphthalene vapor by EPS capsules. The EPS mutant strain showed that
even streaked cells without resuspension did not grow well on MSB with
o
naphthalene vapor at 20 C. Therefore, it was concluded that strain CJ2 cells
were susceptible to naphthalene toxicity and were able to be overcome the
naphthalene toxicity at higher temperatures with high naphthalene vapor
pressure in the presence of EPS capsule.
Keywords: Naphthalene toxicity, P. naphthalenivorans CJ2,
Extracellular polysaccharide, capsule
Alkylbenzene-degrading Rhodococcus sp. strain DK17 is able to utilize
phthalate and terephthalate as growth substrates. The genes encoding the
transformation of phthalate and terephthalate to protocatechuate are
organized as two separate operons, located 6.7 kb away from each other. Interestingly, both the phthalate and terephthalate operons are induced in
response to terephthalate while expression of the terephthalate genes is
undetectable in phthalate -grown cells. In addition to two known plasmids
(380-kb pDK1 and 330-kb pDK2), a third megaplasmid (750-kb pDK3)
was newly identified in DK17. The phthalate and terephthalate operons
are duplicated and are present on both pDK2 and pDK3. RT-PCR
experiments, coupled with sequence analysis, suggest that phthalate and
terephthalate degradation in DK17 proceeds through oxygenation at
carbons 3 and 4 and at carbons 1 and 2 to form 3,4- dihydro-3,4dihydroxyphthalate and 1,2-dihydro-1,2-dihydroxyterephthalate, respectively.
The 3,4-dihydroxyphthalate pathway was further corroborated
through colorometric tests. Apparently, the two dihydrodiol
metabolites are subsequently dehydrogenated and decarboxylated to
form protocatechuate, which is further degraded by a protocatechuate
3,4-dioxygenase as confirmed by a ring-cleavage enzyme assay.
Keywords: Rhodococcus, phthalate/terephthalate operon, gene
duplication, protocatechuate 3,4-dioxygenase
1
178
Novel Aspects of Phthalate and Terephthalate
Degradation by Rhodococcus sp. Strain DK17
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
B037
B039
Development of the Fed-Batch Bioslurry
Reactor for the Bioremediation of Marine
Sediment Contaminated by Polycyclic Aromatic
Hydrocarbons
Hong-Bae JUNG, Kae Kyoung KWON, and Sang-Jin KIM*
Elucidation of Induced Resistance and Growth
Promotion by Volatile Emission of Paenibacillus
polymyxa E681 on Arabidopsis
1
1
Marine Biotechnology Research Center, KORDI
*Corresponding author: h-bjung@kordi.re.kr
Polycyclic aromatic hydrocarbons (PAHs) are a toxic environmental
pollutant, accumulated usually in marine sediments. Due to the
potential hazardous on ecosystem and human, removal of PAHs
from sediments has been great concern. In this study, development
of ex situ bioremediation technique using fed-batch bioslurry reactor
has been conducted for the cleanup of marine sediments polluted by
PAHs. Experimental reactor was consisted of original reactor and
derivative reactors that were receiving part (1/10 (A) or 1/5 (B) of
working volume) of sediment slurry of original reactor after 1 (d1), 2
(d2), and 3 (d3) days incubation. The activity of electron transport
system (ETSA) of original reactor was increased from 5.1±0.1 ㎍
-O2/㎖/hr after 1 day of incubation to 27.4±0.3 ㎍-O2/㎖/hr after 2-3
days of incubation. ETSA of d2 &d3 reactors was similar to that of
original reactor after 2-3 days of incubation. The degradation rate of
PAHs was 81.0-92.7% (A) and 85.1-95.4% (B) at d2 &d3 reactors,
respectively. The degradation rate of PAHs was increased with the
repetition of treatment. The result implies that fed-batch bioslurry
reactor was effective for bioremediation of sediments contaminated
by PAHs. [Supported by Ecotechnopia program]
Keywords: PAHs, marine sediment, bioremediation, fed-batch
bioslurry reactor
2
2
Laboratory of Microbial Genomics, KRIBB, Chemistry and Biochemistry
Department, Texas Tech University, USA, 3Department of Entomology and
Plant Pathology, Auburn University, USA
*Corresponding author: cmryu@kribb.re.kr
Plant growth promoting rhizobacteria (PGPR) in interaction with plant
can elicit plant growth promotion and induced systemic resistance (ISR).
Recently volatile organic compounds (VOCs) from PGPR such as
2,3-butanediol and acetoin was reported bacterial determinants that
involved in plant growth promotion and ISR. We attained that genome
of Paenibacillus polymxya strain E681 encoded whole gene cluster for
production of 2,3-butanediol. In this study, we evaluated the role of
VOCs emitted from P. polymyxa E681 on plant growth and ISR in
Arabidopsis thaliana as a model plant. By using I-plate and microtitre
system, VOCs from strain E681 promoted plant growth in Arabidospis
plants including known volatile hormones such as jasmonic acid and
salicylic acid but did not in ein2.5 mutant Arabidopsis which is defective
on ethylene/cytokinin signaling. For assessment of ISR, we employed
Pseudomoans syringae pv. tomato DC3000 with dipping inoculation of
Arabidopsis seedlings grown in the microtitre plate. Study of ISR by
VOCs of PGPR in promoter-gus-fusion Arabidopsis revealed that
salicylic acid signaling is involved in the induced resistance. Strain E681
produced 12 a. u. for 2,3-butanediol and 47 a. u. for acetoin while negative
control P. fluorescens strain 89B61 4 a. u. and 0.1 a. u. respectively. This
study demonstrates the first report that VOCs produced by P. polymyxa
act an important role on growth promotion and ISR.
Keywords: Plant growth promoting rhizobacteria, volatile organic
compound, Paenibacillus polymxya
B038
B040
A Comprehensive Assessment of Biological
Communities of Polluted Streams for Development
of Integrative Water Quality Index
1
1
1
Jung-Hye CHOI , Se-Eun LEE , Byung-Hyuk KIM , Mi-Young
2
2
1
SONG , Tae-Soo CHON , and Sung-Cheol KOH *
1
2
Choong-Min RYU *, Mohamed A FARAG *, Paul W. PARÉ *,
Seung-Hwan PARK1, and Joseph W. KLOEPPER3
2
Korea Maritime University, Pusan National University
*Corresponding author: skoh@mail.hhu.ac.kr
In Korea, various water utilities (e.g., drinking, industrial,
agricultural, and resort purposes) are often simultaneously required
for the same water resource (river or stream), Hence, a comprehensive
evaluation for water quality status would be necessary. The eventual
goal of this study was to develop an integrated water quality
management system using physicochemical and biological
parameters from an ecosystem. Twelve different streams polluted
with farming, livestock, domestic, agricultural and industrial
wastewater bodies have been monitored in terms of physicochemical
water quality and community analyses. When all the inter-taxa
communities were put together for the SOM analysis, the sites were
patterned as 5 clusters which grouped according to pollution status
and season. This indicated that the combined SOM analysis could
reflect the trend of SOM analysis for each taxa. As a basis for the
development of an integrative water quality management system, a
SOM analysis for the typical biological indices (e.g., diversity and
species richness) was also performed for all the taxa tested. A
representative index for each taxa was first chosen and calculated, and
then an integrative index (I) was developed combining the three
representative indices for the three taxa. Overall the integrative index
(I) had consistently a higher correlation with the indices for other taxa.
Keywords: water quality index, microbial community, algae,
macro-invertebrate, PCR-DGGE, self-organizing map (SOM)
Studies on Cultural Characteristics of Pleurotus
pulmonarius (Fr.) Quel.
1
1
2
YoungHak PARK *, KwangJae LEE *, WonHo LEE , and
1
KyungHee KIM
1
Agricultural Processing Experimental Station, Gangwondo Agricultural
2
Research and Extension Services, Department of Applied Biology, Kangwon
National University
*Corresponding author: pyh1960@chollian.net
P. pulmonarius(Fr.) Quel. is usually from ivory to thin tan and generally smaller than those of P. ostreatus. It is convex to
fan-shaped, and fruit from mid-summer to early fall in natural
environment. P. pulmonarius prefer dead wood of coniferous trees. This
study was carried out to investigate the cultural characteristics of P.
pulmonarius(Fr.) Quel. on the optimal media, temperature, carbon
sources, nitrogen sources and mineral salt. Among various cultural
media used for mycelial growth of P. pulmonarius(Fr.) Quel., Malt
extract yeast extract agar(MYA) was most suitable for mycelial
growth. The optimal temperature for growth was 30℃, and the
mycelia were grown well when Maltose and Yeast extract were
added to media in concentration of 2.0% and 0.4%, respectively.
When the K2HPO4 was added 0.15% to media, the mycelium was
grown most well. Highest mycelial growth was observed when
C/N ratio was 1:1.
Keywords: P. pulmonarius, cultural characteristics, mycelial growth
국제학술대회
179
October 13-14, 2005, Seoul, Korea
B041
B043
Summary of Three Years of High Throughput
Cultivation in the Pacific and Atlantic Ocean
1
2
Ji-Hoon LEE and Hor-Gil HUR*
2
Department of Environmental Science and Engineering, GIST
*Corresponding author: hghur@gist.ac.kr
Jang-Cheon CHO and Stephen J. GIOVANNONI
1
Division of Life and Marine Sciences, Inha University, Department of
Microbiology, Oregon State University, USA
*Corresponding author: chojc@inha.ac.kr
Here we report the comprehensive results of high throughput
culturing (HTC) from seawater. To cultivate novel uncultured marine
microoorganisms, HTC methods based on dilution-to-extinction in
oligotrophic seawater media were developed. One to five cells were
inoculated to each well and incubated at 16ºC for three weeks. The
microbial growth was checked by epifluorescence microscopy using
a custom-made 48-well blotter. Approximately 4,000 individual
wells had been examined during the course of three years. Genomic
DNA was extracted from only 200 µl of cultures and residual cultures
were stored in liquid nitrogen. The 16S rDNA sequences obtained
were aligned in the ARB database and phylogenetic analyses were
carried out. Up to 15% of cells from coastal and pelagic seawaters
were cultured using this method. Among the cultured marine bacteria
are many unique phylogenetic lineages that have been classified a
new phylum, orders, families, and genera. Representatives belonging
to cosmopolitan and previously uncultured bacterial lineages,
including SAR11, OM60, OMG, RCA, OM43, and NAC, were
successfully recovered from the study. Many of isolates cultured in
this method however could not grow on standard marine agar.
Eighteen genomes of novel isolates are now being fully sequenced by
Craig Venter Institute funded by Moore Foundation.
Keywords: Marine, Prokaryotes, Uncultured, Cultivation, Genome
Sequencing
B042
To remove arsenate [As(V)] from the solution, we selected the way
to make As(V) to be insoluble inorganic material. Shewanella sp.
HN-41 was used to precipitate As(V) by making sulfide mineral.
Strain HN-41 reduced both of As(V) and S2O32- in the same incubation
2to As(III) and S to produce arsenic sulfide mineral. As(V) was
reduced to As(III) by Shewanella sp. HN-41. Concentration of As(V)
decreased over time, and correspondingly the concentration of As(III)
showed increase. But the amounts of increased As(III) were not
suitable for the amounts of decreased As(V), which means the
precipitation of As(III) by forming arsenic sulfide. We could also
observe that sulfide increased first, and then decreased, which
explains the participation of sulfide in the precipitation of yellow
colored-arsenic sulfide. The X-ray diffraction (XRD) pattern of the
yellow precipitate, arsenic sulfide is similar to that of mineral orpiment
(As2S3). And the color is appropriate for orpiment mineral. Therefore
we could infer the precipitate is close to orpiment. Then it is likely that
the arsenic sulfide is composed of As(III) from the reduction of As(V)
and S2- from reduction of S2O32- by Shewanella sp. HN-41, because
there is no apparent chemical reductions of the oxidized forms of
2As(V) and S2O3 as in uninoculated controls. The precipitated
materials were characterized by using scanning electron microscopy
(SEM), energy dispersive X-ray spectroscopy (EDX), and X-ray
absorption spectroscopy
Keywords: Iron-reducing bacteria, Shewanella, Arsenic, Thiosulfate,
Reduction, Precipitation
B044
An Attempt to Increase the Snesitivity of BGMK
Cell to Adenovirus
Arsenite Incorporation into Biotransformation
Products of Ferric-oxyhydroxide
Youn Sook JEE*, Hee Suk LEE, Youn Yeon KIM, Kyeong Shik
LEE, and Joo Lae CHO
Ji-Hoon LEE and Hor-Gil HUR*
International Drinking Water Center, Korea Water Resources Corporation
*Corresponding author: ysjee99@kowaco.or.kr
The aim of this study is to ease the detection technique of adenoviruses in environmental sample and to increase of the
sensitivity of BGMK cell to adenoviruses.The traditional cell
culture method, total culturable virus assay, has a disadvantage that
BGMK cell don't detect adenoviruses. Therfore it is important to
increase of the sensitivity of BGMK cell to adenovirusesFor this
purpose, we detected adenoviruses by the cell culuture method using
HeLa cell and it identified by the PCR method. RT-PCR method of
cellular RNA appearde that BGMK cell show to have no adenovirus
receptor. Based on this results, we can propose two method using
another cell with adenovirus receptor or getting BGM cell into
adenovirus receptor. As the first method is inconvenient in time and
cost, the latter is underway.
Keywords: BGMK cell, adenovirus, sensitivity
180
Precipitation of Arsenic Sulfide by Shewanella
sp. HN-41
한국미생물학회연합
Department of Environmental Science and Engineering, GIST
*Corresponding author: hghur@gist.ac.kr
In order to examine if arsenic(III) could be incorporated into β-FeOOH
(akaganeite) during the transformation by the microbial reduction, and
to compare the abilities sorbing and holding As(III) between surface
sorption of β-FeOOH and structural intake of biotransforming β
-FeOOH by Shewanella sp. HN-41, arsenite (0.5 mM) was added to the
β-FeOOH incubations with or without inoculation of HN-41. The final
products of minerals were collected and analyzed for the As contents
which were water-washable, acid-leachable, or totally-digested. When
the particles were washed with water, arsenic(III) was released out more
in control samples of abiotic incubations than in bacterially produced
minerals. But As was eluted out more from the bacterially produced
mineral particles than from the abiotic minerals, when digested with
acids. These results partially explain that As just adsorbs at the surface
of abiotic mineral products, while As is incorporated inside the
structures of the minerals undergoing transformation by bacterial iron
reduction. X-ray diffraction patterns of the abiotic and biotic product
minerals after washing with water or acid-digestion partially explain
that the biotic mineral form was changed after losing As which was
inside the structure of the mineral by acid-digestion, while the changes
are less in abiotic minerals because As mainly existed as adsorbate on
the surface of β-FeOOH.
Keywords: Iron-reducing bacteria, Shewanella, Arsenic, Incorporation,
Sorption, Ferric-oxyhydroxide
2005 International Meeting of the Federation of Korean Microbiological Societies
B045
B047
Biodegradation of Recalcitrant Aromatic Compounds
by Transformed Strain of Mn-repressed Peroxidase
Gene in White Rot Fungus Trametes versicolor
Effects of Cyanobacteria on the Restoration
and Microbial Diversity of Soils Damaged by a
Forest Fire Eun-Hye SHIN, Eun-Hye HWANG, Hyoung-Tae CHOI, and
Hong-Gyu SONG*
Jin-Yong KIM, In-Geun SONG, and Young-Jun KIM*
Division of Biological Sciences, Kangwon National University
*Corresponding author: hgsong@kangwon.ac.kr
White-rot fungi have been known to secrete several ligninolytic
enzymes directly involved not only in the degradation of lignin but
also in the degradation of various recalcitrant aromatic compounds.
Among those enzymes, Mn-repressed peroxidase (MrP) is repressed
by manganese (Mn). MRP expression was dramatically increased
than Mn-dependent peroxidase (MnP) expression by addition of
some aromatic compounds in the culture of Trametes versicolor.
MrP were cloned from T. versicolor, and the transformant MrP1 of
T. versicolor were constructed. The degradation of several
recalcitrant aromatic compounds was tested in the culture of T.
versicolor. T. versicolor MrP1 degraded 87.3% of 50 mg/l
phenanthrene in 6 days of incubation and 50.5% of 2,4,6trinitrotoluene in 4 days. Both degradation rates were much higher
than those of wild type strain. The effect of addition of surfactants
and mediators on the degradation of aromatics by T. versicolor
MrP1 were also examined. This fungal transformant may be utilized
for the bioremediation of recalcitrant aromatic contaminants.
Keywords: Trametes versicolor, transformant, biodegradation,
aromatic compounds
Division of Biotechnology, The Catholic University
*Corresponding author: songig@netian.com
In order to restore the forest soil damaged by a fire, cyanobacteria
which can promote the formation of soil crust was isolated, and their
effects on the burned soil were analyzed. Five different
cyanobacteria were isolated from the site where a fire was broken
out, but the restoring process is poor. Five cyanobacterial strains
(E6-1, E6-2, E6-5, E6-7, 2,3-1) were selected on the basis of the
production of EPS (Extracellular Polymeric Substances) and of the
formation of filament. These strains are found to be either
Leptolyngbya sp. or Oscillatoria sp. by the sequence analysis of 16S
rDNA. To test the ability for the formation of soil crust, selected five
cyanobacteria were inoculated on the burned forest soil and cultured
for eight weeks. Soil aggregates stability (SAS) of the soil treated
with cyanobacteria was higher than the untreated soil. Among the
treated soils, the one treated with E6-1 was the highest. E6-1 strain
also produced the most amount of EPS among all strains tested.
Overall, the forest soil damaged by a fire is expected to be effectively
restored and prevented from soil erosion through the formation of
soil aggregates by treating it with the proper cyanobacteria strains.
Keywords: Cyanobacteria, Soil crust
B046
B048
Inhibitory Efficacy of Lactobacillus Isolated
from the Digestive Tissues and Feces of Pig
Against Pathogenic Bacteria
Heavy Metal Adsorption of Extracellular
Polysaccharide Produced by Photosynthesis
Bacteria
Jung-Yong SUH, In-Geun SONG, Hyo-Sun UHM, and
Young-Jun KIM*
Jeong-Hwa JEONG , Min-Woo YUN , Young-Su BAE ,
3
3
4
Dong-Hyun KIM , Sung-Ho KONG , Eun-Jin LIM , Jong-Yeol
4
1
LEE , and Sang-Seob LEE *
Division of Biotechnology, The Catholic University
*Corresponding author: songig@netian.com
To use as promising probiotic strains for pig, lactic acid bacteria
(LAB) which would possess high survival rates in monogastric gut
and inhibitory effects on animal pathogens were screened from the
gastrointestinal tissue and feces of the pig. Among fifty strains of
LAB that showed high resistance to strong acidity (pH 2.5) and bile
juice (oxgall 0.2%), twelve strains, which have antimicrobial
acitivities against four different pathogenic bacteria (Staphyloccous
aureus, Salmonella typhimurium, Bacillus subtilis and E. coli) were
selected. These are identified as three strains of Lactobacillus brevis,
two of L. plantarum, two of L. arizonensis, L. casei, L acidophilus,
and two Pediococcus sp. strains. Among these twelve strains, two
strains, designated as L. plantarum CJY22 and L. arizonensis CJY3,
showed higher antimicrobial activities and broader spectrum than
those used as controls, such as L. delbruekii subsp. delbrueckii
ATCC 10557, L plantarum ATCC 10771, Pentococcus delbrueckii
ATCC 10557 and Leuconostoc mesenteroides subsp. mesenteroids
ATCC 10770. Further investigation to identify and characterize
these antimicrobial agents are now underway.
Keywords: Probiotic, Lactobacillus
1
1
1
2
2
Department of Biological Engineering, Kyonggi University, Department Soil
Analysis, Gyeonggido Institute of Health and Environment, 3Department of
4
Chemical Engineering, Hanyang University, Beautiful Environmental
Construction Co., Ltd.
*Corresponding author: sslee@kyonggi.ac.kr
There are a lot of mines in about 2,500 places in South Korea. Generally,
over 80% mines are spoiled among them. This has led to increasing
concern about the effects of toxic heavy metals as environmental
pollutants. Extracellular polysaccharides, or exopolymeric substances
(EPS) are produced by bacteria and have an important function in the
removal of heavy metal. In this study cadmium and cupper adsorption from
aqueous solution onto extracellular polysaccharide(EPS) produced by
photosynthesis bacteria was studied. Particularly, the effect of pH,
concentration of EPS and the effects of contact time were considered.
The result, The maximum removal capacities of cadmium and cupper
using the EPS were 91.5%, 89.3%(96.7mg/g, 85.4mg/g), respectively
and both plateau values were gradually reached within 60min. Optimum
adsorption pH values of cadmium and copper were in the range of 8.0,
and 5.0 respectively. Also, when pH condition and amount of EPS were
increased, the highest removal efficiency was discovered. Therefore, It
also indicates the possibility of application of this biosorbent for solving
some of the diverse problems of pollution which affect environment.
Keywords: Heavy Metal, Extracellular Polysaccharide, Photosynthesis
Bacteria, Adsorption
국제학술대회
181
October 13-14, 2005, Seoul, Korea
B049
B051
Screening of Plant Growth Promoting Microorganisms
Isolated from Various Rhizosphere
1
1
2
Sin-Geun LEE , In-Geun SONG , Hyang Burm LEE , and
Young-Jun KIM1*
1
2
Division of Biotechnology, The Catholic University, Department of
Biological Sciences, College of Natural Sciences, Seoul National University
*Corresponding author: songig@netian.com
A screening for plant growth promoting microorganisms was
conducted with 123 fungi and 190 bacteria which were isolated from
various rhizosphere. Salkowski test was performed to select strains
which produced high amount of auxin, and two strains, MF87 and
MF88, which produced 42.09 and 6.34 mg/L of auxin, respectively,
were finally selected and tested for their promoting effects on the
growth and yield of Mungbean and Lettuce. 40 to 50ul of the culture
filtrates from both strains showed similar effect on the rooting
-8
induction of Mungbean to the treatment of 10 M of indole acetic
acid (IAA). On the test of the growth of lettuce seedlings, MF88
promoted the elongation of roots, but MF87 restricted the growth of
roots. PLC plate was applied for the fractionation of auxin-like
materials from both strains using IAA as a standard. MF88 showed
four bands, one of which has similar Rf value (0.78) to that of IAA,
whereas MF87 showed one band that has 0.57 of Rf value. MF87
and MF88 strains were found to be Aspergillus sp. and Phoma sp.,
respectively, through the microscopic morphological analysis.
Keywords: Auxin, Plant-growth promoting Fungi(PGPF)
Effect of Terminal Oxidase Inhibitor (azide) in
Microbial Fuel Cell (MFC) Operation
Yeng Fung CHOO, Jiyoung LEE, Jae Kyung JANG, Gi Su
KANG, Ho Il PARK, Bor Chyan JONG, In Seop CHANG, Kyung
Shik KIM, and Byung Hong KIM*
Bioelectrochemistry Laboratory, Water Environment and Remediation
Research Centre, Korea Institute of Science and Technology
*Corresponding author: bhkim@kist.re.kr
In our previous study, 16S rDNA analyses on acetate enriched MFCs
revealed that various bacteria were identified and this include aerobicand anaerobic respiratory bacteria. Also another study of ours which used
respiratory inhibitors such as azide and cyanide on the MFC performance
showed that these inhibitors inhibits the aerobic respiration and did not
interfere with the electron flow to the electrode. With azide added to
acetate, non-fermentative fuel, we may eliminate the aerobically
respiratory bacteria in the microbial populations and identify the
populations that are contributing to electricity generation. Hence, sodium
azide (0.02mM) was added to acetate-fuel of 100mg/L concentration in
this study. Current data shows that acetate-fed MFCs gave a current
generation of around 4.5± 0.2 mA with a DO concentration of 0.18±
0.02ppm whilst the azide-acetate-fed MFCs gave a current generation of
around 4mA with a DO concentration of 0.08± 0.02ppm. This clearly
shows that azide inhibits the aerobic bacteria that give rise to a higher
oxygen concentration. 16S rDNA library construction work clearly
showed on azide enriched MFC that a major group of deltaproteobacteria
overrides over 98% of the populations. On going experiment, isolating
and identifying the majority bacteria that contribute to electricity
generation would definitely bring an interesting topic for further research.
Keywords: Sodium Azide, terminal oxidase respiratory inhibitors,
MFC
B050
182
B052
Two-electrode System Oxidizing Organic Materials
in Sediment
Factors Influencing the Degradation of Liquid
and Soil-applied Endosulfan Isomers
Gi Su KANG, Yeng Fung CHOO, Bor Chyan JONG, Kyung Shik
KIM, Jae Kyung JANG, Ho Il PARK, In Seop CHANG, and
Byung Hong KIM*
Jung-Bok LEE *, Ho-Yong SOHN , Min-Seb JO , Chung-Sig
3
1
CHOI , and Gi-Seok KWON *
1
1
2
1
2
Bioelectrochemistry Laboratory, Water Environment and Remediation
Research Centre, Korea Institute of Science and Technology
*Corresponding author: bhkim@kist.re.kr
School of Bioresource Sciences, Andong National University, Department of
3
Food and Nutrition, Andong National University, HansBio Co., Andong
National University
*Corresponding author: bio91@andong.ac.kr
We investigated the possibility of stimulating the oxidation of organic
materials in lake sediments through a microbial fuel cell (MFC) system.
Sediment collected from river bed was placed MFC-type bioreactors
with graphite anode at bottom graphite cathode at surface without proton
exchange membrane. Three of MFCs were operated with the connection
of resistor (10 ohm) between anode and cathode electrodes (closed
circuit), and another three MFCs were operated without connection
(open circuit) as control. They were operated over a year with
monitoring potential between the anode and cathode and change in
COD, pH and DO. The current of reactors with closed circuit increased
continuously and reached to 2.1 mA in 6 months. Control reactors
showed a potential over 0.8 V. After 1 year operation, we analyzed the
organics content of sediments. Analyses showed that the closed circuit
MFC consumed more organic materials than that of control. Bacterial
diversity analyses of the 16S rDNA by DGGE showed that the microbial
population in the constructed microcosm is different from the control.
The 16S rDNA analysis showed that diverse bacteria were enriched in
the microcosm including alpha-, betaproteobacteria, among others.
These results indicate that microcosm (electrochemically active
consortia) was developed in the anode compartment of MFCs.
Keywords: organic materials, sediment, MFC, anode electrode,
cathode electrode, closed circuit, microcosm
The chlorinated cyclic sulfate diester endosulfan is a cyclodiene
insecticide possessing a relatively broad spectrum of activity.
Technical-grade endosulfan is a mixture of two stereoisomes, α and
β-endosulfan, in a ratio of 7:3. Generally, both isomers are degraded
by attack at the sulphite group via either oxidation to form the toxic
metabolites endosulfan sulfate, or by hydrosis to form the non toxic
metabolite endosulfan diol. Endosulfan contamination has been
detected in soil, water, air and food products because of its abundant
usage and potential for environmental transport. We have studied the
efficacy of isolated bacterial culture on the biodegradation of water
and soils-applied endosulfan isomers. The influence of factors, such
as Tem, pH, presence of additional carbon source and initial pesticide
load in liquid and soil, on endosulfan isomers. Effect of Tem., pH and
additional glucose could have on the degradation rate of endosulfan
isomers: 80%(30℃), 64%(pH7), 45%, respectively. The amount of
endosulfan recoverd at 0 time were taken as 100%. We isolated
bacterial KE-8 and KS-2P culture have been enriched of organisms
capable if degrading the more persistent endosulfan isomers. And
These strains will be further investigated for their enzymatic reaction
in detoxification of endosulfan isomers. [This work was supported by
a grant(Code:1000520030096000) from BioGreen 21 Program, Rural
Development Administration, Republic of Korea]
Keywords: Insecticide, Endosulfan, Biodegradation
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
B053
B055
Characterization of a Fungus Isolated from
Dwarf Flowering Almond
1
2
Myoung Yong SHIM , Young Jae JEON , and Seong Hwan
KIM2*
1
Institute of Ecological Phytochemistry, Hankyong National University,
Department of Microbiology, Dankook University
Corresponding author: piceae@naver.com
2
*
A fungus isolated from brown rotted fruits of dwarf flowering
almond (Prunus glandulosa Thunb.) in July 2005 at Gongju,
Chungnam, was characterized. With the fungal development on the
fruit of the plant, the rot lesions began with small water-soaked
necrosis and enlarged rapidly. The infection produced a soft-dry rot
and the fungus sporulated abundantly. Then the diseased fruits
shrank and became grayish-black mummies. Based on microscopic
observation of its microstructure and cultural properties on PDA, the
causal fungus was identified as Monilinia sp. The 18S and ITS
ribosomal DNA regions in the Monilinia fungus were amplified by
PCR, and nucleotide sequences in the PCR amplicons were
determined. Search for sequence homology through GenBank
database revealed that the isolated fungus has high sequence
homology with known rDNA sequences of Monilinia species.
Further analyses of its growth physiology and phylogenetic
relationships to other Monilinia species were described.
Keywords: Monilinia sp,, dwarf flowering almond
Biodegradation of Diesel, Kerosene, and Gasoline
by Each Pure Culture in Liquid Medium
1
Byoung-Oh HWANG and Jae-Heon KIM*
Department of Microbiology Dankook University
*Corresponding author: jaehkim1@dankook.ac.kr
In our previous report, the superoxide dismutase containing iron and
zinc(FeZn SOD) of Streptomyces subrutilus P5 has a protective role
against heavy metals in Escherichia coli. Therefore, we produced
the partial gene of FeZn SOD by degenerate PCR and expressed the
insert DNA in E. coli strain M15[pREP4], using the commercial
vector (pQE-30). The protein produced has the N terminal 6x His tag
and formed inclusion bodies in E. coli. We could obtain the protein
only by the denatured purification procedure, but not by the native
procedure. In addition, we should change the composition of the
final buffer to elute the denatured FeZn SOD from the purification
resin, because urea showed a very strong disturbing effect in
quantitative heavy assay. However, the binding assay showed a
weak affinity between the FeZn SOD and heavy metals. A
concentration dependancy in heavy metal binding reaction using
whole cells as well as FeZn SOD are described in this report.
Keywords: Biosorption, Heavy metals, Superoxide dismutase,
Streptomyces subrutilus
2
1
Department of Biological Engineering, Kyonggi University, 2Department of
Environmental Engineering , Kyonggi University, 3Department of Chemical
4
Engineering, Hanyang University, Beautiful Environmental Construction
Co., Ltd., 5Department of Civil and Environmental Engineering, University of
Macau, Macau
*Corresponding author: sslee@kyonggi.ac.kr
Petroleum hydrocarbon pollution of the groundwater and soil has been a
major environmental problems with the industrial development and a lot of
gas stations. Petroleum hydrocarbon are separated three type of commercial
products(diesel, kerosene and gasoline). These commercial products are a
highly complex mixture, containing hundreds of thousands of
hydrocarbons. Several technologies, physical, chemical and biological
technology, have been developed to remove hydrocarbon from groundwater
and soil. Especially, Biological treatment could be a cost-effective methods
to treat contaminated soils and water. In recent, Richard(1999) reported that
microbial consortium degraded 90% of diesel fuel after 50days in liquid
medium. Baryshnikova(2000) showed that pure culture degraded 8~26% of
diesel and they made mixed cultures that two pure cultures or more were
mixed to improve degradation efficiency. Marja(2005) reported that diesel
fuel removal was greater in popular treatment, up to 86% by 28day. In this
study, we isolated bacteria for degrading diesel(16 strains, kerosene(32
strains), and gasoline (10strains). Through the screen test with each pure
culture, we selected high removal bacteria D10 for diesel(1,000ppm) and K6
for kerosene(1,000ppm). D10 removed 67.2% of diesel and K6 removed
94.3% of kerosene for 7days.
Keywords: biodegradation, TPH, Diesel, kerosene, gasoline
B054
Heavy Metals Biosorption of Escherichia coli
Was Increased by a Partial Gene of Iron Zinc
Containing Superoxide Dismutase of Streptomyces
subrutilus P5
1
Min-Woo YUN , Jeong-Hwa JEONG , Sang-Woo AN ,
Soon-Woong CHANG2, Seung-Won SEO3, Sung-Ho KONG3,
4
5
1
Jong-Yeol LEE , Ho-Jae SHIM , and Sang-Seob LEE *
B056
The Fungal Flora of Yungunneung, Hwasung
City, Gyeonggi Province
1
1
1
Yun Hee OH *, Sang Beom KIM , Seong Hwan KIM , Seong
1
2
1
Hwan KIM , Min Woong LEE , U Yoon LEE , and Tae Soo
1
LEE *
1
2
Department of Biology, University of Incheon, Department of Biology,
Dongguk University
*Corresponding author: tslee@incheon.ac.kr
The survey of fungal flora in Yungunneung, Hwasung City,
Gyeonggi province was made from November 1, 2001 to November
7, 2004.One hundred forty three species of higher fungi were
collected from these collection trips. The collected fungi belong to 2
divisions, 3 subdivision, 13 orders, 41 families, 82 genera. Dominant
fungal families were Tricholomataceae and Polyporaceae. On the
respect of mycological resources, edible mushrooms were 60 species,
culturable mushrooms were 8 species, medicinal mushrooms were 8
species, poisonous mushrooms were 18 species, ectomycorrizhal
mushrooms were 20 species and wood-rotting fungi were 70
species. Keywords: edible mushroom, medicinal mushroom, poisonus
mushroom, wood- ratting fungi
국제학술대회
183
October 13-14, 2005, Seoul, Korea
B057
B059
The Survey of Fungal Flora of Incheon Grand
Park, Incheon City 1
1
1
Yun Hee OH *, Sang Beom KIM , Seo Hwan KIM , Kyung Rim
LEE1, Min Woong LEE2, U Yun LEE1, and Tae Soo LEE1*
1
2
Department of Biology, University of Incheon, Department of Biology,
Dongguk University
*Corresponding author: tslee@incheon.ac.kr
The investigation of fungal flora was made in Incheon Grand Park of
Incheon City from May, 2001 to September, 2003.A total of 105
fungal species were collected and these fungi belong to 1 division, 3
orders, 19 families, 44 genera. Dominant fungal families were
Tricholomataceae and Russulaceae. On the respect of mycological
resources, edible mushrooms were 55 species ; culturable mushrooms
were 5 species, poisonous mushrooms were 16 species, medicinal
mushrooms were 4 species, anti-cancer substance containing
mushrooms were 8 species, ectomycorrizhal mushrooms were 19
species and wood-rotting fungi were 50 species. Keywords: edible mushroom, medicinal mushroom, poisonus
mushroom, wood-rotting fungi
B058
Hee-Jung JEONG, Young-Ran KIM, Na-Young KIM, and
Jongseol KIM*
Department of Biological Sciences, University of Ulsan
*Corresponding author: jkim@mail.ulsan.ac.kr
We operated four nitrifying biofilm reactors with synthetic
wastewater of different ammonia and organic carbon loads to
evaluate the relationship between microbial community and
biological nitrogen removal efficiency. In the first experiment, HRT,
DO, and temperature were maintained as 12hr, 4.0mg/L, and 24.0℃,
respectively. In a reactor with 40mg/L ammonia and 100mg/L COD,
the efficiency of ammonia and COD removal was more than 90%.
Whereas the removal efficiency was similar in reactors operated
with 1.5 and 2.0 times higher concentrations, it decreased
significantly with 3.0 times increased concentration. The reactors
with 0.3, 0.5, 0.7 times lower concentrations showed over 90%
removal efficiency. In the second experiment, different ammonia
and organic carbon loads were given by changing the HRTs.
Whereas the removal efficiency was more than 90% in reactors with
the HRTs of 6, 8, and 12hrs, it was less than 80% with 4hr HRT.
When community structures were analyzed through DGGE of 16S
rDNA and band patterns of the reactors were similar except the
reactors with 3.0 times increased concentration and 4 hr HRT. The
sequences of most bands corresponded to the members of β- and γ
-proteobacteria.
B060
The Fungal Flora Survey of Gyeyang Mountain in
Incheon City
1
1
1
Yun Hee OH *, Sang Beom KIM , Seong Hwan KIM , Kyung
1
2
1
1
Rim LEE , Min Woong LEE , U Yun LEE , and Tae Soo LEE *
1
2
Department of Biology, University of Incheon, Department of Biology,
Dongguk University
*Corresponding author: tslee@incheon.ac.kr
Higher fungi were collected at Gyeyang Mountain of Incheon City
from June, 2001 to October, 2003.A total of 115 species of fungi were
collected. These fungi belong to 3 division, 10 orders, 26 families, 50
genera. Dominant fungal families were Tricholomataceae and
Polyporacceae. All the fungi collected in this survey were recorded
species in Korea. On the respect of mycological resources, edible
mushrooms were 68 species, culturable mushrooms were 17 species,
poisonous mushrooms were 13 species, medicinal mushrooms were
8 species, anti-cancer substance containing mushrooms were 5
species, ectomycorrizhal mushrooms were 31 species and
wood-rotting fungi were 62 species. Keywords: edible mushroom, medicinal mushroom, poisonus
mushroom, wood-rotting fungi
184
Microbial Community Structures and Activities
in Nitrifying Biofilm Reactors: Effect of
Ammonia and Organic Carbon Loads
한국미생물학회연합
Distribution and Characteristics of Heterotrophic
and Coliform Bacteria in Groundwater of
Yeungnam Province
Bong-Soo KOH, In-Hwan LEE, Soo-Kyung KIM, and Jongseol
KIM*
Department of Biological Sciences, University of Ulsan
*Corresponding author: jkim@mail.ulsan.ac.kr
To evaluate bacteriological water quality and nutrient concentrations
of groundwater in Yeungnam Province, groundwater was sampled
from 123 locations during summer (June to August, 2003) and 117
locations during winter (December 2003 to January 2004). The median
values of various physicochemical parameters for summer samples
were as follows: pH 7.0, water temperature 21.8oC, NO3--N 1.9㎎/ℓ,
NH3-N 0.007㎎/ℓ, PO43--P 0.025㎎/ℓ, T-P 0.010㎎/ℓ, and CODMn
0.0250㎎/ℓ. For winter samples, the median values of pH and water
temperature were 6.9 and 10.0oC, respectively. In case of heterotrophic
plate count (HPC), the medians of overall samples were 30.0CFU/㎖
for summer and 40.0CFU/㎖ for winter, and those of each
administrative region were as follows: for summer, Kyungnam
45.0CFU/㎖, Kyungbook 30.0CFU/㎖, Daegu 12.5CFU/㎖, Busan
15.0CFU/㎖, and Ulsan 17.5CFU/㎖, and for winter, Kyungnam
27.5CFU/㎖, Kyungbook 70.0CFU/㎖, Daegu 22.5CFU/㎖, Busan
35.0CFU/㎖, and Ulsan 18.0CFU/㎖. Among the 123 locations
sampled for summer, 36.1% were positive for coliform bacteria, while
20.4% were coliform-positive for winter samples. The HPC were
positively correlated with water temperature (rs = 0.342, P<0.01) and
coliform bacteria (rs = 0.253, P<0.01). We could isolate and identify
such genera as Pantoea, Enterobacter, Escherichia, Serratia, Klebsiella,
Citrobacter, Enterobacter from summer samples and Klebsiella,
Citrobacter, Enterobacter, Rahnella, Serratia from winter samples.
Keywords: groundwater, drinking water
2005 International Meeting of the Federation of Korean Microbiological Societies
B061
B063
Adherence of Microorganisms on the Surfaces
Might Enhanced by the Microorganisms Has
High Cell Surface Hydrophobicity
1
1
2
Biotransformation of Methoxychlor by an
Intestinal Bacterium Eubacterium limosum
under Anaerobic Condition Jae Hyun PARK , Kae Kyoung KWON , and Hong Kum LEE *
You-Jin YIM, Jiyoung SEO, and Hor-Gil HUR*
1
Department of Environmental Science and Engineering, GIST
*Corresponding author: hghur@gist.ac.kr
2
Marine Biotechnology Research Center, KORDI, Polar Biotechnology
Center, KOPRI, KORDI
*Corresponding author: hklee@kopri.re.kr
Cell-cell interactions during the process of biofilm formation are
important for the future biofilm architecture. In the present study,
short-term interactions between microorganisms has different cell
surface properties (hydrophobicity (H) and attachment ability on
surface (A)) were investigated using two set of model biofilm
communities consist of 4 strains each. Biofilm formation on acrylic
surfaces was conducted with 50 ml conical tubes containing aged
seawater at room temperature for 3 hours. The amount of microbial
cells adhered on surfaces was determined by measuring decrease of
optical density of seawater and by measuring the amount of crystal
violet stained on surfaces. Strains have high H/A were adhered well
on surfaces (over 90%) but the strains have high A does not
(10~30%). Adherence of microorganisms on surfaces was increased
10~20% when the strains were mixed. Cultures containing strains
have high H/A always showing increased adherence than that of
expected. Strains have high H/A might enhances the adherence of
other strains on surfaces. Further investigation about the factors
concerning on cell-cell interactions is required. [Supported by NRL
for HKLee]
Keywords: Biofilm, Cell-cell interaction, Cell surface hydrophobicity
Methoxychlor, which is a pesticide used as a sutstitute for
DDT , has become environmental concerns due to its strong
potency as an endocrine disrupter. In order to know metabolic
fate of methoxychlor in the intestinal gut, Eubacterium limosum
(ATCC 8486), a strict anaerobe from the human intestinal tract that
is capable of O-demethylation of several compounds, was used as a
model intestinal microbial organism. Eubacterium limosum was
cultured with 100 uM methoxychlor in brain heart infusion (BHI)
broth medium at 37℃ under the anaerobic condition(90% N2, 5% H2
and 5% CO2) and extracted with ethyl acetate at certain intervals.
High performance liquid chromatography was used for the
determination of methoxychlor and its metabolite. After 5 days
incubation, most of methoxychlor was transformed to one
metabolite. Further study is being performed to characterize the
metabolite of methoxychlor produced by Eubacterium limosum by
mass spectrometry and to test for ability of Eubacterium limosum to
metabolize DDT.
Keywords: Eubacterium limosum, Methoxychlor, Biotransformation
B062
B064
Biological Activities and Cultural Characteristics
of an Entomogenous Fungus, Paecilomyces
Tenuipes (Peck) Samson 1
1
1
InPyo HONG *, SungHee NAM , GyooByung SUNG ,
1
1
1
2
HyunBok KIM , IYeon JUNG , PilDon KANG , Hyeon HUR ,
2
and MinWoong LEE
1
2
National Institute of Agricultural Science and Technology, RDA, Department
of Applied Biology, Dongguk University
*Corresponding author: iphong20@rda.go.kr
To develop techniques for the production of P. tenuipes stromata on a large
scale, the infection of P. tenuipes and the growth of stroma were investigated
by silkworm (Bombyx mori) variety. Also, studied about biological activities
of fruiting body formed on silkworm.
Infection rate of the 5th instar larvae of the silkworm with P. tenuipes
was the highest in Yangwonjam, followed by Hachojam, Baegokjam
and Chilbojam in that order. Also, as the inoculation times was increased,
infection rate tended to be raised. The rate of fruiting body formation
of the silkworm pupae infected with P. tenuipes was the highest in
Baegokjam, followed by Yangwonjam and Chilbojam in that order. But,
actually the fruiting body formation of the 5th instar larvae of the silkworm
tested was good in Chilbojam, followed by Yangwonjam and Baegokjam
in that order in 3 times spraying inoculation. Mannitol concentration was
high in the fruiting bodies of the silkworm infected with C. militaris on
a dry weight basis. The fruiting bodies of Yangwonjam and Chilbojam infected
with P. tenuipes had high amount of mannitol, but Baegokjam and Hachojam
had high concentration of glucose.
The most abundant amino acid in the fruiting bodies of the Baegokjam,
Chilbojam and Hachojam infected with P. tenuipes was arginine, while
Yangwonjam was glycine. C. militaris, had a large amount of glycine,
followed by serine.
Occurrence and Community Structure of
Bacteria Resistance to High Concentration of
Sulfamethoxazole in MBR
1
1
2
Jiyoung SEO , You-Jin YIM , and Hor-Gil HUR *
1
Department of Environmental Science and Engineering, GIST, 2Center for
Water Research of GIST
*Corresponding author: jiyoung@gist.ac.kr
Membrane Bioreactor (MBR) technology is becoming an increasingly
popular choice for the treatment of wastewaters. MBR microbial
community structure of resistance to antibiotic sulfamethoxazole
(SMX) was investigated based on 16S rDNA analysis. Sulfamethoxazole
is an anti-bacterial sulfonamide. It prevents the formation of
dihydrofolic acid, a compound that bacteria must be able to make in
order to survive. Twenty different colonies grown on nutrient agar
including 1mM SMX were randomly isolated. Through partial
sequence analysis of PCR-amplified 16S rDNA, SMX resistance
bacteria were divided into seven groups, which were 98%
similarities to Aeromonas caviae, A. hydrophila, Acinetobater
johnsonii, A. haemolyticus, Pseudomonas rhodesiae, P. graminis,
and P. stutzeri, respectively. These isolated bacteria also were tested
resistance to three other sulfa drugs, sulfanilamide, sulfaguanidine,
and sulfadiazine. Antibiotic resistance is a public health concern of
great urgency due to a growing inefficacy of antimicrobial agents to
treat infectious diseases. Therefore, antibiotic resistance could be
considered as an environmental pollution problem.
Keywords: Membrane Bioreactor, Sulfamethoxazole, Resistance
국제학술대회
185
October 13-14, 2005, Seoul, Korea
B065
B067
A Novel Diesel Degrading Bacterium Isolated
from Soils Contaminated with Diesel Oil
Su-Jin LEE, Gun-Young LEE, In-Geun SONG, and Young-Jun
KIM*
Division of Biotechnology, The Catholic University
*Corresponding author: songig@netian.com
Potential hydrocarbon degrading bacteria were screened from the
site artificially polluted with 10,000 ppm of diesel. Among the
isolates, two strains, SJD2 and SJD4, showed higher activities to
degrade diesel on the Bushnell-Hass broth medium containing 2%
of diesel. 16S rDNA sequence analysis revealed that SJD2 and SJD4
were Bacillus fusifomis and B. cereus, respectively. Both strains
were found to be grow in a wide range of temperature between 200C
- 550C, with the best at 300C - 370C. This is also the first report, as
far as we know, that B. fusifomis is capable of degrading diesel. We hope that a new isolate, B. fusifomis, will efficiently conduct
bioremediation at the contaminated sites with petroleum hydrocarbons.
Keywords: Diesel-degrading bacterium, Bacillus fusiformis
Establishment of a Constitutive Expression
System of Atrazine Chlorohydrolase Gene in
Bladyrhizobium japonicum USDA 110
1
1,2
1Department of Environmental Science and Engineering, 2International
Environmental Research Center, GIST
*Corresponding author: nyaong@gist.ac.kr
Bladyrhizobium japonicum USDA 110 is nitrogen fixing bacterium
and a root nodule symbiont of soybean (Glycine max (L.) Merr.).
Nodulation genes of B. japonicum USDA 110 are induced by specific
flavonoids produced by a host soybean and nod box is a promoter of
these genes. Atrazine, one of triazine herbicides, is most widely used
in United States, relatively persistent in soils and detected in
groundwater. AtzA gene (encodes atrazine chlorohydrolase) of
Pseudomonas sp. strain ADP dechlorinates atrazine to hydroxyatrazine.
In this study, we are constructing the plasmid pNod-atz based on
pLAFR3 and containing the 2.7 kb BamHI fragment of atzA gene
downstream of nod box, and it will be transformed to USDA 110.
Genistein, the best inducer of nod box, is going to be used as an inducer
for USDA 110 (pNod-atz) and metabolites of atrazine can be analyzed
using HPLC. Finally this rhizobium will be infected to a host plant. Our
aim is establishment of a constitutive chlorohydrolase expression
system in B. japonicum USDA 110 (pNod-atz) inside nodules, using the
rhizobium-plant signal exchange, without IPTG used in conventional
recombinant plasmids.
Keywords: Bladyrhizobium japonicum USDA 110, nod box,atrazine
chlorohydrolase, rhizobium-plant signal exchage
B066
B068
Improvement in Specific Detection of Vibrio
spp. by Using Species-Specific Primers
1
186
1
Ji-Young RYU , Soo-Jin KIM , and Hor-Gil HUR
1
2
Identification of a Putative Transcriptional
Activator, TbcR from Burkholderia cepacia JS150
1
2
Hyo-Joo BANG , Jin Hwan LEE , Jong Man KIM , and
1
Mal-Nam KIM *
Hyung-Yeel KAHNG * and Jerome KUKOR
1
Department of Biology, Sangmyung University, KORDI
*Corresponding author: jwnme@smu.ac.kr
2
Bacteria of the genus Vibrio are ubiquitous in marine and estuarine
aquatic ecosystems. Vibrio spp. frequently becomes pathogenic when
human beings consume seafood in its raw form. A method should be
developed for rapid and accurate detection of the pathogenic Vibrio
spp.. The detection method for Vibrio spp. includes the culture on the
TCBS selective medium which takes 3~4days, the analysis of the cell
membranous lipids using FAME which requires 3days, the analysis
method of the Vibrio spp. community for which 7 days are needed. In
sharp contrast, 1~2days are enough for the detection method using
species-specific primer. In this study, the last method was attempted
for the detection of 5 strains of Vibrio spp.. Sequence of the 16S-23S
intergenic spacer region of the 5 Vibrio spp. was used to design
species-specific PCR primers. V. parahaemolyticus, V. fluvialis, V.
vulnificus, V. cholerae, and V. proteolyticus were characterized by
400bp, 250bp, 350bp, 600bp, 150bp fragments respectively. The
designed species-specific primers for the 5 strains developed in this
study were specific enough not to affect bacterial species of other
genera. Detection of the 5 strains from fish and shellfishs was carried
out using the designed species-specific primers and the specificity of
the species-specific primers was confirmed against the environmental
samples.
Keyword: species-specific primers
We have previously reported that tbc genes in the locus of 14.3-kb
clone from B. cepacia JS150 are organized into two divergent
operons, tbc1 and tbc2. It has been suggested that tbc2 gene encodes
monooxygenases responsible for degradation of toluene, benzene,
chlorobenzene, trichloroethylene as well as tbc1 gene encodes
cresol/phenol hydroxylases responsible for catabolism of cresol and
phenol. Downstream of tbc2, sequence analysis revealed an ORF
with homology to tbuT, xylR, and similar NtrC-like transcriptional
activators. This putative transcriptional activator was designated
tbcR. Transcriptional and primers extension analyses were used to
demonstrate the role of tbcR in regulating tbc1 and tbc2 expression
from their tandem, divergent promoters. Analysis of gene structure
and expression suggests that expression of tbc1 and tbc2 is activated
by the transcriptional regulator TbcR (This work was supported by
Korea Research Foundation Grant (KRF-2004-041-D00375).
Keywords: transcriptional activator, BTEX, TBC operons
2
한국미생물학회연합
1
Department of Environmental Education, Sunchon National University,
Biotechnology Center for Agriculture and Environment, Rutgers University,USA
*Corresponding author: kahng@sunchon.ac.kr
2005 International Meeting of the Federation of Korean Microbiological Societies
B069
Efflux Pump Gene arsB in Pseudomonas putida
Strain OS-5 Isolated from Arsenic- contaminated
Mining Soil
Jin-Soo CHANG, Sunbaek BANG, and Kyoung-Woong KIM*
Arsenic Geoenvironment Lab (NRL), Department of Environmental Science
and Engineering, GIST
*Corresponding author: kwkim@gist.ac.kr
Pseudomonas putida strain OS-5 (NCBI AY952321), an
arsenic-resistant and arsenite-oxidizing bacterium, was isolated from
arsenic-contaminated soil in the Myoung-bong mine areas. Arsenic
resistance in Pseudomonas putida strain OS-5 was mediated by an
efflux pump gene arsB. The isolated gene from Pseudomonas putida
strain OS-5 is homologous to the arsB, which is a member of the
membrane transport regulatory deoxyribonucleic acid family. The
OS-5 nucleotide sequence analysis revealed that arsB gene of
Pseudomonas putida strain OS-5 had approximately 93% of similarity
by comparison with 16S rRNA gene. The growth of the indigenous
bacteria was inhibited when As(III) concentration was higher than
26mM. On the other hand, the isolated was grown in the presence of
66.7 mM As(V). The arsenite-oxidizing bacteria, strain OS-5, showed
different patterns of substrate for 30 physiological characterizations.
The bacteria strain was grown on solid media with various substrates
such as mannose, melibose, p-n-p-β-galactose, proline nitroanilide,
urea, and several others. Batch experimental results showed that strain
OS-5 oxidized 1 mM of As(III) to As(V) completely within 35 hr. The
arsB gene encoded for a membrane transport regulatory protein was
observed in arsenite-oxidizing Pseudomonas putida strain OS-5.
[Support by grants from Arsenic Geoenvironmental Laboratory
NRL at GIST]
Keywords: Arsenite-oxidizing bacterium, arsB, Pseudomonas
putida, 16S rRNA, Arsenic-contaminated mine.
B070
B071
The Analysis of Microbial Community at Lake
Konstanz Germany
1
2
1
Eun Young SEO , Guede HANS , Jai Kyou HUR , Yong Jeon
KIM1, and Tae Seok AHN1
1
Department of Environmental Science, Kangwon National University,
Institut für Seenforschung, Germany
*Corresponding author: seyel@kangwon.ac.kr
2
Lake Konstanz is the second largest pre-alpine European lake. In the
past, there was concrete bank around shoreline, but now 28km
shoreline has been restored. Hence the stiff concrete bank was
changed into flat slope with vegetation. After restoration, water
quality was improved and aquatic ecosystem was changed. So for
analysing the mechanism of theres changes, microbial community
structure were analyzed with FISH method. Water samples were
taken from littoral zone of restorated and unrestored area of lake
Konstanz and they were analyzed. They were compared with littoral
zone of Swiss. Also, diversity of the restoration in Lake Konstanz
were observed by life observation method with an optical
microscope. We achieved these 27th July, 2005.
Keywords: FISH, restorated area, Lake Konstanz
B072
Diversity and Dominant Members of the
Bacterial Soil Populations in an Acid Forest
Soil
Wastewater Treatment Using Biologically Active
Powdered Carbon and Microfiltration Process Min-Hye CHO* and Kyung-Sook WHANG
Department of Chemical Engineering, Chosun University
*Corresponding author: sibkim@mail.chosun.ac.kr
Department of Biotechnology, Mokwon University
*Corresponding author: kswhang@mokwon.ac.kr
We examined the distribution of aciduric bacterial populations using
two kinds of enumeration media. The number of bacteria enumerated
by a acid medium(pH 4.0) was higher than those enumerated by the
conventional medium(pH 7.0). One hundred forty-two aciduric
bacteria obtained by the isolating procedure from the humus soil.
Analysis of the cellular fatty acid composition is useful for the
classification and identification of various bacterial genera. The
cellular fatty acids of 108 bacterial isolates were analyzed. All strains
were grouped into three cluster according to their fatty acid
composition. Among 108 isolates, 83 isolates(77% of the total
isolates) belong to cluster B identified to Burkholderia suggesting
the dominance of bacteria. All of the strains contained w7cC18:1
(24~36%), C16:0(20~25%) and 2OH-isoC15:0(5~15%) were often
the most abundant fatty acid methyl esters (FAMEs). The strains of
cluster B were identified on the basis of types of hydroxyl acids
found, although these fatty acids usually accounted for less than 10%
of the total peak area in a profile. These hydroxyl fatty acids were
2OH-C16:0, 2OH-C16:1, 2OH-C16:0, 2OH-C18:1, 3OH-C12:0, 3OH-C14:0,
3OH-C15:0, 3OH-C16:0 and 3OH-C17:0. A large proportion of soil
bacterial populations belong to beta-proteobacteria in humus soil.
Keywords: aciduric, humus soil, MIDI, phylogeny
Sun-Il KIM and Sung-Hee ROH
This research is to investigate an innovative new technology for
wastewater using a combined process of biologically active
powdered carbon and microfiltration (BAPC-MF). The continuous
flow bench scale BAPC-MF process studies were conducted for the
treatment of wastewater. It was demonstrated that the BAPC-MF process has several advantages compared to conventional treatment
process in terms of efficency and operational costs. The BAPC-MF process was optimized regard to several parameters including
membrane flux rate, permeate quality, carbon concentration,
temperature, sludge age and biomass concentration. It was found
that the BAPC-MF process is significantly more effective than the
PAC process in treating wastewater. The better removal efficiency
of the BAPC-MF process may be attributed to higher biomass
concentration, lower biofilm thickness, and more favorable
environmental conditions.
Keywords: Microfiltration, BAPC-MF system, PAC
국제학술대회
187
October 13-14, 2005, Seoul, Korea
B073
B075
A Study on the Operating Characteristics
Parameter on BNR Process with Fermented
Leachate of Food Waste
Antialgal Activity of Bacterium Bacillus cereus
on Toxic Cyanobacterium Microcystis aeruginosa
Sun-Il KIM* and Sung-Hee ROH
Department of Life Science, Hanyang University
*Corresponding author: amb618@naver.com
Department of Chemical Engineering, Chosun University
*Corresponding author: sibkim@mail.chosun.ac.kr
In order to obtain the satisfactory treatment water in the biological
wastewater treatment process it is essential that the ratio between the
organic matter concentration and the total nitrogen concentration
(C/N ratio) of the raw water be adequately maintained. In this
research, the effect of external carbon source fermented leachate of
food waste (FLFW) was studied. The effects of FLFW addition into
wastewater with low C/N ratio in SBR process on nitrogen and
phosphorous removal efficiencies were investigated. The removal
efficiencies of NH4+-N and PO43--P were increased 81%~98% and
37%~80% with the addition of FLFW. Although the activity of
nitrogen oxidizing microorganism was decreased when the reaction
temperature was decreased 10℃ lower than that of normal
operation, phosphorous removal efficiency showed independent of
temperature and increased with addition of FLFW. Therefore, the
efficient nutrients removal process can be established when FLFW
and wastewater with low C/N ratio were simultaneously fed into
SBR reactor.
Keywords: SBR, C/N ratio, External carbon source, FLFW
B074
Selection of Fungicide for the Control of Wilt
Disease of Phalaenopsis spp. Caused by
Fusarium oxysporum KS04-3R
1
1
2
Jin-Hee KIM , Jung-Bo SIM , Jin Won KIM , and Se-Chul
3
CHUN
1
Department of Crop Science, College of Life and Environmental Sciences,
2
Konkuk University, Department of Environmental Horticulture, University of
Seoul City, 3Department of Molecular Biotechnology, College of Life and
Environmental Sciences, Konkuk University
*Corresponding author: scchun@konkuk.ac.kr
Wilt disease in basal leaves of Phalaenopsis spp. often occurred in
Namyangju city of Kyunggi-do, causing economic loss to growers.
However, there is no fungicide available at present. Effect of fungicides
was studied in vitro and in vivo on Fusarium oxysporum KS04-3R, which
causes wilt in basal leaves of Phalaenopsis spp. Mycelial plugs (diam.
5mm) of the fungus were inoculated on potato dextrose agar (PDA)
incorporated with 0-500ppm of twenty-five fungicides including
triflumizole(a.i. 30%) and benomyl(a.i. 50%), and incubated at 28oC
until the fungus on the control PDA covered entire an agar plate. The
inhibition of growth was determined at 7 days after inoculation. The
fungus was significantly inhibited by more than 90% on the PDA
amended with 125 and 250ppm of nine fungicides including as prochloraz
manganese complex (a.i. 50%), and acibenzolr-s-methyl mancozeb(a.i.
1% and 48%, respectively). In addition, 125ppm of selected nine
fungicides was applied to naturally infected Phalaenopsis spp. once every
week for 5 times in a greenhouse. Tebuconazole(a.i. 25%) was best to
reduce wilted leaves followed by triflumizole. However, tebuconazole
showed severe phytotoxicity, resulting in no growth of the plants in 4
months period. The result suggested that triflumizole might be a potential
fungicide to control wilt of basal leaves of Phalaenopsis spp.
Keywords: Phalaenopsis, Wilt disease, Fusarium oxysporum,
Fungicide
188
한국미생물학회연합
J-K SEO, B-H KIM, and M-S HAN
We have been screened the algicidal bacteria as relevant bio agents
against toxic cyanobacterium Microcystis aeruginosa using the
double layer agar lawn method. Of 99 isolates, HYS0503-MK62
showed the strong of algicidal activity, which effectively lysed M.
aeruginosa within 10~12 days. The isolate was identified as Bacillus
cereus with 16S rRNA gene sequence data and morphological
characteristics. Among the diverse media as nutrient broth,
peptone-yeast extract, filtered fresh water and modified casitone
medium(MCM), the bacterium cultured in MCM showed the
highest algicidal effect on M. aeruginosa. The isolate HYS0503MK62 gradually increased abundance of cell number when lysed M.
aeruginosa. It suggests that the isolate maybe HYS0503-MK62 use
M. aeruginosa as carbon source. The isolate HYS0503-MK62 also
showed the algicidal activity on several algae in diatom and
cyanobacteria. These results indicate that Bacillus cereus might be a
potential agent for biological control not only Microcystis
aeruginosa but also algal blooms in the eutrophicated fresh waters.
Keywords: algicidal bacteria, Bacillus cereus, Microcystis aeruginosa
2005 International Meeting of the Federation of Korean Microbiological Societies
C001
C003
Activation of the Transcriptional Repressors,
Tup11p and Tup12p, by the LAMMER Protein
Kinase, Lkh1p, in Schizosaccharomyces pombe
Self-Regulation of Aspergillus nidulans veA
Expression
Won Hwa KANG* and Hee-Moon PARK
Hee-Seo KIM*, Miae OH, Xhixing XIE, Houn-Young KIM, Mimi
LEE, and Keon-Sang CHAE
Department of Microbiology, School of Biosciences and Biotechnology,
Chungnam National University
*Corresponding author: yuniya@naver.com
Division of Biological Sciences, and Basic Science Research Institute,
Chonbuk National University
*Corresponding author: chaeks@chonbuk.ac.kr
Previously we had reported the Lkh1p as a negative regulator for
nonsexual flocculation of S. pombe. Through a pull-down assay with
MBP-tagged Lkh1p and subsequent mass spectrometry, we
identified Tup12 as an interacting protein. The interaction between
the Lkh1p and Tup12 was also confirmed by in vitro binding assay.
In contrast to single deletions, double deletion of tup12+and tup11+
showed flocculation of the yeast cells in liquid culture. Transcript
analysis of S. pombe homolog of the S. cerevisiae flocculin gene,
FLO1, revealed up-regulation of the gene expression in the
lkh1+deletion mutant. Assay with the LacZ fused to the downstream
of fructose bisphosphatase(Fbp) promoter, which is negatively
regulated by the Tup11p and Tup12p, showed that the galactosidase
activity in the lkh1+deletion mutant was six-times higher than the
wild type. Our data shown here suggested that the expression of S.
pombe flocculin gene was negatively regulated by the
transcriptional repressors, Tup11 and Tup12, like S. cerevisiae
floccuin genes, FLO1 and FLO11 by the Tup1p. Our data also
indicated that the activity of transcriptional repressors, Tup11p and
Tup12p, was positively regulated by the LAMMER protein kinase,
Lkh1p, in S. pombe. [This work was supported by the grant KOSEF
(R01-2004-000-10082-0)]
Keywords: Lkh1, tup11, Tup12, flocculation, Flo1, Flo11
During the study on the veA gene that is one of the key genes in
sexual development of Aspergillus nidulans, it has been observed
that the veA transcript level is slightly higher in a veA1 mutant than
in a wild type. Therefore, self-regulation of veA expression by VeA
protein (VeAp) was examined. The veA transcript level was much
higher in a veA-null mutant than in a wild type. Furthermore, the veA
gene is transcribed more highly in a veA1 mutant grown at 42℃ than
in a wild type, but equally in a veA1 mutant and in a wild type grown
at 30℃, which was as expected since the veA1 mutation is
temperature-sensitive. Also, illumination of visible light led to
increase in the veA transcript level, which was independent of the
presence of potassium chloride. A. nidulans strain A237 was
transformed with the lacZ reporter plasmids pSH-lacZ-VeA1 to
pSH-lacZ-VeA6 which were constructed by cloning 6 veA
promoters of different sizes into pSH96-lacZ. The b-galactosidase
activities of the 5 transformants are similar at 42°C. However, at
30°C, the b-galactosidase activity of the first strain is much lower
than the others. All of these results clearly indicated that the VeAp
regulates negatively the veA expression and binds to the upstream
region of the veAp.
Key words: Aspergillus nidulans, sexual development, veA
C002
C004
Differential Expression of pbpC during
Morphological Differentiation in Streptomyces
grisues
The A-factor Regulatory Cascade Leading to
Morphological Differentiation in Streptomyces
griseus : eshA Is under the Control of AdpA.
1,2
2
1
1,2
1
3
Hyun-Jung KWON , DongHoon LEE , and Jangyul KWAK *
Kieun LEE *, Sa-Ouk KANG , and Jangyul KWAK
1
1
2
Korea Research Institute of Biology and Biotechnology, Department of
Microbiology, Chungbuk National University
*Corresponding author: eni1110@nate.com
Streptomyces is a gram-positive bacterium that has been intensively
studied for secondary metabolite production and morphological
differentiation. Penicillin-binding proteins are ubiquitous bacterial
enzymes involved in cell wall biosynthesis and are the targets of
b-lactam antibiotics. To identify penicillin-binding proteins that are
required for morphogenesis of S. griseus, we surveyed fourteen
b-lactam antibiotics for their effects on vegetative growth and
sprorulation. Derivatives of 7-aminocephalosporanic acid (7-ACA)
prevented septum formation during sporulation but not during
vegetative growth. Six penicillin-binding proteins were identified
from membrane fractions prepared from growing and sporulating
cultures, when fluorescein-tagged 7-ACA (Flu-ACA) was used to
visualize penicillin-binding proteins. An high molecular PBP that
preferentially bound Flu-ACA accumulated in the membrane
fractions of cell that were undergoing sporulation. Computer
analyses of the 8 kb genomic DNA fragment containing the pbpC
gene encoding the PBP showed that the gene is clustered within
morphological genes identified in other Streptomyces. Transcription
analyses using S1 nuclease protection assays confirmed that the
pbpC were differentially transcribed during submerged sporulation in
the wild-type strain.
Keywords: Streptomyces grieus, Penicillin-binding protein
Laboratory of Biophysics, School of Biological Sciences, Seoul National
2
University, Korea Research Institute of Biology and Biotechnology,
3
Laboratory of Biophysics, School of Korea Research Institute of Biology and
Biotechnology
*Corresponding author: leeki@kribb.re.kr
Genetic complementation experiments were performed to
characterize the genotypes of a group of Class III developmental
mutants of S. griseus, SKK1003, 1004, and 1005. The mutants are
defective in producing streptomycin and aerial mycelia formation as
well as in accumulating EshA, a stress-response factor during
submerged sporulation. The mutants were restored to sporulate and
produce streptomycin by several pieces of the genomic DNA
fragments from the wild-type strain constructed in a low copy number
vector, pXE4. The genetic mapping and sequence determination of
the gene fragments from the mutants revealed that there were a frame
shift mutation and point mutations in the open reading frame of adpA,
which encodes a key transcription regulator required for aerial
mycelium formation and streptomycin production. To test whether
adpA directly reglulates the transcription of eshA, we accomplished
gel mobility shift assay using a recombinant AdpA. The AdpA bound
to the upstream regulatory region of eshA containing two putative
AdpA-binding sites in a concentration-dependent manner. The
binding was also confirmed in an in vivo binding assay system. These
results demonstrated that AdpA activates the transcription of eshA by
direct binding to the upstream regulatory regions.
Keywords: Streptomyces griseus, Morphological differnetiation,
AdpA, eshA, Transcriptional activator
국제학술대회
189
October 13-14, 2005, Seoul, Korea
C005
C007
Changing of Colony Morphology of Paenibacillus
polymyxa E681 by a Metabolite of Penicillium
citrinum
Isolation and Identification of Beauveria bassiana
from Diseased Insects in Korea
1
1
2
Soo-Young PARK , Choong-Min RYU , Choong Hwan LEE ,
Rumi KIM2, and Seung-Hwan PARK1*
1
Laboratory of Microbial Genomics, KRIBB,
Modulator, KRIBB
*Corresponding author: shpark@kribb.re.kr
2
Laboratory of Immune
Mycotoxins are a group of fungal metabolites that not only have
detrimental effect to the health of human and animals but also have
antagonism against bacteria. Among mycotoxins, citrinin produced
by a variety of Penicillium and Aspergillus spp. increase reactive
oxygen species in the human and bacteria resulting in lipid
peroxidation of membrane. Colony morphology of P. polymyxa E681,
a plant root associated bacterium, was changed bald to flat and
translucent forms when a certain fungus grew beside bacterial colony
on the tryptic soy broth agar (TSA). The fungus was identified as P.
citrinum by internal transcribed spacer (ITS) sequences analysis. The
main compound involving in changing colony morphology of strain
E681 was identified as citrinin. We validated the effect of the extracted
citrinin from P. citrinum by using commercial citrinin. Furthermore,
we evaluated effect of citrinin on membrane peroxidation of P.
polymyxa E681. When P. polymyxa E681 was cultured on the TSA
containing 10㎍/ml citrinin, the composition of fatty acid on the cell
membrane of strain E681 was significantly changed. Among fatty
acids of strain E681, palmitic acid was increased as 2.3 folds, while
15:0 anteiso and 17:0 anteiso form was decreased as 0.86 and 0.744
respectively. Our results presented here provide an insight of a fungal
metabolite that affects alteration of bacterial morphology.
Keywords: Paenibacillus polymyxa E681, mycotoxin, citrinin,
Penicillium citrinum
Applied Microbiology Division, NIAST
*Corresponding author: wgkim@rda.go.kr
Beauveria bassiana (Balsamo) Vuill. was isolated from diseased
insects belonging to orders Coleoptera, Diptera, Homoptera,
Orthoptera, etc. which were collected from several mountainous
districts in Korea. Morphological characteristics of the fungus
colonized on the host insects were examined by compound
microscope. Conidia were blastic, hyaline, smooth, globose to
slightly ellipsoidal, and measured 1.5-3.5×1.5-3.0㎛. Conidiophores
branched, globose to flask-shaped, with geniculate raches, and
beared clustered conidiogenous cells which measured 3.0-8.0×
3.0-5.0㎛ . A total of 70 monoconidial isolates was obtained from
the diseased insects. Colonies of the isolates on potato dextrose agar
were shown as white velvet or powder at the early growing stage and
turned to pale yellow or pale red later. The isolates were conserved
in the NIAST to use as a biocontrol organism against insect pests of
various crops. Keywords: Beauveria bassiana, diseased insect, identification,
isolation
C006
C008
SSH-based Analysis of Genes Expressed at
Primordial and Basidiome Stages in Hericium
erinaceum
Strain Selection from Artificial Fruit-Body
Formation of Cordyceps militaris Collected
from Different Area of Korea in 2004
1
190
Wan Gyu KIM*, Soon Ja SEOK, Kang Hyo LEE, Hang Yun
WEON, and Yang Sup KIM
1
2
Han Gyu KO , Hyuk Gu PARK , Seong Hwan KIM , and Won
1
Mok PARK *
Sung-Keun CHOI*, Won-Ho LEE, Je-O YI, Bum-Suck KIM, and
Jae-Mo SUNG
1
School of Life Sciences and Biotechnology, Korea University, Department of
Microbiology and Institute of Basic Science, Dankook University
*Corresponding author: mushroom@korea.ac.kr
Entomopathogenic Fungal Culture Collection, Department of Applied
Biology, Kangwon National University
*Corresponding author: art2321@nate.com
The edible and medicinal mushroom, Hericium erinaceum, has been
used in the Orient to treat various human diseases such as hepatitis,
hypertension, hypercholesterolemia and gastric cancer. Recently,
with the finding of Erinacines, inducers of the synthesis of nerve
growth factor, interest in the cultivation of the mushroom is
gradually increasing. In an effort to understand the properties of
basidioma production at molecular level, we investigated the genes
differentially expressed at different developmental stage in H.
erinaceum. For this aim, suppression subtractive hybridization
(SSH) technique was applied to primordia and basidioma of H.
erinaceum and we obtained 282 SSH cDNA clones from the two
tissues. Regarding the SSH clones, 137 clones were from primordial
library and 145 clones from basidioma library. The size of the insert
in the SSH clones ranged from 90 to 1300 bp with an average of 300
bp for primordial and 370 for basidioma library. BlastX search
through GenBank database showed that about 60% of the cDNA
clones are unknown genes. Comparison of the results of BlastX
search was described.
Keywords: HERICIUM ERINACEUM, Suppression Subtractive
Hybridization
This study reports artificial fruit-body formation of Cordyceps
militaris and selection of superior isolates. Superior isolates
showing BE over 20% were graded as A group. 21% of all the tested
isolates were graded as A group. Stromata size of natural specimen
and their artificial fruit bodies were compared. Size of EFCC
C-11913 was the smallest one with 22mm height, but the fruiting
bodies produced from it was 4 times as tall as the natural one.
Similarly, length of EFCC C-12448 was 39 mm, but the artificial
fruiting bodies were as tall as 106 mm and the BE was the highest,
i.e., 27.97%. BE of fruiting bodies produced using single ascospore
isolates from EFCC C-12448 artificial fruiting bodies was 28.21%.
Thus, the superior isolate was selected based on BE and the
morphological characters.
Keywords: artifical fruit-body, Biological efficiency(BE), single
ascospore isolate, strain selection, Cordyceps militaris
2
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
D001
Production of iso-SOD in Deinococcus grandis, a
UV Resistant Bacterium
Eun Jung PARK and Young Nam LEE*
Division of Biological Sciences, Chungbuk National University
*Corresponding author: ynlee@chungbuk.ac.kr
Deinococcus grandis is a member of Genus Deinococcus,
although it is a gram negative big rod. And, as like other Deinococcal species, this bacterium is an extraordinary resistant to UV, ionizng radiation, and oxidative stress. D. grandis seems to
possess two forms of superoxide dismutase (SOD) which is
indispensableto scavenge superoxide radical generated by a
various of environmental stress including UV, ionizing radiation, oxidative stress, and some toxic chemicals. In order to understand
physiological roles of iso-SODs in D. grandis, production study
of iso-SODs was made upon UV irradiation and external and
internal oxidative stress by measuring enzyme activity and resolution
of the enzmes by PAGE . The SOD activity of D. grandis was
substancially increased during stationary phase and by UV and potassium superoxide treatment . Activity of a slower migrating
SOD on polyacrylamide gel increased during stationary phase and by UV and superoxide radicals, thus this isoform seemed to be
an inducible enzyme. However, a faster migrating SOD was
considered to be a constitutive and major enzyme because constant
level of this SOD activity was observed regardless external and
internal oxidative stresses.
Keywords: Deinococcus grandis, Iso-SOD, Deinococcus
grandis, UV resistant, Oxygen stress,, Inducible, Constitutive
D003
Expression, Purification and Characterization
of Rhodococus equi P2 Medium-Chain-Length
Polyhydroxyalkanoates
Depolymerase
in
Escherichia coli
Ji Hye LIM and Young Ha RHEE
Department of Microbiology, Chungnam National University
*Corresponding author: yhrhee@cnu.ac.kr
A Gram-positive bacterial strain, Rhodococcus equi P2 capable of
degrading Medium-chain-length polyhydroxyalkanoates (MCL-PHAs)
was isolated from a tideland sample and the gene of an extracellular
MCL-PHA depolymerase from the isolate was cloned and sequenced.
The gene (phaZRe) of extracellular MCL-PHA depolymerase of R. equi
P2 consist of an 837 bp open reading frame encoding a protein of 278
amino acids with a deduced molecular mass of 30,692Da. The amino acid
sequence of the PhaZRe had a 98% (274/278) sequence homology with
that of the MCL-PHA depolymerase from Pseudomonas alcaligenes
LB19. The N-terminal regions of the PhaZRe contained many
hydrophobic amino acids, suggesting that substrate binding domain is
located in this region. In contrast, a catalytic triad of the PhaZRe (172Ser,
228Asp, 262His) existed in the C-terminal region, indicating the
presence of a catalytic domain in the C-terminal region. The gene
(phaZRe) was inserted into pBluescript II KS(-) expression vector and
expressed in Escherichia coli DH5a. The expresssd protein in E. coli was
purified from the crude lysate by hydrophobic interaction chromatography
using Octyl-Sepharose CL-4B and subsequently by electroelution. The
enzyme consisted of a monomeric subunit having a molecular mass of 25
kDa, as determined by SDS-PAGE. In this presentation, some of the
biochemical properties of the purified enzyme and comparison with
other MCL-PHA depolymerases will be discussed.
Keywords: Medium-chain-length polyhydroxyalkanoates, polyhydroxyalkanoates
depolymerase, Rhodococcus equi P2
D002
D004
Purification and Characterization of Protease
Produced from Bacillus cereus G9241
Localization of Sup35 Prion Domain near the
Rnq1 Aggregates Enhances the Appearance of
Sup35 Amyloid in Saccharomyces cerevisiae
Hyung Rok PAIK, Mi Sun KIM, Kyung Chul KIM, Chi Nam
SEONG, and Sang Ki CHOI
Department of Biological Sciences, Sunchon National University
*Corresponding author: sangkic@sunchon.ac.kr
A strain producing a potent protease was isolated from wreath shell
obtained in Gwangyang bay. The strain was identified as Bacillus
cereus G9241 based on morphological, physiological and
phylogenical analysis. The protease was purified using ammonium
sulfate precipitation, SP-Sepharose, Hydroxyapatite, and Sephadex
G75-column chromatography. Its molecular mass determined by
SDS-polyacrylamide gel electrophoresis was 77kDa. Peptide Mass
Fingerprinting of purified protein and its database search in NCBI
indicates that pI and mass of this protein deduced from gene was 5.4
and 77.5kDa, respectively. The optimum temperature and pH for
protease activity was 45℃ and 6.8, respectively. The protease was
stable in broad pH range between 5.8 and 7.5. The enzyme activity
was enhanced in the presence of Ca2+ and Mg2+, and was strongly
inhibited by EDTA, indicating that it require divalent metal for
activity. Triton X-100 slightly stimulated the protease activity,
suggesting that the protease could be useful for laundering additives
Keywords: protease, purification, Bacillus cereus, EDTA
Young-Jun CHOE, Yang-Kyun RYU, Hyun-Jin KIM, and
Yeong-Jae SEOK*
Department of Biological Sciences and Institute of Microbiology, Seoul
National University
*Corresponding author: promiseyou@empal.com
Sup35, a translational termination factor in S. cerevisiae,
occasionally accomplishes the conformational change into amyloid
fiber. This results in the depletion of soluble Sup35 in cytoplasm and
causes the read-through of termination codon. The appearance of
Sup35 amyloid can be induced by overexpression of Sup35 and this
induction is dependent on the presence of another amyloid which is
made of Rnq1. It was suggested that Rnq1 amyloid can act as an
imperfect template on which Sup35 protein can fulfill the conformational
change. In this study, we found a new method to induce the
emergence of Sup35 amyloid. When the amyloid forming domain of
Sup35 is fused to Rnq1, it can induce the appearance of Sup35
amyloid even if the expression level is relatively low. Results in this
study confirm the role of Rnq1 amyloid and may shed light on the
mechanism of the appearance of Sup35 amyloid.
Keywords: Sup35 amyloid, protein-protein interaction, yeast prion
국제학술대회
191
October 13-14, 2005, Seoul, Korea
D005
D007
Novel Insoluble Chromogenic Substrate for the
Assay of Endoinulinase
Angiotensin I-Converting Enzyme Inhibitory
Peptide Was Purified from Chungkookjang
1,2
1
Leonid N. TEN *, Wan-Taek IM *, Zubair ASLAM
Sung-Taik LEE1*
1
1,
and
2
Department of Biological Sciences, KAIST, Department of Chemistry,
National University of Uzbekistan, Uzbekistan
*Corresponding author: e_stlee@kaist.ac.kr
Endoinulinases are specific for inulin and hydrolyze the internal β-2,1
fiuctofuranosidic linkages to yield inulo-oligosaccharides, which are
physiologically functional food ingredients because of their various
health-promoting properties. Endoinulinase activity is usually
measured by determining the amount of released reducing sugar from
inulin using DNS method. This assay is not specific for endoinulinase
and it does not offer correct results in the presence of large amounts
of reducing sugars in the crude culture filtrates. New insoluble
chromogenic substrates were synthesized for the assay of endoinulinase.
For this purpose methacrylated inulin (MA-Inulin) was synthesized
by using glycidyl methacrylate and 4- (dimethylamino)-pyridine as
catalyst. Aqueous solutions of MA-Inulin were converted into
cross-linked hydrogels by free radical polymerization. Finally, inulin
hydrogels were dyed by using reactive dyes and diversely colored
insoluble cross-linking inulins were obtained. These dye-labeled
inulins were degraded by inulinase from Aspergillus niger in a timeand concentration-dependent manner and they can be applied as
chromogenic substrates for the assay of endoinulinase. This work
was supported by the Brain Pool Program (2003) and the 21C Frontier
Microbial Genomics and Application Center Program, Ministry of
Science &Technology (Grant MG05-0101-4-0), Republic of Korea.
Keywords: Endoinulinase, Chromogenic Substrate
Department of Biotechnology, Hoseo University
*Corresponding author: hbkim@office.hoseo.ac.kr
Korean fermented soybean, Chungkookjang is known to improve
blood circulation. Its diverse active compounds include Bacillus
protease and peptides. Bacillus protease has fibrinolytic activity.
Formation of small peptides was confirmed by SDS-PAGE. Lys-Pro
was purified by HPLC analysis (0.083 mg/100 g sample). ACE
converts angiotensin I to II, resulting in hypertension. ACE
inhibitory activity of Lys-Pro was determined (IC50=32.1 uM).
Sistolic and diastolic blood pressures dropped by 15, 8 mmHg,
respectively, after a sing administration of 20 g of Chungkookjang.
Chungkookjang is one of promising food helpful in improving blood
circulation. Keywords: ACE inhibitor, Chungkookjang, blood circulation
D006
D008
Characterization of dITP- and XTP-Hydrolyzing
Protein from a Hyperthermophilic Archaeon
Chromate Reductase Activity of QX Protein
Eun Kyoung IM and Ji Hyung CHUNG*
Department of Biotechnology, Hoseo Univerisity
*Corresponding author: hbkim@office.hoseo.ac.kr
Yonsei Research Institute of Aging Science, Yonsei University
*Corresponding author: jhchung@yonsei.ac.kr
Oxidative deamination of DNA is caused by ROS, which converts
DNA base amino groups to keto groups and can trigger abnormal
mutations, resulting in mutagenesis in organisms. In this study, a
non-canonical purine dNTP pyrophosphatase (AfPPase) from a
hyperthermophilic archaeon Archaeoglobus fulgidus, which hydrolyzes
aberrant nucleoside triphosphates, was overexpressed in E. coli,
purified and characterized. For the non-canonical nucleotides,
AfPPase showed remarkably higher activity for XTP and dITP,
suggesting that the 6-keto group of these nucleotides is critical for
the reactivity. Under optimal reaction conditions, the reaction rate
for these substrates was about 120 times that with dGTP. So AfPPase
may enact a significant DNA repair role by the hydrolysis of the
non-canonical nucleotides before they are misincorporated into the
DNA. [This work was supported by the Korea Research Foundation
Grant funded by Korean Government (MOEHRD) (R08-2003000-103898-0).]
Keywords: Hyperthermophile, Mutation, dNTP Pyrophosphatase
192
Hyung Jae YOO, Jae Sung HWANG, and Han Bok KIM*
한국미생물학회연합
Ki-Hyun KIM and Han Bok KIM*
Cr(VI) contamination is prevalent in the environment. Cr(VI) is
toxic, and Cr(III) is less toxic. It is rational to convert Cr(VI) into
Cr(III) using chromate reductase. In this study, a novel chromate
reductase QX was purified. Its basic kinetics (Km and Vmax values,
optimal temperatures) were determined. Also, rapid-scan kinetic
measurements for the enzyem, were determined using a stoppedflow spectrophotometer at 403, 501, 580 nm, respectively. During
the chromate reduction, H2O2 is generated inevitably, which is toxic
to the cell. QX was helpful in defending against H2O2.
Keywords: Chromate reductase, kinetic
2005 International Meeting of the Federation of Korean Microbiological Societies
D009
D011
Enzymatic Characterization of Aromatic
Nitroreductase from White Rot Fungus Irpex
lacteus
Regulation of Histone Deacetylase Gene
Expression in Coprinellus congregatus During
Its Development
Eun-Hye SHIN and Hong-Gyu SONG*
Ju-Hee KEUM* and Hyoung-Tae CHOI
Division of Biological Sciences, Kangwon National University
*Corresponding author: hgsong@kangwon.ac.kr
Department of Microbiology, Kangwon National University
*Corresponding author: selly1818@hotmail.net
White rot fungus Irpex lacteus isolated from Korea can transform
2,4,6-trinitrotoluene (TNT) via two different initial reduction
pathways. Since there has not been a report on fungal enzymes
involved in the initial reduction of nitroaromatic compounds,
characterization of fungal nitroreductase may be very important in
the investigation of precise metabolism of nitroaromatics.
Nitroreductase activity was observed in cell free extract of Irpex
lacteus in aerobic and anaerobic conditions. Its nitroreductase was
purified by phenyl hydrophobic interaction chromatography, DEAE
anion exchange chromatography and size exclusion chromatography.
Two bands were shown in sodium dodecyl sulfate polyacrylamide
gel electrophoresis analysis, but just one band was reported by
non-denaturing polyacrylamide gel electrophoresis. Therefore, this
aromatic nitroreductase seemed to be a dimeric protein. Purified
enzyme has a molecular weight of approximately 50 kDa. Its
optimum pH is 4 and temperature optimum is 20°C. This purified
enzyme utilizes FMN as a cofactor and preferred NADPH rather
than NADH as an electron donor. This enzyme activity was inhibited
by CuSO4, HgCl2,, MnSO4 and dicumarol, diphenyliodonium,
acetate, lactate, benzoate but enzyme activity was increase by FeCl3.
Other enzymatic properties were characterized. Purified enzyme
could transform TNT. This is the first report on the characterization
of membrane bound aromatic nitroreductase of fungi.
Keywords: nitroreductase, white rot fungus, Irpex lacteus
Dynamic histone acetylation has a role in chromatin remodeling and
in the regulation of transcription. Histone deacetylases(HDACs) and
histone acetyltransferases(HATs) catalyze reversible histone
acetylation. Using PCR and RACE-PCR approaches with various
primers based on HDAC-consensus region, we have cloned and
sequenced cDNA fragment and genomic DNA fragment, encoding
sequences of putative histone deacetylase from Coprinellus
congregatus, a mushroom forming basidiomycete. We also have
performed Northern analysis to determine the expression level of
HDAC during its development from monokaryotic and dikaryotic
mycelia, to mushroom cells.
D010
D012
Determination of Gene Expressions Related in
the Development of Coprinellus congregatus
During Its Mushroom Formation
1
1
2
The Increase in Reduced Glutathione Level via
Glutaredoxin 1 Affects the Culmination and
Cell Fate Determination in Dictyostelium
Hye-Yeon PARK *, Ju-Hee KEUM , C. W. CHO , and H. T.
1
CHOI
Chang-Hoon CHOI*, Sun-Young JEONG,
Seong-Jun PARK, and Sa-Ouk KANG
1
Department of Microbiology, Kangwon National University, School of
Biotechnology and Biomedical Science, Inje University
*Corresponding author: librayeon@hotmail.com
Laboratory of Biophysics, School of Biological Sciences, and Institute of
Microbiology, Seoul National University
*Corresponding author: kangsaou@snu.ac.kr
A fruitbody of Coprinellus congregatus, an inky cap, is getting dark
when it matured and rapidly lyse to black liquid in a few hours. Two
monokaryon strains of C. congregatus, Cc16 and Cc44, mated and
cultured in 15 hourslight / 9 hoursdark cycle. A fruitbody formed during
developmental stage, primordium, young mushroom, and matured
mushroom were used for this experiment. Several biochemical
characterizations were performed in each fruitbodies. In order to
determine whether laccase is involved in the mushroom maturation
and autolysis to the black ink, Northern analysis for the laccase gene
was performed. Furthermore, chitinase hydrolyzes the fungal cell
wall component, chitin. Therefore, we have also performed another
Northern analysis for the chitinase involvement during the
mushroom autolysis.
In Dictyostelium glutathione is essential for growth and developmental
process. To better understand the glutathione-dependent redox
signaling on development, we have identified glutaredoxin (Grx1), a
small glutathione-dependent redox protein. Grx1 has a conserved
Cys-Pro-Tyr-Cys sequence within its active site and a glutathionedependent oxidoreductase activity. Its mRNA is highly accumulated
at the mound stage and culmination stage. Overexpression of Grx1
induces the slugs not to culminate but to migrate continuously.
Northern blot analysis reveals that the expressions of prespore and
spore marker genes are markedly reduced in the Grx1-overexpressing
cells, which is consistent with the delayed culmination phenotype.
Grx1 is induced in response to oxidative stress and functions in
defense against oxidants during growth and development. Induction
of Grx1 leads to the increase in reduced glutathione level. The
oxidants, such as menadione and hydrogen peroxide, induced the
culmination of the slugs, suggesting that the increase in reduced
glutathione level given by glutaredoxin 1 may inhibit the culmination
of Dictyostelium. In addition, Grx1- overexpressing cells were
preferentially localized to the prestalk region of the chimeric slugs
with parental cells, indicating that the redox state of the glutathione
affect cell fate determination. Keywords: Glutaredoxin, Glutathione, Slugger phenotype, Cell
fate, Dictyostelium
2
Ji-Sun
KIM,
국제학술대회
193
October 13-14, 2005, Seoul, Korea
D013
Relationship Between ECF Sigma Factor, YlaC,
and Oxidative Stress in Bacillus subtilis
Han Bong RYU*, In Ji SHIN, and Sa Ouk KANG
Laboratory of Biophysics, School of Biological Sciences, and Institute of
Microbiology, Seoul National University
*Corresponding author: hbryu@snu.ac.kr
The ECF sigma factors are found in a wide range of bacteria and
regulate the uptake or secretion of specific molecules or ions and
response to a variety of extracellular stress signals. YlaC was
expressed by oxidative stress such as hydrogen peroxide and
paraquate. The ylaC-overexpressing mutant showed the resistance
to 1 mM hydrogen peroxide, while wild type and disruptant strain
showed a rapid decrease. Northern blot analysis showed that
ylaC-overexpressing mutant increased the ylaC, trxA transcripts and
decreased the sigB, aphC transcripts, while disruptant mutant
increased the aphC transcript. Also, ylaC-overexpressing mutant
increased the sporulation rate and formation of spore. But disruptant
mutant decreased. Northern blot analysis also revealed that
ylaC-overexpressing mutant increased the spo0A transcript, while
disruptant mutant delayed the expression of spo0A. These results
suggest that YlaC provide the cell with resistances to hydrogen
peroxide and may expert physiology effects on the sporulation of B.
subtilis
Keywords: ECF sigma factor, hydrogen peroxide, sporulation, B.
subtilis
D014
Comparison of Phenotypes in Escherichia coli
Strains B and K-12 by Phenotype Microarray
Analysis and in silico Metabolic Pathway Analysis 1
1
2
Sung Ho YOON *, Jeong Im LEE , Choong Hoon LEE ,
Haeyoung JEONG1, and Jihyun F. KIM1
1
Laboratory of Microbial Genomics, Genome Research Center, KRIBB,
Department of Biological Sciences, KAIST
*Corresponding author: moncher@kribb.re.kr
2
Escherichia coli is arguably the best studied microorganism and widely
used in science, medicine, and industry. Almost all laboratory strains of E.
coli are derivatives of wild isolates K-12 or B. Most of the E. coli genetic
and metabolic studies have been done with K-12 or its derivatives. Of the
K-12 strains, E. coli MG1655 has been fully sequenced and its metabolic
network model was developed using constraints-based reconstruction. B
strains show the superior cell growth and foreign protein expression as
compared with K-12 strains. BL21, a derivative of E. coli B, is the most
popular industrial host for overproduction of recombinant proteins on a
large scale. Divergence of genome sequences between E. coli B and K-12
is thought to be <2%. Thus, many phenotypic differences between E. coli
B and K-12 could be accessed by genome-scale metabolic model approach.
We carried out phenotype microarray (PM) experiments of E. coli B
(REL606) and K-12 (MG1655) for accessing phenotypic differences under
a variety of growth conditions. PM tests demonstrated dramatic phenotypic
differences between the two strains. The metabolic model of E. coli K-12
was adjusted to reflect the observed phenotypic characteristics.
Construction of a metabolic network model of E. coli B and in silico
prediction of the capability and characteristics of the strain will be
presented. [Supported by the 21C Frontier Microbial Genomics and
Applications Center Program, Ministry of Science and Technology, Korea]
Keywords: phenotype microarray, E. coli B, E. coli K-12,
metabolic pathway analysis
D016
The Antimicrobial Effects of Fomitopsis
pinicola on Oral Pthogenic Mcroorganisms
Direct Interaction of Enzyme IIAGlc with Adenylyl
Cyclase in Escherichia coli
Yoo Hyun HWANG*, Jae Kyoung LEE, Hee Kuk PARK, Sung
Woo CHOI, and Jeong Weon YOON
Young-Ha PARK *, Byeong R. LEE , Alan PETERKOFSKY ,
1
and Yeong-Jae SEOK
Department of Genetic Engineering and Biotechnology, College of Natural
Science, The University of Suwon
*Corresponding author: jwyoon@suwon.ac.kr
1Department of Biological Sciences and Institute of Microbiology, Seoul
2
3
National University, Department of Biology, Seowon University, Laboratory
of Cell Biology, National Heart, Lung and Blood Institute, USA
*Corresponding author: subzero1@snu.ac.kr
Fomitopsis pinicola has been traditionally used for medicine, the
hot water-extracts of Fomitopsis pinicola for reduce diabetes and
inflammation. Therefore, the aim of this study was to evaluate in
vitro the antimicrobial activity against several oral microorganisms.
The fruiting body of Fomitopsis pinicola were harvested in Nepal,
were dried and powdered in mixer, and extracted twice with
methanol(dried g/20㎖) at 24℃ overnight. The methanol extract
Fomitopsis pinicola(Fp-M) were evaporated and dissolved in
dimethyl sulfoxide by 100㎎/㎖. Antimicrobial activity was carried
out by the minimal inhibitory concentrations(MICs) and short-term
exposure studies with Streptococcus mutans (ATCC 25175),
Streptococcus sanguis (ATCC 10556), Actinomyces viscoccus
(ATCC 15987), Candida albicans (ATCC 10231). The Fp-M was
showing MICs ranging from 0.25(㎍/㎖) against S. sanguis and
0.5(㎍/㎖) against S. mutans respectively. The 5-minute exposure of
S. mutans, S. sanguis, A. viscoccus, and C. albicans to Fp-M (2㎎/
㎖) resulted in a substantial delay in growth. Fp-M exhibited potent
antimicrobial activity against all of the studied oral microorganisms.
It was suggested that F. pinicola may be a valuable antimicrobial
agent to prevent dental caries and periodontal disease.
Keywords: Fomitopsis pinicola, oral microorganism, dental caries,
periodontitis
194
D015
한국미생물학회연합
1
2
3
Protein components of phosphoenolpyruvate: sugar phosphotransferase
system (PTS) play multiple physiological roles besides sugar transport in
Escherichia coli. Enzyme IIAGlc (EIIAGlc), a component of glucose PTS,
also plays several physiological roles including phosphorylationdependent interaction with lactose permease, glycerol kinase, MalK, and
FrsA. Using His-tagging expression vector of the crr gene encoding
Glc
EIIA , we designed protein-fishing experiment to elucidate more
Glc
protein complexomes involving EIIA in vivo. Proteins bound to
Glc
His6-EIIA were separated by 1D-PAGE, and proteins specifically
Glc
bound to EIIA were identified by MALDI-TOF analysis after in-gel
digestion with trypsin. Among the identified proteins, we found adenylyl
cyclase (AC). Previously, IIA Glc of the E. coli PTS has been implicated
by genetic studies as an important regulator of AC activity and it has been
Glc
proposed that the phosphorylated form of IIA is a positive effector of
AC. To confirm the interaction between IIA Glc and AC, AC was
produced associated with a membrane anchor, the two transmembrane
segments of the serine chemoreceptor, Tsr. Membrane-associated
Tsr-AC binds dephospho-IIA Glc , but not phospho-IIA Glc , in the absence
of other soluble proteins; in the presence of PEP and other PTS proteins,
Glc
however, the bound IIA becomes phosphorylated.
Keywords: adenylyl cyclase, phophoenolpyruvate:glucose phosphotransferase
system
2005 International Meeting of the Federation of Korean Microbiological Societies
D017
D019
The Mode of Expression of Cell-Wall-Related
Genes under Cell Wall Stress Conditions in
Saccharomyces cerevisiae
1
2
2
Kook Hyun HYUN , Yeong Man YU , Yong Ju LEE , and Pil Jae
MAENG2
1
Department of Microbiology, School of Bioscience and Biotechnology,
Chungnam National University, 2Department of Microbiology, School of
Bioscience and Biotechnology, Chungnam National University
*Corresponding author: snipershut@nate.com
Cell wall is an important compartment that provides physicochemical protection and determines the shape of cells. In
Saccharomyces cerevisae, weakening of cell wall caused by
mutations in cell wall synthesis-related genes concerning normal cell
wall assembly triggers a compensatory mechanism to ensure cell wall
integrity. Usually, the compensatory mechanism includes increase in
chitin levels caused by compensatory expression of chitin synthetase
genes for restoring cell wall integrity. The aim of this study is to
develop a sensitive and convenient screening system based on the
compensatory activation of transcription of the cell wall synthesisrelated genes,that can be used to examine whether a variety of
chemicals cause cell wall damage or not. We constructed yeast strains
transformed with high copy number plasmids carrying the gene
encoding green fluorescent protein (GFP) under the control of the
promoter of chitin synthase(CHS1), 1,3-ß-D-glucan synthase
(FKS1), or fructose-6-phosphate amidotransferase (GFA1)gene.
By fluorescent assay in 96 well plates , together with quantitative
real-time PCR, we investigated the mode of expression of cell
wall-related genes under cell wall threatening conditions.
Keywords: Saccharomyces cerevisiae, cell-wall-related genes,
screening system, fluorescent assay
D018
Jin-Soo PARK, Seung-Young KIM, and Ki-Bong OH*
School of Agriculural Biotechnology, Seoul National University
*Corresponding author: eva100@nate.com
Aromatic polyketides are widely distributed in bacteria, fungi, and
plants, and many are clinically valuable agents or exhibit other
fascinating biological activities. In bacteria these compounds are
usually biosynthesized by type II polyketide synthases(PKSs), which
minimally consist of the ketoacyl synthase (KSα/KSβ ) heterodimer;
an acyl carrier protein (ACP); and a malonyl-CoA:ACP transperase
(MCAT). In order to detect Streptomyces with PKS gene directly and
exactly, we designed PCR primers based on highly conserved
sequence in KS domain of various type II PKS genes in Streptomyces
species. PCR screening of type II PKS was conducted in genomes of
soil and marine actinomyces. As a result, DNA fragments from 2
strains (KBO0018, KBO0046) were amplified to 700bp size as
wanted. The cultures of these two strains showed potent antimicrobial
activity against various microorganisms. Sequence analysis of the
700bp DNA fragments were identified as highly homologous
sequences of ketosynthase through NCBI Blast searching. KS
domain disruption was conducted to find exactly polyketide
compounds. Compared with a study of wild type's extract, a few
HPLC peaks of disrupted mutants disappeared. The purification and
structure identification of these compounds are now in progress.
Keywords: polyketide, polyketide synthase, gene disruption,
ketoacyl synthase
D020
Antibacterial and Antifungal Activities of the
Essential Oils from the Plant
1,
2
1
Jeong Ho LEE Hye Young YANG , Hyung Kwang KIM , Hong
3
2
Sub LEE , and Soon Kwang HONG *
1
Identification of Type II Polyketide Synthase
Genes from Actinomycetes through PCR and
Identification of Their Products
2
Korea National Aboretum, Department of Biological Science, Myongji
3
University, Ildong Pharmaceutical Co., LTD.
*Corresponding author: psychehy@naver.com
In the present study, we investigated the antibacterial and antifungal
effects of essential oils extracted from Cryptomeria japonica, Pinus
densiflora, Pinus koraiensis, Abies holophylla, and Chamaecyparis
obtusa. The antimicrobial activities of the essential olis against 8
fungi and 13 bacteria were evaluated and the MIC (Minimum
Inhibitory Concentration) values were compared by broth dilution
method. The essential oil from P. koraiensis have powerful
antifungal effects, whereas the essential oils from C. japonica and P.
densiflora show better antibacterial activities. These results indicate
that the essential oils from the plant, can effectively inhibit the
growth of gram-positive and gram-negative bacteria and fungi,
which will broden the applicable field of essential oils as a nontoxic
and environmentally familiar ingredient.
Keywords: essential oil, antibacterial activity, antifungal activity,
MIC
Cloning of the K-252a Biosynthetic Gene Cluster
from Nonomuraea longicatena JCM11136 and
Its Heterologous Expression in Streptomyces
albus G-153
Seung-Young KIM, Jin-Soo PARK, Jee-Yun LEE, and Ki-Bong
OH*
School of Agriculural Biotechnology, Seoul National University
*Corresponding author: bioprobe@paran.com
K-252a , an antitumor antibiotic produced by the bacterium
Nonomuraea longicatena JCM11136, is a prototype of a class of
complex natural products called indolocarbazoles. Several K-252a
analogues, which target protein kinase C, have already entered
clinical trials as anticancer drugs. Using as a probe an internal
fragment of VioB, a Nonomuraea longicatena gene encoding an
indolocarbazole CCA synthase, we isolated a DNA region that
directed the biosynthesis of K-252a when introduced into
Streptomyces albus. The entire K-252a biosynthetic and regulatory
gene cluster spanning 44kb was cloned and sequenced. The gene
cluster consists of 22 ORFs and the amino acid sequence revealed
InkC, V, H, U, S, Q, N, R, L, B, A, M, P, G, O, D, E, T, F, I, J, and
InkK. Heterologous expression of subsets of these genes resulted in
production of K-252a. The cloned genes should help to elucidate the
molecular basis for indolocarbazole biosynthesis and set the stage
for the generation of novel indolocarbazole analogues by genetic
engineering.
Keywords: Indolocarbazole, K-252a, heterologous expression,
CCA synthase
국제학술대회
195
October 13-14, 2005, Seoul, Korea
D021
D023
Cloning and Expression of the Isocitrate Lyase,
a Key Enzyme of Glyoxylate Cycle, of Rice
Blast Fungus Magnaporthe grisea for
Development of Novel Inhibitors
Cloning and Characterization of LipaseEncoding Gene from Marine Bacterium Photobacterium aplysiae GMD509
Sang-Yi PARK , Sang-Jin KIM , and Hyoung-Tae CHOI
3
Seung-Young KIM, Jin-Soo PARK, Jee-Yun LEE, and Ki-Bong
OH*
1
KORDI,
School of Agriculural Biotechnology, Seoul National University
*Corresponding author: bioprobe@paran.com
The glyoxylate cycle can conserve carbons and adequately supply
TCA cycle intermediates for biosynthesis when microorganisms
grow on C2 carbon sources as the sole carbon sources. Recently, it
has been reported that the genes of the glyoxylate cycle are highly
induced when Magnaporthe grisea, an important rice blast fungus,
infected to rice. Isocitrate lyase (ICL1) shows elevated expression
during development of infection structures and cuticle penetration,
and a targeted gene replacement showed that the gene is required for
full virulence by M. grisea. Therefore, ICL1 could be promising
target for the control of fungal infection and develop antifungal
agents. A DNA fragment encoding the ICL1 from M. grisea was
amplified by PCR and constructed a recombinant plasmid with ICL
gene (pGEX 4T-1). The fusion protein was expressed in Escherichia
coli host strain BLR (DE3), and purified with Glutathione affinity
chromatography. The molecular mass of the purified ICL1 was
approximately 60kDa as determined by SDS-PAGE, and the
enzyme activity was directly proportional to incubation time and
enzyme concentration. We also carried out an extensive screening of
ICL1 inhibitors from our chemical and natural products libraries.
The selected compounds should help to prevent the rice blast disease
Keywords: Magnaporthe grisea, glyoxylate cycle, isocitrate lyase,
gene expression
1
Department of Microbiology, Kanwon National University,
Department of Microbiology, Kangwon National University
*Corresponding author: tina_0724@hotmail.com
3
2
Photobacterium aplysiae, a lipolytic marine bacterium isolated from
egg of the sea hare. We cloned lipase-encoding gene from P.
aplysiae by triglyceride lipolytic activity. We have used a modified
shot gun cloning method. pBluescript SK DNA was recombined
with the bacterial DNA ligated to HindIII and these recombinant
plasmids were analysed in E. coli on tributyrin agar plate. We have
picked 1 clone from 760,000 colonies through this method, and this
5.0kb HindIII fragment contained the lipase activity. These are
several ORF such as dienelactone hydrolase, isochorismatase, and
putative DNA binding proteins. We now analyse which DNA
fragment really gives the lipase activity.
Keyword: lipase
D022
D024
Biological Activity of Antarctic Lichen Species
around Korean Antarctic Station (King Sejong
Station)
1
196
2
2
1
Screening of Human and Animal Pathogenic
Bacteria Which Produce the Quorum Sensing
Signals
1
2
2
Hae-Sook Jeon , Sung-Tae YEE , Soon-Ok OH , Kwang-Mi
2
3
1
4
LIM , Young Jin KOH , Jae-Seoun HUR *, Jee Hee KIM , and
4
Hosung CHUNG
Yun-Jung LEE , Jung-Ae KIM , Jung-Kee LEE *, Dae-Kyun
3
3
4
1
PARK , Kun-Soo KIM , Jeong-Weon HUH , and Soo-Ki KIM *
1
Department of Environmental Education, Sunchon National University,
2
Department of Biology, Sunchon National University, 3Department of Applied
4
Biology, Sunchon National University, Korean Polar Research Institute
*Corresponding author: jshur1@sunchon.ac.kr
2
Biological activity of thirteen Antarctic lichen species were examined to
screen a novel bioresource. The lichens were collected around Korean
Antarctic Station (King Sejong Station), King George Island on January
of 2005. Antibacterial activity of the lichen species were examined using
acetone extract of the lichen thalli. Usnea aff. igniaria, U. antarctica, U.
aurantiaco-atra, Lepraria straminea and Sphaeroglobus globosus
showed antibacterial activity against Escherichia coli, Salmonella
anterotidis, Streptococcus mutans, and Helicobacter pylori. Among
them, U. aurantiaco-atra exhibited the strongest antibacterial activity
against all the tested bacteria. Immuno-modulating activity of the lichens
were also determined. Acetone extracts of Placopsis contortuplicata and
Usnea aurantiaco-atra remarkably increased nitric oxide production in
the suspension of macrophage cell line (RAW264.7) in vitro after 48 hr
incubation. There were also apparent induction of IL-6 and TNF-α
synthesis, thus increase in the amounts of cytokines in the cell culture
supernatants. The Antarctic lichens seem to act as a potent immunomodulator causing augmentation of immune cell activity, and thus could
be used as a biological response modifier having possible therapeutic
effects against immunological disorders. Their antibacterial activity
against clinically important bacteria also implies that the lichen
substances can be used as a leading molecule to develop novel antibiotics.
Keywords: Antarctic lichen, antibacterial activity, immunomodulating activity, Usnea aurantiaco-atra
This study was conducted to screen the pathogenic bacteria to
produce quorum sensing signals in 82 strains of both human and
animal pathogenic bacteria. Signal molecules in quorum sensing
were detected from Klebsiella pneumoniae, Pseudomonas aeruginosa,
Listeria monocytogenes, Clostridium perfringens, Enterotoxigenic
E. coli, Pasteurella multocida, Mannheimia haemolytica, Haemopillus
parasuis, Haemopillus somnus, and Streptococcus suis. Using
Vibrio harvey BB886 reporter strain, the luminescence was also
measured to confirm AI (autoinducer) secretion related to quorum
sensing. We examined the effect of inorganic phosphate on the
secretion of quorum sensing signals. High phosphate condition
usually secreted more quorum sensing signals than low phosphate
one. Pasteurella multocida and Mannheimia haemolytica produced
AI when cell growth was reached at the exponential phase. Both
strains secreted several kinds of AI which were detected by C18
reversed-phase TLC analysis.
Keywords: Quorum Sensing, Pathogenic Bacteria, AI(Autoinducer),
Phosphate
한국미생물학회연합
1
Department of Animal Sciences and Environment, Konkuk University,
3
Laboratory of Microbial Genomics, KRIBB, Department of Life Science,
Sogang University, 4Microbiological Inspection Team, Gyeonggido Institute
of Health and Environment
*Corresponding author: lyj0311@hotmail.com
2005 International Meeting of the Federation of Korean Microbiological Societies
D025
D027
Small Interfering RNA Targeting the 2C
Conserved cis-Acting Replication Element of
Enteroviruses Has Generalized Antiviral Effects
1
1
2
Hui Sun LEE , Jeonghyun AHN , Han CHOE , Youngmee
JEE3, Eun-Seok JEON4, Chul Hyun JOO1, Yoo Kyum KIM1, and
1
Heuiran LEE *
1
Departments of Microbiology, University of Ulsan College of Medicine,
Departments of Physiology, University of Ulsan College of Medicine, 3Division
of Enteric and Hepatitis Viruses, Department of Virology, NIH, Korea Center for
Disease Control and Prevention, Ministry of Health and Welfare, 4Department
of Internal Medicine, Sungkyunkwan University School of Medicine
*Corresponding author: heuiran@amc.seoul.kr
2
Nonpolio enteroviruses are a major causative agent of numerous
human diseases and consist of a large number of serotypes with high
genetic instability. We have shown previously that small interfering
RNA (siRNA) targeting CVB3 specific RNA induces antiviral effects
against related enteroviruses, as well as against CVB3 itself. Here we
report a strategy for introducing siRNA as a universal anti-enteroviral
treatment. We developed siRNA design software called CAPSID to
identify highly conserved regions of viral genomes. This software
enabled us to design MET-2C, a siRNA that targets a multi-enteroviral
region located in the 2C cis-acting replication element (cre) of the viral
genome. Treatment of Hela cells with MET-2C significantly
downregulated viral replication and viral cytotoxicity, induced by a
number of reference strains as well as by various clinical isolates. In
conclusion, we showed that our in-house siRNA design program could
be used to design a siRNA targeting a conserved sequence in highly
variable virus genomes. Additionally, our results suggest that
MET-2C siRNA may be a universal and mutation-resistant antiviral
agent in the treatment of nonpolio enteroviruses.
Keywords: nonpolio enterovirus, small interfering RNA, 2C
replication element, CAPSID, anti-enteroviral effect
D026
Growth and Physiolosical Properties of Lactococcus
garvieae tIsolated from River Side Soil.
Su Hee YUN, Youn Jun CHOI, and Doo Hyun PARK*
Department of Biological Engineering, Seokyeong University
*Corresponding author: baakdoo@skuniv.ac.kr
A bacterium isolated from soil separated from riverside of Hangang
was identified by 16s rDNA sequence. The identity was 99 % with L.
garvieae in database of Gene Bank. The isolate was confirmed to be
a hetero fermentation bacterium which was assumed on the basis of
metabolites produced from MRS medium. L. garvieae can reduce
ferric ion to ferrous ion, fumarate to succinate and nitrate to nitrite
coupled to metabolic oxidation of glucose. 24 mM succinate, 12 mM
nitrite and 0.25 mM ferrous were reduced from 20 mM fumarate,
nitrate and ferric ion, respectively for 48 hr. The growth and ferric
ion reduction activity of L. garvieae was higher in anaerobic
condition than aerobic condition. From these results, we propose a
possibility that L. garvieae may have the FNR gene, by which
reduction of fumarate, nitrate and ferric ion may be controlled.
Keywords: Lactococcus garvieae, Fumarate nitrate reductase, ferric
ion reducing-bacterium
D028
Isolation and Identification of a
Hydrogen Using Denitrifying Bacteria
1,2
2
Novel
Haiyan JIN , Doo Hyun PARK , and Yong Keun PARK
1
1
Department of Molecular Cell Biology, School of Life Science and
2
Biotechnology, Korea University, Department of Biological Engineering,
Seokyeong University
*Corresponding author: ykpark@korea.ac.kr
The hydrogen-oxidizing Pseudomonas SKU has unique metabolic
property, which is different from other denitrifying Pseudomonas
sp. Generally, Psuedomonas can dissimilate sugars or organic acids
to carbon dioxide coupling with nitrate reduction to nitrogen under
anaerobic condition. In which sugar or organic acid functions as
both electron donor and carbon source. However, the strain SKU
requires different electron donors, hydrogen, sulfide or sulfur from
organic carbon, which is acetate. When the SKU was cultivated in the
medium with acetate and nitrate as an electron donor and acceptor
under anaerobic nitrogen atmosphere, the bacterium was not grown. The strain SKU is not autotroph but can not be heterotroph because it
can not assimilate carbon dioxide and it can not dissimilate acetate.
Consequently, the SKU is thought to produce free energy and
reducing power from metabolic oxidation of hydrogen coupled to
reduction of nitrate to nitrogen and assimilate acetate into building
blocks by using the free energy and reducing power. On the basis of
the metabolic properties of SKU, we propose that the SKU is a
semihetrotroph.
Keywords: denitrification, hydrogen using, carbon source, energy
source, electron donor, electron acceptor
Physiological Characterization of ButaneOxidizing Achromobacter xylosoxidans
Hye Sun KANG, Ah Young KANG, and Doo Hyun PARK*
Department of Biolosical Engineering, Seokyeong University
*Corresponding author: baakdoo@skuniv.ac.kr
Achromobater xylosoxidans was isolated from mud soil of seashore
of Kanghwa Island and identified by 16S rDNA sequence. The
isolate grew with butane, acetate, butanol and ethanol as a carbon
source under aerobic condition, but did not require organic nitrogen
such as yeast extract or peptone for normal growth. In test with API
50CHL sugar fermentation test kit, no positive reaction by A.
xylosoxidans was observed, which shows that A. xylosoxidans can
not dissimilate sugar or sugar alcohol. Butanol dehydrogenase was
detected by activity staining method of electrophoresis gel. The key
enzymes, malate dehydrogenase and isocitrate dehydrogenase
activity of crude enzyme was 0.352 and 0.30 mM/min.mg protein,
respectively. Theoretically, butane can be metabolically oxidized to
butanol by butane monooxigenase in bacterial cytoplasm, which has
been discovered in the petroleum-degrading bacteria. The butanol
may be degraded into Acetyl-CoA by the b-oxidation and then
dissimilate to carbon dioxide through the TCA cycle. We discovered
+
a method for electrochemical reduction of NAD to NADH without
enzyme catalysis or chemical reducing equivalent. This system may
be applied to oxidation of petroleum gas to petroleum alcohol. We
are composing and testing the electrochemical system for enzymatic
and cellular oxidation of butane to butanol. Keywords: Achromobater xylosoxidans, butane monooxigenase
국제학술대회
197
October 13-14, 2005, Seoul, Korea
D029
Characterization of Acetate-Oxidizing Denitrification
Bacterium
Sun Mi PARK and Ah Young KANG
Cloning and Characterization of Genes Encoding
β-Carotene Diketolases and Hydroxylases from
Marine Bacteria
Department of Biological Engineering, Seokyeong Univsersity
*Corresponding author: sunvictory@naver.com
Yeo-Jin SON *, Eun-Hwa CHOI , Dong-Heon LEE , Duck-Chul
OH2, Jung-Hoon SOHN1, and Eui-Sung CHOI1
A denitrification bacterium was isolated from soil and cultivated in
acetate-nitrate medium. The isolate was identified by 16S rDNA
sequence. This bacterium can grow in medium with acetate as an
electron donor (carbon source) and nitrate as an electron acceptor,
however, was not grown with other carbon source such as glucose,
lactate and pyruvate. We have concentrated on one thing that the
isolate absolutely requires ammonium for growth when cultivated in
acetate-nitrate medium. This shows that the isolate can not
assimilate nitrate to ammonium for biosynthesis of organic nitrogen
compounds such as amino acid and nucleic acid. Consequently, the
isolate can metabolize ammonium, nitrate and acetate as N-source,
electron acceptor and electron donor, respectively. The key
enzymes, malate dehydrogenase and isocitrate dehydrogenase
activity of crude enzyme was 5.8 and 1.84 mM/min.mg protein,
respectively. Theoretically, 3 NADH and 1FADH2 can be produced
from acetate metabolism in TCA cycle and 5 NADH has to be
required for reduction of two nitrate to one nitrogen. By using this
metabolic property, we tried to do stoichiometric balance between
metabolic oxidation of acetate and nitrate reduction to nitrogen.
Keywords: denitrification, acetate-oxidizing
D030
Characterization of Acetate-Oxidizing Denitrification
Bacterium
Sun Mi PARK, Ah Young KANG, and Doo Hyun PARK*
198
D031
1
1
1
2
2
Laboratory Microbial Functions, KRIBB, Department Life Science, Cheju
National University
*Corresponding author: toptop8484@lycos.co.kr
Novel genes coding for the β-carotene diketolase and hydroxylase
were cloned from several marine bacterial isolates. PCR was
performed using degenerate primers based on the recently reported
sequences employing cyanobacterial preference. Sequence analysis
showed that the new genes have 40-50% similarity with the recently
reported genes from Brevundimonas and Nostoc spp. The two genes
(β-carotene diketolase and hydroxylase) were expressed in E. coli
which contains carotenogenic genes (crt E, B, I, and Y), and,
therefore, can produce β-carotene. Expression of these genes was
performed either in a single gene expression mode or in combination
expression mode. The E. coli strain containing diketolase could
produce canthaxanthin and the strain containing both genes
(diketolase and hydroxylase) was found to produce astaxanthin from
β-carotene when the cell extract was analyzed with HPLC.
Keywords: canthaxanthin, astaxanthin, β-carotene diketolase and
hydroxylase, carotenogenic genes (crt E, B, I, and Y)
D032
The Thioredoxin System Controls Terminal
Differentiation through Redox-Modulation of
Vacuolar H+-ATPase in Dictyostelium discoideum
Department of Biological Engineering, Seokyeong University
*Corresponding author: baakdoo@skuniv.ac.kr
Sun-Young JEONG, Chang-Hoon CHOI, Ji-Sun KIM, SeongJun PARK, and Sa-Ouk KANG
A denitrification bacterium was isolated from soil and cultivated in
acetate-nitrate medium. The isolate was identified by 16S rDNA
sequence. This bacterium can grow in medium with acetate as an
electron donor (carbon source) and nitrate as an electron acceptor,
however, was not grown with other carbon source such as glucose,
lactate and pyruvate. We have concentrated on one thing that the
isolate absolutely requires ammonium for growth when cultivated in
acetate-nitrate medium. This shows that the isolate can not
assimilate nitrate to ammonium for biosynthesis of organic nitrogen
compounds such as amino acid and nucleic acid. Consequently, the
isolate can metabolize ammonium, nitrate and acetate as N-source,
electron acceptor and electron donor, respectively. The key
enzymes, malate dehydrogenase and isocitrate dehydrogenase
activity of crude enzyme was 5.8 and 1.84 mM/min.mg protein,
respectively. Theoretically, 3 NADH and 1FADH2 can be produced
from acetate metabolism in TCA cycle and 5 NADH has to be
required for reduction of two nitrate to one nitrogen. By using this
metabolic property, we tried to do stoichiometric balance between
metabolic oxidation of acetate and nitrate reduction to nitrogen.
Keywords: denitrification, acetate dissimilation, nitrate reductase
Laboratory of Biophysics, School of Biological Sciences, and Institute of
Microbiology, Seoul National University
*Corresponding author: jsy99@snu.ac.kr
한국미생물학회연합
The thioredoxin system, consisting of thioredoxin, thioredoxin
reductase and NADPH, has been well-known to be critical for the
redox regulation of protein function and signaling. We investigated
the roles of thioredoxin system by overexpression of thioredoxin
reductase (Trr) in Dictyostelium discoideum. The Trr-overexpressing
strain grew more slowly on bacterial lawn than the parental strain.
Under dark condition of development, Trr-overexpressing strain
showed a slugger phenotype, defined by prolonged migrating slug
stage. Ammonia is believed to be a negative regulator of switching
migrating slug into fruiting body, and its target is known to be acidic
compartments. The Trr1-overexpressing strain was hypersensitive to
ammonia like other slugger mutants, and also had difficulty in
vacuolar acidification. Moreover, the Dictyostelium thioredoxin
system was able to reduce the oxidized A subunit of vacuolar
+
H -ATPase which functions to acidify the intracellular
compartments. These results suggest that thioredoxin system of
Dictyostelium may control the choice between migrating slug and
+
culmination through regulating vacuolar H -ATPase.
Keywords: Thioredoxin, Thioredoxin reductase, Dictyostelium
2005 International Meeting of the Federation of Korean Microbiological Societies
D033
D035
The Homeobox-Containing Protein, Hbx3 Is
Implicated in Tip Formation and Prespore
Differentiation in Dictyostelium discoideum
Identification of Genes in V. vulnificus Inducing
the Expression of a V. fisheri lux Bio-indicator
Ji-Sun KIM, Chang-Hoon CHOI, Sun-Young JEONG, SeongJun PARK, and Sa-Ouk KANG
Department of Life Science, Sogang University
*Corresponding author: kskim@sogang.ac.kr
Laboratory of Biophysics, School of Biological Sciences, and Institute of
Microbiology, Seoul National University
*Corresponding author: ameth03@snu.ac.kr
Vibrio vulnificus produces a cyclic dipeptide signal molecule,
cylic-L-Phe-L-Pro (cFP), which induces the expression of the V.
fischeri lux quorum-sensing bio-indicator in pSB403, and induced
the expression of sets of indigenous genes in the pathogen. In the
effort to determine functions associated with biosynthesis of cFP in
the pathogen, we introduced genomic library clones of V. vulnificus
MO624/O into the Escherichia coli strain MT102, which cannot
produce cFP, harboring pSB403. Among approximately 10,000
clones screened, four clones active on the lux indicator were
selected. Two of the four clones contained cosmid clones sharing
with common genes - vv10183 and vv10184, which encode
asparagine synthase and transcription anti-terminator, respectively.
Both of the genes were required for the expression of the lux gene,
and each of two genes alone could not induce the reporter gene. We
assume that these two genes are associated with the expression of lux
and precise functions of these two genes are in study. [This
research is supported by the 21C Frontier Microbial Genomics and
Applications Center Program.]
Keywords: Vibrio vulnificus, cyclic dipeptide
Homeodomain-containing proteins are transcription factors, important
for development in a variety of organisms. In Dictyostelium, it was
reported that a homeobox gene, hbx1 may control cell-type
differentiation and proportioning between prestalk and prespore cell. We
cloned hbx3, one of many homeobox genes in Dictyostelium discoideum.
When Hbx3 was overexpressed in Dictyostelium, the mutant cells
formed the multi-tipped mounds and finally differentiated into fruiting
bodies with glassy sori. Northern blot analysis in Hbx3-overexpressing
strain showed that the expression of prespore markers, pspA, cotC and
spiA was not detected. Spore viability test also revealed that
Hbx3-overexpressing strain failed to produce spores. These results
suggest that Hbx3-overexpressing cells are defective in prespore
differentiation and sporulation. In order to find out the role of Hbx3 in tip
formation, we analyzed the differentially expressed proteins in
Hbx3-overexpressing cells. Proteomic analysis showed that about 20
spots were differentially expressed in Hbx3-overexpressing cells and
two spots of them were identified as abpB and rdiA. They have been
known to encode an actin bundling protein and a rho GTPase inhibitor,
respectively, and are involved in actin cytoskeleton. We are now
investigating the relationship between these proteins and multi-tip
formation. Taken together, these data suggest that Hbx3 may be
implicated in tip formation and prespore differentiation in Dictyostelium.
Keywords: Homeodomain protein, Dictyostelium, Differentiation
D034
Protein Analysis by Two-Dimensional Gel
Electrophoresis of Carbon Monoxide and
Methanol-Inducible Proteins in Mycobacterium
sp. strain JC1 DSM 3803
Jinho HEO, Hyuk PARK, Sae Woong PARK, and Young Min KIM*
Department of Biology, Yonsei University
*Corresponding author: young547@yonsei.ac.kr
Mycobacterium sp. strain JC1 DSM 3803 is a bacterium that is able to
grow on carbon monoxide (CO) or methanol as a sole source of carbon
and energy. In this study, the proteomics tools, such as two-dimensional
gel electrophoresis and MALDI-TOF, were used for analysis of CO- or
methanol-induced proteins in Mycobacterium sp. strain JC1. The
two-dimensional image of proteins in Mycobacterium sp. strain JC1
grown on glucose was used as a control for the spot detection and the
comparison of protein expression. Among the spots detected
reproducibly after silver staining, eight CO-inducible and five
methanol-inducible spots were identified by MALDI-TOF mass
spectrometry with peptide mass fingerprinting or N-terminal amino acid
sequencing. CutB and CutC, the subunits of CO dehydrogenase, FixA
and FixB, the subunits of electron transfer flavoprotein, glyceraldehyde3-phosphate dehydrogenase, and heat shock protein 65 were increased in
Mycobacterium sp. strain JC1 grown on CO. Interestingly,
methanol:N,N-dimethyl-4-nitrosoaniline oxidoreductase, the key
enzyme for methanol dissimilation, was also slightly increased in cells
grown on CO. Methanol:N,N-dimethyl-4-nitrosoaniline oxidoreductase,
dihydroxyacetone synthase, 3-hexulose-6-phosphate synthase, and
glyceraldehyde-3-phosphate dehydrogenase were increased in cells
grown on methanol.
Keywords: Mycobacterium sp. strain JC1, CO-induced proteins,
methanol-induced proteins
Jung Im SHIM, Dae-Kyun PARK, and Kun-Soo KIM*
D036
Quinone-Induced NADPH-dependent Quinone
Oxidoreductase from Streptomyces seoulensis
Dong-Won KIM and Sa-Ouk KANG
Laboratory of Biophysics, School of Biological Sciences, and Institute of
Microbiology, Seoul National University
*Corresponding author: efence@hanafos.com
Quinone-induced NADPH-dependent quinone oxidoreductase
(NQO) was purified from Streptomyces seoulensis. NQO was
monomeric and had no prosthetic group. NQO could produce
naphthosemiquinone radicals, indicating one-electron transfer.
NQO was not only induced by quinone but also expressed in
response to some thiol-modifying agents. An approximately 4kb
DNA fragment carrying the gene for NQO was cloned in S.
seoulensis. NQO-overexpressed strain exhibited retarded growth,
poor sporulation and resistance to menadione and diamide. S.
coelicolor nqo disruptant was sensitive to menadione and diamide.
But, it had no effect on normal growth and differentiation. Upstream
of S. seoulensis nqo, putative transcription regulator was found.
This gene product homologue of S. coelicolor has a binding affinity
at nqo promoter region. And overexpressed strain of this gene
showed retarded growth, poor sporulation in S. seoulensis and early
actinorhodin production in S. coelicolor. Proteomes of wild type
and mutants were analyzed by two-dimensional gel electrophoresis
Keywords: Streptomyces, quinone
국제학술대회
199
October 13-14, 2005, Seoul, Korea
D037
Functional Analysis of KAR4 Transcription
Factor During Mating and Filamentation Growth
Hyejin KIM and Jinmi KIM*
Department of Microbiology, Chungnam National University
*Corresponding author: hjkim0506@hotmail.com
The KAR4 gene was identified in a screen for new nuclear
fusion-defective mutants. KAR4p is transcription factor in
Saccharomyces serevisiae, that is reguired for the expression of
KAR3, a kinesin gene. In previous studies, KEM1 has been reported
to regulate KAR4 expression and other karyogamy-specific geens in
post-transcription level. KAR4 regulator KEM1 plays a role in nuclear
fusion and filamentous growth of Saccharomyces serevisiae. To
investigate whether or not KAR4 is also involved in filamentous
growth, we transformed KAR4 plasmid to kem1 mutants strain.
KAR4 suppressed the invasive growth defective phenotype in kem1
mutants strain. We also found KAR4 suppressed the filamentous
growth defective phenotype in kem1/kem1 mutants strain. In this
study, KAR4 and KAR3 expression levels during filamentous
growth were also studied.
Keywords: KAR4, KEM1, filamentous growth
D038
Production and Characterization of Laccase
from Formitella fraxinea
Kyunmg Mi PARK, Seok Gun KANG, and Sang-Shin PARK*
Department of Biotechnology, Dongguk University
*Corresponding author: sspark@dongguk.ac.kr
Laccase production from mushroom, Fomitella fraxinea was
studied using various culture media. The highest activity of the
enzyme was obtained in Coriolus versicolor medium(CVM, 2%
dextrose, 0.4% peptone, 0.6% yeast extract 0.6%, 0.046% KH2PO4 ,
0.1% K2HPO4 , and 0.05% MgSO4·7H2O). To optimize the culture
condition on laccase production, the influence of various carbon and
nitrogen sources was investigated in CVM. Native polyacrylamide
gel electrophoresis(PAGE) followed by laccase activity staining
using 2,6-dimethoxyphenol as the substrate was performed to
analyze the laccase under culture conditions studied. Zymogram
analysis of the culture supernatant showed a laccase band with molecular mass of 60 kDa. The enzyme production from was
reached to highest level after the cultivation for 10 days at 25℃ and
pH 9.0. The optimum pH and temperature for the enzyme activity
were 25℃ and pH 10.0.
Keywords: Laccase production, Formitella fraxinea
200
한국미생물학회연합
D039
Isolation and Characterization of N-acetylmuramoyl
-L-alanine amidase (amiB) Gene from Vibrio
anguillarum : Mutation in amiB Gene Affects
Physiological Characteristics Sun Hee AHN, Dong Gyun KIM, Ryoung Hwa KIM, Seung Ha
JEONG, Eun Mi PARK, Hee Jeong CHOI, and In Soo KONG*
Pukyong National University
*Corresponding author: iskong@pknu.ac.kr
A gene encoding the N-acetylmuramoyl-L-alanine amidase, and the
corresponding protein, named amiB was identified from Vibrio
anguillarum. The entire open reading frame (ORF) of amiB gene was
composed of 1722 nucleotides and 573 amino acids. The deduced amino
acid sequence of AmiB shows a modular structure with two main
domains: an N-terminal region exhibiting AmiC domain and a C-terminal
portion contains three highly conserved, continuously repeated LysM
domains. These domain structures are common to autolysins of Vibrio sp.
However, it has been unknown a biochemical characteristic of autolysin
in Vibrio sp. The N-acetylmuramoly-L-alanine amidase is an enzyme
which catalyze the turnover or degradation of peptidoglycan in bacteria.
It has been known that autolysin may contribute to bacterial pathogenesis
by generating inflammatory cell-wall degradation products and by
releasing virulence factors. So, we constructed an amiB mutant, AV3, to
investigate the biochemical function of amiB in V. anguillarum. The
mutant AV3 showed much slower growth rate than wild-type strain. Also,
we observed that the mutant AV3 was formed trimeric and tetrameric
unseparated cell by transmission electron microscopy (TEM). It indicates
that this enzyme is involved in the separation of daughter cells after cell
division.
Keywords: Vibrio anguillarum , N-acetylmuramoyl-L-alanine
amidase
D040
A Cyclic Dipeptide Molecule Produced by
Agrobacterium tumefaciens Induces the Ti
Plasmid Conjugal Transfer
Dae-Kyun PARK, Ko Eun LEE, and Kun-Soo KIM*
Department of Life Science, Sogang University
*Corresponding author: kskim@sogang.ac.kr
Conjugal transfer of Ti plasmids in Agrobacterium tumefaciens is
controlled by a hierarchical regulatory system to sense two
environmental signals. One signal, a subset of opines released from
crown gall tumors in plants induced by the pathogen, serves to induce
production of the second signal, acyl-homoserine lactone, produced by
the bacterium itself. As a result, AHL turns on a quorum-sensing
initiating the expression of the tra operon, leading to the conjugal
transfer of Ti plasmids. Recently, we found that the Agrobacterium
tumefaciens C58 strain produces a cyclic dipeptide, which has been also
identified in Pseudomonas, Vibro, and many other gram-negative
bacteria. We found that the cyclic dipeptide enhanced the conjugal
transfer of Ti plasmid. The compound induced the expression of tra
genes in a concentration-dependent manner. The effect of the cyclic
dipeptide on the expression of traG was synergistic to the effect of 25
nM synthetic agrobacterial autoinducer, and AAI at 250 nM completely
masked the effect of the cyclic dipeptide. These indicated that the cyclic
dipeptide acts as a signal for the expression of tra genes thereby
enhanced the conjugal transfer, and suggested that the molecule
cross-talks with AAI for the induction of tra genes. Two dimensional
analysis of the protein expression showed that cFP modulates numerous
genes in addition to tra in A. tumefaciens C58, suggesting that this signal
molecule plays various roles in the plant pathogen.
Keywords: Agrobacterium, Cyclic dipeptide, conjugal transfer
2005 International Meeting of the Federation of Korean Microbiological Societies
D041
D043
Molecular Characterization of a New Intracellular
Serine Protease of Bacillus megaterium 1
1
1
Type A 1-Deoxy-D-Xylulose 5-Phosphate Synthase
(DXS) of Synechocystis sp. PCC6803
1,2
1
2
Yu Jin JEONG , Mi-Jeong YANG , Eun-Ju KWON , Han Dae
YUN2, and Hoon KIM1*
Wonkeuk KIM *, Junghoon SOHN , Hyongjoo LEE , and
Euisung CHOI1
1
Department of Agricultural Chemistry, Sunchon National University,
Division of Applied Life Science, Gyeongsang National University
*Corresponding author: hoon@sunchon.ac.kr
1
A new Bacillus megaterium gene, ispK, coding for an intracellular
serine protease was cloned into Escherichia coli DH5a by complete
digestion of B. megaterium chromosome with EcoRI. The
transformants were primarily grown on LBS agar plates (LB medium
containing 0.7% skim milk) for about two days. The transformants
were then screened by tooth-picking and streaking to be about 1 cm
length for all about 1,200 transformants. The complete nucleotide
sequence of insert DNA in a positive clone was determined. The
protease gene consisted of an ORF of 999 nucleotides encoding a
protease (IspK) of 332 amino acid residues (36 kDa). The deduced
amino acid sequence showed 49 - 55% similarity with other microbial
intracellular serine proteases. The molecular mass of the enzyme was
estimated to be 36 kDa on a denaturating gel. The enzyme was
activated with Ca2+. The temperature and pH optima of the enzyme
were 65℃ and pH 7.0 in the presence of 5 mM Ca2+, respectively. The
enzyme was inhibited by phenylmethansulfonyl fluoride (PMSF).
The protease retained its activity over 60 min at 50oC. Addition of
CaCl2 to the buffer solution made the purification of the enzyme
convenient due to the increase in enzyme stability and activity.
Keywords: Bacillus megaterium, intracellular serine protease,
IspK, CaCl2, stability
Isoprenoids including carotenoids and quinones form a large,
ubiquitous class of natural products consisting of over 30,000
individual members. They are synthesized by each or both of MEV
pathway (mainly in eucaryotes and archaebactera) and DXP pathway
(mainly in procaryotes). In DXP pathway, first step is catalyzed by
1-deoxy-D-xylulose 5-phosphate synthase(DXS) with pyruvate and
glyceraldehyde 3-phosphate. Interestingly, Rhodobacter capsulatus, a
purple nonsulfur bacterium, has two dxs genes. One, dxsA(ORF 2816)
is located in the photosynthesis cluster, and the other, dxsB(ORF 2895),
is located elsewhere in the chromosome. Synechocystis sp. PCC 6803
has one dxs(sll 1945) whose type was assumed to be A type by a strong
similarity to both transketolase and the E1 component of pyruvate
dehydrogenase involved in pyruvate decarboxylation. Moreover, R.
capsulatus and Synechocystis sp. could grow on the medium containing
fosmidomycin which inhibits 1-deoxy-D-xylulose 5-phosphate
reductase (DXR), the second step of DXP pathway. The dxsA of
Synechocystis sp. was cloned and expressed in E. coli DH5α. E. coli
harboring dxsA gene of Synechocystis sp. grew on medium containing
5 times higher concentration of fosmidomycin. These results suggested
that dxsA of Synechocystis sp. may also be involved in pentosephosphate-pathway as well as DXP pathway.
Keywords: Synechocystis sp., 1-deoxy-D-xylulose 5-phosphate
synthase, DXS
2
D042
2
KRIBB, Department of Food Science and Technology, College of Agriculture
and Life Science, Seoul National University
*Corresponding author: wonkkim@kribb.re.kr
D044
Isolation of a Thermally Stable 24 kDa Endo-1,4beta-xylanase Gene from an Isolate Lactobacillus sp.
BM1, Its Overexpression in Bacillus subtilis WB700
and Biochemical Characterization of the Enzyme
1
1
2
Jongmi PARK , Jongkook RHO , Munhwan CHOI , and
1,2,3
Sungchul YOON
1
Biomaterials Science Laboratory, Division of Applied Life Sciences (BK21),
2
Graduate School, Gyeongsang National University, Biomaterials Science
Laboratory, Environmental Biotechnology National Core Research Center,
3
Gyeongsang National University, Biomaterials Science Laboratory, Division
of Life Science, College of Natural Sciences, Gyeongsang National University
*Corresponding author: kcjha@nate.com
A thermo-tolerant extracellular xylanase was produced from Lactobacillus
sp. BM-1 strain that isolated from a sewage treatment plant in Chinju, Korea.
The optimum growth temperature of the strain was 37℃ and its optimum
growth pH was 7.0. The xylanase gene was identified from a genomic 1.2
kbp BamHⅠrestriction fragment. Nucleotide sequence analysis of this
fragment revealed an open reading frame (ORF) of 684 base pairs encoding
a polypeptide of 228 amino acids. This ORF encoding endo-1,
4-beta-xylanase exhibited a high homology (57% amino acid identity) with
the corresponding proteins of Bacillus subtilis (P18429) and Bacillus
circulans (P09850) endo-1,4-beta-xylanase. A strong promoter, BJ27, was
applied to overexpress the Bacillus sp. BM-1 xylanase gene in Bacillus
subtilis. The shuttle (E. coli-Bacillus subtilis) expression vector, pJH27-xyl,
was designed to contain the open reading frame of the xylanase. The
recombinant plasmid DNA pJH27-xyl was transformed into Bacillus
subtilis WB700. The expressed xylanase was partially purified. Its optimum
temperature for activity was 70℃ and optimum pH was between pH 6.0 and
pH 7.0. The enzyme was fairly stable even at pH 4.0 and 10.0. The activity
of the partially purified enzyme was largely inhibited by acetonitrile and
propanol, while phenylmethylsulfonyl fluoride (PMSF), dithiothreitol
(DTT), toluene and methanol didn’t affect its activity, where the
concentration of the compounds for stability study was equally 30(wt/v)%. Keywords: xylanase, pJH27, Lactobacillus sp.
Influence of Environmental Factors on Growth
and Metabolism of Yeast Cells Isolated from
Kimchi
Youn Jung CHOI, Byung Kwan NA, and Doo Hyun PARK*
Department of Biological Engineering, Seokyeong University
*Corresponding author: baakdoo@skuniv.ac.kr
Two yeast strains were isolated from kimchi prepared for the winter,
o
which was stored for 9 months in refrigerator at 4~5 C. pH of the
kimchi was about 3.5. On the basis of kimchi pH, pH of medium
used for isolation of yeast cells was adjusted to about 3 with glacial
acetic acid. The growth and ethanol productivity of yeast cell was
tested in various pH from 3 to 8 and various concentration of NaCl
from 0 to 9%. Redox potential difference of kimchi according to the
ripening condition or storage period may also be a factor for growth
and metabolism of yeast cells. The growth and ethanol productivity
of yeast were inhibited by NaCl above 5%, and the growth of yeast
was relatively higher at pH 6~7 but ethanol productivity was
relatively higher at pH 5~6. The growth of yeast cells was not
influenced by the redox potential difference but ethanol productivity
was increased at lower potential. From these results, we proposed
that yeast cells growing in kimchi may adapt to the lower pH, NaCl
and low redox potential and its metabolism may be influenced by the
specific environmental factors of kimchi.
Keywords: electrochemical control, bioreactor
국제학술대회
201
October 13-14, 2005, Seoul, Korea
D045
D047
Characterization of Xylanase
Bacillus sp. KD1014 1
Activity
1
of
1
Yu-Jeong KIM , Sung Sun YOON , Sung Hoon KIM , Nguyen
Dinh PHUONG1, Kyung-Dong LEE1, Han Dae YUN2, and Hoon
1*
KIM
1
Department of Agricultural Chemistry, Sunchon National University,
Division of Applied Life Science, Gyeongsang National University
*Corresponding author: hoon@sunchon.ac.kr
2
Bacillus sp. KD1014 showed xylanase activity in LBX medium (LB
medium containing 0.3% xylan). The growth of the strain reached its
maximum after 15 to 18 h at 37℃ and the production of xylanase
reached its maximum after 12 h of growth. The optimum
temperature of the supernatant for the activity was 55℃ and the
optimum pH was pH 5.5 to 6.5. The activity was decreased and
retained 70% and 16% of its original activity at 50℃ and 60℃,
respectively, for 10 min. Activity staining of the supernatant
separated on an SDS-PAGE gel showed a major and a minor band
with molecular masses of 31 and 23 kDa. Thus Bacillus sp. KD1014
produced at least 2 kinds and low molecular weights of xylanase.
D046
Min Woo HYUN, Han Byul JANG, Ji Hwan YUN, and Seong
Hwan KIM*
Department of Microbiology, Dankook University
*Corresponding author: piceae@naver.com
Ophiostoma piceae is one of most dominant sapstaining species. In
previous work we cloned a celluase-like gene to understand nutrient
physiology of the sapstaining fungus. In this study we report cloning
and sequencing of another cellulase gene-like sequence from O.
piceae. For the cloning, degenerate primers were designed based on
information of few known fungal cellobiohydrolase II sequences and
used for PCR amplification of partial sequence of cellobiohydrolase
II gene homolog from the fungal genomic DNA. Through RACE
PCR approach we cloned the full-length sequence of the
cellobiohydrolase II gene homolog. The determined size of the
cloned gene was about 1.2kb, found to be homologous with other
fungal cellulases. Analysis of the deduced protein sequence
suggests that the cloned gene could be cellobiohydrolase II, a family
6 of glycosyl hydrolases that hydrolyse O-glycosyl compounds and
contain N-terminal CBD (cellulose binding domain) and
C-terminal enzyme active site. The putative O. piceae CHB II has
molecular mass of about 40kD. Comparison of the cloned sequence
with the previously cloned cellulase gene-like sequence is presented.
Keywords: Ophiostoma piceae, cellobiohydrolase II gene
D048
Specific Inhibition of Medium-Chain-Length
Polyhydroxyalkanoic Acid (MCL-PHA) Synthesis
by Salicylic Acid in Pseudomonas aeruginosa
BM114 Accumulating a Blend of MCL-PHA and
Short-Chain-Length-PHA
1
2
1
Jihun SHIM *, Munhwan CHOI , Jongkook ROH , Kambizakbari
1
1,2,3
NOGHABI , and Sungchul YOON
1
Biomaterials Science Laboratory, Division of Applied Life Sciences (BK21),
2
Graduate Gyeongsang National University, Biomaterials Science
Laboratory, Environmental Biotechnology National Core Research Center,
3
Gyeongsang National University, Division of Life Science, College of
National Science, Gyeongsang National University
*Corresponding author: simjihun1013@yahoo.co.kr
A bacterium, Pseudomonas aeruginosa BM114, capable of accumulating a blend of MCLand SCL-PHA was isolated. Salicylic acid (SA), without being metabolized, was found to
specifically inhibit only the synthesis of MCL-PHA without affecting cell growth. Acetone
fractionation and characterization of the polymer produced from decanoic acid revealed that
the PHA was a blend of two polymers, P(3HB) and P(3HC-co-3HO-co-3HD). The addition
of 20 mM SA selectively inhibited the synthesis of P(3HC-co-3HO-co-3HD) in
decanoate-grown cells by 83% of the control content in a one-step cultivation. In a
second-step cultivation of nutrient-rich-medium grown cells on decanoate, SA almost
completely blocked the synthesis of MCL-PHA. Growth of P. aeruginosa BM114 on the
medium-chain fatty acids with odd number of carbons produced PHA also containing
3HB-unit as well as 3-hydroxyacids with odd number of carbons, 3HV and higher acids.
Organic solvent fractionation of the polymer produced from undecanoic acid revealed that
the PHA was a mixture of 47.4% P(3HB-co-3HV) and 52.6% P(3HH-co-3-HN-co-3-HU).
Similar to the case of even carboxylic acids, SA inhibited the accumulation of only
MCL-PHA, not SCL-PHA. 2-Bromooctanoic acid (2-BrOA), known as a MCL-PHA
synthesis inhibitor, also showed a similar carbon-source dependent inhibition
characteristics but more sensitively and powerfully than SA. Thus, along with 2-BrOA, SA
could be used to suppress the formation of MCL-PHA in Pseudomonas spp.
Keywords: Pseudomonas aeruginosa, Salicylic acid, MCL-PHA, SCL-PHA
202
Molecular Cloning of Another Cellulase Genelike Sequence from a Sapstaining Fungus
Ophiostoma piceae
한국미생물학회연합
Cloning of Malate Synthase Gene from a
Ophiostoma Fungus
Min Woo HYUN, Sang Do CHA, Yeo Hong YUN, Seung Yeol
SON, and Seong Hwan KIM*
Department of Microbiology, Dankook University
*Corresponding author: piceae@naver.com
Wood discoloration caused by Ophiostoma fungi results in
economic loss in forest product industry. Melanin in Ophiostoma
fungi is known to be responsible for discoloring damage in wood. To
understand the role of carbon in melanin formation in sapstaining
fungi, we have been working on the enzymes used for carbon
metabolism. In this study we report cloning and sequencing of the
gene encoding malate synthase in Ophiostoma piceae, a well known
sapstaining fungus. Based on information of a partial cDNA
sequence of one of O. piceae EST clones that generated from the
fungal cells grown on a cellophane-layered starch-supplemented
medium, the full length sequence of the EST clone was obtained
from the fungal genomic DNA through Annealing Control Primer
(ACP) PCR approach, and its nucleotide sequence was analyzed.
The cloned gene is about 1.6kb in size and has no intron. The
molecular weight of the deduced amino acids sequence is estimated
as about 53KD. Protein motif analysis suggests that potential
microbody targeting signal, proton acceptor site, and proton donor
active sites are present in the deduced malate synthase protein of O.
piceae.
Keywords: Ophiostoma fungi, malate synthase gene
2005 International Meeting of the Federation of Korean Microbiological Societies
D049
D051
Modulation of Aromatic Monomer-Ratio in
Polyhydroxyalkanoic Acid by Salicylic Acid in
Pseudomonas fluorescens BM07 Grown with
Mixtures of Fructose and 11-Phenoxyundecanoic
Acid
B23 Increases Hepatitis B Virus Polymerase
Activity Mihee LEE1*, Jihoon SHIM1, Munhwan CHOI2, Jongkook RHO1,
3,4,5
and Sungchul YOON
School of Biological Sciences, Seoul National University
*Corresponding author: leeseungkeun@dreamwiz.com
1
Biomaterials Science Laboratory, Division of Applied Life Sciences (BK21), Graduate
School, College of Natural Sciences, Gyeongsang National University, 2Biomaterials
Science Laboratory, Environmental
Biotechnology National Core Research Center,
3
Gyeongsang National University, Biomaterials Science Laboratory, Division of Applied
Life Sciences (BK21), Graduate School, Gyeongsang National University, 4Biomaterials
Science Laboratory, Environmental
Biotechnology National Core Research Center,
5
Gyeongsang National University, Biomaterials Science Laboratory, Division of Life
Science, College of Natural Sciences, Gyeongsang National University
*Corresponding author: algmldlqj@nate.com
Salicylic acid (SA) was found to inhibit the synthesis of polyhydroxyalkanoic acid
(PHA) in Pseudomonas fluorescens BM07 as well as to modulate the distribution of
monomer-units comprising the PHA in a concentration dependent manner. For cells
grown with 50 mM fructose plus 5 mM 11-phenoxyundecanoic acid (11-POU) in
which aliphatic PHA synthesis from the fructose was maximally inhibited by
11-POU even in the absence of SA (total amount of the incorporated aliphatic
monomers was only ~10 mol%), the addition of 1.5 mM SA significantly shifted the
major constituting monomer-units to the longer ones: 3-hydroxy-5-phenoxyvalerate,
from 57 at 0 mM SA to 27 mol%; 3-hydroxy-7-phenoxyheptanoate, from 26 to 42
mol%; 3-hydroxy-9- phenoxynonanoate, from 3 to 19 mol%. For cells grown with
50 mM fructose and 3 mM 11-POU where the monomer supplying through PhaG
enzyme catalyzing the transformation of 3-hydroxyacyl-ACP to 3-hydroxyacyl-CoA
by fructose metabolism was partially open, 1.0 mM SA decreased the level of
aliphatic monomers from 67 at 0 mM SA to 33 mol% and increased the level of
aromatic monomers from 33 to 67 mol%. Thus the specific modulation of distribution
of aromatic monomers by SA was possible only under the cometabolism of 11-POU
for PHA accumulation and fructose for cell growth. Thus, to some extents, it seems
possible to change the monomer-unit ratio of the aromatic PHA produced in P.
fluorescens BM07 using the inhibitory effect of SA.
Keywords: Pseudomonas fluorescens, polyhydroxyalkanoic acid,
salicylic acid, 11-phenoxyundecanoic acid
Seungkeun LEE, Sanghun LEE, Hyunsoo KIM, and Guhung
JUNG*
Hepatitis B virus (HBV) core protein plays an important role in viral
nucleic acid synthesis and encapsidation. Previous reports showed
that HBV polymerase is activated in core capsid. In this study, we
analyzed the HBV core binding proteins by using affinity
chromatography. From this analysis, it was found that 38kDa protein
bound to HBV core protein. Through the MALDI-TOF and the
immunoblot analyses, it was revealed that this HBV core binding
protein was human nucleolar phosphoprotein B23 (also known as
nucleophosmin). Biomolecular interaction analysis (BIAcore)
showed that core proteins bound to B23.1, the c-terminal longer
isoform of B23, but not to B23.2. We analyzed interaction between
core capsids and B23.1 by a sucrose density gradient. Our
experiments suggested that B23.1 would affect HBV polymerase
activity in core capsid. To analyze HBV polymerase activity, we
measured DNA Dependent DNA Polymerase (DDDP) and RNA
Dependant DNA Polymerase (RDDP) activities of a purified HBV
polymerase RT domain. The activity of HBV polymerase with
B23.1 was increased by 60%. As a result, we suggested that B23.1
encapsidated into core capsid increase HBV polymerase activity.
Keywords: Hepatitis B virus, HBV core protein, HBV polymerase,
B23
D050
D052
Increased Levels of Ste12, Gpa2, and Cln1proteins
during Filamentous Growth in Saccharomyces
cerevisiae
2-Bromooctanoic Acid Inhibits the Enzyme
3-Hydroxyacyl-ACP:CoA Transferase Channeling
the PHA Monomer Precursors in Pseudomonas spp.
Young-Un PARK* and Jinmi KIM*
Jongkook RHO *, Munhwan CHOI , and Sungchul YOON
Department of Microbiology, Chungnam National University
*Corresponding author: parkyoungun2@naver.com
1
The dimorphic transition from yeast to hyphal form in response to
various external stresses are very much conserved between
Saccharomyces cerevisiae and the pathogenic yeast Candida
albicans. At least conserved two signaling (MAPK, PKA) pathway
and their associated transcription factors regulate yeast filamentous
growth. But, only few genes were regulated at transcriptional level
in yeast filamentous growth. In this study, we were interested in
finding specific mRNAs preferentially regulated at the translation
during the yeast-to-filamentation transition. Based on the fact that
the distribution of transcripts in polysomes reflects the rate of
synthesis of their coresponding proteins, polyribosome fractionation
and RT-PCR analysis were applied to reveal filamentationspecifically translated genes. Here, we show three genes Ste12, Gpa2,
and Cln1 whose transcription are constitutive but protein levels
increased during filamentation. We investigated relationship between
three S. cerevisiae genes, GPA2, STE12, CLN1 whose mRNAs are
activated for translation during the dimorphic transition and singnal
transduction pathway components or mRNA modulating factors.
Keywords: yeast filamentous growth, Ste12, Gpa2, Cln1, polysome
fractionation
1
2
1,2,3
Biomaterials Science Laboratory, Division of Applied Life Sciences(BK21),
2
Graduate School, Gyeongsang National University, Biomaterials Science
Laboratory, Environmental Biotechnology National Core Research Center,
Gyeongsang National University, 3Division of Life Science, College of Natural
Sciences, Gyeongsang National University
*Corresponding author: kirin314@hotmail.com
We identified the phaG (3-hydroxyacyl-ACP:CoA transferase) gene in
Pseudomonas fluorescens BM07. To investigate the role of phaG, we made
phaG mutants from P. fluorescens BM07 and P. putida BM01. The phaG
mutants showed decreased PHA accumulation when grown on fructose
without cell growth inhibition. The inhibition was, however, not observed
in octanoate-grown cells. This is similar to the in vivo inhibition in P.
fluorescens BM07, reported earlier. We compared this result with that for
the wild type strains. P. fluorescens BM07 and P. putida BM01 grown on
fructose accumulated PHA by 33% and 15% of the dry cell weight,
respectively. The addition of 2 mM 2-BrOA to the media decreased the
level of PHA accumulation down to 3% and 2%, respectively, similar to the
phaG mutants. Furthermore, heterologous expression of BM07 phaG in P.
oleovorans, which is originally unable to produce PHA from glucose,
resulted in PHA synthesis (11%) from glucose. The addition of 2 mM
2-BrOA to the pBBR1-07G harboring P. oleovorans growth medium
clearly revealed the inhibition of PHA synthesis by 2-BrOA from glucose
by showing a decrease of PHA accumulation down to about 2.5% while
octanoate-grown cells were not affected. Thus, all data showed that an
inhibiting species derived from 2-BrOA specifically targets the phaG
metabolically linking between fatty acid synthesis and PHA synthesis.
Keywords: P. fluorescens BM07, 2-Bromooctanoic Acid, PHA
국제학술대회
203
October 13-14, 2005, Seoul, Korea
D053
D055
Cloning and Mutant Construction of Escherichia
coli Heat-labile Enterotoxin A and B Subunits
Seung-Youn CHAI, Joon-Hyeong LEE, Yun-Jung LEE, and
Soo-Ki KIM*
Department of Animal Sciences and Environment, Konkuk University
*Corresponding author: chinoakastu@paran.com
Enterotoxigenic Escherichia coli strains causes diarrhea by
producing heat-labile enterotoxin (LT) in human beings and
agricultural animals. Escherichia coli heat-labile enterotoxin has
immunogenic and adjuvant properties when administered mucosally
to animal. Enterotoxin is composed of two functionally distinct
domains which are the enzymatically active A subunit with
ADP-ribosylating activity and the pentameric B subunit. We cloned
the LT-A and LT-B genes by PCR in enterotoxigenic E. coli KCCM
40404 and the strain isolated from Korean cow farm. Both LT-A and
LT-B genes were subcloned into pET22b(+) and pBluescript II (+)
to express their products. For the development of vaccine adjuvant,
the progress of mutant construction will be also discussed. Keywords: mucosal adjuvant, mucosal immunity, Enterotoxin
D054
1
2
1,2,3
Minjeong PARK *, Munhwan CHOI , and Sungchul YOON
1
Biomaterials Science Laboratory, Division of Applied Life Sciences (BK21),
Graduate School, College of Natural Sciences, Gyeongsang National University,
2
Biomaterials Science Laboratory, Environmental Biotechnology National Core
Research Center, College of Natural Sciences, Gyeongsang National University,
3
Biomaterials Science Laboratory, Division of Life Science, College of Natural
Sciences, College of Natural Sciences, Gyeongsang National University
*Corresponding author: alswjd0122@nate.com
Siderophore is an iron chelating compound secreted by microorganisms.
Pseudomonas fluorescence BM07 secreted a yellow-green, water- soluble
fluorescent siderophore, when grown on dicarboxylic acid such as succinic
acid, glutarate and malonic acid (40 mM ~ 70 mM) under 1 mg iron per liter
of which the amount is inevitably present originated from the chemicals in
media preparation. The pigment was purified using XAD-4 chromatography,
followed by CM Sephadex chromatography. The purified pigment was
analyzed by UV/Vis spectroscopy, amino acid analysis, HPLC, LC-MS and
NMR spectroscopy. The UV absorption spectrum of the pigment had
maxima at 388.0 nm and 401.5 nm. An increasing addition of Fe(Ⅲ) shifted
the absorption maximum from 388.0 to 401.5 nm indicating the 401.5 nm
absorption associated with iron binding. The spectral feature of Fe(Ⅲ)–
chelated pigment depended on pH, therefore it was considered to atypical
pyoverdine that has two β-OH-Asp and chromophore as iron binding ligand.
The pigment can also chelate Fe(Ⅱ) and Cu(Ⅱ). The amino acid
composition of the pigment was Glycine, Alanine, Proline and Lysine
(2.8:1.5:1.0:1.4). Thus the pigment is considered to be a pyoverdine. From
more detailed structural data, the structural and biochemical features of the
pigment will be discussed along with the physiological role.
Keywords: fluorescent siderophore, Fe(Ⅲ)–chelated pigment
D056
Protofibril Formation and Amyloidogenesis by
Bacterial Harpins Play Important Roles in
Inducing Rapid Plant Cell Death
1
1
1
Jonghee OH , Jung-Gun KIM , Eunkyung JEON , Chang-Hyuk
1
2
1
1
YOO , Jae Sun MOON , Sangkee RHEE , and Ingyu HWANG
1
School of Agricultural Biotechnology and Center for Agricultural
2
Biomaterials, Seoul National University, Laboratory of Cellular Function
Modulator, KRIBB
*Corresponding author: jake92@snu.ac.kr
Harpins are type III secreted proteins produced by plant pathogenic bacteria
and cause a hypersensitive response when applied externally to appropriate
plant leaves. Several studies have uncovered some of the biochemical
characteristics of harpins. However, the biochemical mechanisms by which
harpins cause plant cell death are largely unclear. Using electron microscopy,
far- and near-UV circular dichroism spectroscopy, and Fourier
transformation infrared spectroscopy, we demonstrate that His6-HpaG
forms biologically active spherical oligomers, protofibrils and β-sheet-rich
fibrils, whereas the null HR mutant HpaG(L50P) does not. In a membrane
permeability assay, HpaG appears to have membrane destabilizing activity. Fibrils of HpaG are considered new kind of amyloid-like protein, based on
positive staining with Congo red to produce green birefringence under
polarized light, increasing protease resistance and β-sheet fibril structure.
Structural and morphological analogies between HpaG and disease-related
amyloidogenic proteins, such as β-amyloid protein (AβP) and prion are
suggestive of possible common biochemical characteristics. Here we show
that the first described Xanthomonas harpin, HpaG forms amyloid-like
fibrils, and that protofibril formation and membrane destabilization activity
play important roles in inducing rapid plant cell death.
Keywords: Harpin, Amyloid, Xanthomonas, Hypersensitive response
204
Biosynthesis and Structural Characterization
of a Pyoverdine-like Pigment Overproduced in
Pseudomonas fluorescens BM07 Grown with
Dicarboxylic Acids
한국미생물학회연합
Isolation and Characterization of Color-Tuning
Mutants in Proteorhodopsin
So-Young KIM, Ah Reum CHOI, Sa-Ryong YOON, and
Kwang-Hwan JUNG*
Department of Life Science and Interdisciplinary Program of Integrated
Biotechnology, Sogang University
*Corresponding author: kjung@sogang.ac.kr
Proteorhodopsin is photoactive 7-transmembrane protein which
uses all-trans retinal as a chromophore. It has been known that a
replacement of single amino acid residue in proteorhodopsin can
alter the photochemical properties and its absorbance. We try to find
critical residues for spectral tuning through the use of random PCR
mutagenesis. We obtained 17 mutant proteins with internal retinal
synthesis system and characterized by the measurement of
absorption spectra, laser-induced difference spectra, and proton
pumping property. Compare with the pumping state of pigment,
absorption maxima of 11 mutants are red shifted and 14 mutants are
blue shifted. The pKa values of these mutants are shifted between
6.0 and 9.2 and most of the mutants have more than two titratable
groups. The result of flash photolysis also showed most of the red
shifted mutants showed the slow down the rate of M decay and O
decay. The residues are spread on the protein except helix A and
about a half of mutations is localized on helices C and D. It means
that helices C and D are important for color tuning. All of these
results suggest that there are several of key residues related to
spectral tuning and each single residue can alter not only spectral
property but also photochemical property.
Keywords: Microbial Rhodopsin, Retinal, Proteobacteria
2005 International Meeting of the Federation of Korean Microbiological Societies
D057
D059
Structural and Functional Domain Identification
of a Transcription Factor BldD from Streptomyces
coelicolor A3(2)
1
2
1
Chang-Jin LEE , Hyung-Sik WON , Jeong-Mok KIM , Bong-Jin
3
1
LEE *, and Sa-Ouk KANG *
1
Laboratory of Biophysics, School of Biological Sciences, and Institute of
2
Microbiology, Seoul National University, Department of Biotechnology,
Division of Life Sciences, College of Biomedical and Health Science, Konkuk
University, 3Research Institute of Pharmaceutical Sciences, College of
Pharmacy, Seoul National University
*Corresponding author: cjlee01@snu.ac.kr
A homodimeric protein BldD is a transcriptional regulator of a
Gram-positive soil bacterium Streptomyces coelicolor. BldD has been
suggested as a transcriptional activator from the phenotypic studies, while
proposed as a repressor from the molecular level studies. Furthermore, no
detailed structural information has been available for BldD, although its
C-terminus has been suggested as a putative DNA-binding site. In the
present work, we clearly identified the domain composition of BldD and
the domains were structurally characterized by circular dichroism and
nuclear magnetic resonance (NMR) spectroscopy. The results certified
that the protein monomer consists of two structural domains and each
domain could be regarded as an independent folding unit that is
structurally and thermodynamically cooperative. Strikingly, it was
revealed by gel permeation chromatography, chemical crosslink, gel
mobility shift, and NMR-monitored DNA-binding experiments, that only
the N-terminal domain is responsible for both the dimerization and DNA
binding of BldD. Thus, the molecular function of the C-terminal domain
remains to be identified. Finally, no or little interaction between the two
domains was evidenced both in the DNA-free and DNA-bound states.
Altogether, the present results establish the first detailed structural data of
BldD and provide fundamental information for its unique and complicated
action mechanism in Streptomyces coelicolor.
Keywords: BldD, Streptomyces coelicolor, transcription factor,
domain identification, DNA binding
D058
Oxidation of Acridine by Laccase of Pycnoporus
cinnabarinus SCH-3
Kyung-Ha YOON* and Hyoun-Su LEE
Division of Life Science, Soonchunhyang University
*Corresponding author: kyungha@sch.ac.kr
This work was performed to investigate whether acridine is
oxidazed by P. cinnabarinus. Transformation of acridine was not
occured when laccase purified from the culture medium of P.
cinnabarinus was reacted with acridine in the sodium tartrate buffer
(pH3.0). However, when 2,2-azino-bis-(3-ethylbenz- thiazoline-6sulfonic acid) (ABTS) was added to the reaction system of purified
enzyme and acridine, transformation of acridine was increased.
About 90% of the substrate was transformed in the presence of 2.0
mM of ABTS after 60 hrs of incubation with laccase. ABTS was
necessary for transformation of acridine by laccase. On the other
hand, when P. cinnabarinus was grown in the culture medium
containing acridine, Laccase activity was gradually increased and
acridine was stoichiometrically transformed to acridone in the
culture medium with culture time. The experimental results suggest
that transformation of acridine in the culture medium of P.
cinnabarinus may be involved by other enzymes except for the
laccase or something to aid laccase activity.
Keywords: P. cinnabarinus, laccase, acridine, acridone, ABTS
D060
Crystallization and Preliminary X-ray Crystallographic
Analysis of a FeZn-SOD from Streptomyces
griceus
Antioxidant Activity of Pigmented Fraction in
Black Colored Rice
Young-Min KIM, In-Kwon KIM, Dong-Won KIM, Chang-Jin LEE,
and Sa-Ouk KANG*
Korea University
*Corresponding author: hichang@korea.ac.kr
Laboratory of Biophysics, School of Biological Sciences and Institute of
Microbiology, Seoul National University
*Corresponding author: esteban1@snu.ac.kr
This study evaluated the antioxidative properties of pigmented fraction
in black colored rice, heukjinjubyeo in vitro. It is known that pigmented
fraction of the black colored rice contains much anthocyanin,
especially cyanidin and peonidin. The pigments scavenged
1,1-diphenyl-2-picrylhydrazyl (DPPH) radical as well as intracellular
reactive oxygen species (ROS). The effect of the pigments on the
viability of human embryonic kidney 293 cells was determined by
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide)
colorimetric method. The result suggested that the radical
scavenging activity of the pigments protected the cell viability of
HEK 293 cells. Furthermore, this pigments reduced the formation of
apoptotic cells induced by H2O2, which was demonstrated by the
decreased number of sub-G1 hypo-diploid cells and apoptotic cell
body formation. All these results suggest that the pigments in black
colored rice, heukjinjubyeo exhibits antioxidant activity.
SODs are ubiquitous metalloenzymes that catalyze the disproportionation
of superoxide to peroxide and molecular oxygen through alternate
oxidation and reduction of their catalytic metal ions. We have recently
purified an intracellular Fe(Zn)-superoxide dismutase encoded by
sodF of Streptomyces griceus. It has been crystallized by
hanging-drop vapor diffusion method at 296 K. A 2.3Å data set has
been collected using synchrotron radiation at Pohang Light Source,
South Korea. The crystal belong to space group P4 with unit cell
parameters a=b=67 Å, c=116.160 Å.
Keywords: superoxide dismutase, X-ray crystrallography, sodF,
Streptomyces griceus
Young-Sam PARK and Hyo-Ihl CHANG*
국제학술대회
205
October 13-14, 2005, Seoul, Korea
D061
D063
ToxJ and LysR-type Regulator ToxR Co-activate
Burkholderia glumae tox Operons Encoding
Toxoflavine Biosynthesis and Transporter in a
Synergistic Manner
1
1
1
Jinwoo KIM , Yongsung KANG , Jae-Eun JEONG , Yunjung
KIM1, Tomohisa NAGAMATSU2, and Ingyu HWANG1*
1,2
1,2
1
Nack-Shick CHOI , Dong-Min CHUNG , Chung Hun RYU ,
Pil Jae MAENG2, and Seung-Ho KIM1
1
2
1
Systemic Proteomics Research Center, KRIBB, Department of Microbiology,
Chungnam National University
*Corresponding author: nschoi@kribb.re.kr
Burkholderia glumae produces a broad-host range phytotoxin, called
toxoflavin, which is a key pathogenicity factor in rice grain rot and
wilt in many field crops. We have previously presented that ToxR, a
LysR-type regulator, regulates both the toxABCDE (toxoflavin
biosynthesis genes) and toxFGHI (toxoflavin transporter genes)
operons in the presence of toxoflavin as a coinducer. In addition,
expression of both operons requires a transcriptional activator, ToxJ,
whose expression is regulated by quorum sensing. We have used gel
shift assay and DNase I footprinting analysis to demonstrate the
regulatory proteins, TofR, ToxJ, and ToxR, bind to the upstream
region of their target promoters. We present biochemical evidence
that TofR, an octanoyl-L-homoserine lactone receptor, binds
upstream of tofI and toxJ promoter regions having lux-box like
sequences. ToxR binds upstream of toxA and toxF promoter regions
having palindromic sequences, T-N11-A, and ToxJ binds to the toxA
and toxF promoter regions. Gel shift analysis and DNase I
footprinting analysis demonstrate that ToxR and ToxJ function
synergistically to activate expression of both tox operons and that the
transcriptional activators exert their effect by directly binding
upstream of their promoter regions. Our biochemical evidence
demonstrates that ToxJ and ToxR co-activate transcription of
toxABCDE and toxFGHI operons in a synergistic manner.
Keywords: Burkholderia glumae, toxoflavin, LysR-type regulator,
ToxR, ToxJ, TofR
Three extracellular proteases (Vpr, peptidase T, and subtilisin) from
the culture supernatant of Bacillus subtilis KCTC 3014 were
identified. All proteins were partially purified by using a
DEAE-cellulose ion exchange column chromatography as a mature
form. Their activities were determined by using zymography and
densitometer. The relative molecular masses of Vpr and peptidase T
(PepT) were determined to be 68 and 48 kDa by SDS-PAGE and
zymography, respectively. However, subtilisin formed a “binding
mode” at the top part of the separating gel. After denaturation by
o
boiling at 100 C for 5 min, its molecular mass was determined to be
29 kDa, whereas its activity was disappeared. The optimal pH of Vpr,
PepT, and subtilisin were at pH 9.0, 6.0-7.0, and 7.0-8.0, respectively.
And, the optimal temperature of Vpr, PepT, and subtilisin were
o
exhibited at 40, 50, and 40 C, respectively. With inhibitor test, Vpr
and subtilisin were classified as a serine protease, and PepT was a
metalloprotease. Interestingly, we newly found that Vpr showed no
enzyme activity on a 2-DE zymogram gel. Three genes, vpr, pepT,
and apr (encoding subtilisin protein), were cloned and their
nucleotide and deduced amino acid sequences were determined. Keywords: Bacillus subtilis KCTC 3014, Mass spectrometry,
PepT, Subtilisin, Vpr, Zymography
2
Seoul National University, Okayama University, Japan
*Corresponding author: pgpr1@snu.ac.kr
D062
D064
Rsa1, a Novel HrpB-Dependent Type II Secretory
Protein, Determines a Host Range of Ralstonia
solanacearum Race 3
1
2
3
Yeonhwa JEONG , Jae Sun MOON , Seungdon LEE , and
1
Ingyu HWANG *
1
2
3
Seoul National University, KRIBB, RDA
*Corresponding author: lucid98@snu.ac.kr
Ralstonia solanacearum causes bacterial wilt in several hundred
plant species. Race identification of the bacterium is based on
pathogenicity in various host plants including solanaceous plants. Race 1 has a broad host range including tomato, tobacco, and pepper,
however race 3 infects only potato and tomato weakly. To identify
genes responsible for host range of R. solanacearum, we transferred
a genomic library of SL2029 (race 3) into SL341 (race 1) by
triparental mating. Among 1,000 transconjugants, we found one
transconjugant that did not induce wilt symptom in pepper. We then
isolated a cosmid clone, pRS1 and subcloned the 0.9-kb PstI/HindIII
fragment conferring host specificity determined by deletion and
mutational analyses. We sequenced the fragment and identified one
possible open reading frame, a rsa1 gene, possibly encoding 110
amino acids. When the rsa1 gene was disrupted by marker-exchange
in SL2029, the mutant was virulent in pepper but much less virulent
in potato. From southern hybridization analysis, we found that most
of race 3 isolates we tested carry the rsa1 gene, however all race 1
isolates we tested do not have any homologous gene. Expression of
rsa1 was regulated by HrpB, and a putative HrpB-binding box is
present in the upstream of the rsa1 gene. This indicates that Rsa1
belongs to the HrpB regulon. Secretion assay indicates that Rsa1 is
secreted probably by Type II secretion systems.
Keywords: Ralstonia solanacearum, Host specificity
206
Identification of Three Extracellular Proteases
from Bacillus subtilis KCTC 3014
한국미생물학회연합
Biochemical Characterization of Monooxygenases
from Microbial Genomes
SuJin KIM*, Myung Hee KIM, Jong Suk LEE, Hyung Pyo
HONG, Tae-Kwang OH, and Choong Hwan LEE
21C Frontier Microbial Genomics and Applications Center, KRIBB
*Corresponding author: sujinkim@kribb.re.kr
Monooxygenases (EC 1.14.13.x) are enzymes that widely used in
various industrial applications because they play a central role in diverse
reactions such as drug detoxification, biodegradation of environmental
pollutants, and biosynthesis of sterols, antibiotics and a wide range of
useful compounds. We have identified four genes encoding
monooxygenases from previously constructed monooxygenase gene
library. Based upon sequence and structure analysis, the enzymes were
classified as alkanesulfonate monooygenase, nitrilotriacetate
monooxygenase, and 4-hydroxybenzoate-3-monooxygenase, respectively
and shown to be significantly different from the previously studied
monooxygenases. Accordingly, this study was initiated to investigate
functions of the monooxygenases. The genes encoding the
monooxygenases were PCR subcloned by using expression vectors
possessing hexahistidine-tag (6´His) or glutathione S-transferase
(GST) and expressed as soluble proteins in E. coli Rosetta-gami™
(DE3) by induction with 0.25 mM IPTG at 18℃ overnight. The
enzymes were homogeneously purified by affinity column
chromatographies and biochemically characterized. Furthermore, the
purified monooxygenases were reacted with their representative
substrates, and the reaction products were analyzed by LC-mass
spectrometry.
Keywords: Monooxygenase, alkanesulfonate, nitrilotriacetate,
4-hydrobenzoate, LC-mass spectrometry
2005 International Meeting of the Federation of Korean Microbiological Societies
D065
Favorable Condition of Mycelial Growth by
Tricholoma matsutake
Won-Ho LEE*, Jae-Mo SUNG, Sung-Keun CHOI, Jeong-Hoon
LEE, and In-Yeup KIM
Entomopathogenic Fungal Culture Collection, Department of Applied
Biology, Kangwon National University
*Corresponding author: art2321@nate.com
The main objectives of this research were to study the cultural and
nutritional characteristics of Tricholoma matsutake and to establish
its liquid culture system. Ectomycorrhizal formation of T. matsutake
was also observed by inoculating liquid spawn in the pine forest. The
last objective of this research was to study the possibility of fruiting
induction of T. matsutake by density control, sprinkle irrigation and
liquid inoculation. Growth and ectomycorrhizal formation of T.
matsutake could be observed up to 12 months after liquid inoculation
in pine forest. Difference in ectomycorrhizal morphology could be
observed between naturally existing and artificially inoculated
mycelia of T. matsutake. Surface of pine roots with natural
mycorrhizae of T. matsutake were black in color and hard in texture,
whereas those with inoculated mycorrhizae were brown and soft.
Morphological characteristics of both natural and artificially
inoculated ectomycorrhiza were similar. Low fruiting of T.
matsutake in 2003, compared to 2002, in spite of artificial
inoculation, density control and irrigation facilities could be due to
relatively higher temperature and low difference between day and
night temperatures in that year.
Keywords: Ectomycorrhiza, Liquid culture system, Liquid spawn,
Tricholoma matsutake
D066
Comparing Methods of Preservation for Artificial
Fruiting Body Production of Cordyceps militaris
Je-O YI, Won-Ho LEE, Young-Hyun LEE, Hye-Kyung JUNG,
and Jae-Mo SUNG
Entomopathogenic Fungal Culture Collection, Department of Applied
Biology, Kangwon National University
*Corresponding author: neoxero@lycos.co.kr
Isolates of Cordyceps militaris were preserved at 4C, -20C, -70C and
liquid nitrogen and tested for fruiting ability after three, six and nine
months of preservation. Glycerol (10%) and dimethyl sulphoxide
(DMSO) (5%) were used as preservatives for preservation al all
temperatures. Isolates were also preserved in distilled water (DW) at 4℃
to use as control. Pre-cooling was done before preserving at freezing
temperatures. Similarly, isolates preserved at freezing temperatures were
thawed at 37C in water bath for 1 min. Liquid inocula of all the isolates
were prepared in SDAY broth and inoculated in brown rice plus pupae
medium for fruiting body production. Isolates preserved at 4C, -20C, -70C
and liquid nitrogen in glycerol (10%) and DMSO (5%) produced higher
weights of fruiting bodies than the isolates preserved in DW after 3 months
of preservation. But, no significant difference was found in weight of
fruiting bodies after six and nine months among the isolates preserved in
DW, glycerol 10% and DMSO 5% at all temperatures. In general, both
fresh and dry weights of fruiting bodies of C. militaris decreased after six
and nine months, when compared to three months. Exceptionally, few
isolates showed complete degeneration of fruiting ability after nine
months of preservation at -70C in DMSO 5%. Continuous preservation of
C. militaris isolates is going on at EFCC to understand the effects of
temperature and duration on fruiting ability.
Keywords: Cordyceps militaris, preservation, temperatures(4C,-20C,
-70C and liquid nitrogen), glycerol (10%), DMSO (5%), distilled water (DW)
D067
Characterization of a Bacillus licheniformis
Mannanase Produced by Recombinant Escherichia
coli
Bueng Wan PARK and Ki-Hong YOON*
School of Food Science and Biotechnology, Woosong University
*Corresponding author: ykh@wsu.ac.kr
Mannanases, which catalyze the random hydrolysis of the β
-D-1,4-mannopyranosyl linkages within the backbone of various
mannan-based polysaccharides, are enzymes useful in food, feed,
paper, and laundry industries. A mannanase gene was cloned in
Escherichia coli from Bacillus licheniformis WL-12. The mannanase
was partially purified from culture filtrate of recombinant E. coli by
a combination of ammonium sulfate fractionation and DEAESepharose column chromatography. The enzyme had a pH optimum
at 6.0 and a temperature at 65℃. The mannanase was capable of
hydrolysing mannotriose, mannotetraose, mannopentaose and
mannohexaose to mannobiose and mannose as major final products. The
enzyme was active on locust bean gum, konjak and guar gum and lichenan,
while it did not exhibit activity towards carboxymethylcellulose, xylan
and para-nitrophenyl-β-mannopyranoside.
Keywords: Bacillus licheniformis, Mannanase, Properties, Recombinant
Escherichia coli
D068
Characterization of Paenibacillus sp Producing
Several Kinds of Extracellular Hydrolytic
Enzymes
Min Ho JUNG, Soo Ho CHOI, Mi-Sung LEE, and Ki-Hong
YOON*
School of Food Science and Biotechnology, Woosong University
*Corresponding author: ykh@wsu.ac.kr
A bacterium producing several kinds of extracellular hydrolytic
enzymes was isolated from soil and was identified as Paenibacillus
sp. on the basis of its biochemical properties and 16S rRNA
sequence. The culture supernatant was used as a crude enzyme for
analyzing its reaction properties with xylan, CMC, locust bean gum,
para-nitropheyl-β-galactoside (pNP-β-galactoside), pNP-β-glucoside,
pNP-β-xyloside and pNP-β-mannoside as substrates. The
hydrolytic activities were detected for all used substrates. It was
found that production of the hydrolytic enzymes was differently
induced according to components added to terrific broth.
Keywords: Paenibacillus isolate, Identification, Hydrolytic enzymes
국제학술대회
207
October 13-14, 2005, Seoul, Korea
D069
Characterization of the Mutated Strains of
Baciullus sp. Hyper-Producing Cellulase
Bueng Wan PARK, Mi-Sung LEE, and Ki-Hong YOON*
School of Food Science and Biotechnology, Woosong University
*Corresponding author: ykh@wsu.ac.kr
In seven mutant strains which were derived from Bacillus sp. 79-23
by gamma-irradiation of the parental spores, effects of carbon sources
were investigated on carboxymethylcellulase (CMCase) production.
By measuring the CMCase activity in the supernatants of bacterial
cultures grown in LB medium containing glucose, xylose, maltose,
lactose, carboxymethylcellulose, starch, rice straw, and wheat bran as
additional carbon sources, it was identified that seven mutants were
comparable to the parent strain. Among the additional carbon sources,
xylose and rice straw affected differently the CMCase production
according to the mutant strains. It was found that the enzyme
production was in association with the growth of parent and mutant
strains. Growth of mutant strains reached the level of parent strain
except mutant 48. Mutant strain 48 was poorly grown and produced
CMCase less than other mutant strains. Especially, spore of mutant 48
was formed at very low level. As the results of them, it was suggested
that mutagenesis of the mutant strains ocurred in different positions
of their chromosomal DNAs, respectively. To examine the
discrepancy between genomes of the mutants, RAPD-PCR was
performed with 2 kinds of oligonucleotides. Patterns of the amplified
DNA fragments was not different between the mutants, though some
fragments was amplified with different amounts.
Keywords: Bacillus sp. 79-23, Mutant strains, Comparison,
Cellulase productivity
D070
High-Throughput Analysis of Physiological
States of Microorganisms by Using Flow
Cytometry: Effects of Anti-Microbial Agents on
the Cell Viability of Bacteria
Heung-Chae JUNG and Jae-Gu PAN*
KRIBB, Laboratory of Microbial Function
*Corresponding author: hcjung@kribb.re.kr
With the increased awareness of the problems associated with the growth
dependent analysis of bacterial populations, direct detection methods of
single cells by FLOW CYTOMETRY have developed a few years ago.
Dye exclusion of propidium iodide and ethidium bromide was the method
of choice to study membrane permeability and thus the viability of the
cells. A lipophilic anion dye, DiBAC4(3) was used as a membrane
potential sensor. By using these different fluorescent dyes, four discrete
physiological states include a) intact reproductive cells, b) intact
de-energized cells (no pump working), c) intact depolarized cells (no
membrane potentials) and d) permeabilized dead cells. In this study, we
investigated how anti-microbial agents affect the physiological states of
bacteria and how they kill the bacteria. For instances, antibiotics, triclosan
(a synthetic agent), and natural compounds showed different bacteriocidal
effects. Antibiotics (ampicillin, kanamycin, and bacitracin) which have
their inhibitory target site in the cells were not able to lyse the cells even
at high concentrations. Natural compounds such as xanthorrhizol and
thymol, and a widely used synthetic anit-microbial, triclosan, rapidly
broke the cell membranes and consequently the cells were killed, which
was concentration-dependent. These results suggest that multi-staining
flow cytometry could be used for characterization of physiological states
of microorganisms based on single cell analysis.
Keywords: flow cytometry, anti-microbial agent, physiological
state, high-throughput
D072
Analysis of Proteomes Regulated by HrpB or
Quormones in Burkholderia glumae
Multiple Triclosan Targets Revealed by Genomic
Enrichment Screening in E. coli
Yongsung KANG, Jinwoo KIM, Hongsup KIM, and Ingyu
HWANG*
Soo-Kyung CHOI, Heung-Chae JUNG, and Jae-Gu PAN
School of Agricultural Biotechnology, Seoul National University
*Corresponding author: yskan99@snu.ac.kr
Many plant bacteria deliver variable numbers of effector proteins
into plant cells during infection. It is thought that expression of these
proteins is regulated by HrpB, an AraC-type transcriptional
activator. Due to the lack of reliable ways to express the effector
proteins in vitro, we engineered the hrpB gene under the control of
trc promoter to be expressed constitutively. We are interested in
bacterial proteomes responding to quorum sensing signal molecules,
quormones because quorum sensing of plant bacteria appears to be
important in coordinating gene expression. We used the twodimensional gel electrophoresis (2DGE) and mass spectrometry
techniques to study and identify proteins of Burkholderia glumae
important for pathogenicity and quormone response. The secreted
proteins from the strain expressing hrpB constitutively were
analyzed to compare the wild type, type III and type II secretion
system mutants. We found that over one hundred proteins induced
by HrpB and most of these proteins are secreted by the type II
secretion systems. The proteome analysis revealed that N-octanoyl
homoserine lactone (C8-HSL) produced by B. glumae significantly
induced or reduced levels of different proteins. Seven up-regulated
proteins and eight down-regulated proteins by C8-HSL were
identified.
Keywords: effector proteins, HrpB, 2DGE, quorum sensing
208
D071
한국미생물학회연합
KRIBB, Laboratory of Microbial Functions
*Corresponding author: jgpan@kribb.re.kr
Triclosan is an antimicrobial agent that is widely used in a variety of
consumer products and acts by inhibiting enoyl-ACP reductase,
FabI, of bacterial fatty acid biosynthesis. As FabI is one of the highly
conserved enzymes, triclosan has been touting as broad spectrum
antimicrobial for more than 30 years on. However, broader use of
any kind of antibacterials should enrich resistant clones. We hereby
investigated systematically the resistance-conferring genomic
clones by competition enrichment cultures followed by microarray
analysis of genomic library DNA. FabI was found as resistanceconferring gene as was expected and, unexpectedly, a lot of other
genes including mar, transporters, and many unknown genes were
also found. Clear differences were observed for clones obtained
from wild-type and imp E. coli strains, indicating that outer
membrane permeability strongly influence selected clones’ phenotype. Taken together, our results show that triclosan has multiple targets
other than FabI and that the selection is strongly biased depending on
the genetic background of E. coli.
Keywords: triclosan, antimicrobial, resistance, genomic screening
2005 International Meeting of the Federation of Korean Microbiological Societies
E001
E003
Characterization of Novel BRM Produced from
Bacillus licheniformis E1
1
2
1
Metabolic Engineering of Escherichia coli for
the Production of L-Ornithine Kun Sub CHUNG *, Joo Young KIM , Sung Wook HONG , and
Bong Ki LEE2
Jae-Yong CHO*, Young-Joon LEE, Ki-Hoon YOON, and
Min-Sang HUR
1
Department of Bioindustry and Technology, Sangji University
*Corresponding author: jycho@sangji.ac.kr
Department of Biological Resources and Technology, Yonsei University,
Department of Microbiology, College of Medicine, Yonsei University
*Corresponding author: kschung@dragon.yonsei.ac.kr
2
In previous study on the functionality of a food, we isolated biologic
response modifier (KfspBRM) from Korean-style fermented soybean
paste (Kfsp) and found out that the KfspBRM enhances specifically
the proliferation and function of B-cells and is not detected in
non-fermented soybean. To isolate the bacteria producing BRM that
was found in Kfsp, and to investigate the chemical and biological
characteristics of the BRM. Bacteria existing in Kfsp were isolated and
their BRM activities were measured. The origin and chemical
composition of BRM produced by the isolate was examined. Bacteria
producing BRM that is similar to KfspBRM specific for B-cells was
isolated from Kfsp, and identified as Bacillus licheniformis E1. The
bacterial BRM (bBRM) was a glycoprotein originated from a slime
layer of B. licheniformis E1 and showed serological response to
anti-KfspBRM. The bBRM appeared to be 1,594 kDa of molecular
weight, and to contain 33%(w/w) of reduced sugar and 4.6%(w/w) of
protein content. It was demonstrated that B. licheniformis E1 isolated
from Kfsp produces a BRM specifically enhancing the proliferation
B-cells but not T-cells, suggesting that this bBRM is a novel
immunomodulator specific for B-cell. The bBRM showed to be
physically, structurally and functionally similar to KfspBRM,
supporting that KfspBRM may be produced by bacteria including B.
licheniformis E1 in fermentation process of soybean paste. Keywords: soybean paste, biologic response modifier(BRM),
Bacillus licheniformis, B-cell-specific BRM
E002
Metabolic engineering efforts aiming at improvement of flux
through an existing pathway and L-ornithine overproduction were
employed in Escherichia coli W3110. The L-ornithine accumulation
on a per cell basis was significantly enhanced when the primary
L-ornithine pathway was optimized both by avoiding the negative
regulatory contour to increase the expression of the genes involved
in the pathway and by preventing conversion of L-ornithine into
citrulline. When the gene for ornithine decarboxylase (speF), one of
at least two decarboxylase that have a possible role in metabolizing
L-ornithine into putrescin, was inactivated to hamper further
metabolism of target product into other metabolite, L-ornithine
production was further enhanced approximately 80% from 33.24
mmol/mg (dry cell weight) to 59.9 mmol/mg (dry cell weight). The
approach described herein might also prove important in other cases
where L-ornithine is limited for increased production of other
primary metabolites of interest.
Keywords: Escherichia coli, Metabolic engineering, L-Ornithine,
Metabolic pathway
E004
Production of β-Hydroxy-β-Methylglutaryl Coenzyme
A Reductase Inhibitor from Bacillus subtilis
LK-12
Metabolic Control Analysis for Increasing
Lycopene Biosynthesis in Escherichia coli
Dae-Hyung LEE, Dae-Hyoung LEE, and Jong-Soo LEE*
Department of Chemical and Biomolecular, KAIST
*Corresponding author: hmwoo@kaist.ac.kr
Department of Life Science and Genetic Engineering, Paichai University
*Corresponding author: biotech8@mail.pcu.ac.kr
β-Hydroxy-β-methylglutaryl coenzyme A reductase(HMG-CoA
reductase, E.C. 1.1.1.34) is a rate-limiting enzyme in endogenous
cholesterol synthesis and catalyzes the reductive deacylation of
HMG-CoA to mevalonate in a two-step reaction. The goal of this
study was to develop a new HMG-CoA reductase inhibitor from
microbial sources for the remedy or prevention of hyperlipemia. A
bacterial strain LK-12 found to produce active intracellular
HMG-CoA reductase inhibitor was isolated from the Korean
traditional Deonjang. It was identified as Bacillus subtilis based on
its 16s rRNA sequence analysis, morphological and physiological
characteristics. The selected strain, Bacillus subtilis LK-12 were
produced high level of antihypertensive angiotensin I-converting
enzyme inhibitor and fibrinolytic substance The optimal culture
conditions for the production of HMG-CoA reductase inhibitor by
Bacillus subtilis LK-12 were investigated. [This study was
supported by Chungnam Agriculture Techno-Park]
Keywords: β-Hydroxy-β-methylglutaryl coenzyme A reductase
inhibitor, Bacillus subtilis LK-12
Han Min WOO and Sang Yup LEE
Metabolic control analysis on kinetic model allows selecting
potential rate-controlling reactions for enhancement of the interesting
flux. Metabolic control analysis is to find rate-controlling steps on
metabolic networks. Metabolic control coefficients and elasticity
represent the normalized partial derivative of the objective flux with
respect to perturbed enzyme activities and concentrations,
respectively. For constraints-based flux analysis does not include
kinetics reactions which represent regulatory information.Kinetics
model containing lumped lycopene pathways with glycolysis and
pentose phosphate pathway of E. coli was constructed. Metabolic control
coefficients were calculated using Jarnac (http://www.sbml.org).
Consequently the dxs gene encoding for 1-deoxy-D-xylulose
5-phosphate synthase recorded the highest flux control coefficient.
Then, dynamic metabolic control analysis was applied after the
overexpression of the dxs gene. It has suggested that 6-phosphofroctokinase
and phosphotransferase system be increased and pyruvate kinase
decreased. It promises metabolic control analysis is useful for lycopene
production in E. coli. This work was financially supported by the Korean
Systems Biology Research program (M10309090000-03B5002-00000)
of the Korean Ministry of Science and Technology (MOST), the
Brain Korea 21 of the Ministry of Education and LG chemicals Chair
Professorship. Hardware for computational analysis supported by the
IBM-SUR program.
Keywords: metabolic engineering, metabolic control analysis,
lycopene, kinetics modelling
국제학술대회
209
October 13-14, 2005, Seoul, Korea
E005
E007
Platform for In silico Metabolic Flux Analysis
1
2
1
Han Min WOO , Dong-Yup LEE *, Hyung Seok CHOI , Tae
Yong KIM1, Hongseok YUN1, and Sang Yup LEE1,2,3
1
2
Department of Chemical and Biomolecular, KAIST, Bioinformatics
Research Center, KAIST, 3BioProcess Engineering Research Center, KAIST
*Corresponding author: hmwoo@kaist.ac.kr
At the heart of this revolution in silico genome-scale metabolic model
has been reconstructed. In order to fully exploit the power of
genome-scale model, a systematic approach employing user-friendly
software is required. we describe the development of an upgraded
MetaFluxNet which allows (1) construction of metabolic models
connected to metabolic databases, (2) calculation of fluxes by
metabolic flux analysis, (3) comparative flux analysis with flux-profile
visualization, (4) the use of metabolic flux analysis markup language
to enable models to be exchanged efficiently, and (5) the exporting of
data from constraints-based flux analysis into various formats.
MetaFluxNet also allows cellular physiology to be predicted and
strategies for strain improvement to be developed from genome-based
information on flux distributions. This integrated software
environment promises to enhance our understanding on metabolic
network at a whole organism level and to establish novel strategies for
improving the properties of organisms for various biotechnological
applications. This work was financially supported by the Korean
Systems Biology Research program of the Korean Ministry of Science
and Technology (MOST), the Brain Korea 21 of the Ministry of
Education and LG chemicals Chair Professorship. Hardware for
computational analysis supported by the IBM-SUR program.
Keywords: MetaFluxNet, metabolic flux analysis, in silico model,
metaboic network
Free Radical Scavengers from the Fruiting
Body of Inonotus xeranticus
1
2
KRIBB, RDA
*Corresponding author: ybs@kribb.re.kr
It has been well known that free radicals are implicated in the
pathogenesis of various diseases such as myocardial and cerebral
ischemia, arteriosclerosis, inflammation, cancer-initiation and aging
processes. There is growing interest in new free radical scavengers
having the potential as protective agents against active oxygenrelated human diseases. As part of our ongoing effort for search of
novel free radical scavengers from wood-rooting basidiomycetes,
three hispidin class antioxidants have been isolated from the
mushroom Inonotus xeranticus. The fruiting body (10 Kg, fresh
weight) of I. xeranticus was extracted with methanol, and the
methanolic extract was partitioned between ethyl acetate and water.
The organic layer was separated by column chromatographies using
Sephadex LH-20 and ODS resins, followed by preparative ODS-TLC
developed with 50% aqueous MeOH to give three antioxidants I-1,
I-2 and I-6. The structures of these compounds were determined by
extensive spectroscopic studies including mass and NMR analysis.
To the best of our knowledge, compounds I-2 and I-6 were novel
hispidin class compounds, and although I-1 was identified as
davallialactone, which was previously isolated from the rhizomes of
Davallia mariesii, this compound was isolated from microbial
resource for the first time. These compounds showed significant
antioxidative activity. Details of structure elucidation and biological
property of these compounds will be presented.
Keywords: Free radical scavenger, Inonotus xeranticus, Hispidin
E008
Bioactive Principles from a Hallucinogenic
Mushroom Gymnopilus spectabilis
1
1
2
Sung-Min CHO , In-Kyoung LEE , Sun-Ja SUK , Wan-Gyu
2
1
1
KIM , Ick-Dong YOO , and Bong-Sik YUN *
2
KRIBB, RDA
*Corresponding author: ybs@kribb.re.kr
Mushrooms have been used as a food and traditional medicine in the
Orient. Mushrooms produce various classes of primary and
secondary metabolites, many that exhibit significant antimicrobial,
antitumor, and antiviral activity. In spite of their potential as a
resource for drug discovery, few bioactive metabolites have been
reported from mushrooms as compared with the higher plants and
microbes. As part of our ongoing efforts for search of biologically
active and chemically novel mushroom metabolites, major metabolic
substances of a hallucinogenic mushroom Gymnopilus spectabilis
were investigated. G. spectabilis is a poisonous mushroom, and its
taxonomic sequence is Subdivision Hymenomycetidae, Class
Agaricales and Family Cortinariaceae. To investigate the major
components of this mushroom, the fruiting body (600 g, dried weight)
was extracted with MeOH, and the methanolic extract was separated
by solvent partition, silica gel and Sephadex LH-20 column
chromatographies and thin layer chromatography. As a result, we have
isolated five major compounds, 4,6-Decadiyne-1,3,8-triol, Ergosta-4,
6,8(14),22-tetraen-3-one, BL V, hispidin and a photochemical
artifact of hispidin. Details of structure determination and biological
activity of these compounds will be presented.
Keywords: Gymnopilus spectabilis, Bioactive substance, Isolation
and chemical structure
210
1
1
E006
1
2
In-Kyoung LEE , Sun-Ja SUK , Jin-Young JUNG , Wan-Gyu
KIM2, and Bong-Sik YUN1*
한국미생물학회연합
Production of 1-Deoxynojirimycin and the Effect
of 1-Deoxynojirimycin in Lowering Blood Glucose
Level in Mice by Bacillus subtilis S10
1
1
1
Yong Seok CHO , Kyung-Don KANG , Young Shik PARK , Jae
1
1
2
Yeon LEE , Kyo Yeol HWANG , Keun KIM , and Su Il
3
SEONG *
1
2
Biotopia Co., Ltd., Department of Bioscience and Biotechnology, The
University of Suwon, 3Department of Life Science, The University of Suwon
*Corresponding author: yscokka@naver.com
1-Deoxynojirimycin (DNJ) has strong inhibitory activity against α
-glucosidase and can be used as drug for the treatment of diabetes. This
study was aimed for production of DNJ using affordable and high
efficient methods : isolation of DNJ-producing strains, identification of
the selected strains, optimization of culture condition with various
carbon, nitrogen, and phosphorus sources, and in vivo experiment using
streptozotocin-induced hyperglycemic mice to check the effect of DNJ
in the lowering blood glucose level. As the results, DNJ-producing
strains were isolated from soils and identified by using16S rDNA
sequence analysis. A strain producing the highest amount of DNJ was
selected and named as Bacillus subtilis S10. DNJ with high purity
was successfully obtained from the culture medium of the strain by
using ion exchange chromatography. In addition, the purified DNJ was
confirmed as an identical compound with DNJ of silkworm Bombyx
mori L. by LC-MS analysis. The optimal culture conditions were
established for the mass production of DNJ by Bacillus subtilis S10.
The result of in vivo experiment using streptozotocin-induced
hyperglycemic mice showed that the concentrated culture medium of
B. subtilis S10 exhibited similar effect of lowering blood glucose
level as acarbose and the concentrate of Bombyx mori L. extract.
Keywords: 1-Deoxynojirimycin (DNJ), α-glucosidase, Bacillus
subtilis, diabetes
2005 International Meeting of the Federation of Korean Microbiological Societies
E009
E011
Purification and Characterization of Phytase
from Bacillus subtilis 92
In-Ho CHU, Sung-Wook HONG, and Kun-Sub CHUNG
Physiological Characterization of Bacillus sp.
HM-6 Screened from Naturally Fermented
Cheongkuk-jang
Department of Biological Resources and Technology, Yonsei University
*Corresponding author: kschung@dragon.yonsei.ac.kr
Kye-Heon OH *, Myeong-Suk JEONG , Chun-Kye PARK ,
Jae-Woo CHUN1, and Sang-Hyun LEE1
Phytase is enzyme which catalyze the hydrolysis of phytic acid into
myo-inositol and inorganic phosphate. This study was carried out to
isolate the phytase producing microorganisms from natural sources.
The isolate CF 92 which was isolated from cattle feces showed the
highest phytase activity. It was identified as Bacillus subtilis, and
designated as Bacillus subtilis CF 92. The production of phytase
from Bacillus subtilis CF 92 reached the high level when grown at
37℃ for 48h in PSL medium. The optimal medium compositions for
enzyme production were 10%(w/v) rice bran extract, 0.1%(w/v)
whey protein powder, 0.02%(w/v) calcium chloride dihydrate and
0.03%(w/v) potassium dihydrogenphosphate. The phytase was
purified by ethanol precipitation, ion-exchange chromatography,
and gel filtration. The purified phytase showed the optimum
temperature of 60℃ and 40% of its original activity remained when
it treated at 80℃ for 30min. Its optimum pH was 7.0 and fairly stable
from pH 4.0-8.0. The enzyme activity was activated in the presence
of EDTA, even though it was inhibited in the presence of metal-ions.
As regards substrate specificity, the phytase had specificity for
polyphosphate compounds such as adenosine triphosphate, sodium
tripolyphosphate, and sodium phytate. The Km and Vmax value for
the sodium phytate was 0.42mM and 4.35μmole/min, respectively.
Keywords: phytase, Bacillus subtilis, substrate specificity
Department of Genetic Engineering, Soonchunhyang University, COSIS BIO
Co., Ltd.
*Corresponding author: kyeheon@sch.ac.kr
1
2
2
1
2
This work was carried out to investigate enzyme activities (e.g.,
alpha-amylase, protease, lipase) of Bacillus sp. HM-6 screened from
naturally fermented soybean, Cheongkuk-jang. The strain HM-6
was compared enzyme activities to Bacillus subtilis isolated from
Japanese natto as a control. Activities of alpha-amylase, protease,
and lipase of strain HM-6 showed 2970, 1399, and 7.2 unit/g,
whereas the activities of a strain isolated from Japanese natto was
2913, 961, and 6.0 unit/g, respectively. In fact, Cheongkuk-jang
fermented by strain HM-6 induced high enzyme activities and better
sensory quality (e.g., thread, color, taste, smell) than that of Japanese
natto. The physiological and biochemical characteristics of strain
HM-6 obtained from the Biolog system and 16S rDNA sequence
were performed. On the basis of the results, HK-6 cells could be
assigned and designated as Bacillus sp. HM-6.
Keywords: cheongkuk-jang, Bacillus sp. HM-6, enzyme activity
E010
E012
Hypolipidemic Effects of Auricularia polytricha
and Cordyceps militaris Mycelia in DietaryInduced Hyperlipidemic Rats
1
2
2
Screening of Edible Mushrooms for the
Production of Lovastatin and Evaluation on
Their HMG CoA Reductase Inhibitory Activity
1
1
2
Byng-Keun YANG *, Yong-Tae JEONG , Guk-Nam KIM , Hun
2
2
JEONG , and Chi-Hyun SONG
Jae Won LEE , Soo Min LEE , Ji Yoon LEE , and In Gyu
1
CHOI *
1
1
Research Center for Processing and Application of Agricultural Products,
2
Daegu University, Department of Biotechnology, Daegu University
*Corresponding author: yangbk@daegu.ac.kr
The hypolipidemic effects of Auricularia polytricha mycelia (APM)
and Cordyceps militaris mycelia (CMM) produced by submerged
cultures in dietary-induced hyperlipidemic rats were investigated.
Hypolipidemic effects were achieved in both the APM- and
CMM-treated group through feeding at the level of 5% daily for 4
weeks. The fed of the APM and CMM substantially reduced the
plasma total and low density lipoprotein cholesterol levels by 16.7%
and 25.5%, and by 19.1% and 32.0%, respectively, when compared
to the control group. The APM and CMM also lowered the plasma
triglyceride level and atherogenic index by 14.2% and 33.9%, and by
17.0% and 46.0%, respectively. It increased the high-density-lipoprotein
(HDL) cholesterol level by as much as 15.3% and 30.0%,
respectively. Furthermore, the liver total cholesterol level was
reduced by 10.8% in APM-treated group, and it was also decreased
by 13.4% in CMM-treated group. [This work was supported by RRC
program of MOST and KOSEF]
Keywords: Auricularia polytricha, Cordyceps militaris, Hypolipidemic
effects, Mycelia
Department of Forest Sciences, College of Agriculture and Life Sciences,
2
Seoul National University, NICEM, College of Agriculture and Life Sciences,
Seoul National University
*Corresponding author: cingyu@snu.ac.kr
Lovastatin is the competitive inhibitor of 3-hydroxy-3-methylglutaryl
-coenzyme A reductase (HMG CoA reductase), the key enzyme in
cholesterol metabolisem that catalizes the reduction of HMG CoA
into mevalonate. It is obtainable by secondary fungal metabolism
from filamentous fungi. For the screening and evaluation, the fruiting
bodies of 6 edible mushrooms were extracted with methanol, and
acetonitrile, and the extracts were tested by TLC, and the quantitative
analysis was evaluated by HPLC. The highest production of
lovastatin among edible mushrooms was obtained from the extract
from fruit body of Pleurotus ostreatus. HMG CoA reductase
inhibitory activities of several extracts from fruit bodies and mycelia
of edible mushrooms were also tested using solubilized microsomal
HMG CoA reductase from yeast. This work was supported by
NICEM in Seoul National University.
Keywords: Lovastatin, HMG CoA reductase, Pleurotus ostreatus
국제학술대회
211
October 13-14, 2005, Seoul, Korea
E013
Antioxidant Effect of Diterpene Analogue
Produced by Streptomyces lincolnensis M-20.
New Secondary Metabolites from a Marine
Actinomycetes sp.
Il-Joong KIM*, Geun-Wan YU, and Kyoung-Ja KIM
Hee Jae SHIN*, Seong-Yun JEONG, Hyi-Seung LEE, Tae Sik
KIM, and Hyun Sun JUNG
Department of Genetic Engineering, Soonchunhyang University
*Corresponding author: blue5365@hotmail.com
Streptomyces lincolnensis M-20 produced puple colored pigment
having antioxidation activity. Antioxidant activity of isolate
compound was measured by lipid peroxidation inhibitory activity
and DPPH radical scavenging activity. This wide spectrum
substance was partially purified from supernantant of Streptomyces
lincolnensis M-20 by butyl alcohol extraction, silica gel column
chromatography and preparative silica gel TLC and the properties of
this substance were investigated. Puple colored pigment was
mixture of five different colored pigments. Yellow colored
substance in butyl alcoholwas active compound. Rf value of this
yellow pigment was 0.2 in chloroform : methanol(20 : 1) developing
solvent and maximum absorbance was at 306 nm. Structural
characterization is carried out by GC/MS. From the GC/MS
libraries, itis estimated as diterpene analogue having MW 308. It
showed blue color by diterpene detection reagent phosphomolybdic
acid(PMA) and ammonuim molybdate(Ⅵ). The srtucture of this
yellow colored substance was elucidated diterpene analogue. This
diterpene analogue has antioxidant activity.
Keywords: antioxdation, peroxidative, diterpene
E014
212
E015
Marine Natural Products Laboratory, KORDI
*Corresponding author: shinhj@kordi.re.kr
Actinomycetes have been the most significant source for compounds
with biological activity and clinical utilities. Though the rate of
discovery of new biologically active compounds from common
soil-derived actinomycetes has been decreased, marine actinomycetes
from various sources represent a new resource for structurally
diverse and biologically active secondary metabolites. In an effort to
search for novel marine natural products, we have collected more
than 3,000 strains of actinomycetes from marine invertebrates,
plants and sediments. Herein we report the results of our chemical
investigation on the biologically active metabolites from marine
actinomycetes and structure determination based on comprehensive
spectroscopic analysis, including 1D and 2D NMR.
Keywords: Acinomycetes, secondary metabolite, NMR
E016
Production of Microbial Siderophore Having
Inhibitory Activity Against Matrix Metalloproteinase
Cholestreol-Lowering Lactic Acid Bacteria
Isolated from Pigs Feces
Kyoung-Ja KIM* and Jong-Soo WOO
Su Won KIM, Hye Myung RYU, Ju Yun CHOI, and Min YOO*
Department of Genetic Engineering, Soonchunhyang University
*Corresponding author: kyoungjakim@hotmail.com
Department of Biology, Keimyung University
*Corresponding author: thebestone@kmu.ac.kr
Matrix metalloproteinases (MMPs) are extracellular enzymes
capable of degrading many extracellular matrix proteins.
Overexpression and activation of MMPs have been linked with a
range of diseases. MMP-2 and MMP-9 have been suggested to play
a role in tumor progression and metastasis. To obtain a novel
matrix metalloproteinase (MMP) inhibitor produced by bacteria, we
have focused on the chelating activity of siderophore. Siderophores
are defined as relatively low molecular weight, ferric ion specific
chelating agents elabolated by bacteria and fungi growing under low
iron stress. Siderophore-producing bacterium was isolated from soil
and the effect of siderophore on MMP-2 activity was assayed by
gelatin zymography. Siderophore was partially purified from
supernatant of microorganism isolated from soil in Oregon State by
methanol extraction and Sephadex LH-20 chromatography.
Partially purified siderophore showed inhibitory activity against
MMP-2.
Keywords: siderophore, matrix metalloproteinase inhibitor, tumor
progression
We have screened the microorganisms from pigs feces for the
development of probiotics which have an anti-cholesterol level. we
could acquire a lot of lactic acid bacteria and experiment oxgal test,
sugar fermentation test. etc. As a result, we obserbed that some of
them were the most tolerant against bile acid and prominent in the
degree of lowering cholesterol level. So we will expand many kind
of lactic acid bacteria from various sources.
Keywords: lactic acid bacteria, cholestreol-lowering, pigs feces
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
E017
E019
Fermentation, Purification and Identification of an
β-Agarase from a Marine Bacterium, Agarivorans
sp. JA-1
Effect of Milk-Fermented Products of Lactic
Acid Bacteria on the Proliferation of MC3T3-E1
Osteoblastic Cells
Jong-Geun JUNG*, Jin-Wook KIM, Song-Ae LEE, Jin-Uk KIM,
Sang-Hyeon LEE, and Jae-Hwa LEE
Eungsuk LEE , Younghoon KIM , Hyun-Sun YUN , Jee-Young
IMM2, Sejong OH3, and Sae Hun KIM1*
Department of Bioscience and Biotechnology, Silla University
*Corresponding author: jjkcfm@nate.com
1
Agarivorans sp. JA-1 is one of the marine bacteria that secreted β
-agarase which catalyze the hydrolysis of agar. β-agarase is unique
in its ability to break down the agarose polysaccharide core made up
of repeating 1,3-linked β-D-galactopyranose and 1,4-linked
3,6-anhydro-α-L-galacto-pyranose into neoagarobiose. In this
study, the effect of cultural conditions on the production of enzyme
in batch culture and purification the β-agarase, extracellular of the
Agarivorans sp., by the fraternity column of the agarose-alginate
bead. In batch culture, production was optimal when strain was
incubated at 30℃ in a marine broth medium with agar and carbon
source. And cultivation broth was comprised the total protein and
enzyme activity at the time. The strain was cultivated in
fermenter(5L) at 30℃, 300rpm and the culture broth was
centrifuged(12,000rpm) to obtain clear supernatant which was used
for all assay. Acknowledgment: The authors wish to acknowledge
the financial support of the Marine Bio 21 made in the program year
2005.
Keyword: Agarivorans sp JA-1
1
1
1
2
Division of Food Science, Korea University, Department of Food and
Nutrition, Kookmin University, 3Department of Animal Science, Chonnam
National University
*Corresponding author: leeusu@gmail.com
Recent studies indicated that fermented dairy products made on effect on
the prevention of osteoporosis. In this study, the effects of milk fermented
products of lactic strains on the osteoblastic cell proliferation and the
subsequent protein expression profiles were investigated. Three strains of
LAB (Lactobacillus casei ATCC 393, Lactobacillus acidophilus ATCC
4356 and Lactococcus lactis KU109) were selected from 33 lactic strains
based on proteolytic activity and evaluate their fermented products on the
proliferation activity of MC3T3-E1 osteoblastic cell. The products of
Lactobacillus casei ATCC 393 showed the most significant increase in
the proliferation activity for MC3T3-E1 cell by MTT assay (130-160%
activity compared to no-serum control), whereas those of other strains did
not show significant effects. Total ten protein spots with three-fold
increase or decrease were detected from MC3T3-E1 cells in the presence
of fermented products of L. casei ATCC 393 compared to no-serum
control by two dimensional gel electrophoresis. These results suggest that
milk fermented products of L. casei ATCC 393 play an important role in
the bone formation by regulation of specific protein expression.
Keywords: Lactic acid bacteria, Osteoblast cell, Two dimesional
electrophoresis
E018
E020
Tolerances of Heat or Acid Stress Adapted
Listeria monocytogenes in Biofilm Against
Hydrogen Peroxide
1
1
1
Antimicrobial Activity of Cultural Metabolites
of Lichen-Forming Fungi Against Plant
Pathogenic Fungi and Food Poisoning Bacteria
1
2
1
Hyun Sun YUN , Younghoon KIM , Eungsuk LEE , Young Mi
1
2
1
CHOI , Sejong OH , and Sae Hun KIM *
Soon-Ok OH , Kwang-Mi LIM , Jae-Seoun HUR *, and Young
3
Jin KOH
1
1
2
Division of Food Science, Korea University, Department of Animal Science,
Chonnam National University
*Corresponding author: christina04@korea.ac.kr
The tolerances of Listeria monocytogenes strains adapted with mild
stress (heat and acid) against hydrogen peroxide (H2O2) were
investigated. The effect of heat and acid stresses on the H2O2 resistances
of Listeria monocytogenes biofilm were observed by distinguishing
protein expressions. When L. monocytogenes were treated with various
heat (30, 40, and 45℃) or acid stresses (pH 4.5, 5.5, and 6.5) for 1 h, cell
viability were not observed significant alteration. Interestingly, the cells
of L. monocytogenes adapted with heat (45℃) and acid (pH 4.5) were
observed the highest resistance to H2O2 (6 and 10%) after formation of
biofilm at glass fiber filter (GFF) for 24 h compared to controls. The heat
and acid adapted biofilms rapidly recovered to the level of 7 log cfu/ml,
whereas L. monocytogenes biofilm controls recovered to the level of 5
log cfu/ml by enrichment step for 24 h. An up or down regulation of
proteins was observed for both heat and acid treatment using
two-dimensional gel electrophoresis (2-DE). The five proteins were
detected that were up-regulated in the pH 4.5 and 45℃. These proteins
regulated heat and acid responses can lead to cross-protection of H2O2
tolerance after biofilm formation. Isolation and identification of these
proteins may assist in developing strategies to influence the survival of
pathogenic bacteria on food-contact surfaces.
Keywords: Listeria monocytogenes, Biofilm, Hydrogen peroxide,
Two dimensional electrophoresis
Department of Environmental Education, Sunchon National University,
3
Department of Biology, Sunchon National University, Department of Applied
Biology, Sunchon National University
*Corresponding author: jshur1@sunchon.ac.kr
2
Antimicrobial activity of 17 lichen-forming fungi (LFF) isolated
form Korean and Chinese lichen species were evaluated against
plant pathogenic fungi of Botrytis cinerea, Pestalotiopsis longiseta
and Rhizoctonia solani, and pathogenic bacteria of Escherichia coli
O-157, Salmonella anterotidis and Streptococcus mutans. The LFF
were cultivated in malt extract broth with peptone and glucose at
o
20 C for 40 days. The cultural extracts were used for antimicrobial
assay with paper disk method. Cultural metabolites produced by the
LFF of Caloplaca flavorubescens, Myelochroa metarevoluta and
Nephromopsis asahina apparently inhibited the mycelial growth of
B. cinerae. All the tested LFF also showed moderate inhibition
activity of the bacterial growth, but their activity was bactericidic.
This study implies that the lichen substances can be produced in a
large scale to develop novel antibiotics. Purification and chemical
identification of the antimicrobial substances will be needed for
further research. Keywords: lichen-forming fungi, culture, antibacterial, antifungal,
mass production
국제학술대회
213
October 13-14, 2005, Seoul, Korea
E021
E023
Improvement of Production Media of
Immunosuppressant Mycophnolic Acid by
Penicillium brevi-compacticum Dierckx ATCC46514
Hyun Seok SHIN and Yong Taik RHO*
Department of Medico Life Science, Youngdong University
*Corresponding author: rhosong@youngdong.ac.kr
Wang-Keun CHOI, Mi-Young SEO, So-Young CHUNG, Byung-Hee
RYU, Joong-Min PARK, Min-Jae SEO, and Jin-Wook KIM*
Mycophenolic acid(MPA) blocking the synthesis of xanthosine
monophosphate is a nonnucleoside inhibitor of inosine
monophosphate dehydrogenase. Therefore mycopholoic acid is a
drug currently used for immunosuppressive agent such as in
transplantation of heart, kidney and liver. And MPA is produced by
fungus Penicillium brevi-compacticum. In this study, the
production media of MPA was developed for the better yield. The
best kinds of carbon sources were chosen and optimal
concentration of carbon source was determined for the highest
production. Molasses is the best carbon source for the industrial
production. Many of various inorganic nitrogen sources and organic
nitrogen sources were tested for the better media composition.
Ammonium nitrate and casamino acid were the best nitrogen
sources as inorganic and organic sources, respectively. And the
effect of surfactant Tween80 on the MPA production was also
studied.
Keywords: mycophenolic acid, immunsuppressant, media optimization
Doosan R&D Center
*Corresponding author: dstkjw@doosan.com
E022
It has been known that rice wine is good for skin beauty especially in
smoothing, detoxifying and regenerating skin. Although it has been reported
that its skin lightening effect is due to the ingredient, kojic acid, other benefits
of it have not been assessed in a systematic way. To investigate whether rice
wine improves the recovery rate of skin barrier function, acute skin barrier
disruption was induced by tape stripping in hairless mice and then
trans-epidermal water loss (TEWL) was measured as one of the indexes of
skin barrier function. Our study shows that in the rice wine concentrate or
RWC EtOH (ethanol extract of rice wine cake)-treated mice, TEWL value
was reduced by 20% compared to the untreated group after 6 hours following
artificial barrier abrogation. After 24 hours, the barrier function was
recovered by 94% in the RWC EtOH-treated mice while only 70% recovery
was observed from the untreated control mice group. To confirm the
influence of rice wine on skin barrier, we employed analysis of stratum
corneum lipid using HPTLC (high-performance thin layer chromatography).
Intracellular lipids in the SC are responsible for the barrier function of
mammalian skin. Ceramide is the main component of the SC lipids. After 24
hrs of barrier disruption, the ceramide level was 1.65 fold higher in the rice
wine-treated group than that of the untreated group.
Keywords: Rice Wine, Skin Barrier, TEWL, HPTLC
E024
Metabolic Engineering of E. coli for the
Production of L-Valine and Its Transcriptome
/Fluxome Analysis
1
1,2
1
Jin Hwan PARK , Kwang Ho LEE , Tae Yong KIM , and Sang
1,3
Yup LEE *
Host Improvement of Saccharomyces cerevisiae
for the Production of Recombinant Proteins
Dong-Hyuk HEO*, Eung-Suek LEE, Chang-Soo CHUN,
Jung-Hoon BAE, Jung-Hoon SOHN, and Eui-Sung CHOI
1
Laboratory of Microbial Functions, KRIBB
*Corresponding author: 78hdh@dreamwiz.com
We constructed L-valine production stain with Escherichia coli W3110 by
targeted genetic modification and identified the effect of the biosynthesis of
L-valine on cell physiology by combined transcriptome and fluxome
analysis. The L-valine-producing strain was constructed by releasing two
regulatory mechanisms, feedback inhibition and attenuation. Two amino
acids alterations were introduced into ilvH to release the feedback inhibition.
The attenuater of ilvGMEDA and ilvBN operon was changed with the strong
tac promoter. ilvA was deleted for the prevention of L-isoleucine synthesis
resulting in increased pyruvate availability for L-valine biosynthesis.
Further improvement of the L-valine-producing strain was achieved by
knocking out leuA and panB thus making more ketoisovalerate available for
the L-valine biosynthesis. Combined transcriptome and fluxome analysis
reveals that an increased pyruvate and ketoisovalerate availability is
essential to direct the flux into the L-valine biosynthesis. Furthermore, target
genes for further metabolic engineering can be selected from the combined
analysis data. Acknowledgements : This work was supported by a grant from
the Korean Ministry of Science and Technology (Korean Systems Biology
Research Grant, M10309020000-03B5002-00000) and by the Brain Korea
21 Project. Further support through the LG Chemicals Chair Professorship
is appreciated.
Numerous eukaryotic proteins have been expressed in S. cerevisiae
using episomal high-copy plasmid harboring a strong inducible
GAL10 promoter with tight regulation. In this system, the industrial
fermentation scale requires a substantial amount of expensive
galactose for protein expression. We constructed several host strains
with modified GAL regulon using genetic engineering. We initially
compared ∆gal1 (galactokinase) mutant strain with wild-type strain.
The ∆gal1 mutant needs less but certain amount of galactose for proper
induction (glucose 2%(w/v), galactose 0.3%(w/v)) than wild-type
(glucose 1%(w/v), galactose 1%(w/v)). Another disadvantage was that
the ∆gal1 mutant showed quite lower growth rate than wild-type. To
overcome these disadvantages, we constructed three strains which
have ∆gal80, ∆gal80∆mig1 and ∆gal80∆mig1∆gal6 genotype. All
strains grew well in YPD (yeast extract 1%(w/v), peptone 2%(w/v),
glucose 2%(w/v)) medium to the level similar to wild-type and GAL10
promoter was fully induced even in complete absence of galactose. To
investigate the heterologous eukaryotic protein expression level in
these strains, we constructed expression vector containing Candida
antarctica lipase B (CalB) and transformed to each strain. A 5ℓ
fermentation study indicated that ∆gal80, ∆gal80∆mig1 and ∆gal80∆
mig1∆gal6 mutant strains are very useful for CalB over-expression.
Keywords: host improvement, Saccharomyces cerevisiae, GAL
regulation
Metabolic and Biomolecular Engineering National Research Laboratory,
2
Department of Chemical and Biomolecular Engineering, KAIST, R&D Center
for Bioproducts, Institute of Science and Biotechnology, CJ Corp.,
3
Department of BioSystems and Bioinformatics Research Center, KAIST
*Corresponding author: jinhwan@kaist.ac.kr
214
Acceleration of Damaged Skin Barrier Recovery
by Cheong-ju (Rice Wine) and Its By-Product
Fermented with Saccharomyces cerevisiae
DSCC 1137
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
E025
E027
Construction of L-threonine Producing Escherichia
coli and Its Transcriptome and Fluxome Analysis
1,2
1
1
Kwang Ho LEE , Jin Hwan PARK , Tae Yong KIM , and Sang
Yup LEE1,3*
Characterization of Succinic Acid Production and
Growth Behaviors of Mannheimia succiniciproducens
MBEL55E in Anaerobic Batch Fermentation
1
1
1,2
Yu-Sin JANG , Hyohak SONG , and Sang Yup LEE *
1
Metabolic and Biomolecular Engineering National Research Laboratory,
Department of Chemical and Biomolecular Engineering, KAIST, 2R&D Center
for Bioproducts, Institute of Science and Biotechnology, CJ Corp, 3Department
of BioSystems and Bioinformatics Research Center, KAIST
*Corresponding author: brilspace@kaist.ac.kr
1
Amino acids have been prominent target-metabolites due to large
commercial demands for flavor enhancers, animal feed, and sweeteners.
L-threonine, one of the essential amino acids, is wildly used as feed- and
food-additives, and various industrial strains that produce L-threonine more
efficiently have been developed by traditional approaches including
deregulation of inhibiting enzymes, elimination of competitive pathways
and amplification of required genes. The previous methodologies have
mostly been based on the trial-and-error type of experiments. However,
elucidation of the factors and bottleneck(s), which are crucial for metabolic
engineering of organisms, remain difficult, mainly due to lack of
information on the entire metabolic fluxes. Therefore, in this study, we
investigated the carbon flux distribution of the central metabolic pathway in
the constructed E.coli strain whose biosynthetic pathways for L-threonine
are strengthened by releasing feedback inhibition and repression. As a
result, we identified crucial factors for the production of L-threonine in the
metabolic pathway of E.coli. Acknowledgements: This work was supported
by a grant from the Korean Ministry of Science and Technology (Korean
Systems Biology Research Grant, M10309020000-03B5002-00000) and
by the Brain Korea 21 Project. Further support through the LG Chemicals
Chair Professorship is appreciated. Keywords: Metabolic engineering, L-threonine, Transcriptome,
fluxome, E.coli
Succinic acid is used either directly as a flavoring agent for food and beverages
or indirectly as an intermediate for many important industrial chemicals such
as n-methylpyrrolidone, 1,4-butanediol, γ-butyrolactone, adipic acid,
tetrahydrofuran, and linear aliphatic esters. Mannheimia succiniciproducens
MBEL55E isolated from the bovine rumen has been known to have an ability
to produce a large amount of succinic acid under anaerobic conditions. The
batch fermentations were performed using a complex medium containing
various concentrations of glucose ranging from 2.5 to 60 g/L in a 5-L reactor
to characterize its growth and succinic acid production behaviors. The reactor
was maintained under an anaerobic condition by flushing with CO2 through
a 0.45 µm membrane since it fixes CO2 for producing succinic acid. The
results show that succinic acid could be produced efficiently with glucose as
a carbon source ranging from 20 to 30 g/L by an anaerobic batch culture of
M. succiniciproducens MBEL55E. Furthermore, this work gave a deep
insight into the process optimization and strain improvement via a metabolic
engineering for the improvement of succinic acid production. [This work was
supported by the Genome-based Integrated Bioprocess Project of the
Ministry of Science and Technology. Further supports by the LG Chem Chair
Professorship, IBM SUR program, Brain Korea 21 project, and by the KOSEF
through the Center for Ultramicrochemical Process Systems are appreciated].
Keywords: batch fermentation, Mannheimia succiniciproducens,
succinic acid
Metabolic and Biomolecular Engineering National Research Laboratory,
Department of Chemical and Biomolecular Engineering and BioProcess
Engineering Research Center, 2Department of BioSystems and Bioinformatics
Research Center, KAIST
*Corresponding author: nall55@kaist.ac.kr
E026
E028
Antibacterial Activities of Polymer Produced
by Bacillus sp. Using Paste-Liquid of Boiled
Soybeans
1
1
2
Se-Won LEE , Jung-Bok LEE , Chung-Sig CHOI , Young-Bae
3
1
KIM , and Gi-Seok KWON *
1
2
School of Bioresource Sciences, Andong National University, HansBio Co.,
3
Andong National University, Nikkyo-Bio. Co.
*Corresponding author: bio91@andong.ac.kr
Chungkookjang, Korean traditional fermented food, is common in
korean meals and Bacillus sp. is usually used in the fermentation of
steamed soybean. For its processing, paste-liquid of boiled soybean is
discarded. We supposed that this water processes high amounts of useful
components. In this study, we investigated on antibacterial activity of the
polymer produced after culturing on paste-liquid of boiled soybeans by
Bacillus spp. We experimented on cell growth with Bacillus licheniformis
KCTC 1026, Bacillus licheniformis KCTC 3045, Bacillus subtilis KCTC
1914, Bacillus pumilus KCTC 10461 and PSW1, KSW3 that was isolated
from chungkookjang. Strains were cultured in boiled paste
soybeans(Semoktae, Daedu, Chungja) at 37℃ for 36hr and controls were
cultured in LB liquid medium. Effect of antibacterial activity was
confirmed with inoculation range of 100~1000㎍ by agar diffusion
method. In antibacterial activity, B. licheniformis KCTC 3045, PSW1
cultured in boiled paste Daedu showed growth inhibition effect against
L. monocytogenes KACC 1950. B. subtilis KCTC 1914, B. licheniformis
KCTC 3045 and PSW1 cultured in boiled paste Semoktae showed
growth inhibition effect against S. typhimurium KCTC 1926. however
strains cultured in LB medium none effect. Further study will be needed
to purify the substances showing antibacterial activity.[This work was
supported by a grant (code : B04-21-004) from Andong-si Technology
＆ Development project for Bio-industry]
Keywords: Chungkookjang, Korean traditional fermented food,
Soybean, antibacterial activity, polymer
Overexpression of fumC Gene Encoding
Fumarase of Mannheimia succiniciproducens
MBEL55E for Succinic Acid Production
1
1
1
1,2
Ji Mahn KIM , Hyohak SONG , Yu-Sin JANG , and Sang Yup LEE *
1
Metabolic and Biomolecular Engineering National Research Laboratory,
Department of Chemical and Biomolecular Engineering and BioProcess
2
Engineering Research Center, Department of BioSystems and Bioinformatics
Research Center, KAIST
*Corresponding author: nall55@kaist.ac.kr
Mannheimia succiniciproducens MBEL55E has been known to have an
ability to produce a large amount of succinic acid. A fumarase has been
known a key enzyme involved in succinic acid production, since it
catalyzes malic acid to fumaric acid, which is directly converted to
succinic acid. In order to improve succinic acid production by enhancing
the formation of fumaric acid, fumC gene in M. succiniciproducens
MBEL55E was overexpressed. The enzyme activity was analyzed by
using cells obtained from batch fermentation. The batch fermentation
was formed in a 5-L reactor by flushing with carbon dioxide, because
CO2 fixation occurs during the whole period of succinic acid formation.
A genetically modified M. succiniciproducens recombinant showed
lower malic acid concentration and higher enzyme activity compared to
those of the wild type strain by 2.0 and 3.0 folds, respectively.
Furthermore, the activity of a wild strain was 3 times higher than that of
E. coli. These observations strongly suggest that M. succiniciproducens
MBEL55E is a promising succinic acid producer and the enhancement
of succinic acid production can be achieved by a genetic manipulation
of M. succiniciproducens. [This work was supported by the
Genome-based Integrated Bioprocess Project of the Ministry of Science
and Technology. Further supports by the LG Chem Chair Professorship,
IBM SUR program, Brain Korea 21 project, and by the KOSEF through
the Center for Ultramicrochemical Process Systems are appreciated].
Keywords: fumaric acid, fumarase, fumarase, malic acid,
Mannheimia succiniciproducens, succinic acid
국제학술대회
215
October 13-14, 2005, Seoul, Korea
E029
E031
Characterization of Malic Enzyme of Mannheimia
succiniciproducens MBEL55E
1
1
1
Jeong Wook LEE , Hyohak SONG , Yu-Sin JANG , and Sang
Yup LEE1,2*
1
Metabolic and Biomolecular Engineering National Research Laboratory,
Department of Chemical and Biomolecular Engineering and BioProcess
Engineering Research Center, 2Department of BioSystems and Bioinformatics
Research Center, KAIST
*Corresponding author: nall55@kaist.ac.kr
Mannheimia succiniciproducens MBEL55E is a very promising succinic
acid producer. A malic enzyme has been known to convert malic acid to
pyruvic acid by carbon dioxide fixation, and also it reconverts pyruvic
acid to malic acid. These acids are very important metabolites for succinic
acid production, because malic acid is further converted to fumaric acid,
which is a precursor of succinic acid, and pyruvic acid is directly
concerned with the formation of by-products such as formic, acetic, lactic
acids, and ethanol. In order to characterize the malic enzyme, maeB gene
in M. succiniciproducens MBEL55E was overexpressed and its activity
was analyzed. The batch fermentation was performed using a complex
medium containing glucose, as a carbon source, in a 5-L reactor under an
anaerobic condition by flushing with CO2. The recombinant strain
showed lower malic acid than that of the wild type strain by 1.6 fold,
indicating, the malic enzyme accelerates the conversion of malic acid to
pyruvic acid. Furthermore, its activity was above 25 times higher
compared to that of E. coli. This works give a knowledge on the efficient
succinic acid production by using M. succiniciproducens.[This work was
supported by the Genome-based Integrated Bioprocess Project of the
Ministry of Science and Technology. Further supports by the LG Chem
Chair Professorship, IBM SUR program, Brain Korea 21 project, and by
the KOSEF through the Center for Ultramicrochemical Process Systems
are appreciated].
Keywords: malic acid, malic enzyme, Mannheimia succiniciproducens,
succinic acid
E030
1,2
1
1
2
1
S. K. LEE , J. C. LEE , D. J. PARK , Y. H. KO , and C. J. KIM *
1
2
KRIBB, Department of Food Science and Biotechnology, Cheju National
University
*Corresponding author: flizzard@naver.com
A010321(A2) and A020645(A4) belonged to Streptomyces species
were selected from domestic soil as antagonistic strains against
potato common scab in Jeju island. In this study, optimal media,
culture conditions and fermentation conditions affecting mycelial
growth and antagonistic activity of A010321(A2) and A020645(A4)
were investigated. GSS medium was used as basic media, various kind of carbon or nitrogen sources were used in media optimization,
C/N ratio and optimal concentration of phosphate were investigated
in flask level. Paper disc diffusion method was used for evaluation of
antagonistic activity, PCV and viable cell counting method were for
mycelial growth. Optimum fermentation conditions for aeration,
rpm, and pH were investigated, respectively.
Keywords: Optimization, Antagonism, Potato common scab
E032
Overexpression of Formate Dehydrogenase for
Succinic Acid Production by Mannheimia
succiniciproducens MBEL55E
1
1
1
Sung Won LIM , Hyohak SONG , Yu-Sin JANG , and Sang Yup
1,2
LEE *
1
Metabolic and Biomolecular Engineering National Research Laboratory,
Department of Chemical and Biomolecular Engineering and BioProcess
Engineering Research Center, 2Department of BioSystems and Bioinformatics
Research Center, KAIST
*Corresponding author: nall55@kaist.ac.kr
Mannheimia succiniciproducens MBEL55E has been known to have
an ability to produce a large amount of succinic acid. However, its concurrent
production of other organic acids, results in the increased costs of purification and
separation. The accumulation of formic acid is generally observed throughout
anaerobic fermentation, and it can be further converted to NADH and carbon
dioxide by a formate dehydrogenase. fdhD and E genes encoding the formate
dehydrogenase in M. succiniciproducens MBEL55E was overexpressed in order
to redirect metabolic flux from formic acid to NADH and carbon dioxide. The
produced NADH and carbon dioxide will be reused for enhancing succinic acid
production by supplying a reducing power and carbon dioxide fixation reactions,
respectively. A genetically engineered recombinant showed the increased
formate dehydrogenase activity more than 3 times compared to its parent strain.
Furthermore, it successfully reduced the accumulation of formic acid in the
culture broth. This study gives an insight into the development of genetically
engineered strains for the efficient production of succinic acid by reducing
by-products such as acetic, lactic and formic acids. [This work was supported by
the Genome-based Integrated Bioprocess Project of the Ministry of Science and
Technology. Further supports by the LG Chem Chair Professorship, IBM SUR
program, Brain Korea 21 project, and by the KOSEF through the Center for
Ultramicrochemical Process Systems are appreciated].
Keywords: formate, formate dehydrogenase, Mannheimia succiniciproducens,
reducing power, succinic acid
216
Optimization for Fermentation of Antagonistic
Strains Against Phytopathogenic Organisms of
Potato Common Scab
한국미생물학회연합
Optimal Conditions for the Production of
Mushroom Mycelium (Agaricus bisporus) in
Submerged Culture
Byungsun CHIO, Kyoungju KIM, and Yonghwi KIM*
Department of Food Science and Technology, Sejong University
*Corresponding author: cbs91@sejong.ac.kr
The nutritional requirements of Agaricus bisporus are rather complex,
but it is important to find less expensive sources of suitable nutrients
in terms of economic value. The objective of the this study was to
determine the optimal culture conditions for mycelial biomass of
Agaricus bisporus in submerged culture. The maximum biomass
was achieved at the temperature of 25℃ and the pH of 6.0 to 6.5 in
flask culture. The initial medium composition for biomass production
was as follow: 24g/L PDB (potato dextrose broth), 10g/L yeast extract,
5g/L malt extract, 5g/L soytone. To examine the effect of carbon
sources on the production of mycelial biomass, various carbon sources
were provided at 20g/L instead of glucose as the carbon source in the
initial medium. The highest level of mycelial biomass was obtained
when sugar cane was used as the carbon source. As a nitrogen source,
soytone gave relatively high mycelial yield and the addition of yeast
extract gave rise to better results. However, the difference in biomass
production according to the addition of malt extract and soytone was
no significant. To minimize the cost of medium, we examined various
carbon sources and inorganic nitrogen sources in 3-liter bioreactor.
The maximum of mycelium cell mass was obtained with medium
containing 20g/L sugar cane, 10g/L sodium nitrate, 5g/L yeast extract.
Keywords: Submerged Culture, Agaricus bisporus, Mushroom
Mycelium
2005 International Meeting of the Federation of Korean Microbiological Societies
E033
E034
Microbial Association and Metabolic Activities
of Lactobacilli Isolated from Kimchi in the
Fermentation Medium Simulating Meat Mixture
1
1,2
Identification of accA1 and accB1 Genes – the
Two Subunits of Acyl–CoA Carboxyltransferase
in Streptomyces toxytricini
1
2
Joo-Yeon LEE , and Benno KUNZ *
Atanas DEMIREV , Ji-Seon LEE , and Doo-Hyun NAM
1
1
Korea Food and Drug Administration, Division of Health Supplement
2
Standard, Institute of Food- and Biotechnology, University of Bonn, Germany
*Corresponding author: foodjooyeon@naver.com
This study was conducted to investigate the utility of lactobacilli isolated
from Kimchi as a substitute for commercial lactic acid starter culture in the
production of fermented sausages. The strains of Lactobacillus brevis,
Lactobacillus curvatus, Lactobacillus plantarum, and Lactobacillus sake
isolated from Kimchi were investigated in combinations for their growth,
souring properties and metabolic activities in the fermentation medium
composed to simulate the substantial conditions of meat mixture. In the two
and three combinations, the growth of L. sake and L. curvatus were
negatively influenced. whereas L. brevis and L. plantarum were the
dominant strains over the fermentation, demonstrating the highest cfu
values of 8.79 log cfu/ml(L. brevis in 3 combination) and 8.56 log cfu/ml(L.
plantarum in 3 combination). The batch of L. curvatus and L. plantarum
involved reached the lowest final pH of 3.13 among the test batches. The
batches containing L. sake or L. brevis indicated typical facultatively
hetero-fermentative characteristics with the acetic acid of more than
2.97% produced from added glucose, whereas L. curvatus and L.
plantarum demonstrated their homo-fermentative characteristics with the
high conversion rate of glucose into lactic acids. As a result, L. plantarum
indicated its potential to be used as a lactic acid starter with its competence
against other lactobacilli, souring property, and homo-fermentative
characteristics.
Keywords: kimchi, lactobacilli, lactic acid starter culture,
fermented sausages
2,3
2
Department of Biotechnology, Yeungnam University, Institute of
3
Biotechnology, Yeungnam University, College of Pharmacy, Yeungnam
University
*Corresponding author: dhnam@yu.ac.kr
Streptomyces toxytricini produces a lipase inhibitor lipstatin via
condensation of a C14 and C8 fatty acid precursors. Molecular
cloning and DNA sequencing of 7.5kb fragment from Streptomyces
toxytricini revealed two putative genes accA1 and accB1 expressed
in the same direction. They are the most likely components of a
carboxyltransferase whose main physiological role is a fatty acid
synthesis. AccB1 (538aa in length) and AccA1 (637aa) showed high
homology to the carboxyltransferase domain of several propionyl
coezyme A (CoA) carboxylases and acyl–CoA carboxyltransferase
of actinomycete origin. accA1 encodes α subunit and accB1 – β
subunit of acyl–CoA carboxyltransferase. However, the real
function of those identified genes is not clear. One possibility is that
α and β subunits of carboxylase play a key role as a unique enzyme
in lipstatin biosynthesis.
Keywords: Streptomyces toxytricini, carboxyltransferase, lipstatin
국제학술대회
217
October 13-14, 2005, Seoul, Korea
F001
F003
Effects of Xanthomonas oryzae pv. oryzae gum
Gene Mutantions on Xanthan Biosynthesis
Jae-Yong CHO, Ki-Hoon YOON, and Min-Sang HUR
Functional Equivalence of Translation Factor
eIF5B from Candida albicans and Saccharomyces
cerevisiae
Department of Bioindustry and Technology, Sangji University
*Corresponding author: jycho@sangji.ac.kr
Eun Ji YANG , Jin-Hee SONG , Kyung Ok JUN , Jung-Ro
PARK1, and Sang Ki CHOI2
1
Xanthomonas oryzae pv. oryzae (Xoo) is a gram-negative bacterium
which is the casual agent of bacterial blight (BB) on rice and is
specific in producing copious amounts of exopolysaccharides (EPS)
named xanthan gum as a virulence factor. Xoo is also the organism
for industrial production of xanthan gum, which has a variety of
applications in the field of recent bioindustry. Genome sequence
analysis of Xoo KACC 10331 provides insight into the Xoo gum
gene cluster composed of 13 open reading frames designated gumB,
-C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, and -N. A cluster of 12 genes
in the Xanthomonas campestris pv. campestris gum gene cluster has
been suggested to encode proteins involved in xanthan gum
biosynthesis. However, no experimental evidence supporting a
similar situation in Xoo has been presented so far. Thus, we decided
to construct a defined set of Xoo gum mutants by using a highly
efficient chromosomal recombination system in order to assign
functions to the products of the Xoo gum genes. In this work, we
present genetic experimental data for assessing a correlation
between a gum gene mutation and xanthan gum biosynthesis. Keywords: Xanthomonas oryzae pv. oryzae, xanthan gum, operon,
chromosomal recombination, mutation
F002
2
1
Department of Food and Nutrition, Sunchon National University,
Department of Biological Sciences, Sunchon National University
*Corresponding author: sangkic@sunchon.ac.kr
2
Prokaryotic translation initiation factor 2 and its ortholog eukaryotic
initiation factor 5B (IF2/eIF5B) is The GTP binding protein (G
protein). eIF5B plays a role in recognition of an AUG codon with
another translation factor eIF2, and promotes joining of the 60S
ribosomal subunit. It has been demonstrated that eIF5B identified in
archaea and human can substitute for its yeast ortholog both in vivo and
in vitro. The FUN12 gene in yeast encodes a protein eIF5B. Deletion
of the FUN12 gene caused a severe slow growth phenotype due to
impaired translation initiation, and recombinant eIF5B restored
translation in extracts prepared from fun12 (Δ eIF5B) strains.To
examine how eIF5B of other organisms functionally resemble each
other and to address how this protein play a role during differentiation,
we chose Oryza sativa, Arabidopsis thaliana, Aspergillus nidulans and
Candida albicans as test organisms. eIF5B cloned from these
organisms by RT-PCR to yeast vectors. This gene expressed in
Saccharomyces cerevisiae either under the control of yeast eIF5B
promoter or under a galactose-inducible GAL promoter in a fun12
strain. Currently we observed that the expression of eIF5B of Candida
albicans partially complemented the slow-growth phenotype of a
fun12 yeast strain. These results reveal that eIF5B in Candida albicans
has close functional relationship with that of Sacharomyces cerevisiae.
Keywords: translation, factor, eIF5B, Saccharomyces cerevisiae
F004
The Control of Organosulfur Utilization Operons
by Spx and YrzC in Bacillus subtilis
1
1
2
Soon-Yong CHOI , Kyung Mi KIM , Kyle N. ERWIN , and Peter
2
ZUBER
1
2
Department of Biotechnology, HanNam University, Department of
Environmental and Biomolecular Systems, OGI, Oregon Health and Science
University, USA
*Corresponding author: sychoi@hannam.ac.kr
Spx protein exerts both positive and negative transcriptional control
over a genome-wide scale in response to oxidative stress. Genes such
as those of the ytmI, yxeL, and ssu operons, which encode proteins that
function in the uptake and desulfurization of organic sulfur
compounds, are repressed when sulfate or cysteine is present as a sole
sulfur source. Both the ytmI operon and the divergently transcribed
ytlI gene, encoding a LysR-type regulator that positively controls the
ytmI operon, are repressed by an Spx-dependent mechanism in
sulfate-containing medium. A recent report has implicated YrzC as
being another negative regulator of the ytmI operon. When yrzC was
inactivated, the expression of the ytmI operon was derepressed in the
presence of either sulfate or cysteine. This is in contrast to an spx
mutant, which still exhibits Cys-dependent transcriptional
repression. We determined that YrzC is a negative regulator for the
expression of ytlI, and hence, indirectly causes repression of the ytmI
operon. When yrzC is expressed from the IPTG-inducible Pspac
promoter in an spx null mutant, the expression of ytmI was repressed
in media containing sulfate. This result suggests that YrzC could bind
to the promoter region of ytlI, thereby repressing the ytlI gene. A
regulatory pathway of sulfate-dependent repression of organosulfur
utilization operons that involves Spx and YrzC is proposed
Keywords: gene expression, organosulfur, spx, gene regulation
218
2
한국미생물학회연합
Characterization of Oxidative Stress-sensing
FinR Homolog in Escherichia coli O157:H7
Yunho LEE and Woojun PARK*
Division of Environmental Science and Ecological Engineering, Korea
University
*Corresponding author: wpark@korea.ac.kr
We have shown that FinR, a LysR-type transcriptional factor divergently
transcribed from the fpr (ferredoxin-NADP+ reductase) gene of
Pseudomonas putida, regulates the expression of the fpr responding to
oxidative stress. Disruption of the fpr and finR result in more oxidative
stress sensitivity and growth inhibition in minimal media.
Transcriptional analysis indicated that the level of fpr expression is
induced by oxidative stress as well as osmotic stress. Analysis of bacterial
genome database has shown that FinR-homologs are present in many
proteobacteria. However, their function has not been characterized.
Genetic organization of an yeiE gene (encoding a putative LysR-type
transcriptional factor) and an yeiH (encoding a putative membrane
protein) in E. coli O157:H7 is the same as that of FinR region. The YeiE
protein has 41% amino acid identity with the FinR. Here, we present the
data that yeiH is induced not by paraquat, hydrogen peroxide, but by
menadione. Surprisingly, loss of yeiE gene caused higher yeiH
expression in the presence of menadione. This suggested that the yeiE
may be a repressor of the yeiH in E. coli O157:H7. Consistent with the
result above, the growth of the yeiE mutant is better than that of wild type
in the presence of high concentration of menadione. Pathogenesis of E.
coli O157:H7 related to the yeiE region will be discussed
Keywords: paraquat, menadione, ferredoxin-NADP+ reductase,
yeiE, yeiH, oxidative stress
2005 International Meeting of the Federation of Korean Microbiological Societies
F005
F007
The Polymorphisms of the Prion-like Protein
Gene (PRND) in Korean Normal Population and
Sporadic Creutzfeldt-Jakob Disease
1
1
2
Byung-Hoon JEONG , Nam-Ho KIM , Jae-Il KIM , Richard I.
CARP2, and Yong-Sun KIM1*
1
2
Ilsong Institute of Life Science, Hallym University, New York State Institute
for Basic Research in Developmental Disabilities
*Corresponding author: bhjeong@hallym.ac.kr
An association between sporadic Creutzfeldt-Jakob disease (CJD) and the
prion-like protein gene (PRND) polymorphism at codon 174 has been
reported in a study of German population, but other studies have failed to
show this linkage. To investigate whether the PRND polymorphisms are
associated with an increased risk for developing sporadic CJD in the
Korean population, we compared the genotype and allele frequencies of
PRND polymorphisms in 95 sporadic CJD patients with those in 102
healthy Koreans. Polymorphisms of PRND gene in Koreans were found
at codons 56, 174 and the 3’ untranslated region (UTR) +28 site. One
heterozygote at codon 56 (P56L) was observed in normal controls, not in
sporadic CJD patients. A strong significant difference of PRND genotype
frequency at codon 174 was found between normal Korean population and
various European populations: German (P = 0.048), British (P = 0.0002),
French (P <0.0001), and Spanish (P <0.0001). In sharp contrast to results
in the German population, our study does not show a significant difference
in PRND genotype (P = 0.486) or allele frequency (P = 0.2447) at codon
174 between sporadic CJD and normal controls. However, a significant
difference was found between sporadic CJD and normal controls in
genotype distribution (P = 0.0058) and in allele frequency (P = 0.0181) at
3’UTR +28. These results suggest that a specific genotype (T/T) or allele
(T) at 3’ UTR +28 of the PRND gene is a risk factor for sporadic CJD.
Keywords: Creutzfeldt-Jakob disease, Doppel, Prion disease
F006
Partial Reconstitution of Hepatitis C Virus RNA
Polymerization by Heterologous Expression of
NS5B Polymerase and Template RNA in
Bacterial Cell
Jong-Ho LEE, Min Soo KIM, Jae Seok PARK, Ji Hye LIM, Yu
Jeong BYUN, and Heejoon MYUNG*
Department of Bioscience and Biotechnology, Hankuk University of Foreign
Studies
*Corresponding author: hjmyung@hufs.ac.kr
The hepatitis C virus is a major etiological agent causing chronic hepatitis
in humans. Our aim was to develop an assay system for the NS5B
polymerase. A plasmid expressing the HCV NS5B polymerase was
maintained with a plasmid containing a reporter gene in an E. coli cell. The
reporter construct contained the HCV 5’ UTR followed by a luciferase gene
with a specific orientation so that a minus-sense transcript containing the
luciferase fused to the 5’ UTR was produced after the initial transcription.
When the HCV NS5B polymerase was expressed in the same cell, the
primary transcript was recognized by the polymerase due to the presence of
the minus-sense 5’ UTR, and a secondary transcript containing a plus-sense
luciferase gene was produced. Thus, a simple luciferase assay was able to
measure the HCV NS5B polymerase activity. The production of minus- and
plus-sense transcripts was confirmed by an RT-PCR, while the production
of HCV NS5B and expression of the reporter luciferase in the bacterial cell
were confirmed by immunofluorescence microscopy. The polymerization
occurred in the absence of any other viral/host factors. Accordingly, this
would appear to be the first study to demonstrate that the heterologous
expression of an animal viral RNA polymerase and its template in a bacterial
cell can partially reconstitute the polymerization reaction.
Keywords: HCV, NS5B polymerase, RNA template, replication,
bacteria
F008
Functional Analysis of Putative Salbostatin
Biosynthetic Genes
Studies on Inhibitory Effect of NS5B-Binding
Peptides for HCV Replication
Woo-Sik CHOI, Seung Ghyu SOHN, Sang Hee LEE,
Soon-Kwang HONG, and Byeong Chul JEONG*
Min Soo KIM, Jong-Ho LEE, Ji Hye LIM, Jae Seok PARK, Yu
Jeong BYUN, and Heejoon MYUNG*
Department of Biological Sciences, Myongji University
*Corresponding author: bcjeong@mju.ac.kr
Department of Bioscience and Biotechnology, Hankuk University of Foreign
Studies
*Corresponding author: hjmyung@hufs.ac.kr
Salbostatin was discovered as a metabolite of Streptomyces albus ATCC
21838, and its structure has a valenamine that is the common structure of
α-glucosidase inhibitor such as Acabose.Salbostatin biosynthetic gene
cluster was cloned to cosmid vector from Streptomyces albus KCTC
9015, using acbC sequence as the prove. The sequence of Salbostatin
biosynthetic gene cluster was similar to that of Acarbose biosynthetic
gene cluster. Each subcloning of Salbostatin biosynthetic genes were
performed by PCR using cloned genes of Salbostatin biosynthasis. The
multistep conversion of sedo-heptulose 7-phosphate to the final cyclitol
moiety was extensively studied in Acarbose biosynthetic steps.
Compared to Acarbose biosynthetic genes, each gene was expected as
sedo-Heptulose 7-phosphate Cyclase, 2-epi-5-epi-valiolone 7-kinase, 2-epi5-epi-valiolone-7-phosphate 2-epimerase, 5-epi-valiolone-7-phosphate
dehydrogenase, 5-epi-valiolol-7-phosphate dehydratase : acbC to salQ,
acbM to salL, acbL to salM, acbO to salO and acbN to salN. A possible
five-step reaction mechanism by these five enzymes was proposed for the
cyclization reaction. For confirmation of the mechanism, SalQ protein
was purified from soluble form. SalQ activity was detected by TLC. The
chemical structure of compound produced sedo-heptulose 7-phosphate
by SalQ is currently under investigation.[Supported by the Driving Force
Project for the Next Generation of Gyeonggi Provincial Government in
Republic of Korea].
Keywords: Streptomyces albus, Salbostatin, sedo-heptulose 7-phosphate
cycl
A 7mer and a C-9-Cmer peptide-displaying phage libraries were
screened against HCV NS5B polymerase. After 3 rounds of
biopanning, two 7mer peptides and one C-9-Cmer peptide were
selected. They showed an inhibitory effect for NS5B in a
biochemical assay using purified enzyme and HCV 3’ UTR RNA
immobilized in the Covalink module. The inhibition was in a
dose-dependent manner. They also showed an inhibitory effect
when present in the cell expressing HCV subgenomic replicon as
determined by realtime PCR analysis of the replicon RNA level. The
interaction between the peptides and NS5B was confirmed by yeast
two hybrid analysis and confocal microscopy analysis after
immunostaining. The deletion mapping analysis revealed the
peptide-binding fraction in NS5B. The inhibitory effect of the
peptides regarding the binding sites on NS5B is discussed.
Keywords: HCV, peptides, phage display, NS5B polymerase
국제학술대회
219
October 13-14, 2005, Seoul, Korea
F009
F011
Production of New Mushroom Hybrids by
Multi-sporous Random Mating
1
1
1
Won-Sik KONG *, In-Yeup KIM , Chang-Sung JHUNE ,
Ki-Wook KWEON1, Young-Bok YOO1, and Kwang-Ho KIM2
1
Applied Microbiology Division, National Agricultural Science and
Technology, RDA, 2Department of Crop Science, Kon Kuk University
*Corresponding author: wskong@rda.go.kr
To provide against an expected international dispute about
mushroom commercial strains, the demands for the domestically
developed ones are increased. In this presentation, the multi-sporous
random mating method for mushroom breeding is introduced, which
is as simple as anyone can apply to make his own favorable mushroom
strain. This method needs no monospore isolation, mating type
determination, microscopic observation and crossing between
monospores. In Flammulina velutipes, many dikaryons were isolated
and cultivated after colonization by random mating with ASI4103
spores. A superior or promising strain could be selected and its
mycelia from the fruitbody could be revived for long term
preservation. And even their parental monokaryons could be
recovered by isolation of asexual spores called oidia. Actually the
oidial isolates showed only two types at the esterase isozyme pattern.
In the case of Pleurotus ostreatus, four different spore mixtures
combined by white or dark gray fruitbody producing strains were
tested to compare with mono-mono mating. Production of dikaryons
by multi-sporous random mating took only 10 days. It was much
shorter than that of mono-mono mating which required 28 days.
Variation range on fruitbody morphology in dikaryons derived from
two methods had no difference except on fruitbody color. Dikaryons
from random mating showed 25%～37.7% of parental type and
62.3%～75% of hybrid type according to mixed combination.
Keywords: Hybrids, Multi-sporous Random Mating, Pleurotus
ostreatus, Flammulina velutipes, Mono-mono Mating
A Large Gene Deletion Causes No Aurofusarin
Production, but Retains Female-Fertility in
Gibberella zeae 1
Myung Ki LEE*, Kyung Hee KIM*, and Ji Young LEE
Korea Food Research Institute
*Corresponding author: microhee@hanmir.com
As many lactic acid bacteria have similar nutritional and growth
requirements, it is very difficult to identify them individually using
conventional methods. Traditional identification methods based on
physiological phenotypes are labor and time consuming. Recently, the
rRNA sequences have been generally accepted for the identification
and phylogenetic analysis. In this, the PCR experiments were carried
out using specific oligonucleotide primer sets based on the metabolic
enzyme gene sequences of various lactic acid bacteria. The PCR
products was confirmed by DNA sequencing. Accordingly, since the
PCR used is simple, specific, and rapid, it will be useful for
monitoring and evaluating lactic acid bacteria succession in the state
of natural flora population found in kimchi. For the designing of
primers, comparative analysis of metabolic enzyme gene sequences
from various lactic acid bacteria was performed. A each of specific
primer set was chosen from the conserved sequences of metabolic
enzyme genes among each of lactic acid bacteria on the genus and
species level. From the result, 45 primer sets were selected for rapid
classification of lactic acid bacteria in kimchi.
Keywords: lactic acid bacteria, PCR
220
한국미생물학회연합
1
1
2
Division of Life Sciences, Soonchunhyang University, School of Agricultural
Biotechnology, Seoul National University
*Corresponding author: sy14@sch.ac.kr
Gibberella zeae is an important pathogen of maize, wheat and rice.
Colonies of G. zeae produce yellow to tan mycelia with the white to
carmine red margins. A screen of insertional mutants of G. zeae generated
using a restriction-enzyme-mediated integration (REMI) procedure led to
the isolation of the mutant R606, which has an albino phenotype.
Outcrossing of R606 to a mat1-1-deleted strain confirmed that the albino
mutation is linked to the insertional vector. The genomic region flanking
5’ of vector insertion site in R606 was identified as a KpnI site 433 bp
upstream of the GIP10 gene (located at contig 1.116 in the G. zeae genome
database), a member of the aurofusarin (red pigment) biosynthesis gene
cluster in G. zeae. However, the genomic region 3’ of the vector insertion
site was matched to the contig 1.120, indicating more than 200-kb
genomic region spanning five contigs was deleted in this mutant during
the REMI mutagenesis. Re-creation of the same mutation using a rescued
plasmid from R606 suggests that the 200-kb deletion is responsible for the
albino phenotype and that a large size of genomic DNA could be
efficiently deleted in G. zeae by double homologous recombination.
Moreover, normal female-fertility of R606, when selfed, is comparable
with the female-sterility of another albino mutant caused by deletion of
PKS12, required for aurofusarin in G. zeae. These two different
phenotypes were confirmed to be genetically linked to each gene deletion.
Keywords: Gibberella zeae, REMI, aurofusarin
F010
Amplified DNA Patterns by Using PCR with
Primers of Metabolic Enzyme Gene of Lactic
Acid Bacteria in Kimchi
1
Hee-Kyoung KIM , Sun-Hee LEE , Sae-Yeon HONG , Yin-Won
LEE2, and Sung-Hwan YUN1*
F012
Staphylococcal Cassette Chromosome mec
(SCCmec) Characterization and Molecular
Analysis for Methicillin-resistant Staphylococcus
aureus Isolated from Bovine Milk in Korea
Nam Hoon KWON1, Kun Taek PARK1, Jin San MOON2, Woo
1
1
1
1
Kyung JUNG , So Hyun KIM , Jun Man KIM , Soon Keun HONG ,
1
3
1
Hye Cheong KOO , Yi Seok JOO , and Yong Ho PARK *
1
Department of Microbiology, College of Veterinary Medicine and School of
2
Agricultural Biotechnology, Seoul National University, Veterinary Pharmacology
Division, National Veterinary Research and Quarantine Service, 3Foreign Animal
Disease Division, National Veterinary Research and Quarantine Service
*Corresponding author: yhp@snu.ac.kr
Methicillin-resistant Stapylococcus aureus (MRSA) is one of the most prevalent
pathogens that cause nosocomial infections. MRSA produces a specific
penicillin-binding protein PBP2a is responsible for the ß-lactam antibiotic
resistance. PBP2a is encoded by the mecA gene carried by a large mobile genetic
element, staphylococcal cassette chromosome mec (SCCmec). SCCmec element
contains the mec gene complex composed of the mecA gene and its regulators, and
the ccr gene complex that encodes site-specific recombinases responsible for the
mobility of SCCmec. Fourteen MRSA and a silent mecA carrying methicillin
susceptible S. aureus (smMSSA) were isolated from milk of cows suspected to
be infected with S. aureus in Korea (isolation rate: 0.18%). SCCmec of 14 strains
was classified as subtype IVg which was designated as new SCCmec type in this
study, and one smMSSA strain was not classified. All the MRSA and smMSSA
isolates showed no multi-drug resistance and had community-acquired MRSA
(CA-MRSA) characteristics. PFGE revealed that all the MRSA isolates belonged
to the same genetic lineage, and MLST analysis showed that MRSA strains had
no genetic relatedness with CA-MRSA which had caused human infection in
Korea. This study suggests that the emergence of CA-MRSA in human infection
might not be correlated with MRSA isolated from bovine milk in Korea.
Keywords: Methicillin-resistant Stapylococcus aureus (MRSA),
Staphylococcal Cassette Chromosome mec (SCCmec), Bovine milk,
community-acquired MRSA (CA-MRSA)
2005 International Meeting of the Federation of Korean Microbiological Societies
F013
F015
Identification of Genetic Differences between
Listeria monocytogenes Epidemic and Environmental
Strains Using Suppression Subtractive Hybridization
and Expression Array Analysis
1
2,3
2
So Hyun KIM , Monica BORUCKI , James REYNOLDS , Edith
OROZCO2, Lisa ORFE2, Deborah DURICKA2, Hye Cheong
1
1
1
1
KOO , Jun Man KIM , Woo Kyung JUNG , and Yong Ho PARK *
1
Department of Microbiology, College of Veterinary Medicine and School of
Agricultural Biotechnology, Seoul National University, 2Animal Disease
3
Research Unit, USDA Agricultural Research Service, College of Veterinary
Medicine, Washington State University, USA
*Corresponding author: yhp@snu.ac.kr
Listeria monocytogenes causes severe disease in humans and invasion of host
tissues is crucial for its pathogenesis. A recent study by Kim et al. (2003)
showed that L. monocytogenes epidemic strains as a group are significantly
more infective than environmental strains in a murine oral infection model.
Interestingly, one epidemic strain had higher infectivity as compared to the
environmental strain in the murine model although the two strains had an
identical PFGE subtype. Therefore, suppression subtractive hybridization was
used to identify genetic differences that might account for the observed
difference in phenotype. Although no regions were detected as unique to the
epidemic strain, 18 regions were unique to the environmental strain, and all
were similar to bacteriophage proteins. A Listeria phage 2389 insertion site
was identified at the 3’ end tRNA-arg gene of the environmental strain where
gene function was reconstituted by phage nucleotides. Whole genome
microarray expression analysis indicated that transcription of two of genes in
the agr locus was significantly down regulated in the environmental stain.
Subsequently a single nucleotide insertion was identified in agrC open reading
frame of the environmental strain which resulted in a severely truncated
protein and appeared to cause a significant decrease in listeriolysin secretion.
Keywords: Listeria monocytogenes, Epidemic strains, Suppression
subtractive hybridization, Phage, agrC
F014
Molecular Analysis of the Novel Intergenic
Transcript in Salmonella typhimurium
Yong Heon LEE*, Bae Hoon KIM, Jin Seok KIM, Ji Hye KIM,
and Yong Keun PARK
School of Life Sciences and Biotechnology, Korea University
*Corresponding author: ykpark@korea.ac.kr
In a previous study, we selected a mutant defective in growth at
acidic pH by transposon (Tn10dTc) mutagenesis. The
Tn10dTc-inserted region is located between the cspH and envE
genes and encodes a novel intergenic transcript, the sequence of
which shows no homology to the E.coli DNA database. Primer
extension and 3’ RACE analyses were performed to identify the 5’
end and the 3’ end of this novel transcript. Here, we show that the
expression of this gene is increased at low pH and this RNA is stable
throughout the time course like other small RNA regulators.
Moreover, the gene encoding this small RNA is expressed from two
independent promoters, the activity of which is related to growth
phase. In conclusion, our results imply that this novel intergenic
transcript may be a small-RNA regulator of acid response.
Keywords: Salmonella typhimurium, small RNA, intergenic
transcript, acid response
F016
Enhancement of Streptomyces griseus Trypsin
(SGT) Production by the Regulatory Genes from
Streptomyces Griseus ATCC 10137
1
1
2
Development of Intra-specific Hybrids by Hyphal
Anastomosis Between Self-Fertile Monokaryotic
Strains in Pleurotus Ostreatus
Mi Soon KIM , Won-Jae CHI , Yong Keun CHANG , and Soon
1
Kwang HONG
In-Yeup KIM*, Ki-Wook KWEON, Won-Sik KONG, Chang-Sung
JHUNE, and Young-Bok YOO
1
Department of BiologicalScience, College of Natural Science, Myongji
2
University, Department of Chemical and Biomolecular Engineering, KAIST
*Corresponding author: pkuirme@nate.com
Applied Microbiology Division, National Agricultural Science and
Technology, RDA
*Corresponding author: wskong@rda.go.kr
Streptomyces griseus is a representative strain in the genus Streptomyces,
because it can produce not only many kinds of secondary metabolites but
also various proteases. The regulatory cascade concerning streptomycin
production and morphological difzferentiation has been extensively
studied in this strain and the involvement of a microbial hormone,
A-factor (2-isocapryloyl-3-R-hydroxy-methyl-γ-butyrolactone), and
some proteases in the regulatory cascade has been reported. SGT is a
bacterial serine protease with greater similarity to a mammalian protease,
trypsin. The sprT gene (Genebank accession No. M64471) encoding S.
griseus trypsin and two putative regulatory genes have been isolated from
S. griseus ATCC 10137 genomic library. To confirm the function of
regulatory genes, the sprT gene was introduced into S. lividans with two
regulatory genes on the same plasmid, and then the trypsin activity and
transcription level of sprT was measured. The trypsin activity of the
transformant was remarkably increased by two regulatory genes. The
transcription of sprT was started earlier and maintained at higher level
during cultivation. Therefore it was concluded that using regulatory
genes would be a very valuable tool for the strain development of high
production. [The research was supported by the Driving Force Project for
the Next Generation of Gyeonggi Provincial Government in Republic of
Korea.]
Keywords: SGT, Trypsin, Streptomyces griseus, Regulatory gene
The main objective of this study was to develop new strains of
Pleurotus ostreatus by the method of mono-mono crossing. The
parental strains used in this study were all Korean commercial
strains, ASI2018, 2183, 2344, 2504, 2706. Spores were collected
from a fruitbody and monokaryotic strains were isolated by serial
dilution of spore suspension. Monokaryotic strains were intra-mated
reciprocally to determine their mating types. Their sexuality
presented tetrapolarity. In each strain, the fruiting ability of
monokaryotic strain was also examined by cultivation in 850 ml
plastic bottle. The fruiting rates of monokaryons derived from
ASI2018, 2183, 2344, 2504, 2706 were 75, 70, 50, 80, 58 percent,
respectively. Mono-mono intra-specific hybrids between
monokaryotic fruiting groups or non-fruiting groups by hyphal
anastomosis were obtained and cultivated in the plastic box filled
with cotton waste to compare their productivity. Result showed that
the yield of hybrids between monokaryotic fruiting group was about
7.7 percent higher than that of non- fruiting group. FinaIly, out of
300 intra-specific hybrids, 25 hybrids were more superior than their
parental strains.
국제학술대회
221
October 13-14, 2005, Seoul, Korea
F017
F019
Genetic Profiles of Salmonella gallinarum
Isolated from Chickens in Korea
1
2
2
Mi Young KANG , Suk Kyung LIM , Jin Mok JUNG , Ha Yean
SONG1, and Hong Bum KOH1*
Sang-Mi RYOU, Ji-Hyun YEOM, Ha-Young GO, and KangSeok LEE*
1
Department of Life Science, Chung-Ang University
*Corresponding author: kangseok@cau.ac.kr
Veterinary Medical Research Center, College of Veterinary Medicine,
Chonnam National University, 2National Veterinary Research and Quarantine
Service
*Corresponding author: hbkoh@chonnam.ac.kr
Fowl typhoid in poultry, caused by S. gallinarum, is a septicemic disease
affecting primarily chikens and turkey. Fowl typhoid has a worldwide
distribution and has been found in almost all poultry-producing areas of
world. The present study was performed to investigate the genotypic
diversities of isolates (epidemiological typing) from jeonnam area in Korea.
Also we analyzed their antimicrobial resistance and detected virulence genes.
hilA gene promoter, component of the Salmonella Pathogenicity Island 1, has
been found in Salmonella serovar typhimurium, being important for the
regulation of type III secretion apparatus genes. We detected hilA gene
sequences in Salmonella serovar Gallinarum by polymerase chain reaction
(PCR). The primers to carry out PCR were designed according to hilA
sequence. 26 isolates of S. gallinarum were detected from liver, spleen and
other lesions past 2 years. They showed 6 different PFGE patterns and 15
antimicrobial resistant phenotypes. hilA gene was identified in all isolates.
The same Gallinarum clone was identified. This clone was identified in
Haman first and next year detected in Gochang, Yeongam, Wanju and Muan
again. These data suggest that the emergence of S. gallinarum probably was
derived from the dissemination of I pattern. hilA gene has been the most
widely studied in S. typhimurium. Our data show that other serovar could have
similar sequences of this kind of virulence genes.
Keywords: Salmonella gallinarum, PFGE, hilA gene, antimicrobial
resistance
F018
Previous work has identified a Streptomyces coelicolor gene, rns,
encoding a 140 kDa protein (RNase ES) that exhibits the
endoribonucleolytic cleavage specificity characteristic of RNase E
and confers viability on and allows the propagation of E. coli cells
lacking RNase E. Here, we identify a putative S. coelicolor 9S rRNA
sequence and sites cleaved by RNase ES. The cleavage of the S.
coelicolor 9S rRNA transcript by RNase ES results in a 5S rRNA
precursor (p5S) that has four and two additional nucleotides at the 5'
end and 3' ends of the mature 5S rRNA, respectively. Nonetheless,
despite the similarities between RNase E and RNase ES, these
enzymes can accurately process 9S rRNA from just their own
bacteria, indicating that these ancient enzymes and the rRNA
segments that they attack appear to have coevolved.
Keywords: RNase E, RNase ES, 9S rRNA, 5S rRNA, p5S, RNA
processing
F020
Pulsed-Field Gel Electrophoresis of QuinoloneResistant Salmonella enterica Serovar Typhimurium
Isolated from Pigs During 2000-2004
1
2
3
Genetic Analysis of a Structural Motif within
the Conserved 530 Stem-loop of Escherichia
coli 16S rRNA
Mi Young KANG , Suk Kyung LIM , Dae Young JEONG , Chul
1
1
Su PARK , and Hong Bum KOH *
Jong-Myung KIM, Hyun-Li KIM, Eun-Kyoung SHIN, Hye-Jeong
HA, and Kang-Seok LEE*
1
Department of Life Science, Chung-Ang University
*Corresponding author: kangseok@cau.ac.kr
Veterinary Medical Research Center, College of Veterinary Medicine,
2
Chonnam National University, National Veterinary Research and Quarantine
Service, 3Chonnam Livestock and Veterinary Research Institute
*Corresponding author: hbkoh@chonnam.ac.kr
Salmonella enterica subsp. enterica serotype typhimurium (S. typhimurium)
is a common cause of salmonellosis among humans and animals in many
countries. S. typhimurium is major zoonotic pathogens worldwide in
animals and humans, it still remains a serious problem. This study was
performed to determine the genotypic diversities of isolates (epidemiological
typing). Also we analyzed their antimicrobial resistance and the presence of
gyrA of quinolone-resistant gene. During 5 years (2000～2004), 48 isolates
of S. typhimurium were detected from mesenteric lymph nodes and feces of
slaughter pigs. They showed 12 different PFGE patterns and 13 antimicrobial
resistant phenotypes. ACSSpSuT resistant phenotypes were showed in 10
isolates (20.83%). The quinolone resistance-determining region of gyrA
gene (313bp) was amplified in all isolates. The same Typhimurium clone
was identified from 2000 throughout 2004. This clone might both persist and
survive in the farmhouse environment or in pigs with sub-clinical infection
which excrete the bacteria. These data suggest that the emergence of S.
typhimurium in 2002 and 2003 probably was derived from the dissemination
of X pattern in 2000.
Keywords: Salmonella typhimurium, PFGE, gyrA gene, antimicrobial
resistance
222
Species-specific Cleavage by RNase E-like
Enzymes in 5S rRNA Maturation
한국미생물학회연합
The 530 stem-loop is a 46 nucleotide stem-loop structure found in all
small-subunit ribosomal RNAs. Phylogenetic and mutational
studies by others suggest the requirement for Watson-Crick
interactions between the nucleotides 505-507 and 524-526 (530
pseudoknot), which are highly conserved. To examine the nature
and functional significance of these interactions, a random
mutagenesis experiment was conducted in which the nucleotides in
the proposed pseudoknot were simultaneously mutated and
functional mutants were selected and analyzed. Genetic analysis
revealed that the particular nucleotide present at each position
except 524 was not exclusively critical to the selection of functional
mutants. It was also indicated that base-pairing interactions between
the positions 505-507 and 524-526 were required for ribosomal
function, while much weaker base-pairing interactions than those of
the wild-type were also allowed for high ribosomal function. Our
results support the hypothesis that the 530 pseudoknot structure may
undergo a "conformational switch" between folded and unfolded
states during certain stages of the protein synthesis process by
interacting with other ligands present in its environment.
Keywords: rRNA, 530 stem-loop, instant evolution, pseudoknot
2005 International Meeting of the Federation of Korean Microbiological Societies
F021
F023
Phylogenetic Relationships Using PCR Finger
Printing for Commercial Strains of Pleurotus
spp.
1
1
2
Ki-Wook KWON , In-Yeup KIM , Hyun-Dong SIN , Won-Sik
KONG1, and Young-Bok YOO1
1
Applied Microbiology Division, National Agricultural Science and
Technology, RDA, 2Department of Environmental Science and Ecological
Engineering, Korea University
*Corresponding author: wskong@rda.go.kr
This study was conducted to determine phylogenetic relationships of
71 commercial strains of Pleurotus spp. using DNA finger printing
based on random amplified polymorphic DNA (RAPD) analysis.
RAPD analysis was carried out using 12 universal rice primers
(URP), operon primer and FGL17 primer. Commercial strains of
Pleurotus spp. were classified into 6 groups: P. ostreatus (62 strains),
P. florida (2 strains), P. sajor-caju (3 strains), P. eryngii (2 strains),
P. abalonus ASI 2072, and Pleurotus sp. ASI 2720. The 62 strains of
P. ostreatus were further classified into 8 groups. FGL17 primer had
a 600 bp specific band on P. ostreatus. This primer was a useful tool
for rapidly detecting and identifying P. ostreatus. There were four
pairs of isolates that had 100% similarity in PCR polymorphic
bands. They were ASI 2180:ASI 2183, ASI 2732:ASI 2733, ASI
2724:ASI 2734 and ASI 2796:ASI 2797.
Keywords: Phylogenetic Relationships, PCR, Pleurotus spp., URP,
Commercial Strains
F022
Oxidative Stress during Toluene Exposure
Cultures of Pseudomonas putida 106
1
2,3
2,4
Ju Soon YOO *, Ja Young MOON *, Dong-Wan KIM *,
Young Kee JEONG5, and Woo Hong JOO6,7*
1
2
Institute of Genetic Engineering Changwon National University, Institute of
Genetic Engineering, 3Department of Biochemistry and Health Sciences,
Changwon National University, 4Department of Microbiology, Changwon
5
National University, Department of Microbiology, Dong-Eui University,
6
Institute of Genetic Engineering, 7Microbiology, Changwon National
University
*Corresponding author: jsyoo701@nate.com
We isolated toluene tolerant Psedomonas putida 106 strain from
wastewater. We have previously studied about the toluene responsible
gene in toluene tolerance bacterial P. putida with 10% toluene at
mRNA level. Several genes were exhibited differential display pattern
in toluene treatment at 0.5 hr to 4 hr. One of the responsible genes,
antioxidant proteins was carried out enzyme activity in order to
stimulation of the expression in protein level. Gene expression of the
antioxidant enzyme, superoxide dismutase(SOD) and catalase(CAT)
was investigated in stationary phase Psedomonas putida 106 during
toluene –induced oxidative stress. Both SOD mRNA levels increased
at 8h exposure to 10% toluene. The cat mRNA induction was no
significantly than sod. However these mRNA levels did not result in
increased protein levels and activities of these enzymes. At 8hr
exposure sample show 8 fold and 15 fold increased than no treatment
time each of SOD and CAT activity. Comparison of toluene exposure
time course and revealed increased activity of antioxidant enzyme,
decreased generation of reactive oxygene species assessed by
2’7’-dichlorofluorescine oxidation.
F024
Gcn4p Degradation Is Occurred by Coordinated
Reaction of Non-ATPase Subunits of 19S
Regulatory Complex
1
2
1
A Pseudomonas aeruginosa PA14 Mutant
Impaired in Competitive Growth Advantage
Upon P. aeruginosa Strains
Kimoon SEONG , Jae-Yung LEE , and Joon KIM *
Yun-Jeong HEO* and You-Hee CHO
1
Department of Life Science, Sogang University
*Corresponding author: youhee@sogang.ac.kr
School of Life Sciences and Biotechnology, Korea University, 2Department of
Biology, Mokpo National University
*Corresponding author: skmhanul@korea.ac.kr
GCN4 is a typical eukaryotic transcriptional activator which is
involved in gene expressions of many amino acids and purine
biosyntheses under stress condition such as nutrient depletion and
UV-irradiation. Gcn4p level is tightly regulated in transcriptional,
translational and post-translational levles. Gcn4p degradation is
occurred in the nucleus by the 26S proteasome and followed by
ubiquitination. Recent data showed that this ubiqutitinated Gcn4p is
recognized by RPN10 of 19S regulatory particle in vitro. In this
study, we investigated whether other non-ATPase subunits of 19S
were involved in the recognition of ubiquitinated Gcn4p and
whether the target gene expression of Gcn4p was affected in each
deletion mutant of non-ATPase subunits of 19S complex in a stress
condition. And we found that degradation of ubiquitinated Gcn4p is
coordinately mediated by non-ATPase subunits of 19S regulatory
complex. The mechanism in degradation of ubiquitinated Gcn4p
will be elucidated further.
Keywords: GCN4, non-ATPase subunits of 19S regulatoty
complex, protein degradation
Pseudomonas aeruginosa PA14 outcompetes several strains
including PAK in mixed planktonic cultures. This competitive
growth advantage is mediated by R-type pyocin that PA14 produces.
We isolated a mutant (115E3) which is highly sensitive to the culture
supernatant of PAK. This mutant retained the killing activity toward
PAK, but was outcompeted in the mixed planktonic cultures with
PAK. 115E3 exhibited reduced swimming and its virulence was
significantly attenuated in plant and mice, but not in Drosophila
melanogaster and Caenorhabidtis elegans models. These phenotypes
are most likely associated with the alterations in lipopolysaccharide
profiles of the 115E3 mutant.
Keywords: pyocin, lipopolysaccharide, Pseudomonas aeruginosa
국제학술대회
223
October 13-14, 2005, Seoul, Korea
F025
F027
Genetic Analysis of Biofilm-Induced Phenotypic
Variations in Pseudomonas aeruginosa PA14
Kelly B. CHOI*, Yun-Jeong HEO, and You-Hee CHO
Regulation of Yeast RPS3 Transcription by Two
Major Yeast Transcription Factors, Gcn4p and
Rap1p
1
1
1
Pseudomonas aeruginosa is a ubiquitous environmental bacterium
and an opportunistic human pathogen capable of forming biofilms
on surfaces as a survival strategy. Here, we studied the colony
morphotypic variations from the laboratory biofilm cultures of
Pseudomonas aeruginosa strain PA14. Two major colony
morphotypes (typical and rugose) were observed from 48 h static
biofilm cultures. We investigated colony variants from various
mutants known to be or predicted to be important in biofilm
formation and found that dsbA, flgK and wspF biofilms exhibited a
single morphotype; only typical for dsbA and flgK vs. only rugose
for wspF. Furthermore, double mutant analyses revealed that wspF
is genetically epistatic to both flgK and dsbA. Based on these, we
propose that the Wsp chemosensory system respond to signals
generated after the initial attachment involving flagella.
Keywords: pseudomonas aeruginosa, biofilm, colony morphotype,
flagella
Laboratory of Biochemistry, School of Life Sciences and Biotechnology,
2
Korea University, Department of Biology, Mokpo National University
*Corresponding author: monm@freecahl.com
RPS3 protein is a component of the ribosomal small subunit and also
has an extra-ribosomal function, an AP endonuclease activity. The
binding site of Rap1p is important for ribosomal protein gene
transcription. By using β-galactosidase reporter system and EMSA,
the UASrpg of RPS3 was identified. Interestingly, RPS3 promoter
region has putative Gcn4p-responsive elements (UASGCRE). This
study revealed that Gcn4p and Rap1p bind to the promoter of RPS3
in vitro. In addition, the physical interactions between Gcn4p and
Rap1p in vitro and in vivo were confirmed. When an amino acid
starvation condition was induced by 3-amino triazole, i.e. 3-AT, or a
rapamycin treatment or post-confluent culture condition, the
transcriptional level of RPS3 appears to be controlled by Gcn4p. In
addition to this it recruits many transcriptional machinery proteins,
when Rap1 acts as transcription activator. This study revealed that
one of these machinery proteins, Esa1, physically associates with
Rap1, and this association decreases under repression conditions of
RPS3 transcription. Therefore, the detailed roles of Rap1, Gcn4 and
Esa1 in the regulation of RPS3 transcription will be discussed.
Keywords: transcription, Rps3, Rap1, Gcn4, Ribosomal gene
F026
F028
A New Temperate Bacteriophage for Pseudomonas
aeruginosa
1
1
2
Proteomic Analysis of Escherichia coli rpoH
Mutant During Post-Irradiation Recovery
1
1
2
2
Yun-Jeong HEO *, Kelly B. CHOI *, Kwan Soo KO , Jae-Hoon
2
1
SONG , and You-Hee CHO
Sangyong LIM , Misong LEE , Minjung KIM , Sangryeol RYU ,
1
and Dongho KIM *
1
1
2
Department of Life Science, Sogang University, Asian-Pacific Research
Foundation for Infectious Diseases and Division of Infectious Diseases,
Samsung Medical Center, Sungkyunkwan University School of Medicine
*Corresponding author: youhee@sogang.ac.kr
A bacteriophage (MP22) was isolated from a clinical strain of
Pseudomonas aeruginosa. It has a coliphage l-like morphology and
a double-stranded DNA genome that approximates 38 kb. Partially
decoded nucleotide sequences indicate that MP22 is closely related
to a temperate, transposable phage, D3112 of the Siphoviridae
family. MP22 requires type IV pili for adsorption to host cells, but
the adsorption is not sufficient for plaque formation on P.
aeruginosa strains.
Keywords: bacteriophage, Pseudomonas aeruginosa, D3112,
type IV pili
224
2
Yoo Jin JOO *, Jae Yung LEE , and Joon KIM
Department of Life Science, Sogang University
*Corresponding author: youhee@sogang.ac.kr
한국미생물학회연합
Advanced Radiation Technology Institute, Korea Atomic Energy Research
2
Institute, Department of Food and Animal Biotechnology, School of
Agricultural Biotechnology, and Center for Agricultural Biomaterials, Seoul
National University
*Corresponding author: fungikim@kaeri.re.kr
In E. coli, a large variety of stress conditions including UV irradiation
induce heat shock response, which is characterized by the synthesis of more
than 20 heat shock proteins under the control of heat shock sigma factor,
RpoH. In this study, the effect of rpoH mutation on post-irradiation recovery
of E. coli was investigated. When E. coli rpoH mutant was exposed to
ionizing radiation (1, 2, and 3 kGy), the lag period before growth resumption
of irradiated mutant strain became longer than irradiated wild type as
increase of irradiation dose. The wild type and rpoH mutant strain were
harvested immediately after exposure to 3 kGy or irradiated cells were
inoculated into fresh LB medium and then harvested for proteomic analysis
using 2-dimensional electrophoresis. Among protein spots showed
significant changes of expression level, 5 protein spots were disappeared
only in the sample prepared from rpoH mutant after re-incubation. Five
proteins were identified as heat inducible lysyl-tRNA synthetase, glutamate
decarboxylase, putative oxidoreductase and protein chain elongation factor
EF-Tu. Of these spots, EF-Tu has been known to be one of proteins induced
by g-irradiation in Deinococcus radiodurans. Concerning the
dose-dependent delay of the growth of rpoH mutant strain, these proteins are
presumably involved in post-irradiation recovery via the rpoH-dependent
regulatory system. Keywords: 2-D electrophoresis, Escherichia coli, gamma irradiation
2005 International Meeting of the Federation of Korean Microbiological Societies
F029
Evaluation of DNA Microarray Approach for
Comparative Genomics Study on Prokaryotic
Genomes
Keum Ok HWANG and Jae Chang CHO*
Department of Envrionment Sciences, Hankuk University of Foreign Studies
*Corresponding author: chojc@hufs.ac.kr
We evaluated the usefulness of DNA microarray for the comparative
genomicstool and tested the validity of the cutoff values for defining
absent genesin test genomes. Three genome-sequenced E. coli
strains K-12, EDL933, andCFT073 were subjected to the
comparative genomic hybridization with DNAmicroarrays covering
almost all ORFs of reference strain K-12, and themicroarray results
were compared to the results obtained from in silicoanalyses of
genome sequences. For defining the K-12 ORFs absent in
testgenomes, we applied and evaluated the cutoff level of -1.
Average sequencesimilarity between ORFs, of which corresponding
spots showed the log-ratioof >-1 was 96.89 ± 4.84. The numbers of
spots showing the log-ratio of <-1 (P <0.05, t-test) were 90 (2.49%)
and 417 (10.63%) for EDL933 genome andCFT073 genome,
respectively. Frequency of false negatives (FN) was ca. 0.2,and the
cutoff level of -1.3 was required to achieve the FN of 0.1.
Averagesequence similarity of the false negative ORFs was 77.76 ±
14.76,indicating that the majority of the false negatives were caused
by highlydivergent genes. We concluded that the microarray is
useful to identifymissing or divergent ORFs in closely related
prokaryotic genomes.
Keywords: Microarray, genome, comparative genomics
F030
F031
Non-AI-2 Quorum-Sensing (QS) Signal Molecule
Produced by Vibrio vulnificus
1
2
1
1
Mi-Ae LEE , Jae-Kyu LIM , Yona CHO , Jong-Bok ROH , and
Kyu-Ho LEE1*
1
2
Department Environ Sciences, Hankuk University Foreign Studies, KORDI
*Corresponding author: khlee@san.hufs.ac.kr
The expression of virulence factors in Vibrio vulnificus is regulated via
cell-density dependent manner. The elastase (VvpE), one of the major
virulence factors, is regulated by autoinducer (AI)-2 synthesized by LuxS.
A putative AI, which was able to induce the vvpE gene expression, was
detected from V. vulnificus spent media. Since this putative AI synthesis
was not influenced by luxS deletion and its addition to V. harveyi BB170
(AI-2 sensor) failed to induce the bioluminescence, it was not believed to
be an AI-2. Instead, this non-AI-2 molecule induced V. harveyi BB886
(sensor for AI-1 [N-acylated homoserine lactone; AHL]). The effort for
its chemical identification was not successful using the standard methods
for AHL. Thus, genetic approach was applied to characterize the nature
of non-AI-2 molecule. The lactonase-deficient mutant of V. vulnificus
was constructed. This mutant showed the same degrees of AI production
and vvpE expression as the wildtype. In addition, overexpressed lactonase
of V. vulnificus did not degrade the putative AI molecule. The BLAST
search of V. vulnificus genomes revealed the orf homologous to the cqsA
of V. cholerae, and the cqsA-null mutant of V. vulnificus was also
constructed. This mutant, however, did not showed any difference in
QS-related phenotypes. Therefore, the non-AI-2 signal molecule of V.
vulnificus is not a typical AHL found in many Vibrio spp., and its synthesis
is independent of CqsA found in V. cholerae.
Keywords: Vibrio vulnificus, Quorum-Sensing, Non-AI-2
F032
Determining the Genome Sizes of Various
Bacterial Species by Modified Pulsed-Field Gel
Electrophoresis
Organization and Regulation of arg operon in
Corynebacterium glutamicum
Yon-Kyoung PARK*, Soohyun YI, Seung-Hwan PARK, and
Jihyun F. KIM
Department of Life Science, Sookmyung Women's University
*Corresponding author: shyim0827@korea.com
Laboratory of Microbial Genomics, Genome Research Center, KRIBB
*Corresponding author: blunight@kribb.re.kr
Gram-positive bacterium Corynebacterium glutamicum is used for
the large-scale production of amino acids. The arg genes were
clustered on the chromosomal DNA and organized in order of
argCJBDFRGH. RT-PCR reactions revealed that there were two
transcripts corresponding to argCJBDFR and to argGH. The
transcription of arg gene expression is regulated by arginine
repressor (ArgR). The argR gene was isolated and characterized.
ArgR protein was encoded by 171 amino acids with a deduced Mr.
18,428Da for monomer and native ArgR protein was 110kDa
meaning that ArgR was a hexamer of equal subunits . ArgR
repressor bound to the promoter region of the argC in the presence of
L-arginine and repressed arginine biosynthetic enzymes in cooperation with L-arginine as the co-repressor. The argR mutant
strain was constructed to investigate function of C. glutamicum
ArgR. The argR mutational analysis shows that C. glutamicm ArgR
controls transcription of the first transcript argCJBDFRGH but not
the second transcript argGH.
Keywords: C. glutamicum, arg operon, regulation
Pulsed-field gel electrophoresis (PFGE) is a technique developed to
resolve extremely large DNA up to megabases in size. For many
wild isolates of bacteria, however, it is often difficult to use due to
the DNA degradation during sample preparation and gel running.
We modified the standard PFGE procedure by changing buffer
conditions to prevent DNA degradation, and tested it with a strain of
Escherichia coli, a lactic acid bacterium isolated from kimchi, and
two species each of paenibacilli and marine bacteria. In particular,
HEPES buffer instead of Tris buffer was used for cell lysis and 200
μM thiourea was added to a running buffer. Dgradation of DNA is
often ascribed to Tris radicals and thiourea is thought to neutralize a
nucleolytic peracid derivative of Tris that is formed at the anode.
With the modified PFGE method, we successfully estimated the
genome sizes of E. coli B REL606, Leuconostoc citreum KM20,
Paenibacillus polymyxa ATCC842, P. polymyxa E681, Hahella
chejuensis KCTC2396 and Kordia algicida OT-1 as 4.4, 2.0, 5.3,
5.5, 7.2 and 4.8 Mb, respectively.
Sei-Hyun YIM* and Myeong-Sok LEE*
국제학술대회
225
October 13-14, 2005, Seoul, Korea
F033
F035
Screening of Oxygen-Responsive Genes using
the hemA Null-Mutant Defective in the First Step
of Heme Biosynthesis in Aspergillus nidulans
Identification of Aspergillus nidulans Genes
Regulated by the Transcription Factor, NsdD
Sun-Hee NOH, Nak-Jung KWON, Kap-Hoon HAN, and
Suhn-Kee CHAE*
Department of Microbiology, School of Biosciences and Biotechnology,
Chungnam National University
*Corresponding author: hmpark@cnu.ac.kr
Department of Biochemistry and Biomed-RRC, Paichai University
*Corresponding author: chae@pcu.ac.kr
Cell senses oxygen concentration changes and activates a hypoxic response
pathway(s), which plays an important role in response to various stresses
induced by low oxygen concentration, when the oxygen concentration is
limited. In eukaryotes, heme plays a central role in oxygen sensing and
promotes transcription of many genes required for oxygen utilization. It is
known that the biosynthesis of heme requires eight enzymes. Among them,
5-aminolevulinate synthase (ALAS) is the first enzyme of heme
biosynthesis. The hemA gene, which encodes ALAS, has been cloned and
sequenced from the filamentous fungus Aspergillus nidulans. HemAp
exhibited high amino-acid similarity to other known ALSA. In this study,
we obtained hemA knock-out mutant. As like other eukaryotes, deletion of
the hemA gene is lethal. But the mutant cells could be rescued by the
supplementation of culture media with 5-aminolevulinic acid (ALA) more
than 100 μM. By using the mutant, it was possible to generate mimicking
heme-deficient (hypoxia) condition by transfer the ΔhemA mutant growing
in ALA supplemented media to no ALA media. Then, the total RNAs from
the two different cultures in a medium with or without ALA were analyzed
for the differentially expressed genes (DEG). The DEGs were re-amplified
and sequenced. The identities of genes isolated will be presented and
discussed. [Supported by ITEP through Biomed RRC]
Keywords: Aspergillus nidulans, Oygen sensing, 5-aminolevulinic
acid, DEG, Hypoxia
F034
Sexual differentiation of A. nidulans is regulated by NsdD (never in
sexual development), which encodes a GATA-type transcription
factor with the type Ⅳb zinc finger DNA-binding domain. Although
expression of the nsdD is detected in the early phase of vegetative
growth, the level increased as sexual development proceeded and
expressed in high level after 30h of sexual induction (SI-30). In order
to identify genes regulated by the NsdD, the DEG analyses were
performed using the RNA preparations from cells of various stages
with wild type and nsdD deletion background. DEG analyses
reveled at least 4 signatures with vegetative cells, 12 with SI-O cells,
and 30 with SI-30 cells. PCR-cloning and sequence analyses of the
11 DEG signatures of the SI-30 revealed one known gene and 12
unknown genes, of which expression might be regulated by the
NsdD. Further analyses for the confirmation and characterization of
these genes will be discussed.
Keywords: Aspergillus nidulans, NsdD, DEG analysis
F036
Combined Global Transcriptome and Proteome
Analysis of Escherichia coli fadR Mutant
1
1
1,2
Jin Hwan PARK , Jeong Wook LEE , and Sang Yup LEE *
1
Metabolic and Biomolecular Engineering National Research Laboratory,
2
Department of Chemical & Biomolecular Engineering, KAIST, Department of
BioSystems and Bioinformatics Research Center, KAIST
*Corresponding author: jinhwan@kaist.ac.kr
Currently, system level approaches using variable omics methods are
available to identify the physiological effect of global regulators. In this
study we examined the regulatory circuit of FadR, global regulator of
E. coli, at both transcriptional and translational level. It negatively
regulates the expression of fatty acid degradative genes, and positively
regulates the expression of fatty acid biosynthetic genes. In this study,
the carbon source-responsive global effect of fadR knockout was
identified by transcriptome and proteome profiling in the presence of
glucose or oleic acid as a carbon source. Growth-associated regulation
of FadR was also identified by clustering genes according to cell
concentration. Interesting result from the proteome data is that AceA
(isocitrate lyase) was not increased at all at translational level in
contrast with the transcriptome result that showed much higher
expression level in fadR knockout mutant. We can conclude that the
different regulatory mechanism exists at translational level.
Acknowledgements : This work was supported by a grant from the
Korean Ministry of Science and Technology (Korean Systems Biology
Research Grant, M10309020000-03B5002-00000) and by the Brain
Korea 21 Project. Further support through the LG Chemicals Chair
Professorship is appreciated.
Keywords: fadR, transcriptome, proteome, global regulator
226
Suk-Yi WOO, Yun-Hee PARK, and Hee-Moon PARK*
한국미생물학회연합
Cloning and Sequencing of Eubacterium A-44
astA Gene, Encoding an Arylsulfate Sulfotransferase
Bomi KIM*, Keun-Sook LEE, Yang-Jin HYUN, Ha-Yeon NOH,
and Dong-Hyun KIM
College of Pharmacy, Kyung Hee University
*Corresponding author: bomi0126@hotmail.com
A gene-encoding arylsulfate sulfotransferase (ASST) was cloned
from a Eubacterium A-44 genomic library. ASST transfers a sulfate
group from phenolic sulfate esters to a phenolic acceptor substrate.
The primers were produced through internal amino acid sequencing
of ASST purified from Eubacterium A-44, and the probe (1.5 kb
fragment) was prepared from the PCR product of the primer. The
astA gene was subcloned into vector pkf3 and sequenced.
Recombinant clone-harbouring astA was directly identified using
the probe. The nucleotide sequencing revealed an open reading
frame (ORF) of 1863 bp encoding a protein of 620 amino acids with
a secretory signal sequence. When searching the database for astA
nucleotide or its deduced amino acid sequence, the sequence of the
astA showed homology of 91% to Eubacterium rectale IIIH
previously reported.
Keywords: sulfotransferase; Eubacterium; gene cloning
2005 International Meeting of the Federation of Korean Microbiological Societies
F037
Streptomyces coelicolor A3(2) Zur Regulate the
Transcription of znuACB, zur Encoding High
Affinity Zn(II) Uptake Transporter and Regulator
Jung Ho SHIN*, So Young OH, and Jung Hye ROE
Laboratory of Molecular Microbiology, School of Biological Sciences, Seoul
National University
*Corresponding author: dicmedic@empal.com
Streptomyces coelicolor A3(2) has four Fur homologs. The rest Fur
homolog is SCO2508. SCO2508 is similar to rather Fur than Zur of B.
subtilis and E. coli. First of all, we closely examined the gene structure
around the SCO2508. Putative metal uptake transporter was located on
the upstream region of SCO2508. This putative metal uptake transporter
was consist of znuA, znuC and znuB. Each gene was similar to znuA
(encoding membrane binding lipoprotein ), znuC (encoding the ATPase)
and znuB (encoding membrane permease) of high affinity Zn(II) uptake
transporter in E. coli. Also, we compaired the gene structure of Zn(II)
uptake system within another bacteria. Zn(II) uptake system of
Mycobacterium leprae and Bifidobacterium longum: BL1128 as well as
S. coelicolor was located on upstream region of Fur homolog, but was not
in E. coli, Salmonella typhi and Yersinia pestis. Next, we made the
mutant(ΔSCO2508) for checking the regulation to znuACB and by itself.
Actually, SCO2508 act as a repressor to znuACB but actively regulate
by itself. Also, we examined metal specificity and optimal concentration
of Zn(II) to regulate of this putative metal uptake transporter and
regulator(SCO2508). From this result, we obtained the fact that this
putative metal uptake transporter is the Zn(II) specific high affinity
uptake transporter and SCO2508 is the Zn(II) specific regulator.
Therefore, we named SCO2508 as Zur.
Keywords: Zn uptake, Streptomyces, Zur
F039
Analysis of Gene Cluster for Salbostatin Biosynthesis
1
2
Yong Hoon CHOENG , Yong Keun CHANG , and Soon Kwang
HONG3*
1
2
Department of Biological Science, Myongji University, Department of
Chemical and Biomolecular Engineering, KAIST, 3Department of Biological
Science, Myongji University
*Corresponding author: skhong@mju.ac.kr
The salbostatin was isolated from the fermentation culture of the polyehter
antibiotic salinomycin producer Streptomyces albus. This component is a
basic, non-reducing pseudodisaccharides which consists of valienamine
linked to 2-amino-1, 5-anhydro-2-deoxy glucitol. Valienamine is known as
a component of the validamycin-type aminoglycoside antibiotics, and of
trehalase inhibitors. To study the biosynthetic pathway of the salbostatin, the
highly conserved gene sequences of the 2-epi-5-epi-valiolone synthase were
used as a probe for related genes in the salbostatin producer Streptomyces
albus KCTC 9015. In this way, about 20Kb DNA was isolated that contains
19 genes putatively involved in the biosynthesis of the salbostatin. These 19
genes were encoding proteins with significant similarity to N-acetylglucosamine6-phosphate deacetylase, Sugar kinase, Trehalose-phosphate synthase,
Transporter, Trehalose-6-phosphate phosphatae, ADP-lucose synthase,
regulatory enzyme, Glycosyltransferase, Trehalose phosphorylase, reductase,
Transporter, 2-epi-5-epi-valiolone 7-kinase, Cyclitol dehydrogenase,
Cyclitol oxidoreduxtase, Valiolone-7-phosphate 2-epimerase, Hydrolase,
2-epi-5-epi-valiolone synthase, NTP-pyrophosphohydrolases, Quinone
oxidoreductase, respectively.[Supported by the driving force project for the
next generation of Gyeonggi provincial government.]
Keywords: salbostatin, trehalase inhibitor, valienamine
F038
F040
Interaction among a Reducer System of SoxR, a
Superoxide Sensor in Escherichia coli.
Molecular Study on the SghC Identified from the
A-factor Deficient Streptomyces griseus HH1
Kyung-Chang LEE*, Kang-Lok LEE*, and Jung-Hye ROE*
Yoon-Hee KIM , Won-Jae CHI , Yong-Hoon CHOENG , and
2
Soon-Kwang HONG *
Laboratory of Molecular Microbiology, School of Biological Sciences and
Institute of Microbiology, Seoul National University
*Corresponding author: lgchang1@snu.ac.kr
We have identified the rsxABCDGE operon (reducer of SoxR) and
the rseC gene in the rpoE-rseABC operon, which affect the SoxR
redox state, with mutant library carrying a chromosomal
soxSp::lacZ fusion. Previous data shows that SoxR reducer system
is composed with RseC and Rsx Proteins. From mutant studies, it is
clear that all components participate in the SoxR reduction. But
specific roles of each protein are unknown. RsxB and RsxC have
ferredoxin motif; which RnfB and RnfC, homologues on
R.capsulatus, have ferredoxins; to transfer electron but other
proteins don’t have well-known motif. And other proteins are almost
membrane proteins except RsxG. These situations suggest that all
components can make complex. Bacterial two hybrid system shows
interaction among RseC and Rsx Proteins. Protein motif search
shows RsxG is a periplasmic protein, which have FMN binding site.
Topology of RsxG is tested with signal-sequence-less PhoA fusion.
Keywords: soxR, rsx operon
1
2
2
1
2
DyneBio Inc, Department of Biological Science, Myongji University
*Corresponding author: skhong@mju.ac.kr
A-factor acts as a switch for aerial mycelium formation and secondary
metabolite formation in Streptomyces griseus. In the present study, one
of the proteins, protien-C, was purified by ion-exchange
chromatography from the A-factor deficient mutant strain, S. griseus
HHI. The Protein-C coding gene was cloned by PCR method and
Southern hybridization. Deduced amino acid sequence from the
nucleotide sequence suggests that protein-C is produced as a precursor,
consisting of an amino-terminal prepro sequence (32amino acids) and
a mature protein (182 amino acids). The gene coding for protein-C was
named as sghC. When the sghC gene was introduced into the S. lividans
TK24 and S. coelicolor A3(2) M145 strain, the production of
actinorhodin and undecyprodiosin was repressed and the degree of
aerial mycellial formation and sporulation was decreased. On the other
hand, aerial mycelium formation and sporulation of S. griseus
IFO13350 were slightly reduced on R2YE solid medium. The alkaline
phophatase activities of S. lividans and S. griseus harboring sghC gene
was increased by 2～ 2.5 fold. Disruption of sghC recoverd aerial
mycelium formation and sporulation of the S. griseus IFO13350. The
aerial mycelium and spore formation of △sghC was slightly increased
on R2YE medium and YMP medium containing 3% glucose or
sucrose. [Supported by the driving force project for the next
generation of Gyeonggi provincial government.]
Keywords: Streptomyces griseus, A-factor, morphological and
physiological differentiation
국제학술대회
227
October 13-14, 2005, Seoul, Korea
F041
F043
The Phosphate Starvation Response of Escherichia
coli Determined by DNA Microarray and Realtime PCR Analyses
1
Jong Hwan BAEK and Sang Yup LEE
2
1
Department of Chemical and Biomolecular Engineering, KAIST,
Department of Chemical and Biomolecular Engineering, Department of
Biosystems and Bioinformatics Research Center, KAIST
*Corresponding author: swcry@kaist.ac.kr
2
When E. coli W3110 strain was grown under phosphate limiting condition,
the changes of global gene expression levels were investigated by using
DNA microarrays. Expectedly, the expressions of some genes relating to
phosphate uptake and metabolism were increased as phosphate was starved,
whereas the expressions of some genes for ribosomal protein and amino acid
metabolism were decreased. The regulated genes could be divided into
temporarily and permanently inducible genes. Especially, real-time PCR
analysis revealed the expressions of phoB and phoR peaked just after
phosphate exhaustion and decreased afterward, suggesting the high stability
of PhoB and PhoR for learning behavior. At the peak point, the phoB- and/or
phoR-dependent regulation was also investigated by comparing the gene
expression levels among the wild type and phoB and/or phoR knock out
mutant strains. Overall, it seems that phoB allele was epistatic to phoR
mutation. And it was found that the genes for phosphate metabolism and
transport system were under phoB- and/or phoR-dependent up-regulation.
Surprisingly, some genes such as amn, tktB, xasA, yibD, ytfK also showed
phoB- and/or phoR-dependent up-regulation and had putative pho box. This
indicates that some genes, which are not directly related with phosphate
assimilation, are controlled by PhoB and/or PhoR, showing the role of PhoB
as a global regulator. [Supported by Korean Systems Biology Research
Grant, LG Chem Chair Professorship, and IBM SUR program]
Keywords: phosphate starvation, phoR-phoB two-component
regulatory system, DNA microarray, real-time PCR
F042
1
1
1
Jeong-Uck PARK , Young-Cheol KWON , Jeong-Won PARK ,
Jin-Su JUN2, Seung-Gyu LEE1, Kyung-Mi KIM1, Mi-Hyun KIM1,
1
1
1
Jung-Soo JOO , Eun-Jin HA , Damir NIZAMUTDINOV ,
1
1
1
Jae-Young SONG , Seung-Chul BAIK , Hyung-Lyun KANG ,
2
1
1,3
Hee-Shang YOUN , Woo-Kon LEE , Myung-Je CHO *, and
1
Kwang-Ho RHEE
1
Department of Microbiology, Gyeongsang National University College of
Medicine, 2Department of Pediatrics, Gyeongsang National University
3
College of Medicine, Research Institute of Life Science, Gyeongsang National
University
*Corresponding author: mjecho@gaechuk.gsnu.ac.kr
The roles of accessory gene products activating the Helicobacter
pylori urease apoprotein were examined by in vivo- and in vitro
activation. The activity of the urease apoprotein increased when it
was expressed with the accessory genes in the following order: ureG
< ureGH < ureFGH < ureEFGH < ureIEFGH. Moreover, stepwise
additions of ureE and ureI to ureFGH resulted in significant
increases in urease activity. The in vivo – and in vitro activation
studies shared that the cooperation of accessory proteins was carried
out by processes in which the UreFGH complex, UreE, and UreI
were involved. Thus, the UreFGH complex may serve to alter the
conformation of the H. pylori urease apoproteion into one that is
more competent to assemble a stable metallocenter, and that
facilitates the cooperative actions.
Keywords: Chymotrypsin Susceptibility, Conformation, Helicobacter
pylori, Urease Accessory Gene
F044
Oxidative Stress Dependent Interaction of Fission
Yeast LAMMER Kinase, Lkh1, with ATPDependent Pre-mRNA Splicing Factor, Prp16
Effect of Secretion and Surface-Display of SipB
N-terminus on Immune Response Against
Heterologous Antigen TTFC
Byoung-Cheol LEE*, Won-Hwa KANG, and Hee-Moon PARK
Jung Im JANG, Jin Seok KIM, Hyeon Guk KIM, Won Cheol
CHOI, Yoo Chang PARK, and Yong Keun PARK*
Department of Microbiology, School of Biosciences and Biotechnology,
Chungnam National University
*Corresponding author: cross872@hotmail.com
Previously, we have reported that Schizosaccharomyces pombe
Lkh1 is a member of LAMMER protein kinase family having
autophosphorylation and dual specificity protein kinase activity, and
is involved in oxidative stress response. Through pull down assay
with MBP-tagged Lkh1, and subsequent Mass spectrometry, we
identified Prp16, which is a ATP-dependent RNA helicase, and a
member of DEAH-box RNA helicase, as an interacting protein.
Prp16 was phosphorylated by Lkh1 in vitro, and interacted with
Lkh1. Interestingly, interaction between Prp16 and Lkh1 in vivo was
dependent on oxidative stress. This result indicated that Lkh1 may
regulate pre-mRNA splicing by modulating Prp16 activity under
oxidative stress. Keywords: Lkh1 ; oxidative stress ; Prp16 ; pre-mRNA splicing
228
The in vivo- and in vitro Activation of Urease
Accessory Genes from Helicobacter pylori
한국미생물학회연합
School of Life Sciences and Biotechnology, Korea University
*Corresponding author: ykpark@korea.ac.kr
It has been known that SipB, one of Salmonella invasion proteins
(Sips), is secreted into the culture supernatant and displayted on
cell-surface via the type III secretion system. In previous study, we
fused tetanus toxin fragment C (TTFC) to N-terminal 160 amino
acids region of SipB (pSFC160). Using ELISA with the mouse
serum immunized by attenuated Salmonella harboring pSFC160, it
was shown that immunized mouse produced more anti-TTFC
specific IgG than unimmunized mouse. Secretion signal of SipB
reside in N-terminal 2~8 amino acids and surface-localization signal
may be existed in N-terminal 100~140 amino acids. Whether
surfac-display make the synergistic effect on presentation of TTFC
to immune system or not, we make SipB40 which possess only N
terminal 40 amino acids and transformed resulting construct
(pSFC40) into the attenuated Salmonella strain. Although normally
expressing and secreting the recombinant SipB40-TTFC protein
into culture supernatant, SFC40 did not directed surface localization
of the SipB40-TTFC by subcellular fractionation.In this study, we
compared the immune response of pSFC160 with pSFC40 using
ELISA and SFC160 was better than SFC40 in vaccine strategy. Keywords: Salmonella, SipB, TTFC, vaccine
2005 International Meeting of the Federation of Korean Microbiological Societies
F045
F047
Cloning and Complete Nucleotide Sequence
Analysis of Ustilago maydis Virus (UmV) SH4-M
and SH8-L dsRNA Segment Isolated in Korea
Isolation and Characterization of Temperate Phage
from Lysogenic Leuconostoc pseudomesenteroides
Su-Ye SUNG*, Ji-Yeon KIM, and Se-Won YIE
School of Life Sciences and Biotechnology, Korea University
*Corresponding author: hichang@korea.ac.kr
Division of Life Sciences, College of Natural Sciences, Kangwon National
University
*Corresponding author: shakeforever@nate.com
Ustilago maydis virus (UmV) is a double-stranded RNA (dsRNA) virus
of the corn smut fungus Ustilago maydis. The UmV genome consists of
three distinct size groups of dsRNA segments: H (heavy), M (medium)
and L (light). UmV SH series (SH1~14) was isolated from Ustilago
maydis from corn smut in Korea. Full-length cDNAs of SH4-M dsRNA
and SH8-L dsRNA were cloned by poly (A) tailing followed by RT-PCR.
UmV SH4-M and SH8-L dsRNA had genomes of 1,008 and 420
nucleotides, respectively. The sequence of SH4-M dsRNA had 111 and
513 nucleotides of 5'- and 3'- untranslated region (UTR) while SH8-L
dsRNA had 87 and 117 nucleotides of 5'- and 3'-UTR, respectively.
Computer analysis of putative open reading frame (ORF) shows that
SH4-M and SH8-L contain a single ORF proteins of 13.7 and 8.0 kDa.
Some UmV subtypes have viral dsRNAs encoding secreted toxins that
kill sensitive cells of the same species and related species. SH4-M show
high nucleotide sequence identity (96%), and high amino acid sequence
identity (86%) with that of P4KP4, UmV subtype P4 strain secreted killer
toxin. Therefore, it appears that SH4-M dsRNA encodes killer toxin
similar to M dsRNA of P4 strain. The L segments of UmV have been
suggested that they are derived from one end of the larger M segments.
However, SH8-L dsRNA does not have any sequence homology with
known nucleotide sequence and amino acid sequence of UmV. The study
of the function of SH8-L dsRNA are in process.
Keywords: Ustiago maydis virus, dsRNA, full-length cDNA
Mi-Hee HWANG and Hyo-Ihl CHANG*
A new temperate phage was induced from Leuconostoc pseudomesenteroides
with Ultraviolet light treatment in about 3 hour after the intracellular replication.
The morpology of the phage was examined by electron microscope. This
bacteriophage has an icosaheadral head of 60.5nm in diameter and a sheathless
noncontractile tail of approximately 144nm in length. Restriction endonuclease
analysis of the genome from the phage showed that it had about a 40Kb
double stranded DNA. Southern hybridization analysis showed that this
phage DNA was incorporated in the host chromosomal DNA as a prophage.
Sequence analysis revealed that 1,071-nucleotide ORF was encoded a
putative site-specific recombinase which has a significant homology with
other recombinases. To characterize the integrase, the gene of putative
integrase was introduced into the pET32a fusing thioredoxin to the 5' end
of the gene, and purified by Ni-NTA regin.
Keywords: bacteriophage, UV induction, integrase
F046
F048
Protein Expression Profiling of Murine Macrophage
Cells Treated with Sub-lethal Dose of the Anthrax
Lethal Toxin
Proteome Reference Map and Comparative
Proteome Analysis of Two Developmental Stages
in Pleurotus ostreatus
Kyoung Hwa JUNG, Gwi-Moon SEO, Seong-Joo KIM, Ji-Chon
KIM, Seon Mi OH, and Young Gyu CHAI*
Joong Ho JOH , Nam Kuk KIM , Jong Hyun LIM , Min Jin SONG ,
2
2
1
Won Sik KONG , Young Bok YOO , and Chang Soo LEE *
Hanyang University Division of Molecular and Life Sciences
*Corresponding author: ygchai@genopia.net
1
Intoxication of murine macrophages (RAW 264.7) with the
sub-lethal dose of the anthrax lethal toxin (LeTx 100 ng/ml) results in
profound alterations in the host cell gene expression. The role of LeTx
in mediating these effects is unknown, largely due to the difficulty in
identifying and assigning function to individual proteins. In this
study, we have used two-dimensional electrophoresis to analyze the
protein profile of murine macrophages treated with the LeTx, and
have coupled this to protein identification using MALDI-TOF mass
spectrometry. Interpretation of the peptide mass fingerprint data has
relied primarily on the ProFound database. Among the differentially
expressed spots, mitogen-activated protein kinase kinase (Mek1) and
glucose-6-phosphate dehydrogenase were increased in the LeTx
treated macrophages. Mek1 acts as a negative element in the signal
transduction pathway, and G6PD plays the role for the protection of
the cells from the hyperproduction of active oxygen. Our results
suggest that this proteomic approach is a useful tool to study protein
expression in intoxicated macrophages and will contribute to the
identification of second substrate for LeTx.
Keywords: anthrax lethal toxin, murine macrophage, proteomics,
cytosolic protein
1
1
1
1
Department of Applied Biochemistry, College of Biomedical and Health
2
Science, Konkuk University, Applied Microbiology Division, National
Institute of Agricultural Science and Technology
*Corresponding author: joh72@kku.ac.kr
During the developmental process of basidiomycetes, dramatic
morphogenetic changes occur. Genetic analyses for developmental
process were initiated early, but the proteome analysis of basidiomycetes
is not sufficient. Two-dimensional electrophoresis (2-DE) and matrix
assisted laser desorption/ionization-time of flight mass spectrometry
(MALDI-TOF MS) were used to analyze proteome of Pleurotus
ostreatus. Proteins isolated from fruiting bodies and mycelia were
separated in immobilized pH gradient (3-10NL) strips and 12.5 %
SDS-polyacrylamide gel. More than 700 protein spots were detected on
each two-dimension gel. Based on peptide mass fingerprinting, more than
56 spots were identified until now. The differentially expressed proteome
between fruiting body and mycelium were also selected. The 74, 66 and
104 spots were significantly expressed during fruiting body, mycelia and
both stages, respectively. Therefore, more than 160 spots including these
specific spots are analyzing now. This study is the first step toward the
establishment of a reference proteome map of P. ostreatus and will
provide genetic and biochemical basis for the developmental studies of
basidiomycetes. All results are exhibited in Bioresourse Information
Center of Konkuk university (AB-BIC, www.ab-bic.com).
Keywords: Pleurotus ostreatus, Proteome map, Mushroom
development
국제학술대회
229
October 13-14, 2005, Seoul, Korea
F049
F051
Cloning and Disruption of a Gene Encoding the
γ–Butyrolactone Autoregulator Receptor from
Streptomyces ambofaciens
Structural and Genetic Analysis of Cyclic
Lipopeptide Surfactin Synthesized by Bacillus
pumilus HY1
Mi-Kyong KIM, Mi-Hyun KIM, Sun-Uk CHOI, Hae-Ryong PARK,
and Yong-Il HWANG*
Su Young HONG , Kye Man CHO , Sun Mi LEE , Young Hee
KIM1, Hoon KIM2, and Han Dae YUN1*
Division of Food Science and Biotechnology, Kyungnam University
*Corresponding author: suchoi@kyungnam.ac.kr
1
A gene encoding a γ-butyrolactone autoregulator receptor, which has
a common activity as DNA-binding transcriptional repressors
controlling secondary metabolism and/or morphological differentiation
in Streptomyces, was cloned from a spiramycin producer,
Streptomyces ambofaciens. PCR using the primers designed from
for the two highly conserved regions of Streptomyces autoregulator
receptors gave a 102- bp band. The sequence of this band had a high
similarity to the expected region of a receptor gene. By genomic
Southern hybridization with the 102-bp insert as a probe, an intact
receptor gene (saaR) was obtained from S. ambofaciens. To clarify
the in vivo function of saaR, construction of saaR-deleted mutant is
now progressing using homologous recombination. To achieve an
effective transformation procedure for S. ambofaciens, we
established an efficient procedure for the conjugal transfer of DNA
from E. coli to S. ambofaciens using a ΦC31-derived integration
vector, pSET152, containing oriT and attP fragments, as well as a
pKC1132-derived homologous recombination vector, containing a
4.7-kb fragment of S. ambofaciens.
Keywords: γ-Butyrolactone autoregulator, Streptomyces ambofaciens,
Spiramycin
1
1
Division of Applied Life Science, Gyeongsang National University,
Department of Agricultural Chemistry, Sunchon National University
*Corresponding author: atpase1123@naver.com
2
Surfactin, a cyclic heptalipopeptide produced by various strains of
bacilli, behaves as a very powerful biosurfactant and possesses
several interesting biological activities. We carried out a hemolysis
zone assay, a surfactin-producing Gram positive bacterium, identified
as Bacillus pumilus HY1, was isolated from the Korean traditional
soybean sauce. Structural analysis showed that the purified surfactin
had five isoforms with protonated masses of m/z 994, 1,008, 1,022,
1,036, and 1,050 using MALDI-TOF MS and ESI-MS/MS. In the
MS/MS analysis, the isolated surfactin had the identical amino acid
sequence (LLVDLL) and hydroxy fatty acids (with 12 to 16 carbons
in length), even though isolated from different Bacillus strains. A
large 32 kb DNA sequence encoding srf operon required for surfactin
biosynthesis was cloned and sequenced using cosmid library from
strain HY1. Three large open reading frames coding for surfactin
synthetases, designated srfA, srfB, and srfC, could be detected. The
srf operon showed >70% identity on the amino acid level to the
corresponding genes of B. subtilis subsp. subtilis 168, B.
amyloliquefaciens FZB42, and B. subtilis KCCM80007.
Keywords: Bacillus pumilus HY1, surfactin, MALDI-TOF MS,
ESI-MS/MS, srf operon
F050
F052
Identification of Quorum Sensing Signalling
Molecules from Bacillus pumilus HY1
1
1
1
Su Young HONG , Kye Man CHO , Sun Mi LEE , Young Hee
1
2
1
KIM , Hoon KIM , and Han Dae YUN *
1
Division of Applied Life Science, Gyeongsang National University,
Department of Agricultural Chemistry, Sunchon National University
*Corresponding author: atpase1123@naver.com
2
Many bacteria produce and respond to signal molecules depending on
high cell density. Natural genetic competence in a few Bacillus species
is controlled by quorum sensing mechanism. We first report the quorum
sensing genes of Bacillus pumilus HY1 isolated from Korean traditional
soybean sauce, and identified the production of ComX pheromone using
ComX-indicator strain. The comQXPA loci of strain HY1 sequenced
and compared the sequences of the quorum-sensing genes from three
closely related bacilli. A comparison of the amino acid sequences
revealed striking variation in the primary structures of ComQ (44-47%
identity), ComX (22-31%), the sensor domain of ComP (47-56%), and
ComA (70-83%) between Bacillus species. Furthermore, we
overexpressed comQ and comX together in Escherichia coli and found
the two amino acid sequence of post-translational modified ComX
pheromone with geranyl pyrophosphate at conserved tryptophan
residue using LC-MS, ESI-MS/MS. These results show that comQ and
comX are required for production of ComX pheromone, the comQXPA
loci of a set of Bacillus species possess a striking polymorphism that
determines specific patterns of both activation and inhibition of the
quorum-sensing response.
Keywords: Bacillus pumilus HY1, quorum sensing, comQXPA,
ComX pheromone
230
1
한국미생물학회연합
Multifunctional and Multidomain Glycosyl
Hydrolases of Endophytic Paenibacillus polymyxa
GS01 from the Interior of Ginseng Roots
1
2
1
Kye Man CHO , Goong Gjung KAHANG , Su Young HONG ,
1
1
1
Sun Mi LEE , Yong Hee KIM , and Han Dae YUN *
1
Division of Applied Life Science, Gyeongsang National University,
Department of Food Science, Chinju National University
*Corresponding author: atpase1123@naver.com
2
Endophytes are viewed as a new source of genes, proteins and
biochemical compounds that may be used to improve industrial
processes. In this study, the gene cel44C was cloned from a ginseng
endophytic Paenibacillus polymyxa GS01. The Cel44C encodes β
-1,4-endoglucanase, β-xylanase, 1,3-1,4-β-D-glucanse (lichenase),
and β-mannase capable of hydrolyzing cellulose, xylan, lichenan, and
mannan under in vitro conditions. The predictedprotein, Cel44C, has
high homology to other bacterial cellulases and mannase and shows
a modular structure containing a catalytic domain of the glycosyl
hydrolase family 44 (GH44) and glycosyl hydrolase family 26
(GH26) and a fibronectin domain type 3 and a cellulose-binding
module type 3 (CBM3). The enzyme was expressed in Escherichia
coli BL21 (DE3) purified to homogeneity, and characterized. The
full-length Cel44C have an optimum at pH 7 (cellulase and lichenase)
and pH 5 (xylanase and mannase), and an optimum at temperature 5
0℃ and the truncated Cel44C have an optimum at pH 7 (lichenase),
pH 6 (xylanase and mannase), and pH 5 (cellulase), and an optimum
at temperature 50℃. A truncated cel44C gene was constructed with
the addition of a nonsense mutation that removes the C-terminal
region of the protein. There truncated Cel44C proteins showed an
enzymatic activity 70 to 200% higher than that of full-length Cel44C.
Keywords: Paenibacillus polymyxa GS01, Cel44C, Ginseng Root
2005 International Meeting of the Federation of Korean Microbiological Societies
F053
F055
Isolation of Endophytic Bacteria and Assessment
of Diversity in the Interior of Ginseng Roots
1
2
2
Goong Gjung KAHANG , Kye Man CHO , Su Young HONG ,
Sun Mi LEE2, Yong Hee KIM2, and Han Dae YUN2*
1
2
Department of Food Science, Chinju National University, Division of
Applied Life Science, Gyeongsang National University
*Corresponding author: atpase1123@naver.com
The composition and diversity of the endophytic bacterial community was
associated with plant roots. Endophytic bacteria are ubiquitous in most
plants and colonies plants without exhibiting pathogenicity. In this study,
the diversity of endophytes associated with the roots of ginseng plants grown
at three different cultivation areas in Korea. The microbial populations were
analyzed the phylogenetic relationships among 16S rDNA sequences by
PCR amplification from genomic DNA extracted from sixty-three colonies.
The phylogenetic analyzes of 16S rDNA sequences showed that the isolates
belonged to three major phylogenetic group: the high G+C gram positive
bacteria (HGCGPB), low G+C gram positive bacteria (LGCGPB), and the
Proteobacteria group. The dominant species of the three different ginseng
growing sites were HGCGPB (Ganghwa, 55.0%), LGCGPB (Geumsan,
45.5%) and Proteobacteria (Jinan, 61.9%). The phylum LGCGPB and
Proteobacteria in sixty-three isolates were related the antifungal activity
against Rhizoctonia solani and hemolytic activity. However, except for
Pectobacterium carotovora in the phylum Proteobacteria, the cellulase,
xylanase and pectinase producing colonies in the endophytic bacteria
belonged to the phylum LGCGPB. Nine colonies of thirteen isolates were
occupied the genus Bacillus (including the genus Paneibacillus) that was
showed the antifungal activity. Thirteen isolates had potentially useful
biological control agent against phytopathogenic fungi.
Keywords: Endophytic bacteria, diversity, 16S rDNA, antifungal
activity
F054
Eun-Kyoung LEE, Youn-Jeong LEE, Jun-Gu CHOI, Ok-Mi
JEONG, Yong-Kuk KWON, Jun-Hun KWON, and Jae-Hong
KIM
Avian Disease Division, National Veterinary Research and Quarantine Service
*Corresponding author: eklee@nvrqs.go.kr
Infectious bronchitis (IB) is an acute, highly contagious disease of
the respiratory and urogenital tracts of chickens caused by a
coronavirus, infectious bronchitis virus (IBV). In Korea, the first
outbreaks of IB were reported in 1986, and nephropathogenic IB
was recognized in 1990. In previous study, Korean IBV isolates
were classified into three genetic groups which included respiratory
type of Korean group 1, nephropathogenic type of Korean group 2
and Massachusetts group. Therefore, inactivated IBV vaccines
including IBV reference strain, M41 and Korean nephropathogenic
strain, KM91 were used in Korea since 1997. In this study, we
analyzed evolution of recent IBV in Korea by phylogenetic analysis.
The phylogenetic trees were constructed based on S1 complete
gene and hypervariable region of S1 gene. In addition to
already-known groups, new group of Korean IBV emerged since
2003. Further study was need to define whether the emergence of
new group was related to isolates in China and Japan or related to
immunologic pressure caused by use of vaccine with Korean
nephropathogenic strain.
Keywords: Infectious bronchitis virus (IBV), nephropathogenic IB,
phylogenetic analysis, S1 complete gene, hypervariable region of S1
gene
F056
The Study about Functional Domain of SipB N-terminal 160 Amino Acids
1
1
1
Hyeon Guk KIM , Jin Seok KIM , Jung Im JANG , Won Cheol
1
1
2
CHOI , Yoo Chang PARK , Sung Ho BANG , and Yong Keun
1
PARK *
1
Phylogenetic Analysis of Avian Infectious
Bronchitis Virus Strains Isolated in Korea
2
School of Life Sciences and Biotechnology, Korea University, Department of
Biology, Hanseo University
*Corresponding author: ykpark@korea.ac.kr
It has been known that SipB, one of invasion proteins encoded in
Salmonella pathogenisity island 1 (SPI-1), is secreted outside cell
and localized on outermembrane. However, a functional domain
essential to surface localization of SipB is not investigated yet. In
this study, it was shown that not only the first ~ 160 amino acids of
SipB N-terminal sequence was directed on outermembrane as like
naïve SipB but also the fragment could direct the recombinant reporter
proteins to outermembrane. The deletion plasmids (containing only
30, 72, 100, 120 and 140 amino acids from N-terminal) showed that
the 100 ~ 140 amino acids fragment of SipB was indispensable to the
localization to outermembrane. Proteinase K susceptibility and
immunofluorescence assay indicated that SipB was not incorporated
into outermembrane but displayed bacterial surface. We also
showed that N-terminal 30~100 amino acid of SipB interacted with
its cognate chaperone, SicA
Keywords: salmonella, sipB, type III secretion
Studies on the Function of Fur and Pathogenesis
of Pseudomonas syringae pv.tabaci
So Young PARK*, Ji Young CHA, Hyun Ju YANG, Jun Seung
LEE, Tae Woo KIM, and Hyung Suk BAIK
Division of Biologial Sciences, Pusan National University
*Corresponding author: hush1111@naver.com
Many bacteria use the low iron concentration in a host as an
important signal to enhance the expression of a wide variety of
bacterial toxin and other virulence determinants. Environmental iron
concentrations coordinately regulate transcription of genes involved
in acquisition and virulence via the ferric uptake regulation (fur)
system. In this study, to investigate the global regulation by Fur in
response to iron in P. syringae. pv. tabaci that causing wild fire
disease in tobacco, we amplified fur gene by PCR using designed
primer and identified it by sequencing. The sequence of the fur gene
revealed a high degree of identity to its homology in other P.
syringae strain. We have constructed an fur deletion mutant through
allelic exchange method and characterized using chrome azurol S
phenotype assays. Physiological studies indicate that fur mutant is
similar to the wild-type strain when they were compared with
growth pattern, toxicity test, pathogenicity test. We compared the
proteome profile of the wild type and its isogenic fur mutant by
Two-dimensional gel electrophoresis grown under iron-rich and
iron-depleted condition. Six major protein were quantitatively
increase in mutant type cells relative to wild type cells while nine
proteins were decrease in mutant under iron-depletion condition. In
addition, ten protein were Fur-independent. We are now investigating
that analyzed the proteins regulated by iron using the MALDI-TOF
mass and purifying Fur.
Keywords: Iron uptake, Fur, Pathogenicity
국제학술대회
231
October 13-14, 2005, Seoul, Korea
F057
Identification and Characterization of the ATPdependent Protease, Lon in Pseudomonas
syringae pv. tabaci 11528
Hyun Ju YANG*, So Young PARK, Ji Young CHA, Jun Seung
LEE, Tae Woo KIM, and Hyung Suk BAIK
Division of Biological Sciences, Pusan National University
*Corresponding author: hush1111@naver.com
Pseudomonas syringae pv. tabaci is a plant pathogen causes wildfire
disease on tobacco and elicits hypersensitive response (HR) in nonhost
plants. Its pathogenicity and host specificity are thought to be determined
by effectors injected into plant cells by type III secretion system (TTSS).
Lon protease functions as a negative regulator of TTSS by degrading
HrpR, activator of hrp regulon. Thus Lon protease is thought to play a
significant role in regulation of P. syringae pathogenesis. In this study,
like other P. syringae strains, TTSS of P. syringae pv. tabaci is expressed
only in hrp-inducing medium similar to plant tissue environment using
hrpA promoter transcriptional fusion. We confirmed that lon of P.
syringae pv. tabaci has 90~93% homology with other P. syringae
strains. As a result of comparison of UV sensitivity, mutant-type
showed increased UV sensitivity relative to wild-type. By conducting β
-galcactosidase activity, hrpA promoter in lon mutant-type is affected
independent of growing conditions in contrast to wild-type. Several
different proteins in wild and mutant-type were acquired from
two-dimensional electrophoresis. Physiological studies indicate that
mutant-type is similar to the wild-type strain when they were made a
comparative study of growth pattern, toxicity test, pathogenicity test.
Purified Lon protease was injected into rabbit for the anti-Lon antibodies
production and it was identified through western blot.
Keywords: Lon protease, pathogenicity
F059
Molecular Detection of Tick-Borne Encephalitis
Virus in Ticks in the South Korea
1
2
1
Division of Arbovirus Department of Virology Korea National Institute of
Health Korea Center for Disease Control and Prevention, 2Department of
3
Parasitology College of Medicine Yonsei University, College of Veterinary
Medicine Jeonbuk University
*Corresponding author: tenksy@hotmail.com
Tick Borne Encephalitis (TBE) virus, like yellow fever, Japanese
encephalitis and dengue viruses, is a flavivirus belonging to the family
Flaviviridae. The virus is transmitted by infected ticks unlikely other
flaviviruses transmitted by mosquito. It is very important disease for the
health because it causes disorder in the central nervous system. The TBE
virus have been reported to two subtypes, western subtype (strain
Neudoerfle) and far-eastern subtype. The latter subtype is known to be
highly pathogenic for humans, with mortality rate of about 30%50%. The vector, Ixodid sp., of this virus have been known to be
distributed in our country. The virus has continuously bee isolated in the
neighboring countries of Japan and China. It is considered that the TBE
virus exists in Korea, but this study has never been examined in Korea. For
the first time, we surveyed the vector of TBE virus from South Korea. We
collected them in the region Gyeonggi-do and Gangwon-do in the months
from April to August, 2005. We pooled together to check respectively
10-20 individuals (about 640 individuals) and found the TBE virus gene
in 7 pools among 51 pools with reverse-transcriptase PCR method. The
nucleotide sequence identities of the gene with far-eastern subtype and
western subtype virus were 97-99%. This study suggests a possible
outbreaking of tick borne encephalitis disease in Korea.
Keywords: Flavivirus, Tick Borne Encephalitis virus, Ixodid,
Reverse-transcriptase PCR
F058
F060
Computational Analysis of Redox-Sensitive
Anti-Sigma Factors in Bacterial Genomes
Expressions of APRO(anti-proliferative) Genes
in Differentiation of Leukemia Cell Lines
Joonseok CHA*, Ji-Yoon SONG, and Jung-Hye ROE
Byoung Ok CHO *, Yong Wook JEONG , Jae We CHO , and
1
Jong Chun PARK
Laboratory of Molecular Microbiology, School of Biological Sciences and
Institute of Microbiology, Seoul National University
*Corresponding author: fender99@snu.ac.kr
In Streptomyces coelicolor, the anti-sigma factor of SigR, RsrA is
oxidized to lead the release of zinc and formation of disulfide bonds to
liberate SigR to express antioxidant exzymes. We performed psi-blast in
NR DB and selected 258 proteins having E-value lower than 1. This
could be divided into two groups, HCC and non-HCC, corresponding to
the presence of HxxxCxxC motif of RsrA. Analysis of neighboring ORFs
of each homolog showed that most of them have putative sigma gene in
its upstream. HMM profiles of both groups showed that Cys11 of RsrA
is highly conserved as cystein or histidine. Non-HCC group showed
relatively heterogeneous feature, which could indicate this group might
be an origin of another group, having more subgroups in itself. We
performed another psi-blast search of RsrA homologs in NR DB with 10
iterations. In this search, the presence of cysteine or histidine around the
25-residue upstream of HCC motif was considered in iterations. 244
proteins were selected and most have Cys/His in similar location. The
proteins were present in 121 eubacteria and phylogenetic distribution
showed that this family is a kind of common regulator in bacterial
genomes. Especialy, they could be easily found in actinobacteria,
proteobacteria and firmicutes. Some organisms have redundant copies of
RsrA homologs which varies in length, which suggested this protein be
a general module functioning in protein-protein interaction and signal
transduction.
Keywords: anti-sigma, RsrA, oxidative stress, zinc, protein family
database
232
1
Su-Yeon KIM , Seok Min YOON , In Yong LEE , Jun Seock
CHE3, Jeong Hee YOO1, Dae Yeon LEE1, Yeong Ui JEONG1,
1
1
Young Hack SHIN , and Young Ran JU
한국미생물학회연합
1
1
2
1
Department of Microbiology, College of Medicine, Seonam University,
Department of Microbiology, School of Medicine, Keimyung University
*Corresponding author: okc1978@hotmail.com
2
Recently there are increasing rates of leukemia in Korea. The key
pathogenesis of leukemia is a defecting of differentiation process of
hematopoietic stem cell. At present APRO(anti-proliferative) genes
were known five types(BTG1, BTG2, BTG3, TOB, TOB2), its function
is anti-proliferation. It was reported that some of APRO genes have
associated with cell differentiation. However it stills unknown APRO
genes are related with differentiation process of blood cells. In this study
we have investigated the expressions of APRO genes in TPA-treated or
retinoic acid-treated HL-60 cell and U937 cell lines. The expression of
BTG2 genes were most increased at 6 h in 32 nM TPA-treated and 1 uM
retinoic acid-treated HL-60 cells, but BTG1 and BTG3 genes were not
increased. The expression of BTG2 genes were most increased at 6 h in
32nM TPA-treated U937 cells, but BTG1 and BTG3 genes were not
increased. However, after 1uM retinoic acid treatment, BTG2 gene
expressions were most increased at 2 h, but BTG1 and BTG3 genes were
not increased. The expression of BTG2 genes were remarkably increased
regardless of types of differentiation inducer. Thus, BTG2 gene may play
a most important role in blood cell differentiation. In conclusion, APRO
genes expression is some different to class of differentiation inducers and
cell lines. However, increased expression of BTG2 genes in common
may be an important basic data on blood cell differentiation research.
Keywords: APRO GENE; BTG; HL60 Cell; U937 Cell; TPA;
Retinoic Acid
2005 International Meeting of the Federation of Korean Microbiological Societies
G001
Investigation of Carbapenemases Produced by
Clinical Isolates of Acinetobacter baumannii
Jae Seok SONG, Seon Ju JANG, Kwang Hoon SUNG, Ki Suk
YANG, Myong Jin HEO, and Sang Hee LEE
Department of Biological Sciences, Myongji University
*Corresponding author: sangheelee@mju.ac.kr
To investigate outbreaks of imipenem-resistant Acinetobacter baumannii
at a University Hospital, we performed antibiotic susceptibility
testing, microbiological tests of carbapenemase activity, pI determination,
transconjugation test, enterobacterial repetitive consensus (ERIC)-PCR,
and DNA sequencing. One outbreak involved seven cases of infection by
A. baumannii producing the OXA-23 beta-lactamase and was caused by
a single ERIC-PCR clone. At the same time, two cases of infection by A.
baumannii producing the IMP-1 beta-lactamase were detected. The
epidemic isolates were characterized by a modified cloverleaf synergy test
and EDTA-disk test synergy test. Isoelectric focusing of crude bacterial
extracts detected nitrocefin-positive bands with pI values of 6.65
(OXA-23) and 9.0 (IMP-1). PCR amplification and characterization of the
amplicons by direct sequencing indicated that the epidemic isolates
carried blaIMP-1 or blaOXA-23 determinant. The epidemic isolates were
characterized by a multidrug resistance phenotype that remained
unchanged over the outbreak, including penicillins, extended-spectrum
cephalosporins, carbapenems, monobactams, and aminoglycosides. This
study shows that the blaIMP-1 or blaOXA-23 resistance determinant may
become an emerging therapeutic problem. [Supported by grant from
BioGreen 21 Program, Rural Development Administration, Republic of
Korea]. Correspondence should be addressed to S.H. Lee (sangheelee@
mju.ac.kr). Keywords: Acinetobacter baumannii, carbapenemase, IMP-1,
OXA-23, ERIC-PCR
G003
Flavonols Inhibit Sortase-mediated Staphylococcus
aureus Binding to Fibrinogen via Clumping Factor
Protein
Jae-Gyu KIM, Tae-Hoon LEE, Su-Jin KIM, Soon-Chun
CHUNG, and Ki-Bong OH*
School of Agricultural Biotechnology, Seoul National University
*Corresponding author: someless@naver.com
The sortase enzymes are a family of Gram-positive transpeptidases
responsible for anchoring surface protein virulence factors to the
peptidoglycan cell wall layer. In Staphylococcus aureus, deletion of
the sortase isoforms results in marked reduction in virulence and
infection potential,making it an important antivirulence target. We
examined the effects of naturally occurring flavonols on
recombinant sortase A (SrtA) and B (SrtB) prepared from S.aureus
and found that these compounds inhibited the activity of sortases,
without exhibiting antibacterial activities. Among the flavonols
tested, morin, myricetin, and quercetin exhibited strong sortase
inhibitory activities (SrtA IC50, 11.3-15.9ug/ml;SrtB IC50, 2,6-11,
7ug/ml). The fibrinogen-binding activity data highlight the potential
of flavonols for the treatment of S.aureus infections via inhibition of
sortase activity
Keywords: sortase, Flavonols, fibrinogen, clumping factor protein
G002
G004
Rapid One Step Detection of Pathogenic
Bacteria Involved in Sexually Transmitted
Diseases (STDs) and Prostatitis by Multiplex
PCR Assay(mPCR) Interfering Property of Live Influenza Vaccine
Virus Protects Mice From Lethal Challenge
Yeon Sun PARK and Young Gon KIM
Deptartment of Biotechnology, College of Engineering, Yonsei University
*Corresponding author: blseong@yonsei.ac.kr
Department of Biology, Chosun University
*Corresponding author: ygnkim@mail.chosun.ac.kr
We developed a multiplex PCR (mPCR) assay for the simultaneous detection
of Chlamymidia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium,
Ureaplasma urealyticum, Pseudomonas aeruginosa and Corynebacterium
spp. in one tube by using multiple specific primers. C. trachomatis-specific
primer CT, N. gonorrhoeae primer NG, Mycoplasma primer MG, Ureaplasma
primer UU, Corynebacterium primer CS, and Pseudomonas primer PA were
used together in the mPCR reaction. Amplification with these primers
produced products of 200, 281, 347, 429, 542 and 1,427 bp, respectively. All
PCR products were easily detected by agarose gel electrophoresis, and the
sequences of specific amplicons were determined. DNA was extracted from
urine samples, collected from patients at a local hospital clinical center, and
the mPCR assay was used to screen for potential pathogenic bacteria.
Resulting amplicons were cloned and sequenced. The resulting sequences matched
exactly with sequences derived from known pathogenic isolates. N.
gonorrhoeae and Corynebacterium, typical causative pathogens for STDs and
prostatitis, were successfully detected using this technique, and, unexpectedly,
P. aeruginosa was also detected. Detection of these additional organisms
may be related to other associated diseases. Assay sensitivity was determined
for purified DNA from six pathogenic laboratory strains. mPCR detected
pathogen DNA at concentrations ranging from approximately 0.018 to 1.899
pM/ml.
Keywords: STD, Prostatitis, multiplex PCR, Clamydomonas, Neiserria,
Mycoplasma, Ureaplasma, Pseudomonas, Corynebacterium
Sang-Uk SEO, Kwang-Hee LEE, and Baik Lin SEONG*
Influenza virus continuously undergoes antigenic change through
frequent mutations in two major surface glycoproteins,
hemagglutinin and neuraminidase, to evade host’s immune response.
As a consequence of these rapid antigenic change, vaccine must be
updated to newly recommended variants. In part of our efforts for
developing live influenza virus vaccine, a cold-adapted(ca) virus was
evaluated for several properties: ca, ts, att phenotype and protective
immune responses. Besides well known prophylactic effect, a
desirable trait for live attenuated vaccine may include potential
therapeutic effect by interfering with wild-type viruses. On the basis
of the ability of the ca virus to suppress wt viral replication, we
examined in mouse infection model if the X-31 ca virus could
interfere with the virulent virus. Prior vaccination with the ca virus
about 1-4 days before challenge with virulent virus or even
simultaneous infection of vaccine virus and virulent virus resulted in
marked improvement in clinical signs associated with influenza
infection. The administration of this ca virus may confer immediate
protection and block further spread of the virulent virus, and could be
considered useful for minimizing morbidity and mortality associated
with natural outbreak or intentional use of influenza virus.
Keywords: Influenza virus, Live attenuated vaccine, Interference,
Cold-adapted virus
국제학술대회
233
October 13-14, 2005, Seoul, Korea
G005
G007
Towards Reconstitution of Influenza RNAdependent RNA Polymerase from Recombinant
Expression System
Comparison of Integron Mediated Aminoglycoside
Resistance among Human, Animal, and
Environmental Isolates of Escherichia coli
Seong-Yeon YOO, Kwang-Hee LEE, and Baik Lin SEONG*
Kyenam LEE, Min-Young LEE, Da-Hye JUNG, and Yeonhee LEE*
Department of Biotechnology, College of Engineering, Yonsei University
*Corresponding author: blseong@yonsei.ac.kr
Department of Biology and Culture Collection of Antimicrobial Resistant
Microbes, Seoul Women's University
*Corresponding author: kyenamlee@paran.com
The RNA-dependent RNA polymerase (RdRp) of influenza virus is
composed of three viral P proteins (PB1, PB2, and PA) and involved in
both transcription and replication of the RNA genome: (i) transcription
of vRNA into mRNA, (ii) replication of vRNA into cRNA and (iii)
replication of cRNA into vRNA. The polymerase controls two distinct
type of RNA synthesis at both initiation and termination, but the
molecular mechanisms involved in the control of transcription and
replication are largely unknown. The role of individual subunits of the
influenza RdRp, have been studied extensively with only limited
success. Moreover, the in vitro reconstitution of the functional RNA
polymerase has not been successful for the lack of sufficient supply of
functional recombinant P proteins. In this study, all three individual
subunits of the influenza RdRp, PB1 and PB2 and PA proteins were
successfully expressed from E. coli as soluble form based on the
2+
cisperone vector system. Ni -NTA chromatography was employed
to purify the three proteins from E. coli. Reconstitution of the P
proteins into functional influenza polymerase complex along with
model RNA template is under investigation with a view to get further
insights into the role of each P protein in viral transcription and
replication. As the first report of soluble expression of influenza
polymerase proteins in bacterial host, the system would provide useful
platform for structure-function analysis of the influenza RdRp.
Keywords: influenza virus, RNA-dependent RNA polymerase,
PB1, PB2, PA
Prevalence and mechanism of aminoglycoside-resistance were studied
with 581 Escherichia coli isolated from human, animal, and
environment between 1997 and 2004. Among 233 human isolates, 82
(35.2%) isolates were resistant to gentamicin and acetyltransferase
gene was detected in 64 (27.5%) isolates. Among 56 integron
containing isolates, class I integron was detected in 42 isolates while
class II integron was detected in 2 isolates. In case of 38 animal isolates,
17 (44.7%) isolates were resistant to gentamicin and adenyltransferase
gene was detected in 15 (39.5%) isolates. Class I integron was detected
in 15 of 16 integron containing isolates while class II integron was not
detected. Among 310 environmental isolates, 23 (7.4%) isolates were
resistant to gentamicin and acetyltransferase gene was detected in 20
(6.5%) isolates. Class I integron was detected in 3 of 19 integron
containing isolates while class II integron was detected in one isolates.
Genes for integron and streptomycin resistance of human, animal, and
environmental isolates were transferred to E. coli J53 Azir with broth
mating experiment. Results showed that aminoglycoside-resistance
was mainly due to modification of aminoglycoside by acetyltransferse
in human and environmental isolates and by adenyltransferase in
animal isolates, and horizontal transfer of resistance gene by integron.
(supported by grants from MAF and MHW)
Keywords: aminoglycoside modifying enzyme, aminoglycosideresistance, integron
G006
G008
Generation of Antibody Library Against Influenza
Viral Proteins
Analysis of Outer Membrane Protein OprD
responsible for Imipenem Resistance in
Pseudomonas aeruginosa
Seung Hee CHO, Kwang-Hee LEE, and Baik Lin SEONG*
Department of Biotechnology, College of Engineering, Yonsei University
*Corresponding author: blseong@yonsei.ac.kr
Influenza viral infection is a major cause of morbidity and the
identification of the type of clinical isolates has important clinical and
epidemiological implications. The frequent outbreak of influenza
epidemics as well as potential pandemic necessitates the development
reliable diagnostic system. The repertoire of influenza viral proteins
and their specific antibodies are required for the establishment of
efficient diagnostic system. The cDNAs of influenza A/WSN/33
virus were cloned into the E. coli cisperone vector, a solubility
enhancer vector system. The influenza viral proteins were
over-expressed in E. coli host as soluble form. The histidine-affinity
2+
tagged fusion proteins were all purified by Ni -affinity
chromatography. Specific antisera were prepared against the purified
proteins in rabbit. Using these specific antibodies, we could detect
each protein in cell lysate from influenza A virus infected cells and
transfected cells. The antibodies against NP from influenza A type
was found to be cross-reactive against other viral strains of the A type,
bus was not reactive to influenza B viruses. Similar specificity was
also observed from the M1 and NS1 antibodies. The purified viral
antigens and the specific antibodies would be useful for developing
and improving the efficiency of the influenza surveillance.
Keywords: influenza virus, diagnosis, anti-influenza virus antibody,
fusion protein.
234
한국미생물학회연합
Jeom Kyu LEE, Yong Sun YOO, Eui Suk SOHN, Kyeong Min
LEE, Jae Il YOO, Yeong Seon LEE, and Bong Su KIM
Division of Antimicrobial Resistant Pathogens, NIH, Korea Center for Disease
Control and Prevention
*Corresponding author: jeomkyu@nih.go.kr
A number of clinical P. aeruginosa isolates have shown reduced
susceptibility or resistance to imipenem (IPM). IPM-resistant strains have
either lost or reduced level of outer membrane porin OprD. This report
assesses the association between mutations in the oprD gene and OprD
levels for IPM resistance. Fifteen IPM-resistant and 3 IPM-susceptible P.
aeruginosa evaluated in this study were isolated from non-tertiary hospitals
in 2003. MICs of IPM and meropenem were determined according to the
criteria of NCCLS. The mutation in oprD gene detected by PCR assays
using oprD-specific primer and sequencing. The mRNA levels of oprD
gene in isolates were obtained by real-time quantitative PCR. Outer
membrane proteins were analyzed by SDS-PAGE. Immunoblotting was
performed with polyclonal rabbit antibody raised against OprD. The amino
acid substitutions within the external loops 2, 3, 5, 6, 7 or 8 in oprD genes
of IPM-resistant isolates were observed. The levels of oprD mRNA in
IPM-resistant isolates with these mutations were reduced compared with
IPM-susceptible isolates. The loss or decreased amount of OprD (45-kDa
protein) was observed in IPM-resistant isolates by SDS-PAGE and
immunoblotting analysis. These results reveal that amino acid alterations
in external loops of oprD gene were associated with the down-regulation of
oprD transcription for IPM resistance, and the decrease or loss of OprD was
caused by the low of oprD transcriptional level. Keywords: Pseudomonas aeruginosa, Imipenem resistance, porin OprD
2005 International Meeting of the Federation of Korean Microbiological Societies
G009
G011
Multi Locus Sequence Typing (MLST) Analysis
of Vibrio cholerae O1 El Tor Isolates Harboring
the Classical CTX Prophage Isolated in Mozambique
Streptococcus pneumoniae ClpL Mediates
Adhesion and Invasion in the Early Stage of
Infection
1
1
1
Jongsik CHUN *, Je Hee LEE , Kyung Ho HAN , Seon Young
CHOI1,2, and Dong Wook KIM2
Le Nhat TU# , Hye-Yoon
JEONG# , Hyog-Young
KWON , A.
2
2
1
David OGUNNIYI , James C PATON , Suhk-Neung PYO ,
1
3
Dong-Kwon RHEE *, and Le Nhat TU
1
1
1
1,2
2
School of Biological Sciences, Seoul National University, International
Vaccine Institute
*Corresponding author: serasera@snu.ac.kr
College of Pharmacy, Sungkyunkwan University, 2School of Molecular and
Biomedical Science, The University of Adelaide, Australia, 3College of
Pharmacy, Sungkyunkwan University#
*Corresponding author: dkrhee@skku.ac.kr
Vibrio cholerae O1 isolates belonging to the Ogawa serotype, El Tor
biotype harboring the classical CTX prophage was first isolated in
Mozambique in 2004. Multi Locus Sequence Typing (MLST) analysis
using nine genetic loci showed that the Mozambique isolates have the
same sequence type in the nine loci examined as O1 El Tor N16961.
Analysis of the CTX prophage in the Mozambique isolates indicated
the there is just one type of rstR in these isolates – the classical CTX
prophage type. We further analyzed the CTX prophage of the
Mozambique isolates and found that ctxB-rstR-rstA-rstB-phs-cep
fragment was PCR-amplified from these isolates. This indicates the
presence of a tandem repeat of classical CTX prophage in the genome
of the Mozambique isolates. The possible origin of these isolates and
the presence of the tandem repeat of the classical prophage in these
isolates implicate the presence of the classical CTX phage which has
not yet been reported. Further studies are required to understand the
origin and the distribution of this unique strain.
Keywords: Vibrio cholerae, Multi Locus Sequence Typing, CTX
prophage
The ClpL has chaperone function and is translocated into cell wall fraction
after exposure of the cells to heat shock (Kwon et al., 2003). In this study, we
examined the effect of the clpL mutation on pathogenesis and the expression
of virulence factors of Streptococcus pneumoniae. Although clpL mutant
produced the same amount of capsular polysaccharide as the wild type, it
could adhere and invade much more efficiently to nasopharyngeal or lung
cells in vitro than the wild type. In addition, after intra-nasal challenge in vivo,
it could colonize and invade mouse nasopharynx and lung more efficiently
than the wild type at the early stage of infection. However, its viability in those
organs was dramatically dropped 96 hours post-infection and in the presence
of penicillin, viability of the clpL mutant was decreased more rapidly than that
of the wild type. Subsequently, the clpL mutant died almost the same rate as
the wild type. To check that ClpL directly binds to the host cell during
infection, pneumococci were treated with ClpL antiserum prior to infection
to A549 cells. However, pretreatment with ClpL antiserum did not prevent
adherence of pneumococci to the host cells. This result indicates that
adherence is not mediated by ClpL. Taken together these results indicate that
clpL mutant could modulate virulence S. pneumoniae at the early stage of
infection. # These authors contributed equally. Supported by KRF 2004.
Keywords: Streptococcus pneumoniae, ClpL, Heat shock protein,
virulence
2
G010
G012
JAK-STAT Signaling Pathway Mediated Astrogliosis
in Brains of Scrapie-infected Mice
1,2
1
I
3
Yeo-Jung NA *, Jae-Kwang JIN , Richard . CARP , and
1,2
Yong-Sun KIM
1
Ilsong Institute of Life Science, Hallym University, 2Department of
Microbiology, College of Medicine, Hallym University, 3New York State
Institute for Basic Research in Developmental Disabilities, USA
*Corresponding author: depudecin@hotmail.com
Scrapie is characterized histologically by astrogliosis in the brains.
However, the mechanisms of astrogliosis occurred by prion infection
are not well understood. Here, we found that the expression levels of
LIF and CNTF known growth factors of Janus kinase (JAK)-signal
transducers and activators of transcription (STAT) signaling pathway
were increased in scrapie-infected brains, compared with control
brains. The expression levels of p-JAK2, p-STAT1, p-STAT3, and
glial fibrillary acidic protein (GFAP) used as astrocyte marker tended
to increase relative to the stage of scrapie development and its strong
expression was observed at 140 days after scrapie strain injection by
Western blot analysis. Moreover, we found that p-STAT1 and
p-STAT3 having function as transcription factor were translocated
into the nucleus in scrapie-infected brains. Immunohistochemically,
p-STAT1 was immunostained with LIF, CNTF, and p-JAK2 in broad
number of reactive astrocytes in scrapie-infected brains. In contrast
with, p-STAT3 was immunostained in a limited region of astrocyte,
but most of expressed p-STAT3 was colocalized with nestin used as
regeneration marker in scrapie-infected brains. Taken together, our
results suggest that activation of JAK2/STAT1 signaling pathway
was mediated the reactive astrocytes, and activation of STAT3 may
be related to astrocyte regeneration in hippocampus region of
scrapie-infected brains.
Keywords: Prion, astrogliosis, JAK-STAT signaling pathway
Serotype and Enzymatic Profile of Cryptococcus
neoformans Isolates from Clinical and
Environmental Sources in Korea
1
1
2
Soo Myung HWANG *, Tae Un KIM , Kwang Seok OH , and
3
Kyungwon LEE
1
Department of Clinical Laboratory Science, Catholic University of Pusan,
Maritime Safety Team, Korea Institute of Maritime and Fisheries Technology,
Department of Laboratory Medicine, Yonsei University College of Medicine
*Corresponding author: smhwang@cup.ac.kr
2
3
Fifty eight C. neoformans strains isolated from clinical and environmental
sources in Korea were examined for their serotypes and extracellular enzyme
activities. Among the 51 strains isolated from clinical sources, 48 strains were
serotype A(94.1%), 2 strains were serotype B(3.92%), and 1 strain was
serotype D(1.96%). All seven environmental strains isolated from pigeon
excreta were identified as serotype A. All isolates of C. neoformans were
positive for the production of extracellular phosphatase and proteinase. In the
API-ZYM system which tested 19 different kinds of enzymes, all 59 isolates
produced alkaline phosphatase(No.2), esterase C4(No.3), esterase lipase C8
(No.4), leucine arylamidase(No.6), acid phosphatase(No.11), naphthol-ASBI-phosphohydrase(No.12), α-glucosidase(No.16), and β-glucosidase(No.17).
39 isolates(67.2%) of C. neoformans produced N-acetyl-α-glucosaminidase
(No. 18). Two isolates, serotype B, and only one serotype A produced α
-glucuronidase(No. 15). Analysis of enzymatic profiles to 21 enzymes
revealed four biotypic patterns among the 58 strains. Out of a total 48 clinical
isolates, serotype A, presented enzymatic pattern I (20.8%) and II (77.1%),
while environmental isolates were pattern I (71.4%) and II (28.6%). Two
serotype B showed pattern IV (100%). The enzymatic patterns of C.
neoformans isolated from clinical and environmental sources represented a
significant relationship with the serotypes.
Keywords: Cryptococcus neoformans, var. gattii, Serotyping, Enzyme
activity, API-ZYM system
국제학술대회
235
October 13-14, 2005, Seoul, Korea
G013
G015
Generation of Monoclonal Antibodies to Putative
Transmembrane Domain of Prion Protein (PrP)
1
1
1
Jin-Kyu CHOI *, Seok-Ju PARK , Yong-Chul JEON ,
Byung-Hoon JEONG1, Jae-Min OH1, Hyun-Pil LEE1, Richard I.
2
1,3
CARP , and Yong-Sun KIM
1
IlSong Institute of Life Science, Hallym Academy of Sciences, Hallym
University, 2New York State Institute for Basic Research in Developmental
3
Disabilities, USA, Department of Microbiology, College of Medicine, Hallym
University
*Corresponding author: jinkyu71@hallym.ac.kr
To develop monoclonal antibodies (mAbs) to react with prion protein
C
Sc
Sc
(PrP ) and abnormal isoform of prion protein (PrP ), PrP was isolated
from brains of 263K scrapie-infected hamsters and used to immunize
PrP knockout mice. The generated hybridomas were screened by
western blot analysis. Two hybridomas, 3F10 and 1C5 (IgG1), were
established. 3F10 has a high affinity for hamster and mouse PrP and may
recognize the residues 137-151. 1C5 recognizes the region 119-130
conserved in mammal species and reacted with hamster, mouse, elk,
bovine, and human PrP. In the immunohistochemical analysis, the
Sc
positive staining for PrP observed presented in the extracellular
compartment of scrapie-infected brains, but not normal brains. The
results demonstrated that the developed mouse mAbs should be prion
protein specific and could recognize an abnormal prion protein in
immunohistochemistry. Therefore, these antibodies could be used in the
analysis of biochemical, structural, and functional properties between
C
Sc
PrP and PrP . Keywords: PrP-specific antibody, Putative transmembrane domain,
Prion protein
Rapid, Accurate and Early Diagnosis of Scrub
Typhus with Lateral Flow Assay Using
Recombinant Antigen of Orientia tsutsugamushi
1
Gyu-Sang LEE* and Jong-Bae KIM
Department of Biomedical Laboratory Science, Yonsei University
*Corresponding author: innkeepers@empal.com
The incidence of resistance to extended spectrum β-lactam antibiotics
is increasing in Wonju city, Korea. Total 57 strains of extended
spectrum β-lactamase (ESBL) producing E. coli and Klebsiella species
were isolated from Wonju Christian Hospital during a 9 month-period
from April to December, 2003. To determine the prevalence and
genotypes of the ESBL producing clinical isolates, antibiotic
susceptibility and ESBL activity test by VITEK system and double
disk synergy (DDS) test, and PCR based genotyping were performed.
Fourteen (82%) isolates of 17 ESBL producing E. coli were found to
have blaTEM gene and 5 (29%) isolates were found to have blaCTX-M
gene by polymerase chain reaction (PCR). Thirty (75%) isolates of 40
ESBL producing Klebsiella species with blaTEM gene, 38 (95%)
isolates with blaSHV gene, and 7 (20%) isolates with blaCTX-M type gene
were also identified. Enterobacterial repetitive intergenic consensus
(ERIC) PCR and similarity index by dendrogram for genetical
similarity to band pattern of each clinical isolates were examined. ESBL producing E. coli were grouped into 6 clusters up to 84% of
similarity index and Klebsiella species were grouped into 12 clusters
up to 76% of similarity index. In conclusion, ESBL producing clinical
isolates were characterized with the results from antimicrobial
resistance pattern and genetical similarity using ERIC PCR.
Keywords: ESBL, MIC, TEM, SHV, CTX-M, PCR, VITEK, ERIC
PCR
236
한국미생물학회연합
1
1
Department of Microbiology, College of Medicine, Hallym University and
2
Immunmed Inc, Department of Microbiology, College of Medicine, Seoul
University
*Corresponding author: zkfmvpeldpa75@hotmail.net
Scrub typhus(tsutsugamushi disease) is one of the febrile illnesses such as
murine typhus, leptospirosis and hemorrhagic fever with renal syndrome.
It is an acute, febrile disease caused by infection with Orientia
tsutsugamushi. It occurs in many areas of the world, especially in Asia and
the Pacific region. The 56kDa major outer membrane protein of Orientia
tsutsugamushi is the immunodominant antigen in human scrub typhus
infections. The gene serially encoding major part of each this protein from
Gilliam, Karp and Kato strain was cloned and expressed in Escherichia
coli. The recombinant antigen(cr56) was used to major antigen in this
study. The gene encoding the imunodominant 56kDa protein from
Kangwon strain and the gene encoding 21kDa protein from Boryong strain
was cloned and expressed in Escherichia coli. The recombinant
antigens(kr56, r21) was used to supplement antigen in this study. We
developed a lateral flow assay(LFA) for the diagnosis of scrub typhus using
chimeric antigen(cr56) and supplement antigen(kr56, r21). This LFA kit
could detect the specific IgG and IgM to Orientia tsutusgamushi in
patient's serum as short as in 5minutes. The results show that the sensitivity
of LFA is 100%, and the specificity is 97.2%. Therefore, theses results
suggest that this LFA kit can be used for rapid, accurate and early diagnosis
for scrub typhus.
Keywords: Orientia tsutsugamushi, Chimeric antigen, Tsutsugamushi
disease, Lateral flow assay
G014
Patterns of Antimicrobial Resistance and Genotyping
of Extended Spectrum β-lactamase(ESBL) Producing
Clinical Isolates in Wonju city, Korea
1
Joo Mi CHUN *, Mi Jeong LEE , Young Jin KIM , Yoon Won
1
2
KIM , and Ik Sang KIM
G016
Increased Expression of Caveolin-3 in the
Brains of Scrapie-infected Mice
1
1
2
Jae-Min OH *, Jae-Kwang JIN , Eun-Young LEE , Jae-Bong
3
4
I
4
1,5
PARK , Jae-Il KIM , Richard . CARP , and Yong-Sun KIM
1
Ilsong Institute of Life Science, Hallym Academy of Sciences, Hallym
2
University, Department of Anatomy, College of Medicine, Chungbuk National
University, 3Department of Biochemistry, College of Medicine, Hallym
4
University, New York State Institute for Basic Research in Development
5
Disabilities, USA, Department of Microbiology, College of Medicine, Hallym
University
*Corresponding author: jmoh76@hallym.ac.kr
Prion diseases are transmissible neurodegenerative disorders in animals and
humans. In previous studies, it has been suggested that the conversion of the
cellular prion proteins (PrPC) to a pathological scrapie isoform (PrPSc) may occur
within caveolae-like domains. It has been reported that caveolins, the major
structural proteins of caveolae, are expressed within cells of central nervous
system (CNS). In the present study, to examine the possible association of
caveolins with the pathogenesis of prion diseases, we investigated the expression
level and the localization of caveolins in the brains of both control and ME7
scrapie-infected mice. Interestingly, immunoreactivity of caveolin-3 was detected
in reactive astrocytes of hippocampal region, and in ependymal cells lining the
lateral ventricles of both control and scrapie-infected mice. Electron microscopy
demonstrated the presence of caveolin-3-positive caveolae structures in reactive
astrocytes, and a large number of lipid-like droplets showed caveolin-3
immunoreactivity along the peripheral region of the droplets in the cytoplasm of
ependymal cells of both control and scrapie-infected mice. Finally, expression
level of caveolin-3 was significantly increased in the brains of infected mice
compare to controls. These results suggests that up-regulation of caveolin-3 is
implicated in the neuropathological changes in scrapie-infected mice and may
provide the basis for understanding the functions of caveolin-3 in the CNS.
Keywords: prion, caveolin, caveolae, reactive astrocyte, ependymal cell
2005 International Meeting of the Federation of Korean Microbiological Societies
G017
G019
Nucleocytoplasmic Shuttling and Regulation of
Gene Expression by the Z-DNA Binding Protein
DLM-1
1
1
1
Hong Thanh PHAM , Mi- Young PARK , Eui Tae KIM , Kyeong
Kyu KIM1, Yang-Kyun KIM2, and Jin-Hyun AHN1*
1
Department of Molecular Cell Biology, Samsung Biomedical Research
Institute, Sungkyunkwan University School of Medicine, 2Department of
Biochemistry, Chung-Ang University School of Medicine
*Corresponding author: jahn@med.skku.ac.kr
DLM-1 is a tumor-associated and interferon-inducible protein that contains
the N-terminal Z-DNA binding domains. Here we investigated subcellular
distribution and regulation of gene expression by human DLM-1. hDLM-1
was distributed primarily as cytoplasmic forms and occasionally as nuclear
foci in IFN-treated cells and in several other transfected cells. However, in
cells treated with leptomycin B, hDLM-1 efficiently accumulated in nuclear
foci, which overlap the subnuclear structures known as the PML oncogenic
domains (PODs). Using deletion analysis, we found that the Zα domain was
responsible for nuclear export, whereas the Zβ domain was required for
nuclear localization. Further mutational analysis of the Zα domain
demonstrated that the integrity of both the leucine-rich sequences and the
bipartite basic sequences was important for nuclear export. We also found
that both GAL4-hDLM-1 and intact hDLM-1 repressed expression of a
reporter gene containing the GAL4 binding sites. In GAL4-fusion assays,
both the N-terminal region containing Zα and Zβ domains and the C-terminal
region were independently capable of repressing the reporter gene. However,
in assays with intact hDLM-1, only the C-terminal region, was shown to be
necessary for repression. Our data suggest that hDLM-1 is a POD-associated
nucleocytoplasmic shuttle protein containing the CRM1-dependent nuclear
export signal, and that it may also play a role in regulating gene expression by
two different mechanisms.
G018
1
1
1
Ji-Hye JEONG , Seung-Chul LEE , Le-Van PHAN , Bok-Soon
MIN2, Kang-Bum LEE1, and Shien-Young KANG1*
1
2
College of Veterinary Medicine, Chungbuk National University, Biologics
Evaluation Department, Korea Food and Drug Administration
*Corresponding author: sykang@chungbuk.ac.kr
Rotaviruses are known to be one of the most important viruses to cause
gastroenteritis and severe diarrhea in young children worldwide. Several
countries including WHO have tried to develop rotavirus vaccines during
last two decades. To confirm the efficacy of rotavirus vaccines
developed in home and/or foreign countries, first of all, detection of
rotavirus and prevalence of rotavirus serotypes in problemed area should
be performed. The objective of this study is to develop and evaluate the
diagnostic method for detection and subgrouping rotaviruses from
fecal samples. For this study, an indirect sandwich enzyme-linked
immunosorbent assay (i-ELISA) was developed using rotavirus groupand subgroup-specific monoclonal antibodies and compared with
commercialiy available rotavirus diagnostic kit. For detection of
rotaviruses, the sensitivity and specificity of developed i-ELISA were
96.4% and 97.7%, respectively. For subgrouping of rotaviruses,
developed i-ELISA differentiated subgroup I from subgroup II with
accuracy. The sensitivity and specificity of developed i-ELISA were
similar with those of RT-PCR/RFLP analysis. The results suggest that
i-ELISA developed in this study is suitable for epidemiological studies
such as prevalence of rotaviral infection and subgrouping.
Keywords: Detection of Rotaviruses, Subgroup, Enzyme-linked
immunosorbent assay(ELISA), Monoclonal antibodies
G020
Production and Inactivation of Vibrio vulnificus
Hemolysin in Cirrhotic Ascites, a Human Ex Vivo
Experimental System
1
Detection and Subgrouping of Human Rotaviruses
from Fecal Samples Using Monoclonal Antibodies
1
1
1
2
3
MH CHOI , RY PARK , HY SUN , CM KIM , YH BAI , SE LEE ,
4
4
4
1
SY KIM , YR KIM , JH RHEE , AND SH SHIN *
1
Research Center for Resistant Cells, Chosun University Medical School,
3
Department of Biology, Chosun University Medical School, Department of
Dental Pharmacology, Chonnam National University, 4Clinical Vaccine R&D
Center, NRL of Molecular Microbial Pathogenesis, Research Institute of Vibrio
Infection and Genome Research Center for Enteropathogenic Bacteria, and
Department of Microbiology, Chonnam National University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
To elucidate the mechanisms about the in vivo-suppression and
inactivation of Vibrio vulnificus hemolysin (VvhA), we used
cirrhotic ascites (CA) as a human ex vivo experimental system. The
vvhA expression was suppressed in the presence of CA, but was
clearly observed, albeit at very low level, even in the whole CA. The
expression of vvhA was suppressed to a further extent via the
addition of glucose into the whole CA. The VvhA produced in the
presence of CA was inactivated via the oligomerization and change
of electrophoretic mobility of VvhA. These results indicated that
vvhA expression was suppressed and the VvhA produced was
inactivated by constituents of CA. Accordingly, only a very small
portion of the VvhA produced in human body fluids may actually
contribute to the pathogenesis of V. vulnificus septicemia. In
addition, we report that CA proved to be quite useful as a human ex
vivo experimental system for the study of V. vulnificus septicemia
[R13-2003-009].
Keywords: Vibrio vulnificus, Hemolysin, ex vivo, Ascites
Interactions among Four Proteins Encoded by
Human Cytomegalovirus UL112-113 Region
Regulate Their Intranuclear Targeting and
Recruitment of UL44 to Pre-replication Foci
1
1
1
Young-Eui KIM , Mi-Young PARK , Myong-Rang SEO ,
1
2
1
Jae-Rin LEE , Chan Hee LEE *, and Jin-Hyun AHN *
1
Department of Molecular Cell Biology, Samsung Biomedical Research
2
Institute, Sungkyunkwan University School of Medicine, Division of Life
Sciences, Chungbuk National University
*Corresponding author: jahn@med.skku.ac.kr
Four proteins of 34, 43, 50, and 84-kDa with common amino termini are
synthesized via alternative splicing from the UL112-113 region of human
cytomegalovirus genome. Although both the UL112 and UL113 loci have been
shown to be required for efficient viral replication, whether the four proteins
play specific roles or cooperate in replication is not understood. Here we present
evidence that the four UL112-113 proteins both self-interact, and interact with
each other. A mapping study of the 84-kDa protein showed that the N-terminal
region from 1 to 125, which is shared in all UL112-113 proteins, was required
for both self-interaction and nuclear localization as foci. Further studies
revealed that, unlike the 43, 50, and 84-kDa proteins, which were distributed as
nuclear punctate forms, the 34-kDa form was located predominantly in the
cytoplasm. However, when all four proteins were coexpressed simultaneously,
all of the UL112-113 proteins were efficiently localized to the PML oncogenic
domains (PODs). We also found that the ability of the UL112-113 proteins to
relocate UL44 (the viral polymerase processivity factor) to the pre-replication
foci relied on self-interaction, and reached maximal levels when the four
proteins were coexpressed. Therefore, our data suggests that interactions
occurring among UL112-113 proteins via their shared N-terminal region are
important to both their intranuclear targeting, and to the recruitment of UL44 to
subnuclear sites for viral replication.
국제학술대회
237
October 13-14, 2005, Seoul, Korea
G021
G023
Upregulation of Cyclooxygenase-2 and TNF-α
Expression in Response to Mycobacterial
Purified Protein Derivatives through TLR2- and
MAPK-dependent Pathways during Active
Pulmonary Tuberculosis
1
1
1
Saet-Byel JUNG *, Chul-Su YANG , Chang-Hwa SONG , Kil-Soo
LEE1, Su-Young KIM1, Ji-Sook LEE2, A-Rum SHIN1, Jae-Hee
1
1
1
1
OH , Yu-Mi KWON , HWa-Jung KIM , Jeong-Kyu PARK ,
2
1
Tae-Hyun PAIK , and Eun-Kyeong JO *
1
Department of Microbiology, College of Medicine, Chungnam National University,
Department of Microbiology, College of Medicine, Konyang National University
*Corresponding author: jtotquf@cnu.ac.kr
2
The MAPK activation is one essential signaling pathway in response to mycobacterial infection.
In this study, we investigated comparative analysis of activation of ERK1/2 and p38 MAPK
pathways and subsequent expression of COX-2 and TNF-α between patients with active pulmonary
tuberculosis(TB) and healthy tuberculin reactors(HTR). The in vitro stimulation with PPD of M.
tuberculosis rapidly phosphorylated the ERK1/2 and p38, and induced COX-2 and TNF expression
in human primary monocytes. The TB patients showed profound enhancement of COX-2, TNF,
and ERK1/2 and p38 MAPK phosphorylation in freshly isolated monocytes and alveolar
macrophages. After stimulation with PPD antigen, monocytes from TB patients showed more
enhanced and sustained pattern of ERK1/2 and p38 MAPK phosphorylation than those from HTR.
Specific inhibition of MEK1-ERK1/2 or p38 MAPK pathways attenuated the PPD-induced
COX-2, and TNF-α expression in monocytes, although TB patients showed less attenuation than
HTR did. The PPD-induced ERK1/2 and p38 phosphorylation is significantly abrogated by specific
antibodies of TLR2, but not TLR4, in human monocytes. Further, a significantly elevated TLR2
expression was found in monocytes and alveolar macrophages from TB patients than those from
control groups. Collectively, these data suggest that up-regulated MAPK activation, at least partly,
via enhanced expression of TLR2, may play an essential role in the increased proinflammatory
responses during early stages of TB.
Keywords: PPD antigen, Mycobacterium tuberculosis, ERK 1/2,
TNF, Interleukin-10, CCL2
Role of Receptor Polymorphism in Syncytium
Induction by Ecotropic Mouse Gammaretroviruses
Jae Hoon JEONG, Eun Hye BAE, Sung-Han PARK, Sang-Min
PARK, Jin Woo PARK, Ji Hyun LIM, and Yong-Tae JUNG*
Department of Microbiology, Dankook University
*Corresponding author: yjung@dku.edu
Cytopathicity was attributed to different amino acid substitutions at
the same critical env residue involved in receptor interaction: S82F
in the Moloney variant Spl574, and S84A in the Friend variant F-S
MLV. Because M. dunni cells carry a variant CAT-1 gene (dCAT-1),
we examined the role of this receptor variant in cytopathicity and host
range. We transfected dCAT-1 or mCAT-1 of NIH 3T3 origin into
cells that are not normally infectable with ecotropic MLVs. Stable
transfectants of ferret (MA139), bat (Tb-1-Lu) or dog (MDCK) cells
expressing dCAT-1 produced syncytia after ecotropic MLV
infection. Syncytium formation did not correlate with susceptibility
to virus infection; virus titers were equivalent in cells transfected with
either CAT-1 gene, and some high titer viruses were poorly
cytopathic. While these results confirm that syncytium formation is
specifically mediated by the dCAT-1 receptor, transfected cells
expressing dCAT-1 differ from M. dunni in that they are infectable by
different MLVs, and they produce syncytia in response to different
MLVs. The possibility that these differences might be due to
post-translational modification of the receptor by glycosylation was
suggested by Western analysis; treatment with glycosylation
inhibitors and the removal of glycosylation sites from dCAT-1 suggest
that N-linked glycans affect receptor function in M. dunni cells but
not in the transfected cells. Keywords: syncytia,dCAT-1, glycosylation
G022
G024
Effects of Temperature and Salinity on the
Production of Vibrio vulnificus Hemolysin
1
1
1
1
1
H. Y. Sun , R. Y. Park , M. H. CHOI , C. M. Kim , Y. H. BAI , S.
2
2
2
1
Y. KIM , Y. R. KIM , J. H. RHEE , and S. H. SHIN *
1
Research Center for Resistant Cells, Chosun University Medical School,
Clinical Vaccine R&D Center, National Research Laboratory of Molecular
Microbial Pathogenesis, Research Institute of Vibrio Infection and Genome
Research Center for Enteropathogenic Bacteria, and Department of
Microbiology, Chonnam National University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
Our previous work revealed that both temperature and salinity
affected the production of V. vulnificus hemolysin (VvhA). In this
study, we investigated the effects of both temperature and salinity
shiftings on the expression of VvhA at transcription and protein
levels, and the effect of toxR mutation on this modulation of VvhA
production. In a merozygotic PvvhA::lacZ transcriptional reporter
strain, the expression of VvhA at both transcription and protein
o
levels was higher in 0.9% salinity and 37 C than in 2.5% salinity and
o
25 C. ToxR mutation down-regulated the expression of VvhA, but
did not affect the expression of VvhA regulated by the changes of
temperature and salinity. These results suggest that V. vulnificus
recognizes the changes of temperature and salinity and modulates
VvhA production via unknown signal transduction systems but not
the ToxRS signal transduction system [R13-2003-009].
Keywords: Vibrio vulnificus, Hemolysin, Temperature, Salinity
238
한국미생물학회연합
Coxsackievirus VP2 Encodes an ITAM Candidate
Which Affects Viral Growth in vitro and in vivo
1,2,3
3
4
Jung-Hyun PARK , Young-Joo CHO , Seong-Joo CHO ,
2
3
3
Cheol-Won YUN , Yeun-Jung KIM , Soo-Young CHUNG ,
3
3
1
Ku-Sun AHN , Dae-Sun KIM , Inho CHO , and Jae-Hwan
3
NAM *
1
Department of Biomedical Science, NIH, 2College of Life Science, Korea
3
University, Department of Biotechnology, The Catholic University of Korea,
4
KIST
*Corresponding author: jhnam@catholic.ac.kr
The immunoreceptor tyrosine based activation motif (ITAM) [Yxx(L/I)x6-8Y
xx(L/I)] sequences in the VP2 region of coxsackievirus (CVB) was found. We
constructed mutant viruses in which phenylalanine was substituted for two
tyrosine residues of ITAM of VP2, Y240F, Y254F, and YYFF. The virus titers
induced from infectious cDNA of mutant Y254F and YYFF were decreased to
5
4
approximately 10 pfu/µg for Y254F, and 10 pfu/µg for YYFF compared with
11
11
10 pfu/µg for wild type virus (WT) and 10 pfu/µg for Y240F. The plaque sizes
of both Y254F and YYFF were less than those of WT and Y240F. As expected,
the growth rate of the mutant viruses, Y254F and YYFF, in HeLa and Jurket cells
were between 1- to 3-fold lower than WT and Y240F on a logarithmic scale. As
6
BALB/c mice were infected ip with 10 pfu of virus/mouse, most mice infected
with mutants survived, whereas most WT-infected mice died within 30 days pi.
Although the virus titer induced from Y240F was similar to WT, mice infected
with Y240F survived even with a reduction in body weight after infection. Based
on the three-dimensional structure of CVB3, we calculated the complete structure
from Cα, showing that Y240 is primarily exposed in water, whereas the hydroxyl
group of Y254 is close to three other oxygen molecules, forming a hydrogen bond.
This observation implies that Y254 has a role in structural stability. Taken
together, these findings suggest that the CVB3 VP2 region contains an ITAM
candidate that may affect viral growth.
Keywords: Coxsackievirus, Immunoreceptor Tyrosine Based Activation Motif
(ITAM), Viral Pathogenesis
2005 International Meeting of the Federation of Korean Microbiological Societies
G025
G027
Dual Regulation of Vibrio vulnificus Hemolysin
(VvhA) Production by Iron
1
1
1
1
2
Vibrio vulnificus Metalloprotease VvpE Is Required
for Swarming Motility
1
1
1
1
2
M. H. CHOI , R. Y. PARK , H. Y. SUN , C. M. KIM , Y. H. BAI , S.
E. LEE3, S. Y. KIM4, Y. R. KIM4, J. H. RHEE4, and S. H. SHIN1*
R. Y. PARK , H. Y. SUN , M. H. CHOI , C. M. KIM , Y. H. BAI ,
S. Y. KIM3, J. H. RHEE3, and S. H. SHIN1*
1
1
Research Center for Resistant Cells, Chosun University Medical School,
Department of Biology, Chosun University Medical School, 3Department of
Dental Pharmacology, Chonnam National University, 4Clinical Vaccine R&D
Center, National Research Laboratory of Molecular Microbial Pathogenesis,
Research Institute of Vibrio Infection and Genome Research Center for
Enteropathogenic Bacteria, and Department of Microbiology, Chonnam
National University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
In the present study, we investigated the effect of iron concentration
and Fur mutation on the transcription of vvhA and the extracellular
production of VVH. The growth of Vibrio vulnificus was stimulated
by iron in a dose-dependent manner. In transcriptional reporter
assay, the transcription of vvhA was iron-repressible in a
dose-dependent manner, and the mutation of fur gene increased the
transcription of vvhA. However, the extracellular production of
VVH was reversed. Under the low iron condition, large amount of
VVH was present within bacterial cells. The addition of iron
stimulated the transcription of pilD gene and the extracellular
secretion of VVH. These results indicate that the expression of vvhA
at the transcriptional level is negatively regulated via Fur by iron,
and the extracellular production of VVH is positively regulated via
the type II general secretion system regulated by iron
[R13-2003-009].
Keywords: Vibrio vulnificus, Hemolysin, Iron
Research Center for Resistant Cells, Chosun University Medical School,
Department of Biology, Chosun University Medical School, 3Clinical Vaccine
R&D Center, National Research Laboratory of Molecular Microbial
Pathogenesis, Research Institute of Vibrio Infection and Genome Research
Center for Enteropathogenic Bacteria, and Department of Microbiology,
Chonnam National University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
In this study, we investigated the effect of metalloprotease (VvpE) on
the swarming motility of Vibrio vulnificus and the expression level of
vvpE gene in swarming V. vulnificus cells. The mutation of vvpE
decreased swarming motility and the complementation of vvpE
recovered swarming motility to the level of wild type strain. The
transcription of vvpE was more stimulated in swarming V. vulnificus
cells than in not-swarming V. vulnificus cells, and in the central area
than in the peripheral area of swarming. The mutation of luxS
decreased the transcription of vvpE in swarming V. vulnificus cells.
These results indicate that the metalloprotease is required for
swarming motility of V. vulnificus and the expression of the
metalloprotease is upregulated in swarming V. vulnificus cells via a
quorum-sensing system [R13-2003-009].
Keywords: Vibrio vulnificus, Protease, Swarming, Quorum sensing
G026
G028
Identification of Viral Genes Required for
Neuronal Tropism of Murine Gammaherpesvirus
68 Using STM
1
2
2
Decreased Production of VvhA but Increased
Production of the Unknown Factor (s) in
Swarming Vibrio vulnificus Cells
1
1
1
1
1
Sungbum KIM *, Seungmin Hwang , Ting-Ting WU , Ren
2
1
1
SUN , Moon Jung Song *, and Sangmi Lee *
M. Y. KIM , R. Y. PARK , M. H. CHOI , H. Y. SUN , C. M. KIM ,
2
3
3
1
Y. H. BAI , S. Y. KIM , J. H. RHEE , and S. H. SHIN *
1
1
Department of Microbiology, College of Medicine, Hallym University,
Department of Genetic Engineering, College of Natural Science, UCLA
University, USA
*Corresponding author: bioksb@nate.com
2
Gammaherpesviruses including Epstein-Barr virus and Kaposi’s
sarcoma-associated herpesvirus are important human pathogens as
they are involved in tumor developments. Furthermore, recent
reports argue that herpesviruses may be associated with diverse
neurological diseases. Murine gammaherpsvirus 68 (MHV-68)
infects multiple organs and shows different pathological outcomes,
suggesting viral tropism is an important factor to determine viral
pathogenesis. However, molecular and cellular mechanisms
underlying tissue-dependent pathogenesis of gammaherpesviruses
are poorly understood. We used a recombinant virus expressing
enhanced green fluorescence protein (MHV-68/EGFP) to monitor
MHV-68 infection. Upon incubation of MHV-68/EGFP with
neuronal cells, we found that MHV-68 established either lytic or
latent infection, depending on the origin of cells. Viral genes for
neuronal tropism are currently under investigation using a mutant
library of MHV-68.
Keywords: MHV-68, STM, EGFP, MHV-68/EGFP
Research Center for Resistant Cells, Chosun University Medical School,
3
Department of Biology, Chosun University Medical School, Clinical Vaccine
R&D Center, National Research Laboratory of Molecular Microbial
Pathogenesis, Research Institute of Vibrio Infection and Genome Research
Center for Enteropathogenic Bacteria, and Department of Microbiology,
Chonnam National University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
Hemolysin (VvhA) is the most potent exotoxin produced by Vibrio
vulnificus. In this study, we attempted to determine the expression
level of vvhA gene in swarming V. vulnificus cells. The vvhA
mutation did not affect the expression of swarming phenotype of V.
vulnificus. The vvhA-transcriptional activity was lower in swarming
cells than that in non-swarming cells or planktonic cells. The
mutation of luxS increased the transcription of vvhA in swarming
cells. Interestingly, unknown factor (s) causing greenish black
discoloration on human blood agar was produced concomitantly
with swarming of V. vulnificus. Our results indicate that the
production of VvhA is down-regulated via a quorum-sensing
system, but production of the factor (s) is up-regulated in swarming
V. vulnificus cells [R13-2005-009].
Keywords: Vibrio vulnificus, Hemolysin, Swarming, Quorum
sensing
국제학술대회
239
October 13-14, 2005, Seoul, Korea
G029
G031
Molecular Characterization of Antimicrobial
Resistance in the Genetically Related Shigella
sonnei Isolates in Korea
Sung Yong SEOL, Yong Tae KIM, Jae Young OH, Young Sook
JEONG, Hee Young KANG, Dong Chan MOON, Jungmin KIM,
Yoo Chul LEE, Dong Taek CHO, and Je Chul LEE
Department of Microbiology, Kyungpook National University School of
Medicine
*Corresponding author: syseol@knu.ac.kr
Antimicrobial resistance of 150 Shigella sonnei isolates obtained in Korea
during the period 1991 to 2000 was characterized. Resistant to commonly
prescribed antibiotics such as ampicillin was annually increased in S. sonnei
isolates during the outbreak period 1998 to 2000. Resistance to ampicillin
was mediated by conjugative R-plasmids carrying blaTEM-1 from different
sources. S. sonnei isolates were highly resistant to traditional antibiotics
such as trimethoprim (100%), streptomycin (100%), sulfamethoxazole
(95%), tetracycline (95%), and nalidixic acid (79%). All S. sonnei isolates
carried Tn7 in the chromosome, which was responsible for resistance to
trimethoprim and streptomycin. The 8.4 kb of non-transferable R-plasmid
carrying tetA, strA-strB, and sul1 was found in 94% of the S. sonnei isolates.
The combinations of 3 genetic repertoires, conjugative R-plasmids, Tn7,
and the 8.4 kb of non-transferable R-plasmid, could fully cover resistance
to the antibiotics used for treatment of shigellosis. In conclusion, S. sonnei
acquired antimicrobial resistance to commonly prescribed antibiotics
through the horizontal transfer of conjugative R-plasmids when they were
confronted with antibiotic selective pressures, while the genetic stability of
transposon and non-transferable R-plasmid was responsible for resistance
to traditional antibiotics.
Keywords: Shigella sonnei, Antimicrobial resistance, Transposon,
R-plasmid
G030
1
1
1
1
2
R. Y. PARK , H. Y. SUN , M. H. CHOI , C. M. KIM , Y. H. BAI ,
and S. H. SHIN1*
1
Research Center for Resistant Cells, Chosun University Medical School,
Department of Biology, Chosun University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
Most strains, but not all, of Vibrio vulnificus form green colonies on
TCBS agar which is commonly used as a differential and selective
medium for Vibrio species. When we screened 250 clinical and
environmental strains of V. vulnificus, 9 strains showed yellow
colonies on the agar. All the strains showing yellow colonies were
genetically identified as V. vulnificus by the presence of specific
vvhA gene amplified by PCR. API 20E test was unable to identify
these strains as V. vulnificus due to their ability to ferment sucrose.
Except for the ability to ferment sucrose, these strains did not show
remarkable differences in other phenotypes from strains showing
typical green colonies. Through this study, we proved the presence of
sucrose-fermentable V. vulnificus strains showing yellow colonies
on the TCBS agar and incapable of identifying using API 20E test.
Therefore, it is desirable that genetic identification approaches
should be combined with conventional culture and biochemical
methods in order to rule out or identify V. vulnificus. [R13-2003-009].
Keywords: Vibrio vulnificus, TCBS agar, Sucrose
G032
Molecular Epidemiology of Group A Human
Rotavirus Isolated in Korea, July 2002 through
June 2004
1
2
3
Jung Oak Kang *, Jung Soo Kim , Paul Kilgore , Batmunkh
3
4
5
6
Nyambat , Jeonguk Kim , Hun Suk Suh , Yeomin Yoon ,
7
8
9
10
Sookjin Jang , Chulhun Chang , Sukwoo Choi , Mi-Na Kim ,
11
11
11
Jon Gentsch , Joseph Bresee , and Roger Glass
Identification of Low Molecular Weight Proteins
of Helicobacter pylori Strain 26695
Eun Jin Ha, Jeong Won Park, Seung Gyu Lee, Young Chul
Kwon, Jae Young Song, Jeong Uck Park, Hyung Lyun Kang,
Seung Chul Baik, Myung Je Cho*, and Kwang Ho Rhee
1
Department of Microbiology, Gyeongsang National University College of
Medicine
*Corresponding author: mjecho@gaechuk.gsnu.ac.kr
Background: Recent studies on rotavirus genotypes show that rotavirus is both more diverse and more adept at
change than previously thought. The objective of this study was to characterize the Gand P genotypes of rotavirus
strains collected fromnationwide hospitals and a field site (Jeongeub) in Korea. Material and Methods: From
July 2002 through June 2004, 920 strains of rotavirus from 8 Korean Rotavirus Strain Surveillance Network
(KRSSN) hospitals and 427 from Jeongeub district were characterized by RT-PCR. Results: 1. In KRSSN,
globally common genotypes constituted 54%. The most prevalent strain was G4P[6] (26%), followed by G3P[8]
(23%), G2P[4] (17%), and G1P[8] (13%). G9P[8] strains were detected first time in Korea. G4P[6], appeared and
peaked earlier than the usual winter epidemic of the common strains. Eighty eight percent of the G4P[6] was
isolated from newborns. The distribution of genotypes was different by hospital and by year. 2. In Jeongeub,
globally common genotypes constituted 65%. The most prevalent strain was G3P[8] (37%), followed by G9P[8]
(20%), G1P[8] (14%), and G2P[4] (12%). The newly emerging strain, G9P[8], constituted major strain (44%)
in the 02/03 season, but not detected at all in the 03/04 season. Conclusion: Globally uncommon G4P[6] was a
prevalent in Korea for the last 2 years and globally emerging strain G9P[8] was also emergent recently in Korea.
We confirmed that the prevalent strains are quite different depending on regions or by time
Keywords: rotavirus, genotype, Korea
Investigations of low molecular weight (LMW) proteins and peptides are
increasingly exploiting the developing technologies and methodologies of
proteomics with the hope of discovering better indicators of the onset or
progression of diseases. About 50% below than 15,000 Da proteins in size
were reported hypothetical proteins in the genome sequence data of
Helicobacter pylori strain 26695. Preparation of LMW proteins were carried
out by the elution from SDS-PAGE gel. After electrophoresis, the gel
containing LMW proteins was cut into pieces, according to pre-stained
protein size marker, and these pieces were ground in an extraction buffer and
liquid nitrogen. The eluted proteins were isolated from gels, followed by
cleaning and precipitation, and the precipitated proteins were applied to
two-dimensional electrophoresis and protein identification with
MALDI-TOF-MS. As a result, the 2-DE profile of H. pylori LMW proteins
was displayed and proteins such as hypothetical protein HP0268, hypothetical
protein HP1358, cupper ion binding protein, thioredoxin were newly
identified. In addition, this result suggest that proteins eluted from
SDS-PAGE gels could be efficiently seperated by using 2-DE and several
hypothetical proteins was conformed to real proteins expressing from
authentic genes. Analysis of LMW proteins may provide diagnostic
candidates in the bacterial infection and relative disease.
Keywords: Proteomics, Low molecular weight protein, MALDI-TOF-MS
Department of Laboratory Medicine, Hanyang University College of
2
Medicine, Department of Pediatrics, Chonbuk National University College of
3
4
Medicine, International Vaccine Institute, Departments of Laboratory
Medicine, University of Ulsan and Gangnung Asan Hospital, 5Daegu Catholic
6
7
University Hospital, Cheju National University Hospital, Chosun University
8
9
Hospital, Pusan National University Hospital, Eulji University Hospital,
10
University of Ulsan and Asan Medical Center, 11Centers for Disease Control
and Prevention, USA
*Corresponding author: jokang@hanyang.ac.kr
240
Vibrio vulnificus Showing Yellow Colony on
TCBS (Thiosulfate-Citrate-Bile-Sucrose) Agar
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
G033
G035
The Different Aspects of Viral Replication
According to the Existence of Hantavirus
Nucleocapsid Protein in Early Stage of Infection
1,2
2
1
2
Sun-Whan PARK , Hak KIM , Byoung-Yoon AHN , and Pyung-Woo LEE *
1
Establishment of Stable Cell Line Expressing
Hantavirus Nucleocapsid Protein
1
1,2
1
Hak KIM , Sun-Whan PARK , Ki-Joon SONG , and Pyung-Woo
LEE1*
Laboratory of Molecular Virology, Graduate School of Biotechnology, Korea
University, 2Laboratory of Virology, Department of Microbiology, College of
Medicine, Korea University
*Corresponding author: htn76@korea.ac.kr
Laboratory of Virology, Department of Microbiology, College of Medicine,
Korea University, 2Laboratory of Molecular Virology, Graduate School of
Biotechnology, Korea University
*Corresponding author: gold_hak@korea.ac.kr
Hantaviruses are a member of Bunyaviridae, which have negative sense
strand three segmented RNA genome and viral RNAs are encapsidated with
viral nucleocapsid protein (Np). Hantaviral Np seems to be actively involved
in transcription, replication, and also in packaging of RNPs into virus
particles.To investigate the role of Np in viral replication, we fractionated the
cells infected with Hantaan virus using anti-Np antibody. As a result, we
found that the infected cells showed 4 kinds of viral RNAs such as positive
and negative sense RNAs, genomic and anti-genomic RNAs. Genomic and
anti-genomic RNAs are bound with viral Np. Both of genomic and
anti-genomic RNAs exist as full sequence, and form pan-handle structure and
dimers. Additionally, truncated viral mRNA is more efficient for translation.
To investigate different phase of viral replication by Np existence, first of all,
we constructed stable cell line expressing Hantaan virus nucleocapsid protein.
This cell and Vero E-6 cell were infected with Hantaan virus and then were
analyzed RNAs, proteins and progeny viruses. By comparing replication
aspects in two types of cells, we could find that the viral replication is occurred
lately in Np expressing cells. The study is in progress to clarify the reason why
viral replication is occurred lately in Np expressing cell and these results
suggest that Np may play an important role in viral replication. Supported by
grants from KRF (E00109)
Keywords: Hantavirus, Nucleocapsid protein, Replication, Bunyaviridae,
negative sense, RNA genome
Hantavirus has tripartite (L, M, and S) negative sense genome which
is one of the principal characters of family Bunyaviridea. The
nucleocapid protein(NP) of Hantavirus plays an important role in
replication of viral genomic RNA with RNA-dependant
RNA-polymerase. However, the function of NP is not well known.
We established stable cell line expressing Hantavirus to investigate
function of NP and Hantaviral replication mechanism as well. Using
‘pcDNA3.1/His A’ as vector and ‘NP expressing region’ as insert,
we prepared cDNA. The plasmid was transfected to Vero E6 cell,
and then G-418 was used for selection factor. Each survivor cell was
examined by Western blotting analysis and was clarified specific
reactivity with ‘HTN polyclonal Ab’, ‘Anti-HTN NP monoclonal
Ab’ and ‘Anti-Xpress monoclonal Ab’. As the result, the selected
cells expressed NP stably for longer passages showing
establishment of the stable cell line expressing NP. Further, this cell
line will be used for following investigation on functional study of
NP in replication of Hantavirus. Supported by grants for
KRF(E00109)
Keywords: Hantavirus, Bunyaviridae, Stable cell line
1
G034
G036
In vitro Susceptibility Study of Filamentous
Fungi to Xanthorrhizol Isolated from Java
Turmeric (Curcuma xanthorrhiza Roxb.)
1,2
3
1,3
Yaya RUKAYADI , Jeong Han CHOO , and Jae-Kwan HWANG *
1
Bioproducts Research Center, Yonsei University, 2Biopharmaca Research
3
Center, Bogor Agricultural University, Indonesia, Department of Biotechnology,
Yonsei University
*Corresponding author: jkhwang@yonsei.ac.kr
The interest on the in vitro susceptibility to natural antifungal has
recently increased. In this study, comparative MIC and MFC of
xanthorrhizol (XTZ) isolated from java turmeric (Curcuma
xanthorrhiza Roxb.) with amphotericin B (AMB) were carried out in
6 isolates of filamentous fungi using the CLSI microbroth methods
described in M38-A. MIC of XTZ (1 µg/ml) was lower against
Aspergillus flavus and A. fumigatus compared to that of AMB (2.5 µ
g/ml), but the MFC of XTZ (5 µg/ml) was higher than that of AMB
(4 µg/ml). Susceptibility to XTZ and AMB was found the same for
the A. niger, Rhizopus oryzae and Trichophyton mentagrophytes
(MIC, 1 µg/ml; MFC, 2.5 µg/ml). AMB was found more susceptible
against Fusarium oxysporum compared to XTZ. MIC and MFC of
AMB were 1 µg/ml and 2.5 µg/ml, respectively, while MIC and MFC
of XTZ were 5 µg/ml and 7.5 µg/ml, respectively. Generally,
susceptibility of XTZ against 6 kinds of filamentous fungal was
comparable with the commercial antifungal, AMB. Thus,
xanthorrhizol can be recommended to be developed as a natural
antifungal agent.
Keywords: Xanthorrhizol, Filamentous fungi, Anti-fungal activity
Inhibition of Bacterial Quorum Sensing by
Vanilla Extract and Vanillin
1
2,3
1,2
Jeong Han CHOO , Yaya RUKAYADI , and Jae-Kwan HWANG
1
2
Department of Biotechnology, Yonsei University, Bioproducts Research
3
Center, Yonsei University, Biopharmaca Research Center, Bogor Agricultural
University, Indonesia
*Corresponding author: jkhwang@yonsei.ac.kr
Bacterial cell-to-cell signaling, termed as quorum sensing, has been
shown to greatly contribute to pathogenesis in humans and other
organisms. The purpose of this study was to search for a novel
quorum sensing inhibitor and analyze its activity. 75% methanol
extracts of tropical medicinal plants were screened for quorum
sensing inhibitory activity using the Tn-5 mutant, Chromobacterium
violaceum CV026. Results of the screening showed that the extract
of vanilla beans (Vanilla planifolia Andrews) significantly reduced
violacein production in a concentration-dependent manner,
indicating inhibition of quorum sensing. Further studies revealed
that the activity of vanilla was due to its main component, known as
the widely consumed flavor compound vanillin. Vanillin also
inhibited violacein production by C. violaceum CV026 in a
concentration-dependent manner. The activity of vanillin was
further confirmed using the Pseudomonas aeruginosa model.
Vanillin inhibited pyocyanin production as well as superoxide
dismutase and chitinase activities in Ps. aeruginosa. The results of
this study suggest that vanilla and vanillin may be applied in medical
use to prevent bacterial pathogenesis.
Keywords: Quorum sensing, Vanilla, Vanillin, Chromobacterium
violaceum CV026, Pseudomonas aeruginosa
국제학술대회
241
October 13-14, 2005, Seoul, Korea
G037
G039
The ORF49 Gene Product of Murine Gammaherpesvirus
68, a Homolog of BRRF1 of Epstein-Barr Virus,
Enhances Viral Replication in Cooperation with
Replication Transcription Activator
Molecular Analysis of the Isoniazid-Resistance
Related Genes in Mycobacterium Tuberculosis
Isolated from Korea
Sangmi LEE*, Min-Soo KIM, and Moon Jung SONG
Department of Microbiology, College of Medicine Hallym University
*Corresponding author: freemi88@hallym.ac.kr
Department of Biomedical Laboratory Science, College of Health Sciences,
Yonsei University
*Corresponding author: aquz0708@nate.com
Human gammaherpesviruses, including Epstein-Barr virus (EBV) and
Kaposi's Sarcoma-associated herpesvirus (KSHV), are associated with
tumors. All herpesviruses have distinct life cycle phases: lytic replication
and latency. The regulation in the switch of life cycles in
gammaherpesvirueses is critical to understand their pathogenesis. The
BRRF1 gene product (Na) of EBV was recently reported to cooperate with
RTA (BRLF1) to induce an efficient lytic EBV infection, suggesting its
critical roles in the switch of the life cycle. However, the function of a
BRRF1 homologue of MHV-68, ORF49 is unknown. MHV-68 ORF49
protein tagged with FLAG at the N-terminus was found in the nucleus of 293
and Vero cells. ORF49null and RTAnull viruses were generated by
random-transposon mutagenesis. The ORF49null virus showed attenuated
growth in BHK21, suggesting an important function of ORF49 in virus
replication. The growth of RTAnull was further enhanced by ORF49 in the
presence of RTA, indicating functional cooperation of ORF49 with RTA
in the context of virus genome. Co-transfection of ORF49 further enhanced
the ability of RTA to activate downstream target promoter, suggesting a
possible mechanism for ORF49 cooperation with RTA. In conclusion, our
results showed that ORF49 plays an important role in regulation of viral life
cycle in gammahepresviruses.
Keywords: EBV,KSHV,MHV-68,RTA,ORF49,life cycle
The present study investigated the prevalence and diagnostic potential of the
most commonly reported mutations associated with isoniazid
(INH)-resistance, such as katG, inhA promoter region, oxyR-ahpC intergenic
region, and kasA gene, in 50 INH-resistant and 23 INH-susceptible clinical
isolates of M. tuberculosis. Mutation affecting katG (codon 315), inhA
promotor region (15 upstream of the start codon·(UPS), 8UPS), oxyR-ahpC
intergenic region, kasA gene, and none mutations were found in 64, 16, 8, 0,
and 12% of 50 INH-resistant isolates, whereas they were found in 0, 0, 0, 4,
16% of susceptible strains, respectively. Mutation at the katG gene were found
at codon 315 alone (4%), at codon 463 alone (38%), and both at codon 315 and
463 (58%) in 50 INH-resistant isolates. However while mutations at codon 315
were only detected in INH-resistant isolates, mutations at codon 463 were
detected in isoniazid-susceptible isolates. In case of inhA, mutations at 15UPS,
8UPS were detected only in INH-resistant isolates, whereas mutations at 102,
103, 112, 115, and 125 UPS were detected only in INH-susceptible isolates.
Mutations at oxyR-ahpC intergenic region were detected only in INH-resistant
isolates, not INH- susceptible isolates. In case of kasA, there were no mutations
in INH-resistant isolates. In total, 88% of INH-resistant isolates could be
identified by analysis of three loci : katG 315, the inhA promototer region
(15UPS, 8UPS), and the oxyR-ahpC intergenic region.
Keywords: Isoniazid
G038
G040
Cloning, Sequencing and Expression of HilA
Protein Gene in Salmonella enterica Serovar
Typhi
1
2
1
Ji Young MOON , Moo Hyung LEE , and Yung Bu KIM *
1
Departments of Microbiology and Immunology, College of Medicine, Pusan
2
National University, Departments of Oral Anatomy, College of Dentistry,
Pusan National University
*Corresponding author: ybkim@pusan.ac.kr
Salmonella enterica serovar Typhi (S. typhi) invasion genes are necessary for bacterial
invasion of intestinal epithelial cells and are thought to allow salmonellae to enter and
cross the intestinal epithelium during infection. Penetration of intestinal epithelial cells
by S. typhi requires the expression of invasion genes, encodes a type III secretion
system (TTSS) within a Salmonella pathogenicity island 1 (SPI1) that is located at
centrosome 63 of its chromosome. TTSS gene transcription is activated in response to
environmental signals and requires transcriptional regulators encoded within (HilA)
and outside (SirA) SPI1. Expression of components and substrates of this system is
subject to complex regulatory mechanisms. The hilA regulator encodes an
OmpR/ToxR family transcriptional regulator that activates the expression of invasion
genes in response to both environmental and genetic regulatory factors. Recently, work
from several laboratories has highlighted that regulation of hilA expression is a key
point for controlling expression of the invasive phenotype. In this study, the HilA
protein gene of S. typhi in clinical samples was cloned and sequenced and its properties
were analyzed. The hilA gene was subcloned into pGEM-T vector and sequenced. The
open reading frame consisted of 1662 bp, encoded a 61.5-kDa HilA protein of 553
amino acids. When searching the database for astA nucleotide or its deduced amino
acid sequence, the sequence of the astA showed homology of 99% to S. typhi previously
reported. The hilA gene of S. typhi was amplified by PCR and constructed a
recombinant plasmid with hilA gene (pGEX 4T-1 expression vector). The fusion
protein was expressed in Escherichia coli host strain BL21 (codon +), and purified with
Glutathione affinity chromatography. The molecular mass of the purified HilA was
approximately 60kDa as determined by SDS-PAGE. The results of this study facilitate
the study of the functions of the HilA protein of S. typhi.
242
Joo Hwan HWANG
한국미생물학회연합
Low pH-dependant Endocytosis of Rubella Virus
1,2,3
1,2,3
2,3
Mi-Young JUNG *, Eun Jin CHO , Sun-Ho KEE , Luck Ju
2,3
2,3
2,3
BAEK , Kwangsook PARK , Jin-Won SONG , and Ki-Joon
2,3
SONG
1
2
Korea University Graduate School, The Institute for Viral Diseases, 3Bank
for Pathogenic Viruses, Department Of Microbiology, College of Medicine,
Korea University
*Corresponding author: maeng1227@korea.ac.kr
Rubella virus (RV) is an enveloped positive stranded RNA virus of the family
Togaviridae. In previous research, Semliki Forest Virus (SFV), an enveloped
alphavirus of the family Togaviridae, has been reported that they use
clathrin-coated vesicles to entry and acidic endosomal pH induce the fusion
activity of SFV during virus infection into host cells.The cellular entry
mechanism of RV and low-pH dependent membrane fusion in culture cells
were studied. Specific endocytic pathway was inhibited using various drugs,
and immunoblotting and immunofluorescent assay were conducted. The cells
treated with cationic amphiphilic drugs(CADs) such as chlorpromazine were
inhibited RV infection, suggesting that clathrin-dependent endocytosis is to
be major RV infection pathway. To characterize the endocytic transport in
cytoplasm of cells, VeroE6 and BHK-21 cells were treated with
lysosomotropic weak base agents such as the vacuolar H+-ATPase inhibitor
bafilomycin A1, chloroquin and ammonium chloride to block endosomal
acidification. These agents in both VeroE6 and BHK-21 cells inhibited RV
infection, and microtubules and actin cytoskeletal integrity were also
important for RV infection.Taken together, these results suggest that RV
infectious entry is mediated by clathrin-dependent endocytosis into VeroE6
cells, and showed low-pH dependently of RV infection in both VeroE6 and
BHK-21 cells.
Keywords: rubella virus, Endocytosis, low-pH
2005 International Meeting of the Federation of Korean Microbiological Societies
G041
G043
Ciprofloxacin-resistance Mutations in the gyrA,
gyrB and parC Genes of Clinical Isolates of
Acinetobacter baumannii
New Virulence Factors Identified from Pseudomonas
aeruginosa-Drosophila melanogaster Interaction
Yong Sun YOO, Jeom Kyu LEE, Eui Suk SOHN, Yeong Seon
LEE, and Bong Su KIM
Department of Life Science, Sogang University
*Corresponding author: youhee@sogang.ac.kr
Division of Antimicrobial Resistant Pathogens, NIH, Korea Center for Disease
Control and Prevention
*Corresponding author: jeomkyu@nih.go.kr
Pseudomonas aeruginosa is an important opportunistic human
pathogen that pathogenically interacts with diversified
non-mammalian hosts including plants, insects and nematodes.
Here, we exploited the killing of the fruit fly, Drosophila
melanogaster by P. aeruginosa as an assay to screen a library of
random TnphoA transposon mutants of P. aeruginosa strain PA14.
Out of 2,000 mutants tested, ten were identified with attenuated
virulence in D. melanogaster and 8 of them (80%) exhibited
significantly reduced virulence in murine peritonitis model. Genetic
analysis of those mutants revealed the TnphoA insertions in PA5489
(dsbA), PA3703 (wspF), PA2424 (pvdI), PA0253, PA0369, PA2077,
PA2113, and PA2002 genes and in an intergenic region between
PA1928 (rimJ) and PA1929, as well as in a gene located within the
13th variable segment. These results demonstrate that D.
melanogaster can be used for an in vivo high throughput screen to
identify novel virulence factors involved in P. aeruginosa
pathogenesis.
Keywords: Pseudomonas aeruginosa, Drosophila melanogaster,
random transposon mutagenesis, virulence
Increasing numbers of fluoroquinoloes (FQ)-resistant isolates of
Acinetobacter baumannii have become a clinically significant problem.
The FQ resistance in A. baumannii is associated with alterations in gyrA and
parC genes but the association of mutations in gyrB gene is not well
understood. This report assesses the mutations in gyrA, gyrB and parC
genes for FQ resistance in A. baumannii isolates and the correlation
between the FQ MIC and the numbers of mutations in gyrA, gyrB and parC
genes. A total of 79 ciprofloxacin (CIP)-resistant A. baumannii isolates
were recovered from in non-tertiary hospitals in Korea. The MICs of CIP,
gatifloxacin, gemifloxacin and levofloxacin was measured for using the
agar dilution method according to NCCLS guidelines. To investigate the
mutations in gyrA, gyrB and parC genes, PCR assays and sequencing were
carried out. All of CIP-resistant isolates possessed the substitution of Leu
for Ser83 in gyrA gene. Double mutation, Ser83→Leu in gyrA gene and
Ser80→Leu in parC gene, was the most frequently detected among these
isolates. A novel mutation with the substitution of Asp for Glu479 in gyrB
gene was identified. The MICs of CIP for isolates with double mutation
were higher than the isolates with single mutation. These results estimated
that MICs level of CIP was correlated with the number of mutations in gyrA,
gyrB or parC genes among A. baumannii isolates and a novel mutaion of
gyrB gene was suggested to be responsible for FQ resistance.
Keywords: Acinetobacter baumannii, ciprofloxacin resistance, gyrA,
gyrB, parC
G042
Shin-Young Park*, Kelly B. Choi, Yun-Jeong Heo, and You-Hee Cho
G044
Four Cases of Q Fever in Febrile Individuals in
Korea
Vibrio cholerae Is Pathogenic Toward Drosophila
melanogaster
M. H. KANG, Y. S. CHOI, J. H. KIM, B. C. LEE, S. H. PARK, S.
K. SHIM, K. J. HWANG, and M. Y. PARK
Shin-Young PARK*, Yun-Jeong HEO, Kun-Soo KIM, and
You-Hee CHO
Division of Rickettsial and Zoonotic Diseases, KNIH
*Corresponding author: yschoi83@yahoo.co.kr
Department of Life Science, Sogang University
*Corresponding author: youhee@sogang.ac.kr
Q fever is a zoonosis caused by Coxiella burnetii. But Q fever lacks clinical
specificity and may present as acute or chronic disease. Therefore,
serological confirmation and detection of C.burnetii DNA are necessary to
assess laboratory diagnosis. Although several clinical cases and distribution
of antibody of Q fever were reported since 1990, nobody has been reported
to provide serological and molecular evidence thereafter from febrile patient
in Korea. The aim of this work is to illustrate results of serological
confirmation and detection of C. burnetii DNA. Four cases were diagnosed
using MIFA and 27kDa OMP encoding gene (com-1, 438bp) of C.burnetii
was detected by nested PCR. The patients were three men and one woman
and had not a history of animal contact. Clinical manifestations were high
fever and chills over the 20 days. The results of serology tests, one case was
IgG antibody titers for phase II antigen were increased from 1:1024 to
1:2048 and IgM titers were decreased from 1:1024 to 1:256. The IgG titers of
the others cases was 1:1024, 1:512, 1:256. And the IgM titers of 2 cases were
1:320 and 1 case was 1:80. Furthermore, all of the 4 cases were amplified to
the com-1 genes. PCR–RFLP patterns by Sal I and sequence blast showed
that identical patterns to that of C.burnetii. This study is the first serologic
and molecular evidence by laboratory diagnosis about Q fever and provides
useful information for optimization and standardization of Q fever diagnosis
in Korea
Keywords: Q fever Coxiella burnetii laboratory diagnosis
Infections of Drosophila melanogaster with V. cholerae killed the
flies (100% mortality) within 20 h, proliferating in the fly bodies as
a result of systemic infection. Avirulent preinfection by V. vulnificus
restricted the subsequent virulent infection by V. cholerae. While the
transcript levels of antimicrobial peptides (AMPs) were elevated by
V. vulnificus infection, the immediate transcriptions of AMPs, most
notably, Attacin A were not elevated by V. cholerae infection. The
ectopic expression of Attacin A and Metchnikowin enhanced the
survival of D. melanogaster upon V. cholerae infection as well.
These results suggest that AMPs may be important in response to
infections by Vibrio species and that the signaling pathways to
govern their expression might be the target for V. cholerae virulence
factors to elude the resistance mechanisms of D. melanogaster
innate immunity.
Keywords: Drosophila melanogaster, antimicrobial peptides, Vibrio
species, innate immunity
국제학술대회
243
October 13-14, 2005, Seoul, Korea
G045
G047
Distribution of mec Genes and Analysis of
Diversity of Mutation in the mecI gene and mecA
Promoter Region of Animal Methicillinresistant Staphylococcus aureus Strains
Preliminary Characterization of the Type III
Secretion Systems in Hahella chejuensis
John Hwa LEE
Genome Research Center, KRIBB
*Corresponding author: dossage@nate.com
College of Veterinary Medicine, Chonbuk National University
*Corresponding author: johnhlee@chonbuk.ac.kr
Methicillin (oxacillin)-resistant Staphylococcus aureus (MRSA) are major
clinical and epidemiological pathogens in the veterinary field. From 2001 to
2005, various specimens from cattle, pigs and chickens were collected and
examined for the presence of MRSA. The isolates from the 19 specimens were
tested for the presence of the mecA gene. Methicillin resistance was confirmed
by determining the MICs for these isolates. Among these 19 mecA-positive
isolates, 16 were consistently found to be resistant to methicillin. The mecR1
gene was found in all 19 mecA-positive S. aureus, and mecI was also detected
in 15 of the mecA-positive S. aureus. The mecI gene had an identical sequence
to the reference sequence in 9 of the 15 mecI-positive isolates. Three of the
other six isolates had a C to T substitution at nucleotide 202, and one had a G
to T substitution at nucleotide 43. These have been previously identified in
MRSA from humans, which shows that the transmission of human MRSA to
animals is plausible. Two isolates from chickens contained an addition of C
at position 23. This type of genetic change in MRSA has not been reported
elsewhere. In all 15 mecI-positive MRSA, the sequence of the mec
promoter/operator region was identical to the reference sequence. This
suggests other mechanisms for overcoming the repression of resistance
caused by mecI, beyond the simple product interaction between the mecA,
mecR1 and mecI genes.
Keywords: MRSA, Animal, Mec Gene, Mutation
Jeong-Im Lee*, Choong-Min Ryu, Ho-Young Kang, Seung-Hwan
Park, And Jihyun F. Kim
Many bacterial pathogens of plants and animals employ type III secretion
systems (TTSSs) to deliver effector proteins into the host cell. Among them,
certain pathogenic bacteria cause disease symptoms in the compatible hosts
while elicit the programmed cell death in incompatible hosts. The newly
completed genome sequence of a bacterium Hahella chejuensis, isolated
from marine sediment in Cheju Island, Korea, contains two TTSS gene
clusters that are similar to that of Yersinia spp. We evaluated whether these
H. chejuensis TTSSs of an animal pathogen type can function in plants.
Nicotiana benthamiana was chosen as a plant model system to study H.
chejuensis TTSSs. Infiltration of bacterial suspension of H. chejuensis at
OD600nm= 1 elicited necrosis similar to that of a typical hypersensitive
response (HR) on the leaf of N. benthamiana within 24 hours. It was
dependent on bacterial growth stage; it appeared when bacterial suspension
at early stationary phase was infiltrated. The necrosis by H. chejuensis was
suppressed when the strains are grown in a medium containing 0.5% glucose.
A previous study reported that AvrPto, an HR effector known for ability to
interact with Pto, may suppress the HR. We observed that H. chejuensis
containing AvrPto suppresses elicitation of the HR in N. benthamiana. Taken
together, these results suggest that the TTSSs of H. chejuensis is functional
and responsible for induction of the HR-like necrosis in N. benthamiana.
Keywords: type III secretion system, Hahella chejuensis, hypersensitive
response
G046
Prevalence and characteristics of Enterohaemorrhagic
Escherichia coli O26 and O111 from Cattle in Korea
John Hwa LEE and Hyun-Suk LEE
Characterization and Transcriptional Analysis
of Gene Clusters for a Type IV Secretion System
in Orientia tsutsugamushi
College of Veterinary Medicine, Chonbuk National University
*Corresponding author: johnhlee@chonbuk.ac.kr
Jeong-Eun GOO , Ji-Hyun YUN , Myung-Sik CHOI , Ik-Sang
2,3
1,2
KIM , and Young-Sang KOH *
Enterohemorrhagic Escherichia coli is recognized as an important
cause of diarrhea, hemorrhagic colitis and hemolytic-uremic
syndrome worldwide. EHEC O26 and O111 are the most frequently
isolated serotypes from human enterohemorrhagic Escherichia coli
infections in Korea. Cattle are probably the major sources of EHEC
O26 and O111. In this study, we investigated the prevalence of
EHEC O26 and O111 in fecal samples of cattle in Korea from April
2002 to March 2004. Of 809 samples, 54 (6.67%), 37 (4.57%), and
16 (1.98%) were positive for O26, O111, and both O26 and O111.
Most of the EHEC O26 and O111 strains were isolated from May to
October of each year. PCR analysis of EHEC virulence markers
revealed that most of EHEC O26 and O111 isolates were positive for
EhlyA, eaeA and stx1 and/or stx2. Cytotoxicity analysis revealed that
many of the isolates showed high cytotoxicity on Vero cells. Our
data suggest that the majority of Korean EHEC O26 and O111
isolates from cattle can cause serious diseases in humans.
Keywords: EHEC, Prevalence, genetic characteristic, cattle
244
G048
한국미생물학회연합
1,2
1,2
2,3
1
Department of Microbiology, Cheju National University College of Medicine,
Microbial Genome Center for Skin Infections, Seoul National University,
Department of Microbiology and Immunology, Seoul National University
College of Medicine
*Corresponding author: yskoh7@cheju.ac.kr
2
3
Orientia tsutsugamushi, the causative agent of scrub typhus
(tsutsugamushi disease), is an obligate intracellular bacterium that
freely replicates in the host cytoplasm and cause febrile systemic
illness in humans. Genomic sequence data indicate that 11 genes
(virB3, B4, B6, B8, B9, B10, B11, and virD4) encoding products that
are similar to components of the bacterial type IV secretion system
(T4SS) are located on separate contiguous 65 kb and 23 kb regions
of the Orientia chromosome. It was found that several vir genes
(virB4, virB10 and virD4) for T4SS were expressed by Orientia
during host infection in cell culture (L929 fibroblast and J774A.1
macrophage cell lines) and murine hosts. Transcripts for these genes
increased as early as 1 hr, peaked at 6 hr, and returned to baseline
preinfection levels by 12 hr after infection in J774A.1 macrophage
cell lines. Temporal regulation of gene expression may be associated
with intracellular survival and pathogenesis of this pathogen in host
cells.
Keywords: Orientia tsutsugamushi, Type IV Secretion System,
Transcriptional Regulation
2005 International Meeting of the Federation of Korean Microbiological Societies
G049
G051
Genetic Analysis of the Coxsackievirus B2 and
B3 Isolated from Clinical and Environmental
Samples during 1999 to 2005
Secretory Responses in Intestinal Epithelial
Cells Stimulated with Bacteroides fragilis
Enterotoxin through Cyclooxygenase-2 Pathway
Jiyoung HONG, Misoon KIM, Kangbum LEE, Wooyoung CHOI,
Jeehee KIM, and Youngmee JEE
Young Mee YOON, Jin Young LEE, and Jung Mogg KIM
Division of Enteric and Hepatitis Viruses, Department of Virology, NIH,
Korea Center for Disease Control and Prevention
*Corresponding author: hongzee22@hotmail.com
We isolated coxsackievirus B2 (CVB2) and CVB3 from clinical and
environmental samples in Korea during 1999 to 2005. To investigate the
genomic characteristics of these isolates, we determined the nucleotide
sequences of the 5' UTR and VP1 region of CVB2 and CVB3. 33 samples
from patients with aseptic meningitis or myocarditis and environmental water
from 1999 to 2005 were analyzed by RT-PCR of 5'UTR and VP1 regions
followed nucleotide sequencing. The analysis of serotype by neutralization
test were well correlated with the genotype based on the sequence analysis of
a partial VP1 region of Korean isolates . The nucleotide homologies of the
partial 5'UTR and VP1 of CVB2 were 86~89% and 82~84% with Ohio-1
strain (GenBank AF085363), respectively. Comparison of 5'UTR of CVB3
Korean isolates with CVB3-CO (nonvirulent strain, GenBank AF169665)
and Nancy (virulent strain, GenBank M33854) revealed homologies of
85~87% and 89~92%, respectively while the VP1 region of CVB3 Korean
isolates with CVB3-CO (nonvirulent strain, GenBank AF169666) and Nancy
(virulent strain, GenBank M33854) showed homologies of 77~85% and
74~99%, respectively. Seventeen CVB3 Korean isolates clustered together
with the nonvirulent CVB3-CO strain and one CVB3 isolate (01-CAR) from
an acute myocarditis patient was more closely related to virulent Nancy strain
than to other 17 CVB3 Korean isolates from patients with aseptic meningitis .
Keywords: Coxsackievirus B, sequencing, VP1, 5'UTR
G050
Department of Microbiology, Hanyang University College of Medicine
*Corresponding author: jungmogg@hanyang.ac.kr
Background: Bacteroides fragilis produces an approximately 20 kDa
heat-labile toxin (BFT) which is known to be associated with diarrhea. To
determine if cyclooxygenase (COX)-2, via NF-κB activation, can contribute
to BFT-induced diarrhea, the relationship between COX-2 expression and
fluid secretion in BFT-stimulated human intestinal epithelial cells was
examined. Methods: COX-2 expression in normal and BFT-stimulated
intestinal epithelial cells was determined using immunoblot analyses,
quantitative RT-PCR and luciferase assays. Prostaglandin E 2(PGE2) levels
were measured using enzyme immunoassays, and chloride secretion was
tested in polarized T84 cell monolayers using Ussing chambers. To assess the
pathophysiologic relevance of COX-2, studies were also performed in mice
bearing disruptions in COX-2 or NF-κB. Results: BFT stimulation increased
the expression of COX-2, but not COX-1, in human intestinal epithelial cells.
PGE levels were increased in parallel with COX-2 expression, and,
conversely, PGE2 production was significantly inhibited when COX-2 or NFκB activities were suppressed using siRNA for COX-2 or a retrovirus
encoding an IκBα superrepressor. The released PGE2 caused an increase in
chloride secretion in polarized T84 epithelial cell monolayers. Conclusions:
These results suggest that the diarrheal response to BFT stimulation of
intestinal epithelial cells is mediated by the secretion of PGE2, through NF-κ
B activation and the up-regulation of COX-2.
Keywords: Bacteroides fragilis enterotoxin, cyclooxygenase-2, intestinal
epithelial cells, prostaglandin E2
G052
Nuclear Factor-kappaB Activation Pathway Is
Essential for Chemokine Gene Expression in
Clostridium difficile Toxin A-Stimulated Intestinal
Epithelial Cells
Stimulation of Human Umbilical Cord Vein
Endothelial Cells with Bacteroides fragilis
Enterotoxin Upregulates Expression of
Intercellular Adhesion Molecule-1
Jin Young LEE, Young Mee YOON, and Jung Mogg KIM
Hyun Chuol Roh, Young Mee Yoon, Jin Young Lee, and Jung
Mogg Kim
Department of Microbiology, Hanyang University College of Medicine
*Corresponding author: jungmogg@hanyang.ac.kr
Intestinal epithelial cells are known to upregulate the expression of
several chemokine genes in response to stimulation with bacterial
toxin. However, there has been little understanding on the cellular
mechanisms of C. difficile toxin A-induced mucosal inflammation. In
this study, we investigated whether NF-κB could regulate chemokine
gene expression in intestinal epithelial cells. Toxin A increased signals
of NF-κB complexes containing p65/p50 heterodimers and p65/p65
homodimers. In contrast, toxin A decreased IκBα signals. Inhibition
of NF-κB downregulated the expression of IL-8, GRO-α, and MCP-1
in toxin A-stimulated HT-29 cells. Toxin A stimulation increased the
signals of phosphorylated IKKα/β and NIK, whereas suppression of
IKK or NIK inhibited the up-regulated transcription of downstream
target gene IL-8 and MCP-1 of NF-κB. In addition, costimulation with
toxin A and IFN-γ synergistically increased IL-8 secretion. These
results suggest that an NF-κB pathway is required for chemokine gene
expression in intestinal epithelial cells exposed to toxin A.
Keywords: C. difficile toxin A, chemokine, intestinal epithelial cells,
NF-kappaB
Department of Microbiology, Hanyang University College of Medicine
*Corresponding author: jungmogg@hanyang.ac.kr
Background: The acute host response to stimulation with toxigenic
Bacteroides fragilis is characterized by an accumulation of neutrophils in the
lamina propria. We have already demonstrated that B. fragilis enterotoxin
(BFT)-stimulated intestinal epithelial cells play an important role in the
recruitment of inflammatory cells through the secretion of chemokines.
However, little is known regarding the expression, by endothelial cells, of
molecules that are involved in infiltration of neutrophils following BFT
stimulation. Methods: After human umbilical vein endothelial cells
(HUVEC) were treated with BFT, expression of ICAM-1 was determined by
quantitative RT-PCR, flow cytometric analysis, and confocal microscopy.
Results: BFT up-regulated the expression of ICAM-1 mRNA on HUVEC.
The expression of ICAM-1 was dependent on the concentration of BFT.
Consistent with this, molecules of ICAM-1 were increased in the surface of
HUVEC in response to BFT stimulation. In addition, BFT also activated NF-κ
B signals in HUVEC. Furthermore, the suppression of NF-κB significantly
attenuated the increased BFT expression in BFT-stimulated HUVEC.
Conclusion: These results suggest that BFT released by pathogenic B. fragilis
can enhance interactions of leukocytes with the endothelium through NF-κ
B-dependent mechanism, thus inducing an inflammatory process.
Keywords: B. fragilis enterotoxin, human umbilical vein endothelial cells,
ICAM-1
국제학술대회
245
October 13-14, 2005, Seoul, Korea
G053
G055
Genetic Diversity of Hepatitis C Viruses from
Intravenous Drug Users in Korea
1
1
1
2
Sunok KIM *, Sujin KANG , Soyeon KIM , Daejin KIM ,
Younghun CHUN3, Jooshil LEE1, Haewol CHO1, and Youngmee
1
JEE
1
Division of Enteric and Hepatitis Viruses, NIH, Korea Center for Disease
Control and Prevention, 2The Catholic University of Korea Holy Family
3
Hospital, Incheon Chamsarang Hospital
*Corresponding author: ymeejee@nih.go.kr
As the prevalence of HCV infection has increased over the last 20 years, the
prevalence of HCV genotype has also changed. The aim of this study was to
investigate the quasispecies heterogeneity of hepatitis C virus(HCV) from
intravenous drugusers in Korea. Genetic diversity of hepatitis C virus(HCV)
detected from 31 intravenous drug users was analysed in 31 Korean patients
by using a genotype-specific probe-based assay(Inno-LiPA HCVII;
INNOGENETICS, Ghent, Belgium). Among 51 serum samples of
intervenous drug users which were collected by the Catholic University of
Korea Holy Family Hospital, 31 samples were proved to be HCV RNA
positives. In this study, viral RNA was extracted from serum using TRIZOL
and reverse transcription was performed. We performed first and nested
PCR to amplify hypervariable region of HCV. Hypervariable region of E2 of
HCV was then subcloned and sequenced. Sequence homology of genotype
1b and 2a of HCV ranged 80% and 65%, respectively. Phylogenetic analysis
indicated that HCV variants would be clustered by each individual.
Heterogeneous quasispecies were detected in most subjects. It is suggested
that the high level of quasispesies of HCV is consistent with the transmission
of multiple infectious particles. Needle sharing among intravenous
drugusers appears to be predominant route of transmission of multiple viral
variants, which contributes to evolution of quasispecies of HCV.
Keywords: HCV, hypervariable region, quasispecies
Construction and Characterization of a Stable
Full-Length Ecotropic MuLV Molecular Clone
for Studying the Genetic Determinant of Fv1
Restriction
Eun Hye BAE, Jae Hoon JEONG, Sung-Han PARK, Sang-Min
PARK, Jin Woo PARK, In Chul BAEK, and Yong-Tae JUNG*
Department of Microbiology, Dankook University
*Corresponding author: yjung@dku.edu
A number of genes which affect the susceptibility of mice to infection by
retroviruses have been described. One of the most interesting of these genes
is Fv1(Friend virus susceptibility 1), which acts at a stage in the retoviral
life-cycle following virus entry into the cell but prior to integration and
formation of proviral structures. The host range subgroups of the
mouse-tropic MuLVs can be further subclassified as N-tropic if they replicate
best in Fv1n cells (NIH-swiss strain), B-tropic if they replicate best in Fv1b
n
b
cells (BALB/c ), or NB-tropic if they grow equally well in Fv1 or Fv1
0
cells. A phenotypically null allele, designated Fv1 has been identified in
certain wild cell line sensitive to all retrovirus. Genetic evidence suggests that
the target for restriction is the viral capsid (CA) protein encoded by the viral
gag gene. In an effort to further characterize this resistance, we constructed
stable infectious molecular clones of AKV type N-tropic MLVs which
0
generate high titer of progeny virus in infected SC-1 cells(Fv1 ). Attempts to
define more precisely which of the preintegration steps is affected Fv1 action,
we used site specific mutagenesis in CAgag of AKV molecular clone. Our
experiments showed reduction in the amounts of freshly synthesized viral
DNA in the cytoplasm of SC-1 cells expressing Fv1n , therefore the
Fv1-mediated block to infection in murine cells occur before completion of
reverse transcription.
Keywords: Fv1, resistance, full length molecular clone
G054
G056
Rapid and Sensitive Method Using Multiplex
Real-Time PCR for Diagnosis of Infections by
Hantaan virus, Seoul virus and Puumala virus
Quantitative Analysis of Representative Proteome
Components and Clustering of Helicobacter pylori
Clinical Strains
Sung-Sil Moon1,2*, Jin-Won Song1,2, Ki-Joon Song1,2, and
Luck Ju Baek1,2
Jeong-Won PARK , Kyung-Mi KIM , Jung-Soo JOO , Damir
1
1
2
NIZAMUTDINOV , Mi-Hyun KANG , Jin-Su JUN , Hee-Shang
2
1
1
YOUN , Woo-Kon LEE , and Kwang-Ho RHEE *
1
2
Department of Microbiology, College of Medicine, Institute for Viral
Diseases, Bank for Pathogenic Viruse, Korea University
*Corresponding author: sincery@korea.ac.kr
Hemorrhagic fever with renal syndrome (HFRS) is a disease cause
by viruses of the family Bunyaviridae, genus Hantavirus, world
wide, several different human pathogenic hantaviruses are known.
The subtypes Hantaan (HTN), Suchong (SC), Muju (MJ) and Seoul
(SEO) cause moderate to severe HFRS in Korea. Laboratory
diagnosis of Hantavirus infections is generally performed by
immunofluorescent (IF) assays and reverse transcriptase PCR
(RT-PCR). The methods are not a rapid diagnostic test, and have
been shown to be less sensitive and specific, and therefore, its
clinical value is limited. A rapid real-time multiplex PCR assay was
developed for the detection of subtype of Hantaviruses (HTNV,
SEOV and PUUV) in a one-tube multiplex reaction which used
TaqMan probe to discriminate the pathogens. The application of
real-time PCR to clinical samples increases the sensitivity for HFRS
diagnosis. In addition, results can be obtained within 2hr, which
increases clinical relevance. Therefore, use of this real-time PCR
assay would improve patient management and infection control.
Keywords: Hantavirus, Real-time PCR
246
한국미생물학회연합
1
1
1
1
Department of Microbiology, Gyeongsang National University College of
Medicine, 2Department of Pediatrics, Gyeongsang National University
College of Medicine
*Corresponding author: khrhee@gaechuk.gsnu.ac.kr
Representative of proteome components from 71 clinical isolates of
Helicobacter pylori, were analyzed quantitatively to determine the
relationships among clinical isolates and whether the protein expression
levels were associated with specific gastric diseases. Proteomic analysis of
whole-cell protein solutions of H. pylori isolates identified 10 representative
proteins cytotoxin-associated antigen, urease beta subunit (UreB), heat shock
protein 60, elongation factors (EF-Tu and EF-P), inorganic pyrophosphatase,
superoxide dismutase, 26 kilodalton antigen, adhesin-thiol peroxidase,
flavodoxin and one unidentified spot. The spot intensities were measured and
compared among the isolates and specific disease patterns. The intensity
values of all target spots were highly variable among all of the clinical isolates.
However the expression levels of UreB, EF-P, and the unidentified protein
differed significantly according to the gastric disease corresponding to the
clinical isolates. Hierarchical agglomerative cluster analysis generated a
dendrogram with clusters indicative of chronic gastritis/gastric cancers or
gastric/duodenal ulcers. These results indicate that quantitative analysis of
proteome components is a feasible method identifying disease-associated
proteins and clustering clinical strains of H. pylori.
Keywords: H. pylori, Proteomics, MALDI-TOF-MS
2005 International Meeting of the Federation of Korean Microbiological Societies
G057
G059
Cell Density-Dependent Regulation of Quorum
Sensing System of Escherichia coli, Pseudomonas
aeruginosa and Staphylococcus aureus
1
1
2
3
Ha-Yan LEE , Mi-Hye LEE , Kyong-Ran PECK , Ji-Youl LEE ,
and Sang-Seob LEE1*
1
2
Biological Engineering, Kyonggi University, School of Medicine, Sungkyunkwan
University, 3Urology, School of Medicine
*Corresponding author: sslee@kyounggi.ac.kr
Quorum sensing is a cell-density-dependent bacterial intercellular
signaling mechanism that enables bacteria to coordinate the expression of
certain genes. The purpose of this study is gene regulation anaylsis of
quorum sensing mechanism related biofilm forming by mRNA
expression rate. We have done quantitative analysis of mRNA expression
of gene related autoinducer synthesis. This quantitative analysis was
measured by competitive RT-PCR. First, we cloned lasI and rhlI in
Pseudomonas aeruginosa, ygaG in Escherichia coli and luxS in
Staphylococcus aureus. Then competitor genes of each target genes were
cloned. Second, we prepared for samples. One is single cell samples. We
cultured each stains in LB broths. And then we spreaded culture of each
strains on LB agar plates and incubated at 37℃. And we scraped up the
cells according to sampling times. The other is mixed cell samples. We
mixed three strains, incubated in 37℃ and scraped up the cells. Third,
we purified total RNA from bacteria used by lysozyme. And then
analyzed mRNA expression of lasI, rhlI, ygaG and luxS by using the direct
competitive RT-PCR.
Keywords: competitive RT-PCR, Quorum sensing, Pseudomonas
aeruginosa, Escherichia coli, Staphylococcus aureus
G058
The Quantitative Analysis of mRNA Expression
Levels of ygaG, lasI, rhlI and luxS Gene Involved in
Quorum Sensing in Infected Foley Catheter by
Using Competitive RT-PCR.
1
1
1
2
Mi-Hye LEE , Ha-Yan LEE , Sang-Seob LEE *, Ji-Youl LEE ,
and Kyong-Ran PECK3
1
2
Department of Biological Engineering Kyonggi University, Department of
Urology, School of Medicine, The Catholic University of Korea, 3School of
Medicine, Sungkyunkwan University
*Corresponding author: sslee@kyonggi.ac.kr
Catheter-associated urinary tract infection (CA-UTI), which is frequently
occurring in the patients with indwelling Foley Catheter, can cause higher
mortality in immune deficient patients. On catheter matrix, CA-UTI is form
with biofilm by infected bacteria when catheter matrix is filled with host
proteins and microbial percolations. Formation of biofilm is involved quorum
sensing mechanism between infected bacteria and it has resistant to immune
system of host and antibiotics. These properties of biofilm prevent to
treatment of CA-UTI with antibiotics. Therefore, we need to study of quorum
sensing mechanism and it's related bacteria. In this study, we have done
quantitative analysis of mRNA expression of autoinducer synthesis related
gene. Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus
were isolated from infected catheters. We detected ygaG(from E. coli),
lasI(from P. aeruginosa), rhlI(from P. aeruginosa), luxS (from S. aureus)
genes and these competitors were previously made to perform competitive
RT-PCR. Then, we executed direct competitive RT-PCR for quantitative
analysis of mRNA which was isolated from infected catheter of patients in the
unit area.
Keywords: Quorum sensing, competitive RT-PCR, ygaG, luxS, rhlI, lasI
gene, autoinducer synthesis
G060
Characterization of Trichosporon yeasts Isolated
from Toenails
1
2
1
Kye Seung JANG , Hee Jun KANG , Wook Ha PARK , Sua
1
1
PYO , and Seong Hwan KIM *
1
2
Department of Microbiology, Dankook University, Asan Middle School
*Corresponding author: piceae@naver.com
The genus Trichosporon includes 25 species of basidiomycetous
yeasts. Some species of the genus are implicated in infectious or
allergic diseases. In Korea, Trichosporon beigelii has been
considered as a causal agent of tedia pedis. However, in recent
treatment of the genus Trichosporon, T. beigelii is abandoned in
favor of T. asahii, T. cutaneum, T. mucoides, T. ovoides, and T. inkin.
Consequently, we need to define that among the five Trichosporon
species what species can replace Trichosporon beigelii in Korea.
Thus, we isolated two basidiomycetous yeasts from toenails of two
middle school students and characterized them. On SD media, the
yeasts produced abundant and well-developed pseudohyphae and
hyphae. Blastoconidia are unicellular and variable in shape. For
molecular comparison, we determined nucleotide sequences of the
internal transcribed spacers (ITS1 and ITS2), 5.8S and intergenic
spacer 1 (IGS1) regions in ribosomal DNA. Together with the
results of growth test at 37℃ and physiological tests, taxonomic
position of the two Trichosporon yeasts was discussed.
Keywords: Trichosporon, tedia pedis
Increased Cell Adherence in PER-1-producing
Acinetobacter baumannii
1
2
1
1
Hee Woo LEE *, Yu-Mi LIM , Kyung-Min JUNG , Je Chul LEE ,
1
1
1
Yoo Chul LEE , Sung Yong SEOL , Dong Taek CHO , and
1
Jungmin KIM *
1
Department of Microbiology, Kyungpook National University, School of
Medicine, 2Department of Microbiology, Dankook University, School of
Medicine
*Corresponding author: dinks@snu.ac.kr
Acinetobacter baumannii, an important nosocomial pathogen, is usually
found on various surfaces in the hospital environment and causes severe
infections in patients of intensive care units. Recently, the dissemination of
PER-1 extended-spectrum β-lactamase among A. baumannii isolates was
reported in Korean hospitals. We investigated the presence and the
association of various virulence determinants in 23 A. baumannii isolates, of
which 12 were blaPER-1 positive. Virulence tests were the adherence to
polystyrene, formation of biofilm and NCI-H292 cell adhesion and genomic
DNAs were analyzed by pulse-field gel electrophoresis (PFGE). Although all
23 isolates were able to attach to polystyrene disk and to form biofilm, A.
baumannii strains with blaPER-1 showed significantly increased adherence to
NCI-H292 cells compared with those without blaPER-1. PFGE analysis showed
that 12 blaPER-1-positive strains were very closely related with each other but
not with blaPER-1-negative strains. Currently, we are doing proteomic analysis
with two isolates, high (blaPER-1-positive) or low (blaPER-1-positive) adherence
to NCI-H292 cells, to investigate proteins involved in cell adherence of A.
baumannii strains. This study indicates the existence of a relation between
blaPER-1 and cell adhesion in A. baumannii isolates and this could be a reason
for why blaPER-1-positive A. baumannii strains are spreading rapidly and
maintained continuously in the hospital environment.
Keywords: Acinetobacter baumannii, PER-1, adherence, formation of
biofilm
국제학술대회
247
October 13-14, 2005, Seoul, Korea
G061
G063
Identification of Nuclear Localization Signal
(NLS) in Omp38 of Acinetobacter baumannii
1
2
3
Chul Hee CHOI *, Sung Hee HYUN , Soon Ae KIM , Yoo Chul
LEE1, Jungmin KIM1, Sung Yong SEOL1, Dong Taek CHO1,
1
and Je Chul LEE
1
Department of Microbiology, Kyungpook National University School of
Medicine, 2Department of Biochemistry, Eulji University School of Medicine,
3
Department of Pharmacology, Eulji University School of Medicine
*Corresponding author: jsa465@hotmail.com
Outer membrane protein38 (Omp38) is a major Omp of Acinetobacter
baumannii. We previously demonstrated that Omp38 induced apoptosis of
epithelial cells and was responsible for invasion of bacteria to epithelial cells
and serum resistance. Nuclear localization signals (NLSs) are short stretches
rich in basic amino acid residues, lysine (K) and arginine (R). The putative
NLS, KTKEGRAMNRR, was observed in the C-terminal region of Omp38.
The putative NLS regions were also observed in major Omps of Acinetobacter
species, but not in Omps of other pathogenic Gram (-) bacteria. Nuclear
translocation of Omp38 was confirmed by monitoring the localization of
Omp38-EGFP fusion proteins in COS-7 cells, HEp-2 cells, and NCI-H292
cells. Transient transfection assay with the small fragment containing NLS
(amino acids 229-356) showed that the fusion proteins were observed in
nuclei, while the EGFP fusion proteins of N-terminal region (amino acid
1-102) or amino acids 103-228 did not enter to nuclei. Omp38-EGFP fusion
proteins were highly cytotoxic in COS-7 cells, which was accordance with the
results of apoptosis of epithelial cells by treatment of Omp38. These results
indicate that Omp38 enters to nucleus through the short stretches of basic
amino acid in C-terminal region and now we investigate the functional
characterization of Omp38 in the nucleus of eukaryotic cells.
Keywords: Acinetobacter baumannii, Nuclear localization signal,
Apoptosis, Outer membrane protein
G062
248
Characterization and Detection of Mutations in
gyrA, ParC and ParE Genes in CiprofloxacinResistant Isolates of Escherichia coli from 1994
to 2004
Dong Chan MOON*, Jae Young OH, Jungmin KIM, Je Chul
LEE, Yoo Chul LEE, Dong Taek CHO, and Sung Yong SEOL*
Department of Microbiology, Kyungpook National University, School of
Medicine
*Corresponding author: ansehdcks@naver.com
A total of 1,280 isolates of E. coli collected from 1994 to 2004 in a university
hospital were examined for the frequency and degree of ciprofloxacinresistance, relation with resistance to other antimicrobial agent, and mutation
in quinolone resistance determining regions (QRDRs) of gyrA, gyrB, ParC,
and ParE. Ciprofloxacin resistance was observed in 264 of 1,280 isolates and
the frequency of ciprofloxacin-resistance was raised from less than 12% in
1994-1995 to near 20% in 1996-2004. Multi-drug resistance (more than 3
drugs among ampicillin, trimethoprim, sulfisoxazole, gentamycin,
kanamycin) was significantly higher in ciprofloxacin-resistant E. coli
(75.4%, 199 of 264 isolates) than in ciprofloxacin-susceptible E. coli (29.5%,
300 of 1,016 isolates) but extended-spectrm β-lactams (cefotaxime,
ceftazidime, aztreonam, cefoxitin, cefepime)-resistance was not. Sequencing
of the PCR amplified products of the QRDRs in randomly selected 59
ciprofloxacin-resistant isolates showed that all isolates tested carried double
mutations in gyrA at codon 83 and 87 and at least one parC mutation at codon
57, 78, 80, 84 and/or 108. The most prevalent pattern was the S83L mutation
and the mutation at codon 87 from an aspartate to an asparagine (D87N) of
GyrA plus a mutation from a serine to an isoleucine (S80I) at codon 80 of ParC
plus a mutation from a serine to an alanine (S458A) at codon 458 of ParE (14
of 59 isolates).
Keywords: Escherichia coli, gyrA, ParC, ParE, ciprofloxacin-resistant
G064
Predominance of ST5 and ST239 Clones with
the Presence of Diversity within SCCmec in
Methicillin-resistant S. aureus in Korean hospitals
The armA Aminoglycoside Resistance Methylase
Gene and blaCTX-M Gene Are on the Same
Self-Transmissible Plasmid
Hwa Yoon CHA, Jong Sook JIN, Jungmin KIM, Je Chul LEE,
Yoo Chul LEE, Sung Yong SEOL, and Dong Taek CHO*
Hee Young KANG, Yu Mi LIM, Je Chul LEE, Yoo Chul LEE,
Sung Yong SEOL, Dong Taek CHO, and Jungmin KIM*
Department of Microbiology, Kyungpook National University, School of
Medicine
*Corresponding author: khy7734@nate.com
Department of Microbiology, Kyungpook National University, School of
Medicine
*Corresponding author: khy7734@nate.com
Staphylococcus aureus is one of the most important pathogens in nosocomial
and community-acquired infections. Since the first detection of
methicillin-resistant S. aureus (MRSA) in 1961, MRSA has spread
worldwide and contributes to cause serious problems in a clinical setting. To
analyze the MRSA clones circulating in Korean hospitals, a total of 425
non-duplicate MRSA isolates obtained from 2001 to 2005 in hospitals were
characterized using multilocus sequence typing (MLST), spa typing and
SCCmec typing. From the MLST study, the most prevalent clonal type was
ST5/ST5 slv (ST221) (51%, 216 of 425 isolates) and the second was
ST239/ST239 slv (ST344) (36%, 154 of 425 isolates) . Other four types, ST1,
ST72, ST254/ST254 slv (ST345) and ST89, were detected in 30, 15, 8 and 2
isolates, respectively. From SCCmec typing, 262 (62%) of 425 possessed one
of the previously reported SCCmec types such as type II, III, IIIA, IV or IVA
but various kinds of novel SCCmec variants were detected in 163 isolates,
which were grouped into the followings: (i) ST5, spa type TJMBMDMGMK
or TJMBBMDMGMK, and one of five SCCmec type II variants (IIa to IIe)
(152 isolates); (ii) ST254/ST254 slv (ST345), non-detectable spa and
SCCmec IV variant (8 isolates); (iii) ST89, spa type Y2EJCMBPB, and
SCCmec IIe or IIf (2 isolates); (iv) ST239, spa type WGKAOMQ, and
SCCmec III variant (1 isolate). This study revealed the predominance of ST5
and ST239 clones with the presence of diversity within SCCmec.
Keywords: Methicillin-resistant Staphylococcus aureus, Multilocus
sequence typing, spa typing, SCCmec typing
The armA (aminoglycoside resistance methylase) gene is of clinical
importance since it confers high-level resistance to all the clinically available
aminoglycosides except streptomycin. The armA gene was initially found in
Klebsiella pneumoniae which also carrying the extended-spectrum β
-lactamase CTX-M-3, which confer resistance to all ß-lactams with the
exception of carbapenems. In a previous study, dissemination of CTX-M-3
was found in Enterobacteriaceae and all CTX-M-3 producing isolates
showed high-level broad-spectrum aminoglycoside-resistance. Therefore,
We examined the presence of armA gene in 17 CTX-M-3-producing isolates
of Citrobacter freundii, Escherichia coli, K. pneumoniae, and Serratia
marcescens. The armA gene was detected in all 17 blaCTX-M-3 carrying isolates
and blaCTX-M3 and armA were co-transferred by conjugation. Southern
hybridization with the probes for blaCTX-M-3 and armA revealed that both genes
were located on the same plasmid. These data indicated the presence of armA
on self-transferable plasmid together with blaCTX-M-3, supporting the notion
that the spread of blaCTX-M-3 with armA results from conjugation, and
dissemination of aminoglycoside resistance by 16S rRNA methylation in
enterobacteria. The spread of blaCTX-M-3 with armA on the same plasmid in
Enterobacteriaceae is particularly troublesome because it severely restricts
the therapeutic options for infection by these bacteria.
Keywords: CTX-M-3 ESBL, aminoglycoside resistance, armA,
Enterobacteriaceae
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
G065
G067
Peripheral Blood Mononuclear Cell Responses
to Low Molecular Weight Antigen MTB12 of
Mycobacterium tuberculosis in Human Tuberculosis
1
2
2
Ji-Sook LEE , Eun-Kyeong JO *, Saet-Byel JUNG , Chul-Su
YANG2, Chang-Hwa SONG2, Hwa-Jung KIM2, Jeong-Kyu
2
1
PARK , and Tae-Hyun PAIK
1
Department of Microbiology, College of Medicine, Konyang University,
Department of Microbiology, College of Medicine, Chungnam National
University
*Corresponding author: charles514@lycos.co.kr
Murine Acquired Immunodeficiency Syndrome
(MAIDS) Model Is a Useful Tool For Screening
Herb Extracts Showing Antiviral Effect
1
2
1
Yun-Hee YANG , Ki-Moon PARK , and Joo-Sung YANG *
1
2
Department of Genetic Engineering, Sungkyunkwan University, Department
of Food Science and Biotechnology, Sungkyunkwan University
*Corresponding author: jsyang@skku.edu
2
MTB12(CFP-2) protein constitutes a major component of the Mycobacterium tuberculosis
culture supernatant, and appears to be as abundant as the well-characterized culture filtrate
proteins, antigen (Ag) 85B complex. In this study, we purified the native MTB12 protein and
investigated the profile of cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)- , and
IL-6] present in peripheral blood mononuclear cells (PBMCs) or monocyte-derived
macrophages (MDMs) of early tuberculosis (TB) patients after stimulation with the MTB12
protein or 30-kDa Ag of M. tuberculosis. The data were compared with data obtained from
healthy tuberculin reactors (HTR). In HTR, the MTB12 Ag elicited less cytokine productions
[IFN-γ, TNF- , and IL-6] by PBMCs than the 30-kDa Ag did. Although the mean production
of IFN-γ and TNF- induced by MTB12 were significantly decreased compared to those
by the 30-kDa Ag in PBMCs from TB patients, the MTB12-induced IL-6 production was as
high as those by stimulation with the 30-kDa in MDMs. After 2 months of anti-TB therapy,
the mean IFN-γ and IL-12 p40 production was significantly increased in PBMC from TB
patients who were followed up. Our findings suggest that MTB12 protein from the culture
supernatant of M. tuberculosis induces less T cell immunoreactivities for PBMCs in both
HTR and TB patients, although it has comparable activities to elicit the proinflammatory
responses by human MDMs from TB patients.
Keywords: MTB12, active pulmonary tuberculosis, Mycobacterium tuberculosis, IFN-γ,
TNF- , IL-6
G066
G068
Treatment of Small Interfering RNA (siRNA)
Targeting West Nile Virus (WNV) Capsid (Cp)
Regulates Viral Progeny Release
1
1
1
Joong-Bin PARK , Yun-Hee YANG , Sang-Woo KIM , Jin-Ju
2
2
2
2
NAH , Young-Joon KO , Yong-Joo KIM , Kang-Seuk CHOI ,
2
3
1
Yiseok JOO , Jaewhan SONG , and Joo-Sung YANG *
1
Oriental medicinal herb extracts (OHE) are known to have strong
antioxidative characteristics in foods. An OHE has been reported to have an
anticancer activity previously. Therefore, we were interested in testing a
potential antiviral activity of the RW and performed experiments to show a
strong inhibitory effect on pathogenesis of Murine Leukemia virus (MuLV).
In vitro experiments, infection of MuLV to SC-1 cell resulted in formation of
giant syncytia that could be enumerated as a plaque by XC plaque assay. The
number of syncytia from the cell culture treated with the OHE before and after
the MuLV infection was approximately 40 % decreased than that of
non-treated culture. To determine whether the OHE is effective in post-entry
event, presence of provirus was identified by genomic DNA PCR amplifying
gag gene. To examine effects on progeny release, virus titration in tissue
culture supernatant using XC plaque assay was set up. To investigate whether
the OHE prevent mice from being immunodeficient, the OHE was orally
administered before and after MuLV infection with different dose. Since the
OHE reduced cytopathic effect in cell culture, we expect that the OHE may
be able to suppress clinical symptoms such as splenomegaly and lower CD4+
T cell counts in the mice. Therefore, the OHE could be useful reagents for
treating MuLV/MAIDS and further HIV/AIDS. Moreover, this screening
system is a useful tool for screening antiviral therapeutics candidates.
[Supported by KRF grant]
Keywords: Retrovirus, MuLV, MAIDS, Antiviral reagents screeing system
2
Department of Genetic Engineering, Sungkyunkwan University, National
Veterinary Research and Quarantine Service, 3Department of Food Science
and Biotechnology, Sungkyunkwan University
*Corresponding author: jsyang@skku.edu
WNV is a single-stranded RNA virus in the family Flaviviridae. WNV causes fatal
encephalitis in humans as well as other vertebrates. So far specific antiviral
therapeutics for WNV infection has not been developed. Therefore, strategies for
regulating viral progeny release were investigated. WNV Cp is a cytopathogenic
protein inducing apoptosis and inflammation. To determine whether Cp-specific
siRNAs can interfere WNV replication, translation or its pathogenisity, several
reagents such as chemically synthesized siRNA duplexes, siRNA-RFP plasmids,
or stably siRNA transformed cell lines were analyzed. The level of interference
was evaluated by Cp-induced apoptosis as determined by Caspase-3 assay and
viral progeny production as determined by plaque formation assay. Moreover,
degree of WNV RNA replication was determined by RT-PCR for Cp gene.
Apoptosis in siRNA treated and then WNV infected cells was decreased about
50% compared to that of non-infected cells. To determine whether this inhibition
is Cp gene-specific, RT-PCR with total RNA was performed. Data also presented
that Cp message expression was about 50% decreased. A reduction of viral
progeny release is under investigation using plaque formation assay. Cp-specific
siRNA treatment maybe efficient strategy to inhibit not only replication,
translation but also release viral progeny. Our data suggest potential preventive
and further protective therapeutics for WNV-induced brain encephalitis.
[Supported by KOSEF grant]
Keywords: Flavivirus, West Nile virus, Capsid protein, siRNA, Apoptosis
Human Cytomegalovirus-Induced Accumulation
of Histone Deacetylase 2 at Immediate-Early IE2
Protein Domains and Viral Replication Sites
Jung-Jin PARK and Jin-Hyun AHN*
Department of Molecular Cell Biology, Sungkyunkwan University School of
Medicine
*Corresponding author: jahn@med.skku.ac.kr
In human cytomegalovirus (HCMV)-infected cells, the input viral
genomes are deposited to the periphery of nuclear structures referred
to as PML oncogenic domains (PODs) or nuclear domain 10
(ND10). These sites are also targeted by the major immediate-early
IE2 protein, which acts as a strong transactivator of lytic cycle genes,
and provide the sites where immediate-early transcription occurs.
We found that formation of the nuclear foci containing histone
deacetylase 2 (HDAC2) was induced in HCMV-infected cells at
very early times and these HDAC2 foci were colocalized with the
IE2 domains. HDAC2 also accumulated at viral DNA
pre-replication sites and replication compartments at late times after
infection. IE2 alone was able to induce the HDAC2 foci in
transfected cells and HDAC2 was found to be associated with IE2 in
HCMV-infected cells. Both the protein level and the deacetylase
activity of HDAC2 were increased during infection and this
correlated with accumulation of IE2. In cells infected with a
recombinant adenovirus, IE2 was responsible for both HDAC2
stabilization and the increased deacetylase activity. Our data suggest
that HCMV may regulate HDAC2 stability and local concentration
at both the IE2 domains and viral replication sites.
국제학술대회
249
October 13-14, 2005, Seoul, Korea
G069
G071
Highly Pathogenic Avian Influenza in Magpie
(Pica pica sericea), South Korea
Y. K. KWON*, Y. J. LEE, J. G. CHOI, S. J. JOH, M. C. KIM, E.
K. LEE, O. M. JEONG, J. H. KWON, and J. H. KIM
Jun Ho KIM*
Avian Disease Division, National Veterinary Research and Quarantine
Service
*Corresponding author: kwonyk@nvrqs.go.kr
Depatement of Biomedical Laboratory Science, College of Health Science,
Yonsei University
*Corresponding author: scent1016@nate.com
Highly pathogenic avian influenza (HPAI) is an extremely infectious,
systemic viral disease of birds that produces high mortality and
morbidity. HPAI was diagnosed in the three dead magpies (Pica pica
sericea) submitted to the National Veterinary Research and
Quarantine Service. At necropsy, the prominent lesions were
multiple mottled necrosis of the pancreas with enlargement of the
livers and spleens. Microscopically, there were severely necrotized
pancreatitis and lymphocytic meningoencephalitis. Influenza viral
antigen was also detected in areas closely associated with
histopathologic lesions. AIV was isolated from cecal tonsils and feces
of the magpies. The isolated virus was identified as a highly
pathogenic H5N1, with hemagglutinin proteolytic cleavage site
deduced amino acid sequence of QREKRKKR/GLFGAIAG. In
order to determine the pathogenicity of the isolate, eight 6-week-old
specific pathogen free chickens were inoculated intravenously with
the virus, and all birds died within 24 hours after inoculation. This is
the first report of an outbreak of HPAI in the Magpies.
Keywords: Pathogenic Avian Influenza, H5N1, Korea, Magpie.
Fluoroquinolones have antimycobacterial activities that possibly contribute
a pivotal role in the second-line drug regimens used in the treatment of
multidrug-resistant tuberculosis Resistance to fluoroquinolone is associated
with mutation in a gyrA gene. The major objective of this study was to
invetigate mutation rate at gyrA among fluoroquinolne resistant M.
tuberculosis isolated in Korea. For this study, we obtained 43
fluoroquinolone-resistant M.tuberculosis clinical isolates from Masan
National TB Hospital, and a DNA fragment of 320 bp, region of gyrA,
corresponding to the fluoroquinolone resistance-determining region was
amplified by PCR. The amplification product was used as the template in
direct nucleotide sequencing. Twenty-four of the 43 isolates had mutations at
this position resulting in a total of five different types of amino acid changes.
Five isolates contained a mutation at codon 90. Four isolates had mutations
at codon 91. Two isolates contained a mutation at codon 88. From these results
and papers, we designed oligonucleotide probe based on quinolone resistance
detergent region(QRDR) of the gyrA for dot-blot hybridization. To evalutat
this probe, Dot-blot hybridization was carried out using other 60 clinical
isolates. From these results, we could conclude the rate of mutations present
in gyrA among fluoroquinolne resistant M.tuberculosisin Korea is similar to
the general rates of mutations found throughout the world and this probe is
very available.
Keywords: M.tuberculosis, Fluoroquinolone, gyrA gene
G070
G072
Vibrio vulnificus Metalloprotease VvpE Has No
Effect on the Vulnibactin-Mediated Iron-Uptake
from Human Transferrin
1
1
1
1
2
C. M. KIM , J. H. PARK , H. Y. SUN , R. Y. PARK , Y. H. BAI ,
3
3
3
1
P. Y. RYU , S. Y. KIM , J. H. RHEE , and S. H. SHIN *
1
Research Center for Resistant Cells, Chosun University Medical School,
2
3
Department of Biology, Chosun University Medical School, Clinical Vaccine
R&D Center, National Research Laboratory of Molecular Microbial
Pathogenesis, Research Institute of Vibrio Infection and Genome Research
Center for Enteropathogenic Bacteria, and Department of Microbiology,
Chonnam National University Medical School
*Corresponding author: shsin@chosun.ac.kr
Our previous study demonstrated that Vibrio vulnificus metalloprotease
VvpE had no direct effect on the iron-assimilation from human transferrin by
using a vvpE-insertional mutant. In this study, to reconfirm this in accordance
with molecular Koch’s postulates, we used a vvpE deletional-mutant and its
complementation strain, a fur-deletional mutant, and a venB-insertional
mutant. The mutation of the vvpE gene did not affect the production of
vulnibactin, the iron-assimilation from human transferrin, and the growth on
transferrin-bound iron. In contrast, the mutation of the fur gene facilitated the
iron-assimilation from transferrin via the derepressed production of
vulnibactin. The mutation of the venB gene encoding an enzyme for
vulnibactin synthesis inhibited the iron-assimilation from transferrin and the
growth on transferrin-bound iron. The mutations of the fur gene and the venB
gene did not affect the protease production. These results demonstrate that
vulnibactin is essential for the iron-assimilation from transferrin and the V.
vulnificus metalloprotease has no direct effect on facilitating the
vulnibactin-mediated iron-assimilation from transferrin in spite of its ability
to destroy transferrin [R13-2003-009].
Keywords: Vibrio vulnificus, Metalloprotease, Vulnibactin, Transferrin,
Iron
250
Analysis of gyrA Mutation in FluoroquinoloneResistant Mycobacterium tuberculosis Clinical
Isolates in Korea
한국미생물학회연합
Identification of Enterococcus faecalis Grown on
MSB Medium Selective for Mutans Streptococci
So Young YOO, Hwa-Sook KIM, Min-Jeong KIM, Soon-Nang
PARK, Sook-Jin KANG, and Joong-Ki KOOK*
College of Dentistry, Chosun University
*Corresponding author: jkkook@chosun.ac.kr
Mitis-salivarius sucrose bacitracin (MSB) medium is widely used in
the selective isolation of mutans streptococci (MS), a designation for
a group of oral cariogenic species. Recently, we have isolated 3
bacterial strains grown on MSB agar from human dental plaques.
The 3 strains exhibited biochemical characteristics similar to those
of the biotype IV of MS, with the exception that they manifested a
positive reaction for arginine deaminase. The objective of this study
was to identify and characterize these 3 clinical isolates. The bacteria
were identified with biochemical tests as well as by 16S rDNA
cloning and sequencing methods. In order to conduct a comparison
of the susceptibility to 9 antibiotics between the type strains and
clinically isolated strains, the minimal inhibitory concentrations
were determined using broth dilution assays. The results identified
all of our 3 clinical isolates as E. faecalis. All E. faecalis strains were
found to be susceptible to penicillin G, amoxicillin, augmentin, and
vancomycin, but were resistant to ciprofloxacin, cefuroxim axetil,
and clindamycin. Our findings indicate that E. faecalis is capable of
growth on MSB agar, and suggest that the MSB medium be
improved so that only MS should be recoverable on the medium, as
originally devised for their selection.
Keywords: Enterococcus faecalis, MSB medium, 16S rDNA
2005 International Meeting of the Federation of Korean Microbiological Societies
G073
G075
Identification and Detection of Streptococcus
anginosus Using Species-specific 16S rDNA
Primers
Reactive Oxygen Species Mediated by Activation of
p38 MAPK Is Crucial for Cell Death of Jurkat T-Cell
Line Induced by Vibrio vulnificus
Joong-Ki KOOK*, So Young YOO, Hwa-Sook KIM, Min Jung
KIM, Soon-Nang PARK, and Sook-Jin KANG
Woo-Hyang KIM *, Kyu-Ho LEE , Heu-Ran LEE , and
Soon-Jung PARK1*
College of Dentistry, Chosun University
*Corresponding author: jkkook@chosun.ac.kr
1
This study was undertaken to develop PCR primers for the
identification and detection of Streptococcus anginosus using
species-specific forward and reverse primers. These primers
targeted the variable regions of the 16S ribosomal RNA coding gene
(rDNA). The primer specificity was tested against 12 S. anginosus
strains and 6 different species (10 strains) of oral bacteria. The
primer sensitivity was determined by testing serial dilutions of the
purified genomic DNA of S. anginosus ATCC 33397T. The data
showed that species-specific amplicons were obtained from all the S.
anginosus strains tested, which was not observed in the other
species. The PCR could detect as little as 0.4 pg of the chromosomal
DNA from S. anginosus. This suggests that the PCR primers are
highly sensitive and applicable to the detection and identification of
S. anginosus.
Keywords: Identification, PCR primer, Streptococcus anginosus,
16S rDNA
1
2
3
Department of Parasitology and Institute of Tropical Medicine, The Brain
Korea 21 Project, Yonsei University College of Medicine, 2Department of
Environmental Science, Hankuk University of Foreign Studies, 3Department of
Microbiology, Ulsan University College of Medicine
*Corresponding author: sjpark615@yumc.yonsei.ac.kr
Vibrio vulnificus is a gram-negative pathogenic bacterium causing primary
septicemia, necrotizing wound infection, and gastroenteritis, especially in
humans with a heavy alcohol drinking habit or hepatic diseases. The pathogen
frequently causes fatal septicemia with a rapid progress, resulting in a
mortality rate of 50% within a few days. Little is known about the interaction
between the host cells and V. vulnificus. In this study, nature of cell death and
intercellular signaling pathways triggered by V. vulnificus were defined in
Jurkat T-cell. The host cell incubated with V. vulnificus was examined under
transmission electron microscope showing a necrotic pattern of cell death.
Infection of V. vulnificus to Jurkat cell did not result in an activation of any
of three caspases (caspase-9, -3, and -8), and did not produce DNA
fragmentation. In addition, V. vulnificus induced activation of MAPK
pathway including p38 and ERK as well as production of reactive oxygen
species (ROS). Treatment of the host cells with DPI (flavoprotein inhibitor of
NADPH oxidase) abolished ROS generation as well as phosphorylation of
p38 MAPK, which were happened in the host cells upon an exposure to V.
vulnificus. Our data suggest that the mechanism mediating the death of Jurkat
T-cell by V. vulnificus includes ROS production and consequent activation of
the p38 MAPK
Keywords: Vibrio vulnificus, caspases, p38 MAPK, ERK, DPI, ROS
G074
G076
Analysis of Influenza Virus Isolated in Seoul
1999-2005 to Predict Future Subtypes
1
1
1
Antiviral Effect of Plant Derived Natural
Compounds Against Influenza Viruses
1
1
2
Young Ok HWANG *, Mi Ok SONG , Jae In LEE , and Dong
2
Hee LEE
Woo-Jin SHIN , Kwang-Hee LEE , Myung-Hwan PARK , and
1
Baik-Lin SEONG *
1
Seoul Metropolitan Government Research Institude of Public Health and
2
Enviroment, Department of Life Sciences, University of Seoul
*Corresponding author: hyoung611@bcline.com
1
Influenza exerts a serious threat to the human health in temperate climate
zones with significant rates of mortality and hospitalization during
winter. In this study, 293 influenza viruses were isolated from 2324
clinical specimens, which were collected from patients with Influenza
like illness (ILI) in Seoul from 1999 through 2005. ILI incidence was the
highest in the infant younger than 2 years as 36.4% while the rate of virus
isolation was the highest, 24.0% at the 7-19 age group. Among isolated
293 influenza viruses four of them were identified as A/H3N2 type in
1999-2000 season, seven of them were A/H3N2 in 2000-2001 season,
fourteen of them were A/H1N1 in 2001-2002 season, forty nine of them
were A/H3N2 in 2002-2003 season, eighty three were subtyped for
influenza A/H3N2, forty one for influenza B viruses in 2003-2004 season
and ninety five were subtyped as influenza A/H3N2, nine for influenza B
viruses in 2004-2005 season. During the 1999-2005 influenz seasons,
influenza A(H3N2) was the most frequently isolated infuenza virus
type/subtype. Influenza viruses were collected 36.6% at Nowon-Gu,
17.2% at Dongjak-Gu and 10.7% at Seocho-Gu etc. and the isolate rate
of virus had the area difference. These findings may contribute towards
the recommondation on the influenza vaccine formulation and
development of influenza control measure based on the analyzed data.
Keywords: Influenza virus, ILI, A/H3N2, A/H1N1, Subtype
We have tested Korean medicinal plant extracts (plant A to J) for
antiviral activity against influenza viruses. Among ten different
extracts tested, one was shown to be highly effective against
influenza A virus and influenza B virus in MDCK cell cultures. The
extract also exhibited strong inhibitory effect on avian influenza
virus as tested in embryonated hen’s eggs. The antiviral effect of the
plant extract on influenza A virus as tested by plaque reduction assay
was about 30ug/ml of 50% inhibitory concentration (IC50) for both
influenza A and B viruses. The extract also exhibited virucidal effect
at concentration of 3.9ug/ml against influenza A and B viruses when
tested by mixing the virus and extract before plaque assay. Similarly,
the extract was shown to have virucidal effect against avian
influenza virus in embryonated hen’s eggs at concentration of
1.95ug/ml. It exhibited hemagglutination inhibition effect against all
three types of viruses tested, including influenza A, B viruses and
avian influenza virus, suggesting that the major target for the
observed antiviral effect is viral surface antigen and/or the viral
membrane. The antiviral activity observed in vitro is being further
extended in vivo using mouse infection model and the data will be
presented.
Keywords: Influenza virus, Medicinal plant extracts, Antiviral
Department of Biotechnology, College of Engineering, Yonsei University,
Ambo Research Institute
*Corresponding author: blseong@yonsei.ac.kr
2
국제학술대회
251
October 13-14, 2005, Seoul, Korea
G077
G079
Can Lethal H5N1 Influenza Viruses from 2004
Vietnam and Thailand Infection and Transmit
Pig to Pigs?
1
2
3
Young-Ki CHOI , Tien Dzung NGUYEN , Hiroichi OZAKI ,
Richard J. WEBBY4, Pilaipan PUTHAVATHANA5, Chantanee
6
7
4
BURANATHAL , N.T.H HANH , and Robert G. WEBSTER
1
Chungbuk National University, Medical School, 2Department of Virology,
National Institute of Veterinary Research, Ministry of Agriculture and Rural
3
Development, Vietnam, Division of Project Research, Creative Research
Initiative, Hokkaido University, Japan, 4Division of Virology, Department of
Infectious Diseases, St. Jude Children's Research Hospital, USA 5Department
6
of Microbiology, Sriraj Hospital, Thailand, Department of Livestock
Development, National Institute of Animal Health, Thailand, 7Virology
Department, National Institute of Hygiene and Epidemiology, Vietnam
*Corresponding author: choiki55@chungbuk.ac.kr
Pigs are susceptible to infection with both avian and human influenza viruses,
as well as their own swine viruses, and thus serve as the intermediary for
human and avian virus reassortment. To investigate seroepidemiological
evidence of avian H5N1 influenza virus transmission to pigs, we investigated
the sero-prevalences of H5N1/04 influenza virus in pigs from Vietnam. Of the
3175 pig sera tested, 8 sera (0.25%) were positive to avian H5N1/04 viruses
by hemagglutination inhibition (HI) assay and Western blot analysis. We also
tested the potential of replication and transmission of the 2004 H5N1 viruses
in Asia in pigs. All viruses tested replicated in the respiratory tract, but none
was transmitted to contact pigs in the same isolators. Viral titers in nasal
swabs peaked on day 2 after inoculation at 3.33 to 3.75 log10 EID50/0.1 ml and
viruses can persist in respiratory tracts of pigs for at least 6 days after
inoculation. Our findings indicate that pigs could be infected but these viruses
do not spread readily between pigs in experimental conditions.
Keywords: avian influenza, H5N1, pig, transmission, serology
G078
Hyun-Ju KIM*, Hong KIM, Hyun-Ah DO, Ho-Suk MUN,
Chang-Yong CHA, Yoon-Hoh KOOK, and Bum-Joon KIM*
Department of Microbiology and Liver Research Institute, College of
Medicine, Seoul National University
*Corresponding author: kbumjoon@snu.ac.kr
A novel duplex PCR method which can differentiate Mycobacterium
tuberculosis and Nontuberculosis mycobacteria (NTM) strains by
amplifying hsp65 DNAs of different size, 195 bp and 515 bp,
respectively, was developed and applied to 54 mycobacteria
reference and 170 clinical isolates. The duplex PCR could
differentiate all the strains into two groups, showing 100% sensitivity
and specificity. Furthermore, a duplex PCR-restriction analysis
(DPRA) and direct sequencing protocols for the differentiation of
NTM strains into species or subspecies level was also developed on
the basis of hsp65 DNA sequences previously reported (H. Kim et al.,
Int J Sys Bacteriol. 55: 1649-1656) and applied to 103 NTM clinical
isolates. All NTMs could be clearly differentiated into species or
subspecies level by subsequent procedure (PRA or direct sequencing)
of 515 bp duplex PCR amplicons. Our results suggest that these
duplex PCR targeting hsp65 DNA linked protocols are simple and
feasible method for the identification of mycobacteria culture isolates
at the species level.
Keywords: duplex PCR, hsp65, M. tuberculosis, Nontuberculous
mycobacteria
G080
Analysis of ATP Synthase Beta on Macrophages
Infected with Bacillus anthracis Sterne Spores
Novel Polymerase Activity of Hepatitis B virus
DNA Polymerase
Gwi-Moon SEO*, Kyoung Hwa JUNG*, Seung-Joo KIM*,
Ji-Chon KIM*, and Young Gyu CHAI*
Hee-Young KIM, and Kyoungmin KIM
Division of Molecular and Life Sciences, Hanyang University
*Corresponding author: gmseo@ihanyang.ac.kr
Anthrax is an infectious disease caused by toxigenic strains of the
gram-positive bacterium Bacillus anthracis, which is mainly present
in the environment in the form of highly resistant spores. In this
study, we performed a proteomic analysis and carried out to
identify different expressed mitochondrial proteins on mouse
macrophages infected with the spores of B. anthracis Sterne. We
identified several mitochondrial proteins on mouse macrophages
infected with B. anthracis spores. The analysis revealed that the
ATP synthase beta subunits are found to up-regulation. The ATP
synthase beta subunits assumes the increase on mouse
macrophages by the infection and proliferation of B. anthracis
Sterne spores. Finally the B. anthracis spore will be survival
within the mouse macrophages.
Keywords: Anthrax, Bacillus anthracis, Spore, ATP synthase beta,
Macrophage, Mitochondria
252
Differentiation of Mycobacterial Species by
Duplex PCR Targeting hsp65 Gene Linked
Restriction Analysis and Direct Sequencing
한국미생물학회연합
Department of Microbiology, Ajou University School of Medicine
*Corresponding author: hee_young_kim@hotmail.com
Hepadnavirus DNA polymerase functions DNA synthesis from RNA or
DNA template ,and acts as athe primer for minus-strand DNA synthesis. From
the previous study, we found that the priming- deficient mutant polymerase
of HBV has the ability to can synthesize oligomers (presumably nascent
minus-strand DNA) in the absence ofwithout covalent linkage between
terminal protein of HBV polymerase and the first deoxynucleotide,
suggesting the primer independent initiation of HBV polymerase. This raises
a very important question that HBV polymerase may have the feature of RNA
polymerase. In this study, the RNA polymerase activity of HBV DNA
polymerase was explored by testing the NTP incorporation capacities. Phe
436, the bulky amino acid, at supposedly dNTP binding cleft of HBV
polymerase was changed to Gly or Val, the smaller amino acids. NTPs
incorporation capacity of HBV DNA polymerase mutants was tested by
32
endogenous polymerase assay with P-labeled ATP from isolated core
particles. HBV polymerase mutants can incorporate NTPs, even though
authentic DNA synthesis was not detected. Our results indicate that the bulky
amino acid in dNTP binding cleft acts as a steric gate in dNTP selections and
the single amino acid substitution can blur property of DNA polymerase in the
direction of RNA polymerase. Taken together, we suggest that HBV DNA
polymerase has the RNA polymerase activity with the single amino acid
substitution.
Keywords: hepatitis B virus polymerase, dNTP binding cleft, NTP
incorporation
2005 International Meeting of the Federation of Korean Microbiological Societies
G081
G083
Antimicrobial Mechanisms and Cytotoxicity of
Nanoparticulate Silver
1
1
1
Hee-Jeong KANG , Young-Hee KIM , Ki-Ho LEE , Kwang-Kjune
KO1, Byong-Taek LEE2, and Ho-Yeon SONG1
1
Department of Microbiology, College of Medicine, Soonchunhyang
University, 2School of Advanced Materials Engineering, Engineering College,
Kongju National University,
*Corresponding author: songmic@sch.ac.kr
Silver is one of the most universal antimicrobial substances.
Nano-technogy enables us to expand the surface area of silver
particles markedly. Nanoparticulate silver was successfully
produced less than 10 nm in size. The anti-microbial activity of
nanosilver for several pathogenic bacteria was examined. Nanosilver
showed excellent antibacterial activities against Salmonella typhi,
Escherichia coil, Pseudomonas aeruginosa around 1 ppm and
Staphylococcus aureus and Mycobacterium tuberculosis around 10
ppm. Nanosilver induced severe morphologic changes in cell wall
and cytoplasm of bacteria (by SEM and TEM). To confirm the
toxicity of nanosilver, four groups of mice (60 mice in total) have
been fed with nanosilver suspension (0, 10, 30, 100 ppm) for 6
months. Their behaviors, weight changes, blood chemistries and
pathologic findings were compared with control. The weight
changes, and hematologic and pathologic findings among four
groups showed similar results although the mice had been fed 100
ppm of nanosilver suspension for 6 months.
Keywords: Silver nanoparticle, Antimicrobial mechanism, Cytotoxicity
G082
Involvement of MAPK Pathway in HCMV Induced
COX-2 Expression
Hyun-Ah YI and Chan-Hee LEE
Division of Life Sciences, Biotechnology Research Institute, Chungbuk
National University
*Corresponding author: chlee@chungbuk.ac.kr
Proinflammatory immune reactions to human cytomegalovirus
(HCMV)-infection have been associated with diseases such as
atherosclerosis, retinitis, pneumonitis, and colitis. Cyclooxygenase (COX)-2
plays an important role in proinflammatory reaction and HCMV is known to
induce COX-2 gene expression. In HCMV infection, however, little has been
known for the mechanisms on the HCMV-induced COX-2 gene expression.
In this study, we report the evidence suggesting a role of MEK/ERK pathway
in HCMV-induced COX-2 gene expression. HCMV stimulated the
expression of intercellular adhesion molecule (ICAM)-1, one of the
consequences of proinflammatory response, on the surface of human foreskin
fibroblast (HFF) cells. COX-2 expression was up-regulated by HCMV and
this process did not require viral gene expression. COX-2 inhibition by NS398
was shown to decrease ICAM-1 up-regulation by HCMV. Western blot
analysis revealed that extracellular signal regulated kinase (ERK) 1/2 was
activated during HCMV infection. When HCMV-infected HFF cells were
treated with PD98059 or U0126, MAPK kinase (MEK) specific inhibitors,
both the HCMV-induced COX-2 expression and ERK activation were
inhibited. NF-kB transcription factor assay revealed that NF-kB was involved
HCMV induced COX-2 expression in the down stream MAPK pathway.
Therefore, it was concluded that MEK/ERK pathway and NF-kB may
participate in the COX-2 mediated proinflammatory response to HCMV
infection.
Keywords: HCMV, COX-2, MEK/ERK pathway, proinflammatory
response
The Expression and the Polymerase Activities
of Chimeric DNA Polymerase of Human and
Duck Hepatitis B Virus
Gil Soon PARK and Kyongmin KIM*
Department of Microbiology, Ajou University School of Medicine
*Corresponding author: gillja2003@hotmail.com
Hepatitis B Virus (HBV) DNA polymerase is a multifunctional protein with
four domains, terminal protein (TP), spacer, reverse transcriptase (RT), and
RNaseH. Since HBV DNA polymerase is very unstable and rapidly
degradable, it has been very difficult to study the biochemical aspect of HBV
DNA polymerase. In the present study, rabbit polyclonal antiserum specific
for each domain of HBV DNA polymerase was produced. All chimeras
produced RNAs of chimeric DNA polymerases, and 2.4 and 2.1 Kbp of
surface RNAs. The distribution of HBV polymerase was exclusively
localized in cytoplasm. There were no significant co-localization between
HBV polymerase and various organelle markers such as endoplasmic
reticulum, Golgi complex and peroxisome. We analyzed chimeric DNA
polymerases of HBV and DHBV, to identify the motifs or regions of HBV
DNA polymerase that are necessary for pgRNA encapsidation and
replication. When 27 amino acids of C-terminus of RNaseH domain of HBV
DNA polymerase was substituted into the corresponding region of DHBV,
this chimeric DNA polymerase was not compatible for encapsidation.
However when we substituted three to eight amino acids in the same
C-terminus of RNaseH domain, all of the chimeric DNA polymerases were
compatible for encapsidation and some of them even support the HBV DNA
replication. By analyzing these chimeric polymerases, we can identify the
functional region of HBV DNA polymerase that is necessary for pgRNA
encapsidation and replication. Keywords: Hepatitis B Virus DNA polymerase, chimera, encapsidation,
replication
G084
Molecular Characterization of Vancomycinresistant Enterococcus faecium Isolates from
Non-Tertiary Hospitals and Tertiary Hospitals
Young Hee JUNG, Yeong Seon LEE, Jae Oh AHN, Ha Rhim
LEE, Jeom Kyu LEE, Jae Il YOO, and Bong Su KIM
Department of Microbiology, KCDC
*Corresponding author: yhjung60@paran.com
Background Resently, because of gradually increasing of vanA-type VRE.
faecium in hospitals, we investigated the prevalence and genetic
characterization of VRE in E. faecium collected from non-tertiary and
tertiary hospitals. Methods We collected 54 E. faecium of 3 tertiary
hospitals and 168 E. faecium of non-tertiary hospitals through 2 clinical
centers from Jan. to Nov. 2003. Detection of resistant genes, esp gene, and
vanA gene cluster in VRE. faecium were performed by PCR. Molecular
epidemiology was done by PFGE. Results Rates of resistance to
tetracycline and streptomycin were 29.8%, 51.8% in 168 E. faecium isolates
collected from non-tertiary hospitals and 7.4%, 44.4% in 54 E. faecium from
tertiary hospitals, respectively. E. faecium isolates from tertiary hospitals and
non-tertiary hospitals were resistant to vancomycin in 25.9% and 11.9%,
respectively. vanA-type VRE. faecium have esp gene to over 80%. Of 34
strains, there was no original Tn1546, 8 strains were inserted IS1542 in orf2
and IS1216V in vanX-vanY intergenic region, 21 strains were left-end deleted,
2 strains were right-end deleted, and 3 strains were left-end and right-end
deleted. PFGE patterns of these isolates were manifested in 32 different
types.Conclusion Prevalence of vanA-type VRE in E. faecium was 11.9% in
non-tertiary hospitals and 25.9% in tertiary hospitals. Frequency of esp gene
was over 80%. Transposon and PFGE type were very diverse.
Keywords: VRE, vanA gene cluster, esp
국제학술대회
253
October 13-14, 2005, Seoul, Korea
G085
Identification of Induced and Repressed Genes
in RAW 264.7 Cells by Salmonella Infections
Jungah SONG*, Hyunju KIM*, Hyon E CHOY*, and Yeongjin
HONG*
A Novel Cis-acting Element of the Porcine
Reproductive and Respiratory Syndrome Virus
Required for Viral Replication Localizes in the
C-terminus of the ORF7 Protein-coding Region
Genomic Research Center for Enteropathogenic Bacteria and Department of
Microbiology, Chonnam National University Medical School
*Corresponding author: yjhong@chonnam.ac.kr
Yu-Jeong CHOI, Sang-Im YUN, Jeong-Min KIM, Byung-Hak
SONG, Jun-Sun PARK, Ah-Yong JEONG, and Young-Min
LEE*
The bacterial pathogen Salmonella typhimurium triggers various signals that
lead to cytoskeletal and nuclear responses required for pathogenesis to host
cells. Using 22,571 human cDNA chip Detweiler et. al. have previously
reported that 68 genes were induced in human U937 myeloid leukemia cells
by Salmonella infection. Here, we investigated differentially expressed
genes by Salmonella infection using a modified differential display PCR
(DD-PCR), to identity which genes were induced or repressed in the host
cells. From the DD-PCR results, we found that 8 genes (MIP-1a, Cxcl10,
Mcm2, pdcd1, Mum111, Actg1, and Tmsb4x) were induced and 6 genes
(Psmd3, Copz1, SR-B1, Lyzs, Oasl1 and hypothetical) were repressed.
However, these results were not consistent with Detweiler’s report except
for MIP-1α. Thus, we carried out northern hybridization to confirm the
mRNA expression level of those genes affected by Salmonella
infection.Among the induced genes, the expression of Cxcl10 gene was
highly induced and suddenly decreased (12.1 fold at 1hr vs. 5.1-fold at 4hr).
By contrast, expression of Lys gene was decreased serial 0.7-fold at 1hr to
0.3-fold at 4hr. It was observed that most of induced genes were closely
related to the production of inflammatory protein, perturbation of cell cycle,
induction of program cell death, and cytoskeleton rearrangement and
decreased genes would be closely related to bacterial infection and
intracellular survival.
Keywords: Salmonella infection, MIP-alpha, Cxcl10
Department of Microbiology, College of Medicine and Medical Research
Institute, Chungbuk National University
*Corresponding author: ymlee@chungbuk.ac.kr
G086
The porcine reproductive and respiratory syndrome virus (PRRSV), one of the most
common and economically significant infectious agents in the swine industry
worldwide, belongs to the family Arteriviridae in the order Nidovirales. PRRSV is a
small-enveloped virus with a positive-sense, single-stranded RNA genome of »15 kb
in length. Using the viral replicon system of PRRSV, we have identified a novel
cis-acting element of PRRSV required for RNA replication. Of 11 viral replicons
containing various deletions in the genome of PRRSV, only 3 vrial replicons
(PRRSV/RepLuc S8, PRRSV/RepLuc MB and PRRSV/RepLuc ME) found to be
replication-competent, whereas the other 8 replicons were replication-incompetent. It has coincidentally been noticed that all three replication-competent viral replicons
found to express the ORF7 protein in the RNA-transfected cells, but not in the cells
transfected with the replication-incompetent viral replicon RNAs. To examine the
possibility that expression of the ORF7 protein is required for PRRSV replication, all
PRRSV viral replicon RNAs were transiently co-transfected with the infectious
PRRSV viral RNAs to provide the ORF7 protein in trans. This revealed that the
replication-incompetent viral replicon RNAs were still replication-incompetent when
the ORF7 protein was provided in trans. Thus, our findings revealed that the novel
cis-acting element of PRRSV appeared to be localized in the C-terminus of the ORF7
protein-coding region.
Keywords: Reverse genetics system, Porcine reproductive and respiratory syndrome
virus, Viral replicons, cis-acting element, trans-complementation
G088
Identification and Functional Characterization of
Common Surface Proteins in Oral Spirochetes Associated with Periodontitis
Half an Hour Enabling You Confirm SARS
Infection: Development of High-speed Real-Time
PCR Detection Method for SARS-CoV
Hye-Kyoung JUN, Young-Mi KANG, Sung-Hoon LEE, and
Bong-Kyu CHOI*
Sang-Hoon HAN*, Do-Bu LEE, Hye-Min LEE, and Byoung-Su
YOON
Seoul National University, College of Dentistry, Oromaxillofacial Infection
and Immunity
*Corresponding author: bongchoi@snu.ac.kr
Department of Biology, College of Natural Science, Kyonggi University
*Corresponding author: youthanathia@hanafos.com
Oral spirochetes are composed of enormously heterogeneous Treponema
species and some of them are implicated in the etiology of periodontitis In
addition, Treponema denticola, the most intensively studied species among
oral spirochetes, was detected in atherosclerotic lesions and in pre-term
low-birth-weight mothers. Tp92 of T. pallidum, a surface protein, has been
reported to have opsonic potential and to induce a protective immune response
against syphilis, T. pallidum infection. The objective of this study was to
identify the Tp92 homolog genes in representative oral spirochetes and to
analyze host-stimulating activity using the recombinant proteins expressed in
E. coli. The Tp92 homolog genes were identified in the representative oral
spirochetes like T. denticola, Treponema lecithinolyticum, and Treponema
socranskii. The amino acid sequences translated from the nucleotide
sequences of the genes showed high homology with Tp92 (>55%). The
proteins encoding Tp92 homologs were successfully expressed in E. coli. The
recombinant Tp92 homologs stimulated the expression of the mRNA
expression of IL-6, IL-8, and MMP-9 in human gingival fibroblasts. In
conclusions, Tp92 homologs are common proteins localized in the surface of
oral spirochetes. Further functional tests, examining cytopathological effects
on host cells, opsonic potential, and protective potential using mouse abscess
model, will be necessary to understand the role of the protein and to be used
as an immunogen.
Keywords: Periodontitis, Oral spirochetes, Surface proteins, Tp92 homologs
254
G087
한국미생물학회연합
Our laboratory has developed several detection methods based on
PCR detection system since the outbreak of SARS. More recently,
we focused on reducing the detection time of the disease. With
Samsung Techwin GenSpectorTM (TMC-1000) Real-Time PCR
machine, equipped a reaction module (heating 10℃/sec, cooling
5℃/sec), we were able to complement the above defects even with
Syber-Green as a detection dye. In the experiment with artificially
synthesized DNA template of SARS CoV, there was an outstanding
result that detection of 294bp PCR product, comparatively long
sized product in Real-Time PCR, was reliably confirmed at 33
minutes. In addition, the minimum concentration of template DNA
was about 300 copies in 1 ul of final reaction volume. This result is
the fastest detection time comparing with those of predeveloped
SARS PCR detection methods from our laboratory. From this result,
shortening the detection time in SARS diagnosis could induce a
strong advance in this field.
Keywords: SARS, Real-Time PCR, High-speed, PCR Detection,
Syber-Green, GenSpector
2005 International Meeting of the Federation of Korean Microbiological Societies
G089
G091
Specific Gene Expression Patterns in Liver
Cirrhosis
Novel Real Time PCR Method for Detection of
Plasmodium vivax
Department of Chemistry, Dongguk University,
Bundang Jesaeng General Hospital
*Corresponding author: skim@dongguk.edu
Soyoun KIM*, Yeonah KI, Jungeun LEE, Seram LEE, and
Hyosun LEE
1
2
Hepatology Center,
Liver cirrhosis (LC) is a complex disease that can develop into
hepatocellular carcinoma (HCC). In an effort to investigate genetic
differences between LC and HCC, we used cDNA microarray
analysis to characterize the gene expression profiles in LC and HCC
tissues. Consistent differences were observed among the expression
patterns in LC, HCC, and normal liver tissues. Interestingly,
theexpression patterns of LC without tumor association (LCT) were
also readily distinguished from those of LC tissues near hepatic tumor
tissues (near-tumor tissue, NTT). Moreover, 25 cirrhosis-specific
genes could be used to divide the NTT samples into two groups:
inflammatory active cirrhosis (NTTa) and in.ammatory inactive
cirrhosis (NTTi). We found that NTTa samples showed gene
expression patterns similar to those of the LCT and HCC groups,
whereas the expression patterns of the NTTi group weresignificantly
different from those of the LCT, NTTa, and HCC groups. Finally, we
selected two of the 25 LC-specific genes and showed that these
markers could be used to successfully discriminate among the
different LC subtypes. Collectively, these novel results allow the
identification of new genetic subgroups of LC and provide new
candidate genes for use as early markers for active cirrhosis and HCC.
Keywords: Liver cirrhosis; Hepatocellular carcinoma; cDNA
microarray; Real-time RT-PCR; Cirrhosis markers
G090
PCR Detection of Deformed Wing Virus Infection
in Honeybees, in Korea
Hye Min LEE, Sang Hoon HAN, Do Bu LEE, and Byong Su
YOON*
Department of Chemistry, Dongguk University
*Corresponding author: skim@dongguk.edu
Malaria is a re-emerging infectious disease that is spreading to areas where
it had been eradicated. several other methods have been introduced for
quantitative detection of malaria parasites. Real time PCR has recently been
used to detect several human malaria parasites. 18S rRNA sequences from
malaria parasites have been amplified using Taqman real time PCR assay.
Here, a SYBR Green-based real time quantitative PCR assay for the detection
of malaria parasite-especially, Plasmodium vivax - was applied for the
evaluation of 26 bloodsamples from Korean malaria patients. Even though
SYBR Green-based real time PCR is easier and cheaper than Taqman-based
assay, SYBR Green-based assay cannot be used because 18S rRNA cannot
be specifically amplified using 1 primer set. Therefore, we used DBP gene
sequences which is specific for the SYBR Green based assays. We amplified
the DBP gene from the 26 blood samples of malaria patients using SYBR
Green based assay and obtained the copy numbers of DBP genes for each
sample. Using the resultant titer from DBP based SYBERGreen assay with
ACTB reference gene, we compared the results from our study with the titer
from Taqman based assay. We found that our results showed identical
tendency with the results of 18S rRNA Taqman assay,especially in lower titer
range. Thus, our DBP gene-utilized real time assay can detect Plasmodium
vivax in Korean patient group semi-quantitatively and easily.
Keywords: Malaria, Plasmodium vivax, quantitative real time PCR, SYBR
Green-based real time assay
G092
PAF-R, Ang1, and Tie2 Involved in the Regulation of
Vascular Permeability in Hantaan Virus Infected
Human Umbilical Vein Endothelial Cells
Ji-Young HWANG and Ho-Sun PARK*
Department of Biology, College of Natural Science, Kyonggi University
*Corresponding author: hmengel@yahoo.co.kr
Department of Microbiology, College of Medicine, Yeungnam University
*Corresponding author: hspark@med.yu.ac.kr
Deformed wing virus(DWV) causes wing deformities and
premature death for adult honeybee. Although a lot of honeybees
have those symptoms, the reports about the infection of the virus
have not been presented in Korea yet. Rapid detection system may
be need for hive management, because DWV generally persists as a
latent infection with no apparent symptoms and there is no available
control method for this disease yet. In this study, our laboratory
confirmed the presence of the virus using PCR detection method
from adult honeybees that have wing deformities. PCR product size
is 479 base pairs by a specific primer set. Through T-vector cloning,
the sequence of the PCR product was analyzed. This PCR detection
method is expected to improve the control method of DWV in
honeybee.
Keywords: Deformed Wing Virus, PCR detection Method
Change of vascular permeability is a common feature of Hemorrhagic fever
with renal syndrome (HFRS) caused by Hantaan virus (HTNV) but the
mechanism is unclear yet. The goal of this study was to find the molecular
mechanism of vascular permeability shown in HFRS. Platelet activating
factor (PAF) affects diverse biologic functions such as vascular permeability,
adhesion of leukocytes to the endothelium and reduction of cardiac output.
Ang1, an agonist for the Tie2 receptor, prevents the permeability-inducing
effects of PAF and VEGF. We evaluated the permeability of HTNV-infected
human umbilical vein endothelial cells (HuVECs) and HTNV non-infected
HuVECs (control HuVECs) by the leakage of horseradish peroxidase. It was
increased in HTNV-infected HuVECs than control HuVECs at 2 days post
infection. We measured the expression of PAF receptor (PAF-R) in
HTNV-infected HuVECs instead of PAF, because PAF is rapidly degraded
by PAF-acetylhydrolase (PAF-AH) in vivo. The cell surface expression of
PAF-R, when detected by FACS analysis, was increased in 6 fold in
HTNV-infected HuVECs than control at 2 days post infection. The mRNA
expression of PAF-R was increased in HTNV-infected HuVECs than control
when detected by RT-PCR. The mRNA expression of Ang1 and Tie2 receptor
were reduced in HTNV-infected HuVECs than control HuVECs. Those
results suggested that the changes of PAF-R, Ang1 and Tie2 might be related
with the increased permeability of Hantaan virus infected endothelial cells.
Keywords: hantaan virus, angiopoietin, platelet activating factor
receptor, tie2
국제학술대회
255
October 13-14, 2005, Seoul, Korea
G093
G095
High Sensitive, Accurate Diagnostic Protein Chips
for Hepatitis C
1
1
2
2
Soyoun KIM *, Jungeun LEE , Kapno LEE , Jeogah KWON ,
Seram LEE1, Hyosun LEE1, and Yeonah KI1
1
2
Department of Chemistry, Dongguk University, Diagnostic Division, Kuro
Korea University Hospital
*Corresponding author: skim@dongguk.edu
The hepatitis C is the major infectious disease caused by hepatitis C
virus (HCV) and over three hundred millions are infected all over the
world. ELISA method is being used as a major hospital diagnosis
since it is quick, inexpensive and automated in a process. However,
ELISA method needs to be confirmed by complicate PCR or LIBA
tests because there are problems in false positive, negative detection
of HCV. In this research, sol-gel utilized 3D protein chip technology
was used.96 well plates were used for substrates of protein chip. The
optimal sol-gel formulation for 96 well plate-type protein chips were
screened and selected by adhesiveness during washing, spot
morphology, gelation time, optical transparency, low autofluorescence, the yield of immobilization and sensitivity of
immunoassay detection. This selected formulation applied to develop
96 well type protein chip, whose each well contains different HCV
markers. By comparison with ELISA test, this study confirmed that
our diagnostic protein chip has good correlation to ELISA. In
addition, the false positive in ELISA tests turned out to be negative in
our protein chip without further tests. Also, the sensitivity of protein
chip was 1000 times better than that of ELISA tests and by dilution
test, we had quantitative results from HCV protein chip.
Keywords: hepatitis C virus (HCV), ELISA, sol-gel protein chip
G094
Advanced Technique to Detect Virus Using
Samsung GeneSpector
1
1
2
Min-Seok PARK *, Ji-Hyoun SHIN , Sang-Chul RHO , and
1
Chan-Hee LEE
1
2
Division of Life Sciences, Chungbuk National University, CoreBio
*Corresponding author: chlee@chungbuk.ac.kr
PCR(polymerase chain reaction) is useful tool to detect virus from
various samples, for example, blood and blood products. It is
possible to detect viruses even if there are very small amount of
viruses. However, it has some drawbacks. Thus, many reports have
suggested advanced technologies to detect virus with accuracy,
safety and fast. Here, we suggest an advanced technology using
Samsung GeneSpector TMC-1000 (VER2.0) system based on real
time PCR on the chip. The machine has CPU inside it. The CPU
controls and monitors temperature accurately. Most PCR machines,
which are used in most laboratories, have ramping temperure
problem. Sometimes it is possible to make wrong products, leading
to wrong interpretation. However, GeneSpector monitors
temperature 1,000 times per second and wrong interpretation could
be minimized. A new detection methodology requires less sample
volume than conventional PCR machines. Although it is sometimes
hard to detect PCR products on agarose gel, the Samsung
GeneSpector monitors amout of amplicon instantly and
consistently. Therefore, Samsung GeneSpector would provide a
new viral detection way and saves sample, time, and labor.
Keywords: PCR, GeneSpector
256
한국미생물학회연합
HCMV-Induced Proinflammatory Response in
Monocyte Cell Lines.
Ji-Young CHUN, Hyun-A YI, and Chan-Hee LEE
Division of Life Sciences, Chungbuk National University
*Corresponding author: chlee@chungbuk.ac.kr
Human cytomegalovirus (HCMV) is a member of the Herpesvirus family
that causes disease and mortality in immunocompromised individuals.
The biological properties of herpesviruses to establish latency the
presence of viral DNA in the inflammatory desease lesions, suggest the
possible role of herpesviruses in the development of inflammatory
deseases. Several reports suggested a relationship between HCMV and
inflammtion in fibroblasts, and endothelial cells. Although monocytes
are critical players in the inflammation, relation between monocytes and
HCMV- induced inflammatory response has been elusive. Here, we
report that HCMV may induce inflammatory response in the monocytes
as well as damaged cells by HCMV. Intercellular adhesion molecules
(ICAM-1) are the molecule to help attachment of monocytes to cells of
the vascular tissue. Flow cytometry analysis revealed that ICAM-1 was
upregulated by HCMV in monocyte cell line, HL-60 and THP-1.
Cyclooxygenase-2 (COX-2) plays an important role in proinflamatory
immune reaction processes as well. Western blot analysis revealed that
HCMV stimulated COX-2 expression in the monocyte cell lines.
Therefore, the HCMV induced proinflamatory response appear in the
monocyte cell lines.
Keywords: HCMV, proinflammation and monocyte cell line
2005 International Meeting of the Federation of Korean Microbiological Societies
H001
H003
Effect of Lactic Acid Bacteria on the Enhancement
of the Production of Nitric Oxide and TNF- α in
RAW 264.7 Macrophage Cell
1
2
2
So Hee PARK *, Myung Jun CHUNG , Soo Dong KIM , Dae Heoun
BAEK2, and Nam Joo HA1
1
2
Department of Pharmacy, Sahmyook University, Cellbiotech, Co., Ltd.
*Corresponding author: roee81@nate.com
The immune reinforcement of the probiotic lactic acid bacteria
Lactobacillus acidophilus, Streptococcus thermophilus and Bifidobacterium
bifidum, was studied in RAW 264.7 cell line treated with different
dose(3.1,6.25,12.5,25,50,100 ㎕/㎖) of the bacteria. RAW 264.7
cell line was used here as a macrophage model to assess the effects of
lactic acid bacteria on the production nitric oxide(NO), cytokine
tumor necrosis factor(TNF)- α and cell morphological changes.
Three lactic acid bacteria largely affected production of NO, TNF- α
in dose-dependent manner in 24 or 48h cultures lactic acid bacteria
and cell morphological changes were largely affected by lactic acid
bacteria. Especially, Bifidobacterium bifidum differentially stimulated
the production of NO and TNF- α. NO production was increased by
Bifidobacterium bifidum 25 ㎕/㎖ more than LPS (20ng/ ㎖) control
and TNF- α was increased by Bifidobacterium bifidum 6.25 ㎕/㎖
more than LPS(10ng/ ㎖) control. The in vitro approaches employed
here should be useful in further characterization of the effects of
lactic acid bacteria and systemic immunity.
Keywords: Lactic acid bacteria, Macrophage, Nitric oxide, TNF- α
Biochemical Characteristics of New Anti-Viral
Cytokine, VSF Secreted from Fused Splenocytes
1
2
Young Jin KIM , Jee In AHN , and Yoon Won KIM
1
1
Department of Microbiology, College of Medicine, Hallym University and
2
ImmuneMed Inc., Department of Molecular and Cellular Biology, School of
Medicine, Sungkyunkwan University
*Corresponding author: j020721@nate.com
Virus-Suppressing Factor (VSF) is secreted from fused splenocytes of
mice infected with one variant of encephalomyocarditis D virus
(EMC-DV) and has been found to inhibit the infection of several
viruses including EMC virus, mengovirus, influenza virus, human
immunodeficiency virus, human cytomegalovirus and vesicular stomatitis
virus in vivo and in vitro. The VSF reacted to several kinds of cell lines
for antiviral activities and did not viruses. We have purified VSF
using by various open column chromatography method. Molecular
masses of the peptides of VSF were about 55, 31, 30, and 25kDa by
SDS-PAGE. Thus, VSF appeared to be comprised of four peptide
subunits. No subunit of VSF seems to be glycosylated due to absence
of migration shift on SDS-PAGE after treatment with endoglycosidase
H. Amino acid sequence was analyzed by N-terminal and internal
sequencing, and MALDI-TOF. VSF got many new sequences
different from the known proteins. RT-PCR and EIA were performed
to investigate expression and secretion of specific known antiviral
cytokine in the hybridoma secreting VSF protein. The VSF might not a
known antiviral cytokine. The antiviral activity of VSF was synergistic
with interferon γ. VSF was presumed to be a new antiviral cytokine by
the biochemical and genetic characteristics and biological activities.
This VSF protein may be a cytokine to provide a pharmaceutical
composition for prevention and treatment of viral infections.
Keywords: antiviral, cytokines, innate immunity, interferon
H002
H004
Mycobacterial 38-kDa Glycolipoprotein Antigen
Activates the MAPK Pathway and Release of
proinflammatory Cytokines through Toll-like
Receptor (TLR) 2 and 4 in Human Monocytes
Antiviral Activities of Flavonol Glycosides Against
Porcine Coroanvirus
1
2
Saet-Byel JUNG *, Chang-Hwa SONG1, Tae-Hyun PAIK , Ji2
1
1
1
Sook LEE , Chul-Su YANG , Su-Young KIM , Kil-Soo LEE ,
1
1
1
A-Rum SHIN , Jae-Hee OH , Yu-Mi KWON , Hwa-Jung KIM1,
1
1
Jeong-Kyu PARK , and Eun-Kyeong JO *
1
Department of Microbiology, College of Medicine, Chungnam National University,
2
Department of Microbiology, College of Medicine, Konyang University
*Corresponding author: jtotquf@cnu.ac.kr
Despite that the 38-kDa glycolipoprotein of Mycobacterium tuberculosis (M.
tbc) H37Rv has been known to evoke prominent cellular and humoral
immune responses in human tuberculosis (TB), little is known about the
intracellular signaling mechanisms involved in the 38-kDa-induced
cytokine responses. In this study, we investigated the role of TLR and
MAPK pathways involved in the purified 38-kDa antigen-induced TNF-α
and interleukin (IL)-6 expression in human primary monocytes. The
purified 38-kDa glycolipoprotein was shown as a strong inducer of TNF-α
and IL-6 in human primary monocytes. MAPK [extracellular signalregulated kinase (ERK) 1/2 and p38] are rapidly phosphorylated in human
monocytes stimulated with the 38-kDa antigen. Both p38 and ERK 1/2
activation are essential for 38-kDa-induced TNF-α and IL-6 production.
The inhibition of TLR2, and partly TLR4, by specific antibodies
significantly abrogated the 38-kDa-induced secretion of TNF-α and IL-6
in human monocytes. Moreover, the specific antibodies for TLR2 or TLR4
inhibited ERK 1/2 and p38 activation by the 38-kDa protein applied to
HEK293 cells overexpressing TLR2 or TLR4, as well as human monocytes.
Collectively, these data demonstrate that 38-kDa glycolipoprotein, acting
through both TLR2 and TLR4, induces the activation of the ERK 1/2 and
p38 MAPK pathway, which in turn mediates an essential role for the early
inflammatory immune responses during human TB.
Keywords: 38-kDa glycolipoprotein, ERK, p38 MAPK, Mycobacterium
tuberculosis, TNF- , IL-6
1
1
2
Hwa-Jung CHOI , Young-Joon AHN , Jin-Hee KIM , Choong2
3
2
Hwan Lee LEE , Man-Bae KIM , and Dur-Han KWON *
1
2
Seoul National University, Korea Research Institute of Bioscience and
3
Biotechnology, Kyung Nam Agriculture Reseach Center
*Corresponding author: dhkwon@kribb.re.kr
Coronaviruses cause severe respiratory or gastroenteritis disease in
poultry, avian and human. SARS is caused by a novel coronavirus
known as SARS-CoV. But effective treatment for SARS or other
coronaviruses is not developed. The objective of this study is
evaluated antiviral activity of natural compounds against porcine
epidemic diarrhea virus (PEDV), a member of coronavirus.Antiviral
activities of natural compounds existed in many plants were
investigated. Flavonol glycoside isolated from Houttuynia cordata,
was showed strongest activity against PEDV, and higher antiviral
activity compared with current antiviral drugs. This compound
could play an important role in SARS therapy.
Keywords: Coronaviruses, SARS, porcine epidemic diarrhea virus,
Antiviral activities, Houttuynia cordata
국제학술대회
257
October 13-14, 2005, Seoul, Korea
H005
H007
Immunostimulatory Oligodeoxynucleotide Originated
from Mycobacterium bovis Chromosomal DNA
Involvement of PKCζ in the Development of
Airway Hyperreactivity and Inflammation in a
Mouse Model of Asthma
1
2
3
Keun-Wook LEE , Jinwon JUNG , Younghee LEE , Doo-Sik
KIM2, and Hyung-Joo KWON1*
1
Department of Microbiology, College of Medicine, Hallym University,
Department of Biochemistry, College of Science, Yonsei University,
Department of Biochemistry, College of Natural Science, Chungbuk National
University
*Corresponding author: hjookwon@hallym.ac.kr
2
3
Bacterial DNA has a variety of immunostimulatory activity such as
B cell and natural killer cell activation, induction of interferon
(IFN)-γ and induction of Th1 type immune responses, whereas
mammalian DNA does not. To evaluate the genomic DNA sequences
of M. bovis having immunostimulatory activity, we designed a series of
synthetic 20 base length phosphodiester backbone oligodeoxynucleoptides
(ODNs) containing CpG-motifs (MB-ODNs) based on computerassisted analysis of the genome of M. bovis. We screened
immunostimulatory MB-ODNs that induce the activation of the NFκB-responsive IL-8 promoter in RAW 264.7 cells. Experimental
analyses demonstrate the existence of potent CpG-DNA in the M.
bovis genomic DNA that has functional effects as a Th1 responsive
adjuvant and activates the transcription factors NF-κB. We found
that both CpG motif and the context of the sequence surrounding the
CpG motif are important for the immunostimulatory effects. The
identification of the potent immunostimulatory DNA sequence in
native bacterial genome may give insights to the optimal sequence
for well-controlled immune responses.
Keywords: CpG-DNA, Mycobacterium bovis, NF-κB, adjuvant
1
1
1
2
Department of Biological Science, Jeonju University, Department of
Pediatrics, Presbyterian Medical Center
*Corresponding author: sangyun@jj.ac.kr
PKC is a complex family consisting of many types of isoenzymes of
which PKCζ, an atypical isoform, has been implicated in the regulation
of apoptosis and NF-κB, as well as in the control of T-dependent
responses. In this study, we investigated the functional role of PKCζ in
the pathogenesis of allergic asthma. A mouse model of allergic asthma
was induced by repeated sensitization (50 μg) followed by intranasal
challenge (1 mg) with OVA. When PKCζ inhibitor was instilled before
OVA challenge, their AHR was significantly reduced in comparison to
control mice given PBS. Moreover, numbers of eosinophils in
bronchoalveolar lavage fluid (BALF) was also significantly lowered
by PKCζ inhibitor, suggesting that PKCζ pathway is involved in AHR
and airway inflammation. Levels of IL-4 and TNFα in BALF were also
markedly decreased while IFNγ level was less predominantly lowered
by PKCζ inhibitor. Level of tissue inhibitor of metalloproteinase-1 in
BALF was significantly elevated, whereas not altered in serum. However,
cytokine production by peribronchial lymph node cells, serum MMP-9
and IgE levels were not affected. These results indicate that
intratracheal instillation of PKCζ inhibitor downregulates local, allergen
-specific response and inflammation and subsequently leads to
attenuation of allergic manifestations. Collectively, data from the current
study showed that PKCζ is involved in the development of allergic
asthma, suggesting that PKCζ can be a therapeutic target in asthma.
Keywords: asthma, PKCζ, airway hyperreactivity, airway inflammation
H006
H008
Genetic Adjuvant CD154 Enhances Protective
Immunity of gD DNA Vaccine to Aujeszky's Disease
Nuclear Localization Signal of Outer Membrane
Protein of Acinetobacter baumannii
1
1
1
Ji Da CHOI *, Joong Bok LEE , Chang Seon SONG , In Su
1
2
1
CHOI , Bang Hun HYUN , and Seung Yong PARK
1
Laboratory of Infectious Disease and Immunology, Konkuk University,
National Veterinary Research and Quarantine Service
*Corresponding author: apjida@konkuk.ac.kr
2
It has been known that DNA vaccination is a promising strategy for
preventing infectious diseases. Previously, we developed a DNA
vaccine against Aujeszky's disease virus (ADV), where gD gene was
cloned into the pMYK vector. However, there remained suspicion
that the level of which was so high as to protect target animals from
AD. In order to enhance its immunogenicity, we have exploited the
CD154, which strongly interacts with CD40 on professional antigen
presenting cells, thereby increasing the immune responses. We
constructed a recombinant DNA in the backbone of pMYK vector by
inserting cDNA encoding CD154 in frame into the downstream of gD
gene that was truncated by deletion of transmembrane domain
(pMYKtgD-CD154). To verify whether expected protein was
produced, pMYKtgD-CD154 was transfected into 3T3 cell lines, and
the detection of chimeric protein was confirmed from culture
supernatants. pMYKtgD-CD154 was injected intradermally into the
mice, and the inducing immune responses were compared with those
of other DNA vaccines. Serum neutralizing antibody titers of a group
of pMYKtgD-CD154 were higher than that of other groups. When the mice
were challenged with lethal dose of ADV, CD154- coimmunized
animals exhibited a significantly enhanced survival rate. These
results demonstrate that molecular adjuvant CD154 in DNA
vaccination may enhance humoral and protective immunity.
Keywords: DNA vaccine; Aujeszky's disease virus; Aujeszky's
disease; CD154; Molecular Targeting
258
1
Jeong-Su DO , Youn-Hwa CHOI , Hyo-Jung SEO , Kang Seo
PARK2, and Sang-Yun NAM1*
한국미생물학회연합
1
2
3
4
Jungha KIM , JeChul LEE , SoonAe KIM , Seongkyu LEE ,
5
7
1
Jaesun CHUN , Sangsun KANG6, Jinkyu KIM , Jungha KIM ,
1
and Sounghee HYUN *
1
Department of Clinical Laboratory Science, Eulji University, School of
Medicine, 2Department of Microbiology, Kyungpook National University
3
School of Medicine, Department of Pharmacology, Eulji University, School of
4
Medicine, Department of Biochemistry, Eulji University, School of Medicine,
5
Department of Biological Science, Korean national University of Education,
6
7
School of Science Education, Chungbuk National University, Korea Atomic
Energy Research Institute
*Corresponding author: hyunsh@eulji.ac.kr
Acinetobacter baumannii, an opportunistic pathogen, is well known
to cause a wide spectrum of nosocomial infections particularly in
intensive care units. The outer membrane protein (Omp) plays an
important role in the diffusion properties of the outer membrane of
A, baumannii. The cloned outer membrane protein was localized to
the nucleus and induced apoptosis in COS-1 cells. We also noticed
the putative nuclear localization sequences (nls) in Omp. Thus, our
study indicate that Omp has true NLS site with the NLS deletion
mutant and deduce the function of Omp in nucleus.
Keywords: Acinetobacter, NLS, Omp
2005 International Meeting of the Federation of Korean Microbiological Societies
H009
H011
Negative Regulation of Mycobacteria-induced
IL-23 p40/p19 Expression by PI3-K, ERK1/2, and
mTOR/ S6K1 Pathways in Human Macrophages
via Toll-like Receptor 2
1
2
1
Chul-Su YANG , Ji-Sook LEE , Chang-Hwa SONG , Saet-Byel
JUNG1, Jae-Hee OH1, Yu-Mi KWON1, A-Rum SHIN1, Su-Young
1
1
1
1
KIM , Kil-Soo LEE , Hwa-Jung KIM , Jeong-Kyu PARK , Tae1
1
Hyun Paik PAIK , and Eun-Kyeong JO *
1
Department of Microbiology, College of Medicine, Chungnam National University,
Department of Microbiology, College of Medicine, Konyang University
*Corresponding author: charles514@lycos.co.kr
2
The heterodimeric cytokine IL-23 p19/p40, mainly produced by activated
macrophages, has an important role for Th1 immunity, however, little is
known about the intracellular signaling mechanisms underlying the
regulation of IL-23 p19/p40 expression during human tuberculosis. Here we
show that PI3K, mammalian target of rapamycin (mTOR)/70-kDa
ribosomal S6 kinase 1 (S6K1), and ERK 1/2 pathways negatively regulate
the expression of IL-23 p19/p40 by human monocyte-derived macrophages
(MDM) in response to Mycobacterium tuberculosis (M. tbc). In contrast, p38
MAPK pathway was essential for M. tbc-induced expression of IL-23
p19/p40 in human MDMs. The inhibition of PI3K-Akt pathway significantly
abrogated the M. tbc-induced ERK 1/2 and S6K1, but (activate) not p38
MAPK, phosphorylation. There was no interdependence between ERK 1/2
and p70 S6K1 pathways. Although the inhibition of PI3K pathway downregulated the IL-10 expression in a dose-dependent manner, ablation of
mTOR/p70 S6K1 or ERK 1/2 activity unaffected IL-10 expression in human
MDMs. Further, we found the PI3K and p38 MAPK differentially regulate the
M. tbc-induced IL-23 activation in a Toll-like receptor (TLR) 2-dependent
manner in human MDMs. Collectively, these studies provide new insight
into the role of PI3K, and its downstream ERK 1/2 and p70 S6K1 pathways,
which may contribute a negative regulation of IL-23 in APC via TLR2.
Keywords: mTOR/S6K1, IL-12, TNF-α, Mycobacterium tuberculosis, PI
3-K, TLR2
Neutralizing Antibody Titer in Test Samples Using
Pseudotyped Human Papillomavirus Genotype
16 Virus-like Particle
1
1
2
Jung-Ah CHOI , Sang-Woo KIM , Joo-Young KIM , Sue Nie
PARK3, and Joo-Sung YANG1*
1
2
Department of Genetic Engineering, Sungkyunkwan University, Research Institute
and Hospital, National Cancer Center, 3Korea Food and Drug Administration
*Corresponding author: jsyang@skku.edu
HPV has been know as the most crucial pathogen for a cervical cancer.
Cervical cancer is the major cause of death in women of reproductive age in
parts of the developing world. In fact, cervical cancer patients are 100%
positive for HPV infection, development of prevention strategies as well as
reliable diagnostic strategies are very important. Therefore, we focused on
an optimization and a standardization of methodology to evaluate humoral
immune response against HPV16 L1 marker. To enhance the sensitivity of
the assay and allow quantitation of the virus particles in samples, a
neutralization assay using VLPs encapsidating secreted enhanced alkaline
phosphatase(SEAP) reporter gene was produced. To produce pseudotyped
VLPs, HPV16 L1, L2, SEAP plasmids were transfected and cell lysates
were harvested after 48hr of transfection. Purified VLPs by Opti-prep
density gradient ultracentrifugation were confirmed by monoclonal Ab as
determined by Western blot analysis and neutralization assay. Moreover,
antigenic B cell epitopes of HPV16L1 were predicted and synthesized for
polyclonal Abs production. These Abs also showed neutralizing activity
over 60 % with the purified VLP. To verify our NAb assay system is
applicable to test human sera samples, HPV positive patients sera were
analyzed. The sera have positive NAb activities against SEAP VLP.
Therefore, our pseudotyped VLP NAb assay system is a suitable diagnostic
tool for screening of HPV infection in human.[Supported by KFDA grant]
Keywords: Human Papillomavirus 16, Virus-like Particle, Pseudotyped
Virion, Neutralizing Antibody Assay
H010
H012
Immune Stimulating Activity in Exo-Biopolymer
from Submerged Culture of Lentinus edodes
with Rice Bran
1
1
1
Hwa-Young KIM , Jae-Taek HAN , Seong-Gil HONG , Sung-Bum
1
2
3
1
YANG , Kwang-Soon SHIN , Hyung-Joo SUH , Mi-Hyoun PARK *,
1
and Sung-Joo HWANG
Proinflammatory Cytokine Profiles in West Nile
Virus Capsid Protein Expressing Brain Cells
1
1
2
Yun-Hee YANG , Joong-Bin PARK , Young-Joon KO , Jin-Ju
2
2
2
2
NAH , Yong-Joo KIM , Kang-Seuk CHOI , Yiseok JOO ,
3
4
1
Jaewhan SONG , Suhk-Neung PYO , and Joo-Sung YANG *
1
2
Erom R&D Center, Department of Food Science and Biotechnology,
Kyonggi University, 3Department of Food and Nutrition, College of Health
Sciences, Korea University
*Corresponding author: mhpark@erom.co.kr
Department of Genetic Engineering, Sungkyunkwan University, National
Veterinary Research and Quarantine Service, 3Department of Food Scinece
4
and Biotechnology, Sungkyunkwan University, College of Pharmacy,
Sungkyunkwan University
*Corresponding author: jsyang@skku.edu
The present study purposed to examine the effect of substance
extracted from rice bran using the mycelia of Lentinus edodes on the
enhancement of immunological activity. According to the result of
measuring the in vitro macrophage activity of the exo-biopolymer
from submerged culture of Lentinus edodes with rice bran(SLRB), it
had activity similar to LPS and the activity was dose-dependent.
According to the result of measuring splenocyte proliferation, the
exo-biopolymer increased proliferation by around 1.4-fold compared
to the control group. We measured the proliferation of bone marrow
cells to measure gut immunity and, according to the result,
proliferation increased by 109% compared to the control group and
was similar to that of LPS. In order to examine the enhancement of
immunological activity in vivo, we orally administered the exobiopolymer (25, 50, 250mg/kg bw) to C3H/He mice, measured the
macrophage activity and found that the activity was higher than that
of the control group at concentration of 50, 250mg/kg. Accordingly,
the exo-biopolymer from SLRB is considered usable as BRM in order
to keep health from immunological diseases.
Keywords: rice bran, Lentinus edodes, immune stimulating activity
West Nile virus (WNV) causes cerebral inflammation and stimulates
inflammatory cytokine expression. Glial cells mount immunocyte
recruitment to focal sites of viral infection within the central nervous
system (CNS) and synchronize immune cell functions through a
regulated network of cytokines. We investigated the production of
proinflammatory cytokines (IL-1β, IL-6, TNF-α) or inflammation
mediating proteins such as iNOS, and COX-2 in response to transfer
of WNV-Capsid (Cp) in glial, microglial cells, and neuroblastoma
cells as determined by RT-PCR and Western blot analysis. These
cytokines mediate inflammation and other mediators are directly
associated with the inflammation. Each message level of cytokines
and mediators was slightly different per each cell type and level of
IL-1beta, iNOS, and COX-2 in Cp-transfered cells were more
elevated than those in normal cells. Even though level of the cytokines
was slightly increased, this result made it possible to suppose that
WNV-Cp has an influence on the expression of inflammatory
cytokines or mediators, and ultimately determinants for brain
encephalitis. [Supported by KOSEF grant]
Keywords: Flavivirus, West Nile virus, Capsid protein,
Inflammation, Cytokines
1
2
국제학술대회
259
October 13-14, 2005, Seoul, Korea
H013
H015
Effects of Electrolyzed Alkaline Reduced Water
on Echinostoma hortense Infection and Immune
Response in C57BL/6 Mice
1
2
2
2
1
1
1
Dan JIN , Eun Ju YANG , Soo Jung LIM , Yong Suk RYANG ,
Sun Gu LEE3, Dong Huei KIM4, Young Kun DEUNG4, Seung
5
5
Kyu PARK , and Kyu Jae LEE *
Hyun-Ouk SONG , Jae-Sook RYU *, Myoung-Hee AHN , DukYoung MIN1, and Myeong Heon SHIN2
1
2
Department of Microbiology, Wonju College of Medicine, Yonsei University,
2
Department of Biomedical Laboratory Science and Institute of Health Science,
3
College of Health Science, Yonsei University, Department of Pathology,
Oriental Medical College, SangJi University, 4Department of Basic Sciences,
Wonju College of Medicine, Yonsei University, 5Department of Parasitology and
Institutes for Basic Medical Science Wonju College of Medicine, Yonsei University
*Corresponding author: jindan0104@empal.com
Electrolyzed Alkaline Reduced Water (ERW) is produced at the cathode by
electrolysis of water containing electrolytes. It had been known that the ERW
containing high amount of hydrogen molecules scavenges the noxious free
radicals generated in organisms. In this study, we investigated the effect of the
ERW on immunofunction of the intestinal mucosa and the serum cytokine
levels in C57BL/6 mice fed on the ERW after oral infection with Echinostoma
hortense. The level of the worm recovery was higher in the experimental group
than in the control group, but no statistical difference. The numbers of the mast
cells and goblet cells were significantly lower in the experimental group than
in the control group (p< 0.001). The levels of the Th1 cytokines (IL-6, IL-1β,
IFN-γ, TNF-α, and IL-2) and the Th2 cytokines (IL-4, IL-5, IL-10, and IL-13)
in the serum were not significantly different. In conclusion, this study
demonstrates that the ERW inhibits hyperplasia of the goblet cells and
increase in the number of the mast cells in the intestinal mucosa. Also, the
expulsion of adult worms was more delayed in the experimental group than in
the control group. We suppose that the results are come from the
immunomodulatory effect of the ERW on intestinal mucosa in the mice.
Keywords: electrolyzed alkaline reduced water (ERW), Echinostoma
hortense, goblet cell, mast cell, cytokine
H014
FK506-binding Protein 51 Gene Is Inducible in
Chickens Infected with a Virulent Newcastle
Disease Virus and an Infectious Bronchitis Virus
260
Trichomonas vaginalis Induce Human Neutrophil
Apoptosis by Reactive Oxygen Species- Mediated
p38 MAPK Activation
1
Department of Parasitology, Hanyang University College of Medicine,
Department of Parasitology, Yonsei University College of Medicine
*Corresponding author: 94agape@hanyang.ac.kr
Neutrophils are known to be observed at the vaginal discharges of women
infected with Trichomonas vaginalis, a common cause of human STD. The aim
of our study was to determine whether the neutrophil apoptosis induced by
trichomonads is related with reactive oxygen species (ROS), and MAPK
signaling pathway. When human neutrophils were cultured with live T.
vaginalis T016 isolate, CD16 receptor shedding and decreased MMP
showing apoptosis were shown after 3 h and 12 h incubation, respectively.
Cleaved caspase-3 was also observed after 3 h incubation with T. vaginalis.
ROS of neutrophils cultured with trichomonads was significantly increased
at 90 min, and this alteration was inhibited by NADPH oxidase inhibitor,
diphenyleneiodonium chloride (DPI) pretreatment. DPI also inhibited
increase of CD16 receptor shedding, cleavage of caspase-3, and decrease
of MMP. To investigate involvement of signaling pathway associated with
neutrophil apoptosis by T. vaginalis, activity of p38 MAPK was examined
by western blot. p38 MAPK was strongly activated initially at 1 h
cultivation and increased gradually, and the activity was reduced by DPI
treatment. However, ROS generation in neutrophils was not affected by
p38 MAPK inhibitor. These results suggest that ROS generation of
neutrophils and activation of p38 MAPK by the increased ROS were
involved in T. vaginalis-induced neutrophil apoptosis.
Keywords: Neutrophil, Trichomonas vaginalis, Apoptosis, ROS, MAPKinase
H016
Regulation of Hepatitis C Virus Replicon by the
Interferon-Induced Protein Kinase PKR
Mijin KIM, Yoonhee SEO, Giyoun NA, and Yongbum KOO*
Shi Nae KWON, Se Hoon PARK, Jungsuh Park KIM, and
Byung Yoon AHN
School of Biotechnology and Biomedical Science, Inje University
*Corresponding author: mbkooyb@inje.ac.kr
School of Life Science and Biotechnology, Korea University
*Corresponding author: punky@korea.ac.kr
FK506-binding protein family proteins (FKBPs) have been thought
to be regulators of immune reactions. Since immune reactions are
induced by specific antigens, the expression of regulators of immune
reactions may be regulated by antigens. We used a newcastle disease
virus (NDV), a strong inducer of immune reactions, to test whether
any of the FKBPs(FKBP12, FKBP12.5, FKBP25, FKBP51, FKBP52)
are inducible in NDV infected chickens. We performed semiquantitative
RT-PCR and found that only FKBP51 was strongly induced in all 12
tissues of NDV-infected chickens. We also tested whether this
induction is specific to NDV infection or it is a general response to
infections. Chickens were infected with an avian bronchitis virus or
Salmonella pullorum. FKBP51 was induced only in cecal tonsil of
chickens infected with ABV while no induction was recognized in
any of 12 tissues of chickens infected with Salmonella pullorum.
These results suggest that the regulation of FKBP51 expression has
evolved to be induced by infections, although the tissue-specific
induction patterns and extent of induction varies depending on the
pathogens. This work was supported by Biogreen21 research
grant(2005) from Rural Development Administration, Korea
Keywords: FK506-binding protein 51, newcastle disease virus
Despite the worldwide use of interferon (IFN) therapy for the
treatment of hepatitis C virus (HCV) infection, little is known how
IFN and/or its cellular effector molecules inhibit viral replication.
Interferon itself is not directly anti-viral, but is an effective inducer
of cellular anti-viral proteins such as the double-stranded RNA
dependent protein kinase (PKR). PKR inhibits cellular protein
synthesis through phosphorylation of translation initiation factor
eIF2, thereby mediating antiviral and anti-proliferative action of type
I IFNs. Studies on HCV replication have been advanced by recent
establishment of replicon systems that consist of subgenomic HCV
RNA coding for the viral nonstructural proteins and the neomycin
resistance gene as a selectable marker. We established a human
hepatoma Huh7 cell line with a siRNA construct that suppressed
PKR expression by up to 90% without affecting other IFN-inducible
cell proteins, and asked how the HCV replicon was affected in this
cell. Immunoblot analysis of the viral proteins indicated that the
inhibitory effect of IFN on HCV replicon was relieved by 30-40%.
Furthermore, formation of neomycin-resistant foci increased by more
than 100% compared to the control cell. These results demonstrate
the inhibitory role of PKR on HCV replication.
Keywords: HCV, PKR, Interferon
한국미생물학회연합
2005 International Meeting of the Federation of Korean Microbiological Societies
H017
A Promoter Polymorphisms in Monocyte
Chemoattractant Protein-1 Increases Susceptibility
to Pulmonary Tuberculosis in Koreans
1
1
1
2
Jae-Hee OH *, Yumi KWON , Chul-Su YANG , Ji-Sook LEE ,
Eun-Kyeong JO1, Hwa-Jung KIM1, Jeong-Kyu PARK1, Tae2
1
Hyun PAIK , and Chang-Hwa SONG
1
Department of Microbiology, College of Medicine, Chungnam National
University, 2Department of Microbiology, College of Medicine, Konyang
University
*Corresponding author: skuly@nate.com
We analyzed single nucleotide polymorphisms (SNPs) in monocyte
chemoattractant protein (MCP)-1 gene in 129 tuberculosis cases and
162 healthy controls. In control subjects, genotypes of MCP-1 gene
were in Hardy-Weinberg (HW) equilibrium. The allele G of the
MCP-1 gene was strongly associated with tuberculosis, compared to
healthy controls, with a significant χ2= 32.279 (p= 0.0001), and an
odds ratio 0.381 (95% confidence interval 0.272-0.533). Carriers of
MCP-1 genotypes AG and GG were significantly overrepresented
among tuberculosis cases, compared to healthy controls. The odds
ratio for heterozygous AG in tuberculosis cases versus healthy
controls was 2.8 and strongly increased to 6.9 for the comparison of
homozygous GG. These data suggest the polymorphism at the
MCP-1 in individuals homozygous for the (-2518) G allele could
contribute to their increased risk of developing tuberculosis in
Korea.
Keywords: SNPs, MCP-1, pulmonary tuberculosis
국제학술대회
261
October 13-14, 2005, Seoul, Korea
I001
I003
Improved Method for the Recovery of Poliovirus
from Oyster
1
2
2
1
2
2
Sookhee HA , Gun-Jo WOO , Hyo-Sun KWAK , and Weon
Sang CHOI1*
Yeon Jae KANG , Su-Jin KU , Donghwan KWAK , and Sang
Yup LEE1,3*
1
Department of Biotechnology, Dongguk University, Korea Food and Drug
Administration
*Corresponding author: weonsang@dongguk.ac.kr
1
Molecular detection using polymerase chain reaction (PCR) to
detect virus in contaminated oysters can be hindered by the low virus
recoveries during the concentration process and by natural PCR
inhibitors in oyster. A method for concentrating and detecting virus
in oysters has been developed. Poliovirus was adsorbed to the oyster
tissue homogenates and was eluted with buffers and then concentrated
by polyethylene glycol (PEG) precipitation, chloroform extraction,
and second PEG precipitation. The amount of virus was evaluated
by poliovirus plaque assay. For detection, the viral RNA in oyster
was examined using one-step RT-PCR after extraction with TRIzol.
Nested PCR improves the efficiency of this procedure. Overall, this
procedure could remove the inhibitors sufficently to allow the virus
to be detected in oysters.
Keywords: detection of virus, oyster
A Biosensor is an analytical device utilizing biological elements
integrated with a transducer that converts biological expressions into a
measurable signal. The development of immobilization technologies
for stabilizing biomolecules and coating with them on the surface is
a significant factor in biosensor construction. Since the research of
whole cell biosensor has been increased, it is important to develop the
method of cell immobilization on the transducer surface. Gold
binding polypeptide(GBP) which was developed in an E. coli
cell-surface display system is known to interact with gold surface.
This study show bacterial cell immobilization on the gold surface by
GBP displayed on the E.coli cell surface using SPR analysis. This
work was supported by the R&D program for Regional Development,
which is sponsored by the Korea Ministry of Commerce, Industry and
Energy
Keywords: biosensor, cell immobilization, gold binding polypeptide
2
I002
2
Department of Chemical and Biomolecular Engineering, KAIST, KMAC,
Department of BioSystems, KAIST
*Corresponding author: yj7444@kaist.ac.kr
3
I004
Thermotolerant Agarase Producing Novel Marine
Agarivorans Species
1
2
2
Geun-Tae PARK , Dong-Geun LEE , Nam Young KIM , Eo-Jin
2
2
2
2
LEE , Jong-Geun JUNG , Jae-Hwa LEE , Jong-Myung HA ,
2
3
2
Bae Jin HA , Moon-Soo HEO , and Sang-Hyeon LEE *
1
Research and University-Industry Cooperation, Pusan National University,
Department of Bioscience and Biotechnology, College of Engineering, Silla
3
University, Faculty of Marine Sciences, Cheju National University
*Corresponding author: slee@silla.ac.kr
2
Novel marine bacterium, producing thermotolerant agarase, was
isolated from north-eastern sea of Jeju island. Isolated novel
bacterium was named Agarivorans sp. JA-1 based on biochemical
and morphological characteristics and 16S rDNA gene. Agarase was
produced as growth-related and expressed regardless of agar presence.
Agarase activity was optimal at pH 8 in 50 mM Glycine- NaOH buffer
and 40℃. Enzymatic activity was maintained over 80% at 60℃ and
70% at 80℃, which is thermotolerant. Hence isolated novel
Agarivorans sp. JA-1 strain and its beta-agarase could be used for
the production of functional oligosaccharide from agar in solution
state. Acknowledgement: This work was supported by the Marine and
Extreme Genome Research Center Program, Ministry of Maritime
Affairs &Fisheries, Republic of Korea.
Keywords: marine, Agarivorans, agarase, thermotorelant enzyme
262
Cell Immobilization by Gold Binding Polypeptide
Displayed on the E. coli Cell Surface
한국미생물학회연합
Bio-data Analysis System that Use Datamining
and WebServices for Microarray
Yo Han CHOI*, Ho Il LEE*, Kyong Oh YOON, and Hyun Seok
PARK
Macrogen BI Institute
*Corresponding author: hani9999@nate.com
Researches on the huge province of genes are rapidly ongoing due to
the development of the Microarray Chip and those data from the
researches bring diverse analysis systems of the genes of the chip.
However, the current systems for chip analysis only offer the
incipient stage of the analytic information about the genes and don't
cope with the rapid change of the Microarray Chip. For those
reasons, this treatise researches and develops the new paradigm of
Microarray Chip using the Datamining analysis technology to
analyze the complicated and huge chip data more logically and
precisely than before and also mutual correlation analysis technique
which can analyze various genetic information using Webservices.
Keywords: WebServices, microarray, datamining, dna chip
2005 International Meeting of the Federation of Korean Microbiological Societies
I005
I007
Overexpression and Characterization of an α
-Amylase from the Hyperthermophilic Archaeon
Thermococcus sp. NA1
1
1
1,
1
Jae Kyu LIM , Hyun Sook LEE , Yun Jae KIM Jeong Ho JEON ,
Kyu Ho LEE2, Sung Gyun KANG1*, and Jung-Hyun LEE1*
1
2
Korean Ocean Research and Development Institute, Department of
Environmental Science and Engineering, Hankuk University of Foreign Studies
*Corresponding author: jacky41@freechal.com
Genomic analysis of a hyperthermophilic archaeon Thermococcus NA1.
revealed the presence of an open reading frame consisting of 1,377 bp
similar to GH13 α-amylases from the members of the genus Thermococcus
and Pyrococcus. This amylolytic enzyme, designated TNA1_amyl
(Thermococcus sp. NA1 amylase), was cloned and expressed in
Escherichia coli. The recombinant protein was secreted to the culture
supernatant and the intracellular expression of the mature form of the
protein was accomplished based on the N-terminal sequencing data of the
cleavage site of signal peptide. The purified protein by His-tagged affinity
chromatography was highly thermostable with an optimum temperature of
80°C, and an optimum pH of 5.5 to 6.0. More than 50% of the maximum
activity was retained in the range between pH 4.0 and pH 7.0. The enzyme
displayed thermostability with half-life (t1/2) of 10 min at 90 ℃, and the
thermostability of TNA1_amyl was significantly enhanced in the presence
of calcium with half-life (t1/2) of 153 min at 90 ℃. The substrate specificity
of TNA1_amyl suggests that it possesses characteristics of an α-amylase.
Like typical α-amylases, TNA1_ amyl hydrolyzed maltooligosaccharides
and starch, not pullulan to produce mainly G2 to G7. The similarity analysis
among hyperthermophile α-amylase showed that C152 and C153 might be
critical for thermostability and optimum temperature.
Keywords: alpha-amylase, Thermococcus sp. Archaeota
I006
Transcript and Protein Level Analysis of Crossregulation in Phosphate Starvation Response
in Escherichia coli
1
1
Yeon Jae KANG , Jong Hwan BAEK , and Sang Yup LEE
1
1,2
2
Department of Chemical and Biomolecular Engineerin, KAIST, Department
of BioSystems, KAIST
*Corresponding author: yj7444@kaist.ac.kr
Phosphorus is the essential cellular element as major building blocks
of various biomolecules, and its metabolism is closely related with
diverse metabolic pathways including central carbon metabolism.
Escherichia coli has a PhoR-PhoB two-component regulatory system
to detect and respond to environmental phosphate concentration.
Additionally, this system is connected to other regulatory systems
and cross-regulated with them. In this study, the multiple controls of
pho regulon and cross-regulation were investigated at transcript and
protein levels using DNA microarray followed by real-time PCR
analysis and fluorescence resonance energy transfer (FRET) analysis.
From this study, the interactions among PhoB, PhoR, PhoU, and
CreC could be revealed in phosphate limiting condition in E. coli, and
this is valuable to understand the celluar physiology relating to the
cross-regulation of phosphate starvation response. This work was
supported by the Korean Systems Biology Research Grant
(M10309020000-03B5002-00000) from the Ministry of Science and
Technology. Further supports by the BK21 program and LG
Chemicals Chair Professorship are greatly appreciated.
Keywords: PhoR-PhoB two-component regulatory system, crossregulation, DNA microarray, real-time PCR, FRET
I008
Tyrosinase Inhibitor from the Flowers of Impatiens
balsamina
1
2
3
4
Development of Reusable Microfluidic Immunoassay
for Detection of Escherichia coli O157:H7
Young-Hee LIM *, In-Whan KIM , Jung-Ju SEO , Sung-Ran KIM ,
5
and Jeong Keun KIM
Yoon Sun YANG, Nae Yoon LEE, Youn Sang KIM, and Sungsu
PARK*
1
Division of Nano Science, Ewha Womans University
*Corresponding author: geneys@ewhain.net
Department of Clinical Laboratory Science, College of Health Sciences,
2
Korea University, Department of Food and Nutrition, College of Health
Sciences, Korea University, 3Hazardous Substance Research Team, Korea
4
5
Basic Science Institute, Korea Food Research Institute, Department of
Chemical Engineering, Korea Polytechnic University
*Corresponding author: yhlim@korhealth.ac.kr
Kaempferol was isolated and identified from the methanol extract of
the flowers of Impatiens balsamina. Kaempferol showed inhibitory
activity against mushroom tyrosinase with an ID50 value of 0.038
mM. The inhibition kinetics analyzed by a Lineweaver-Burk plot
found kaempferol to be a competitive inhibitor of the mushroom
tyrosinase with a Ki value of 0.025 mM. In addition to tyrosinase
inhibiting activity, it strongly inhibited the melanin production of
Streptomyces bikiniensis without the inhibition of cells growth,
showing a dose-dependant inhibition of melanin formation.
Keywords: Tyrosinase, Inhibitor, Streptomyces bikiniensis
We report development of microfluidic immunoassay for detection
of E. coli O157, a foodborne pathogen causing serious illnesses in
many countries including Korea. The microfluidic device (1 x 1 cm)
was fabricated with a glass slide and PDMS by transferring a pattern
of a microchannel (length: 0.5 cm, widths: 200 ~ 90 μm, height: 800
μm) from a master mold to the polymer and later binding the
patterned polymer to a glass slide. Protein A-conjugated agarose
beads (diameter: 45~160 μm) were introduced into the microchannel
through its entrance (width 200 μm) and packed into two identical
microchambers (diameter: 1.7 mm, height: 800 μm) of the microchannel
due to its outlet (width: 90 μm) smaller than the beads. Rabbit
antibodies (α-E. coli O157:H7), E. coli O157:H7, alkaline phosphataseconjugated goat antibodies (α-E. coli O157:H7) and chromogenic
substrate were introduced into the michrochannel by flow in a
sequential manner. Since Protein A beads can be regenerated for
multiple use and detection can be instantaneously observed with
naked eyes, this microfluidic immunassay is useful for reusable
detection of various antigens in the fields which cannot carry
sophiscated detection equipments. Keywords: E. coli O157:H7, microfluidic device
국제학술대회
263
October 13-14, 2005, Seoul, Korea
I009
I011
Identification and Growth Activity to Bifidobacterium
spp. of Locust Bean Gum Hydrolysates by
Trichoderma harzianum β-mannanase
1
The Preparation of Crystalline β-1,4- Mannotriose
from Poonac Using the Enzyme System and Yeast
Fermentation
1
Gwi Gun PARK* and Yu Jin KIM *
Gwi Gun PARK * and Hideyuki KOBAYASHI
Department of Food and Bioengineering, Kyungwon University
*Corresponding author: ggpark@kyungwon.ac.kr
1
This study was performed to elucidate substrate specificity to the locust
bean gum galactomannan by Trichoderma harzianum β-mannanase.
The medium composition for enzyme production were determined 3%
cellulose, 3% corn steep liquor, 1% KH2PO4, 0.2% (NH4)2SO4, and
incubated for 115hr at 28℃. The β-mannanase exhibited maximum
activity at pH 4.5 and 60℃. Locust bean gum galactomannan was
hydrolyzed by the β-mannanase, and then hydrolysates separated by
activated carbon column chromatography. The main hydrolysates were
composed of D.P 4 and 7 galactosyl mannooligosaccharides by TLC.
For the elucidate the structure of D.P 4 and 7 oligosaccharides,
methylation analysis was performed. D.P 4 and 7 were identified as
M-M-M-M and M-MG-M-MG-M (G- and M-represent α-1,6-Dgalactosidic and β-1, 4-mannosidic linkages, respectively). To investigate
the effects of locust bean gum galactosyl mannooligosaccharides on the
in vitro growth of B. longum, B. bifidum, B. infantis, and B. breve,
Bifidobacterium spp. were cultivated individually on the modifiedMRS medium containing carbon source such as D.P 4 and 7 galactosyl
mannooligosaccharides, respectively. B. longum grew up 3.4-fold and
4.3-fold more effectively by the replacement of D.P 4 and 7 galactosyl
mannooligosaccharides as the carbon source in a comparasion of
standard MRS.
Keywords: Trichoderma harzianum, Beta-mannanase, Bifidobacterium
spp.
Department of Food and Bioengineering, Kyungwon University, 2Biological
Function Division, National Food Research Institute, Japan
*Corresponding author: ggpark@kyungwon.ac.kr
β-1,4-Mannotriose was prepared by the enzymatic hydrolysis of
poonac and the subsequent elimination of monosaccharides and
disaccharide from the resultant hydrolysate with a yeast. The enzyme
system hydrolyzed poonac and produced monosaccharides,
disaccharide and β-1,4-mannotriose without other oligomers at the
final stage of the reaction. Poonac (50 g) was hydrolyzed at 50℃ and
pH 6 for 48 hr with the crude enzyme solution (500 ml) from
Trichoderma harzianum. By the elimination of monosaccharides and
disaccharide from the hydrolysis products with a yeast (Candida
guilliermondii), 10.5 g of crystalline β-1,4-Mannotriose was
obtained without the use of chromatographic techniques. After 48
hours of yeast cultivation, the total sugar content fell from 4.8% to
3.4%, and the average degree of polymerization rose from 2.5 to 3.2.
Keywords: Beta-1,4-mannotriose, Yeast fermentation, Candida
guilliermondii
I010
Separation, Preparation and Structure Identification
of Copra Cake Hydrolysates by Xylogone sphaerospora
β-mannanase
Gwi Gun PARK* and So Hyun SHIN
Department of Food and Bioengineering, Kyungwon University
*Corresponding author: ggpark@kyungwon.ac.kr
For the elucidation of substrate specificity to the copra cake by
Xylogone sphaerospora β-mannanase, the enzymeatic hydrolysate
after 24 hr of reaction was heated in a boiling water bath for 10 min,
and then centrifuged to remove the insoluble materials from
hydrolysates. The major hydrolysates were D.P 4 and 6 galactosyl
mannooligosaccharides. For the separation of glactosyl mannooligosaccharides, the supernatant solution of 150ml was put on a first
activated carbon column. The column was then washed with 5 ℓ of
water to remove mannose and salts. The oligosaccharides in the
column were eluted by a liner gradient of 0~50% etahanol, at the flow
rate of 250ml per hour. The sugar composition in each fraction tubes
was examined by TLC and Bio-LC analysis. For the elucidate the
structure of D.P 4 and 6 galactosyl mannooligosaccharides, methylation
analysis performed. To investigate the effects of copra cake galactosyl
mannooligosaccharides on the in vitro growth of B. longum, B.
bifidum, B. infantis, and B. breve, Bifidobacterium spp. were
cultivated individually on the modified-MRS medium containing carbon
source such as D.P 4 and 6 galactosyl mannooligosaccharides,
respectively.
Keywords: Xylogone sphaerospora, Beta-mannanase, Identification
of oligosaccharide
264
한국미생물학회연합
2
I012
Degradation of the Lignocellulosic Biomass by
Brown-rot Fungus Fomitopsis palustris
1
1
2
Yee-Kyoung KIM , Jeong-Jun YOON , Chang-Jun CHA , Young1
1
Kyoon KIM , and Yeong-Suk KIM
1
2
Department of Forest Products, Kookmin University, Department of
Biotechnology, Chung-Ang University
*Corresponding author: kimyk@kookmin.ac.kr
Agricultural wastes and forest products contain a high level of
lignocellulose that can be converted to sources for energy by
microorganisms. Among these microorganisms, brown-rot fungi are
known to be effective in degradading lignocellulosic compounds and
expected to play an important role in utilizing of biomass for energy. Some
brown-rot fungi are different from other basidiomycetes in that they can
vigorously decompose wood cellulosic compounds without a need of
removing lignin. This study investigated how lignocellulosic substrates
such as wood substance by species, recycled paper, and corrugated
cardboard might be decomposed by brown-rot fungus Fomitopsis
palustris. We specially tried to characterize the major glycosylhydrolases
which were produced by the fungi in the cultures of test lignocellulosic
substrates. Furthermore, the crystallinity of the lignocellulosic materials
was measured by powder X-Ray Diffractometry to evaluate the degree of
crystallinity according to incubation times. The results showed that F.
palustris in the test of lignocellulosic substrates produced exoglucanase
along with endoglucanases, although this fungus has long been thought to
lack the cellulases that degrade crystalline cellulose. We also found that
this fungus could decompose vigorously both crystalline and amorphous
type cellulose in wood. This study suggests further research needs for
wood degradation by F. palustris when it is cultured in various kind of
lignocellulosic biomass.
Keywords: lignocellulosic biomass, degradation, brown-rot fungus,
Fomitopsis palustris
2005 International Meeting of the Federation of Korean Microbiological Societies
I013
I015
Enhanced Oral Salmonella Vaccine Delivery
Based on Polysaccharide Coated Microparticle
1
1
1
1
Hee Sam NA *, Hyun-Chul LEE , You-Jin LIM , Xiao-Ying JIN ,
Hyon-E CHOY1, Jong-Suk OH1, Sun-Sik CHUNG1, Chang-Moon
2
2
LEE , and Ki -Young LEE
1
Department of Microbiology and Immunology, Chonnam National University
Medical School, 2Faculty of Applied Chemical Engineering, Chonnam National
University
*Corresponding author: heesamy@naver.com
Microparticle delivery systems for oral vaccine offer a number of significant
advantages over systemic delivery and are receiving considerable attentions.
Polymeric delivery systems can be designed to enhance the efficacy of
mucosally administered vaccines in a number of ways; they can protect
antigens from degradation, concentrate them in one area of the mucosal
tissue for better absorption, extend their residence time in the body, or target
them to sites of antigen uptake (e.g., Peyer's patches in the gut). In this study,
GFP-expressing Salmonella typhimurium (ST)-loaded microparticles were
prepared with liposomes and then coated with O-palmitoylcurdlan (OPC),
O-palmitoylpullulan (OPP) chitosan, mannan or scleroglucan. The
synthesized microparticles and their delivery was evaluated by confocal and
fluorescent microscopy following oral administration. Delivery of STloaded microparticles to Peyer's patches or mesenteric lymph nodes was
significantly enhanced in OPC and chitosan-coated group. However, serum
IgA was significantly increased by ST-loaded scleroglucan or OPC coatedliposomes. Vaccination with ST-loaded OPC or scleroglucan coated
liposomes protected mice from wild type Salmonella typhimurium
challenge. These results indicate that modified polysaccharide (such as OPC
or scleroglucan) can be used as a potential adjuvant for effective oral
immunization.
Keywords: Salmonella, Microparticle, oral vaccine, polysaccharide,
liposome
Bacillus subtilis Proteases Facilitate Swarming
and Siderophore-Mediated Iron-Uptake via Proteolytic
Cleavage of Transferrin 1
1
1
1
2
R. Y. PARK , H. Y. SUN , M. H. CHOI , C. M. KIM , Y. H. BAI ,
and S. H. SHIN1*
1
Research Center for Resistant Cells, Chosun University Medical School,
Department of Biology, Chosun University Medical School
*Corresponding author: shsin@chosun.ac.kr
2
We isolated a highly serine metalloprotease-producing Bacillus
subtilis strain named PRY, which was identified by biochemical
characteristics and ribosomal DNA sequencing. The PRY strain
exhibited a robust swarming behavior and was able to efficiently
digest human transferrin concomitantly with the production of
catechol-siderophore in the exponential growth phase. The growth of
PRY was in proportion to increased iron availability resulting from
transferrin destruction. These findings indicate that B. subtilis
proteases can play an important role in the pathogenesis of human
infections by facilitating siderophore-mediated iron-uptake from
transferrin and swarming motility [R-13-2003-009]. Keywords: Bacillus subtilis, Protease, Transferrin, Iron, Swarming
I014
I016
Cloning, Expression and Characterization of
Dextranase Gene (lsd11) of Lipomyces starkeyi
Using Pichia pastoris
1
2
3
Ji-Young PARK , Hee-Kyoung KANG , Xing-Ji JIN , Joon-Seob
4
5
1
6
AHN , Seung-Hee NAM , Young-Hwan MOON , Do-Won KIM ,
7
8,9,10
Seung-Heuk KIM , and Doman KIM
*
1
Department of Material Chemical and Biochemical Engineering, Chonnam
National University, 2Engineering Research Institute, Chonnam National
3
University, Department of Fine Chemical Engineering, Chonnam National
4
University, Department of Molecular Biotechnology, Chonnam National
University, 5School of Biological Sciences and Technology, Chonnam
6
7
National University, Kangnung National University, Lifenza Co. Ldt.,
8
School of Biological Sciences and Technology, and The Research Institute for
Catalysis, Chonnam National University, 9Biology Research Center for
10
Industrial Accelerator, Dongshin University, Institute of Bioindustrial
Technology, Chonnam National University
*Corresponding author: dmkim@chonnam.ac.kr
A lsd1 gene encoding an extracellular dextranase was isolated from the
genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase
(lsd1) after codon optimization for gene expression with Pichia pastoris
system. It was directly cloned from Taq polymerase-amplifide PCR by
using TA cloning methods. A open reading frame of lsd11 gene was 1,827
bp and it was inserted into the pPIC3.5K expression vector. The lsd11
gene fragment encoding a mature protein of 608 amino acids was
expressed in the methylotrophic yeast P. pastoris by controling the
alcohol oxidase-1 (AOX1) promoter. The recombinant lsd11 was
optimized by using the shake-flask expression and upscaled using
fermentation technology. Over 8 μg/ml of active dextranase was obtained
after induction by methanol.
Keywords: dextranase, Lipomyces starkeyi, Pichia pastoris, expression,
fermentation
Roles of Amino-acids in Dextransucrase Activity
from Leuconostoc mesenteroides DSRN
Seung-Hee NAM1, Jin-Ha LEE2, Hee-Kyoung KANG3, MyYoung SEO4, Ji-Young PARK4, Do-won KIM5, Suk-Sang CHANG6,
and Doman KIM3,7,8*
1
School of Biological Sciences and Technology, Chonnam National University,
3
Engineering Research Institute, Chonnam National University, School of
Biological Sciences and Technology and Research Institute for Catalysis,
Chonnam National University, 4Department of Materials and Biochemical
5
Engineering, Chonnam National University, Kangnung National University,
6
7
Kangnung, Pohang Accelerator Laboratory, Pohang University, Institute of
Bioindustrial Technology, Chonnam National University, 8Biology Research
Center for Industrial Accelerators, Dongshin University
*Corresponding author: dmkim@chonnam.ac.kr
2
In addition to catalyzing the dextran synthesis from sucrose as a primary reaction,
dextransucrase catalyzes the transfer of glucose from sucrose to the other
carbohydrates like maltose as a secondary reaction. We obtained new
dextransucrase gene (DSRN) favoring secondary reaction mostly after exposure
of Ultrasoft X-rays to Leuconostoc mesenteroides B-742CB. Seven nucleotides
of the parent gene (DSR742) were changed in the nucleotide sequence that resulted
in a 30 amino acid deletion in the N-terminus. Four nucleotides were changed in
the open reading frame (ORF) including S196F, N346Y, T395L, and T980P. In
order to identify four amino acids function related to high oligosaccharide
synthesis, single amino acid was independently recovered to DSR742 and cloned
to express the enzyme in BL21DE3 harboring pET28a(+) expression vector.
Properties of DSRN mutants with DSRB- 742CB dextransucrase were compared
by the acceptor reaction, synthesis and hydrolysis of dextran. Recovery from 346
or 980 positions resulted in completely suppressed oligosaccharide and dextran
synthesis compared to DSRN. This result indicates that these sites are considered
as essential for catalysis process.
Keywords: Dextransucrase, Mutation, Acceptor Reactions, Leuconostoc
mesenteroides
국제학술대회
265
October 13-14, 2005, Seoul, Korea
I017
I019
Expression of Protease, Actinidin, of Wild Kiwi
Fruit from Bacillus subtilis
Development of β-Lactamase Fragment Complementation (BFC) System for Protein-protein Interaction
Nam Keun LEE, Mi Seon JANG, and Young Tae HAHM*
Jong-Hwa PARK, Jung Ho BACK, and Ye Sun HAN*
Department of Biotechnology, Chung Ang University
*Corresponding author: ythahm@cau.ac.kr
Department of Advanced Technology Fusion, Konkuk University
*Corresponding author: yshan@konkuk.ac.kr
Actinidin (EC 3.4.22.14), cysteine protease, is one of the proteases
existed in kiwi fruit and belongs to enzyme of the papain family.
Actinidin gene from wild kiwi fruit was isolated, characterized and
expressed in Escherichia coli and Bacillus subtilis. Actinidin
expressed in E. coli was, however, detected as inclusion body and its
enzyme activity was very low level. To optimize the production of
active actinidin, Bacillus secretion systems with pWB980 contained
P43, a constitutively expressed promoter, and B. subtilis WB600,
extracellular proteases deficient strain, as host cell has been used.
Keywords: actinidin, expression, Bacillus subtilis
The applicability of TEM-1 β-lactamase fragment complementation
(BFC) system was investigated to develop a strategy for the screening of
protein-protein interaction in microorganisms. Bait and prey plasmid of
BFC system were separately constructed by the subcloning of a
C-terminal (residues 198~290) and an N-terminal fragment (residues
24~196) of β-lactamase containing a connecting linker to plasmids
containing different antibiotic resistance genes (kanamycin and
chloramphenicol). To test β-lactamase complementation induced by
these two plasimds, human Fas-associated death domain (hFADD) and
hFas death domain (hFasDD), which have been known to interact each
other, were cloned to bait and prey plasmid by an N-terminal fusion to a
C-terminal fragment and a C-terminal fusion to an N-terminal fragment of
β-lactamase, respectively. Co- transformant of bait and prey plasmid
containing hFADD and hFasDD showed an ampicillin resistant
phenotype. β-lactamase activity produced by the complementation could
be observed by the color change of nitrocefin, which changes from yellow
to red when hydrolyzed by b-lactamase. In this study, we confirmed that
BFC system had a possibility to screen proteins interacting with
interesting proteins in microorganisms. We are currently trying to screen
proteins interacting with human DNA glycosylases from the cDNA
library, which is constructed from DNA damaging induced cells.
Keywords: β-lactamase fragment complementation, hFADD, hFasDD,
nitrocefin, DNA glycosylases
I018
I020
Effects of Bacillus licheniformis Administration
on Fecal Microflora and Putrefactive Metabolites
in Healthy Adults
1
1
2
3
Jin Wook KIM *, Hee Hyun LEE , Kyoung Dong JUN , Hyun JOO ,
1
and Jae Hwa LEE
1
2
Department of Bioscience and Biotechnology, Silla University, Research and
3
Development Center, Binex Co. Ltd., Department of Physiology, College of
Medicine, Inje University
*Corresponding author: kimjk8@lycos.co.kr
Bacillus licheniformis is one of the lactic ferments isolated from
kochujang and soybean paste. It's useful material for improvement of
fecal microflora. To investigate the effect of Bacillus licheniformis
administration on fecal microflora and putrefactive metabolites in
human. Bacillus licheniformis was administrated to twenty-three
healthy subjects (15 men, 8 women) three times a day for 2 weeks. Fecal
samples were collected before (1st and 2nd weeks, control), during (3rd
and 4th weeks), and 2weeks after the administration. The following
microbial groups were evaluated in the feces : aerobic and anaerobic
bacteria, Bacillus licheniformis, Yeast, Staphylococcus, Pseudomonas,
Coliform, Lactobacillus, total lactic acid bacteria, Salmonella,
Bifidobacterium, and Clostridium perfringens. Fecal concentrations of
Bacillus licheniformis, Lactobacillus, total lactic acid bacteria were
increased in all subjects, compared to the control, from the 3rd week after
the administration of the probiotic. Total anaerobic bacteria, Salmonella
were significant reduced from the 3rd week of administration. Despite
the absence of a statistical significance, the putrefactive metabolites
(ammonia, indole, skatole, and ρ-cresol) tended to be lower during and
after the test periods than the base line. These results show that this
probiotic preparation is able to colonize the intestine, and suggest that
it may be useful as a beneficial probiotic in humans.
Keywords: Bacillus licheniformis, probiotics, fecal microflora,
putrefactive metabolites
266
한국미생물학회연합
NAD-Dependent Enzyme Electrodes for Biosensor
Application
1
2
Sei-Eok YUN *, Abhay VAZE , and James RUSLING
2
1
Department of Food Science and Technology, Chonbuk National University
Department of Chemistry, University of Connecticut, USA
*Corresponding author: seyun@chonbuk.ac.kr
2
The vast majority of redox enzymes are NAD(P)/NAD(P)Hdependent biocatalysts, and their electrical contacting with the
electrode reveals fundamental difficulties that prevent their broad use
in biosensor applications. Electrical contact of redox enzymes with
electrode surfaces is a key process in making of enzyme electrodes for
bioelectronics and biosensor applications. It was aimed to assemble
an integrated, electrically-contacted, NAD-dependent enzyme electrodes,
since the organization of electrocatalyst-NAD(P)-enzyme assemblies
in nondiffusional, electrically-wired configurations is essential.
Carbon cloth is covalently coupled by 5,5'-dithiobis (2-nitrobenzoic
acid) or pyrroloquinoline quinone, followed by covalent linkage
of NAD to the electrocatalyst. Affinity binding of NAD-dependent
enzymes to the electrocatalyst-NAD-modified carbon cloth electrode
is done by immesing the modified electrode in the enzyme solution
for a while, followed by cross-linking of the enzyme. This system was
proved to work in the Tris buffer solution to which substrate was added,
requiring further study to increase the response. Keywords: NAD-dependent enzyme, biosensor, electrocatalystNAD-enzyme assembly, modified carbon cloth electrode
2005 International Meeting of the Federation of Korean Microbiological Societies
I021
I023
A Bacillus licheniformis Isolate Producing a
Thermostable Mannanase and Its Molecular
Breeding
1
1
2
Hyun-Hee LEE , Hee-Su YANG , Do-Young YUM , Han-Sick
KAWK3, Jung-Kee LEE1, and Seung-Hwan PARK1*
1
2
Laboratory of Microbial Genomics, KRIBB, R&D Center, inBioNET Corp.,
Department of Life Science and Genetic Engineering, Paichai University
*Corresponding author: soohee0506@hotmail.com
3
We screened thermophilic bacteria having mannanase activity from soil.
An isolate HS04 which was identified as Bacillus licheniformis was
selected due to greater activity of mannanase production. The mannanase
produced by strain HS04 was stable at 60°C. The amino acid sequence
deduced from the cloned gene was matched with that of B. licheniformis
DSM13. However, the mannanse activity of strain HS04 was higher than
that of strain DSM13. To improve the productivity of mannanase by
strain HS04, an expression vector that consisted of the mannanse gene
of strain HS04 following cry3 promoter and mRNA stabilizer was
constructed. The vector was transformed into strain HS04. The
productivity of mannanase from the recombinant strain was increased
as 3 folds than that of strain HS04. We also carried out mini-Tn10
mutagenesis of strain HS04 resulting in obtaining over 6,000 mutants.
Among mutants, we selected two mutants that showed significant
improvement on production of mannanase. The mini-Tn10 insertional
sites on the genome of HS04 were sequenced as sacB and ykvZ . The
function of sacB and ykvZ was confirmed by single cross-over deletion
mutation. In addition, multi-copy expression vector containing sacB or
ykvZ with each indigenous promoter was transformed into mini-Tn10
mutants. Collectively, we demonstrated that mannanase production of
B. licheniformis strain HS04 was improved by molecular breeding.
Keywords: mannanase, Bacillus licheniformis, sacB, ykvZ
I022
Agrobacterium tumefaciens-Mediated Transformation
of Monascus ruber
1
1
2
Yun Jung YANG *, Sung-Yoon HONG , Hee-Sool RHO ,
Yong-Hwan LEE2, and Inhyung LEE1
1
2
Food and Life Sciences Major, Kookmin University, School of Agricultural
Biotechnology, Seoul National University
*Corresponding author: yj-ideal@nate.com
Agrobacterium tumefaciens-mediated transformation (ATMT) was
successfully applied to the ascomycete fungus Monascus ruber
using the binary vector pBHt2 containing the hygromycin B
phosphotransferase gene (hph gene) as a selection marker. The pretreatments of Agrobacterium tumefaciens AGL-1 cells containing
pBHt2 during induction with acetosyringone (AS) produced more
transformants than without AS, however, there was no significant
difference. In contrast, the presence of AS in co- cultivation medium
(CM) was imperative for transformation. The optimum co-cultivation
time was 84 hr with the efficiency of 600 to 1,000 transformants
6
when 1×10 spores of M. ruber were used. The stability of transformants
was over 95% after several generations in the presence of
hygromycin B (100㎍/㎖). The presence of the hph gene was
confirmed by PCR and Southern blot analysis of randomly selected
transformants. The development of transformation system by ATMT
will enable us to study M. ruber genetically for understanding of
biosynthesis of secondary metabolites.
Keywords: Agrobacterium tumefaciens-mediated transformation,
Monascus ruber, hygromycin B
I024
Characterization of dUTPase from the Hyperthermophilic
Archaeon Thermococcus sp. NA1
Vibrio vulnificus Biofilm Formation : Identification
of Genes that Code for Biofilm Phenotypes
Yona CHO, Hyun Sook LEE, Yun Jae KIM, Jae kyu LIM, Sung
Gyun KANG, and Jung-Hyun LEE*
Hyun-Ju KIM, Kyu-Yong YEON, Yoon-Ju KIM, Jin-Young DOH,
Young-Jun YOON, and Jung-Wan KIM*
KORDI
*Corresponding author: yona_cho@yahoo.com
Department of Biology, University of Incheon
*Corresponding author: kjw5864@incheon.ac.kr
As a part of genomic research of the Thermococcus sp. NA1 (TNA1),
the hyperthermophilic archaea collected from a deep-sea
hydrothermal vent area, a gene whose deduced amino acid sequence
had high similarity to the gene encoding the dUTPase from
Pyrococcus woesei and P. furiosus (83% identity and 93% similarity)
was revealed. The dUTPase encoding gene comprises open reading
frame of 468 bp with valine at the N-terminus, corresponding to
polypeptides of 156 amino acids with predicted molecular mass of
17,679 Da and pI of 8.1. To characterize the dUTPase, the gene was
cloned and overexpressed in Escherichia coli Rosetta(DE3)pLysS
with the pET-24a(+) vector system. The His6-tagged TNA1 dUTPase
was purified on Co2+-IMAC-Sepharose, dialyzed, and the enzyme
activity was investigated. TNA1 dUTPase was capable of converting
dUTP to dUMP and inorganic phosphate and enhanced the PCR in a
broader range of target lengths in combination with TNA1 DNA
polymerase. TNA1 dUTPase also increased the yield of products
amplified with other archaeal DNA polymerases.
Keywords: Thermococcus sp. NA1, dUTPase
Biofilm formation of Vibrio vulnificus V1 strain on abiotic surfaces
has been shown to be affected by phosphate concentration and carbon
source, especially being repressed by glucose. In this study, the genes
involved in biofilm formation of the bacteria were identified using
mini-Tn5 mutagenesis via conjugation. Although the conjugation
efficiency was pretty low and inconsistent, more than one thousand
mutants were screened for their capability to form biofilm on PVC and
borosilicate glass and several mutants were selected for identifcation
of the genes that were likely to be related to the phenotype.
Sequencing analysis of the DNA fragments amplified by arbitraryPCR indicated that mutants M17 and M41 forming much less biofilm
than wild-type were mutated at the phosphoribosylaminoimidazole
succinocarboxamide synthase gene and the N-acetylglutamate
synthase gene, respectively. Mutnat M20 that formed much more
biofilm than wild-type had the Tn5 inserted at the locus encoding a
homologue of cqsA or aminotransferase. Other mutants had insertions
at loci that code for hypothetical proteins. Pleiotrophic effects of these
mutations are under investigation in the laboratory. (This study was
supported by KRF)
Keywords: Vibrio vulnificus, biofilm, mutagenesis
국제학술대회
267
October 13-14, 2005, Seoul, Korea
I025
I027
Enzymatic Reactions in Bicontinuous microemulsion
System 1
1
2
1
1
2
Sathishkumar M. , Eun-Seon JEONG , Sung-Phil MUN , and SeiEok YUN1*
Jong-Deog KIM , Jong-Kwon LIM , Min-Yong KIM , Tai-Sun
SHIN3, Hyo-Jin SEO1, Eun-Ok KIM1, and Se-Young LEE1
1
Department of Biotechnology, Yosu National University, Department of
Refigeration Engineering, Yosu National University, 3Department of Food
Science and Nutrition, Yosu National University
*Corresponding author: pasteur@yosu.ac.kr
Department of Food Science and Technology,Chonbuk National University,
Department of Wood Science and Technology,Chonbuk National University
*Corresponding author: seyun@chonbuk.ac.kr
2
There is a growing interest in using enzymes in the presence of varying
concentrations of nonaquous media for bioorganic reactions. The
problem encountered in these reactions are too low yields since only a
small amount of both enzyme and hydrophilic substrate can dissolve in
the systems. Taking into account of such disadvantages in using
solid-liquid systems, biphasic systems, and W/O microemulsion systems,
bicontinuous microemulsion system was employed as a reaction
medium. This type of microemulsion does not contain droplets but
features continuous oil and water phases interwined in dynamic
extended networks which is used only in electrocatalytic reactions. As
a model reaction, β-glucosidase-catalyzed synthesis of n-hexyl-β-Dglucoside through the condensation of glucose and n-hexanol was
investigated. Kinetic study for the β-glucosidase catalyzed hydrolysis of
n-hexyl-β-D-glucoside was also performed. Maximum synthesis of 4%
n-hexyl-β-D-glucoside was achieved at around 24 hours. However,
n-hexyl-β-D-glucoside was hydrolysed up to 70 % at 5-hour reaction,
suggesting that bicontinuous microemulsion system is more applicable
to the enzymatic hydrolysis.
Keywords: Bicontinuous microemulsion, Enzymatic reaction, Nonaquous
medium, n-hexyl-β-D-glucoside
I026
1
2
Angiogenesis is a process of forming new vasculogenesis from blood
vessels, in the case of cancer, the new vessels allow tumor cells to escape
into the circulation and lodge in other organs like tumor metastases. For
inhibition of angiogenesis caused cancer, five different kinds of named λ
-bacteria bearing higher antioxidative capacity were isolated from the
intestines of fusiform fish, and the supernatant of fermented from λ28bacterium was performed by salting out, dialyzing and freeze drying. And
this protein-like material from λ-28 bacterium(PLM-28) was executed size
exclusion chromatography with a fraction collector. PLM-28 was identified
with MW 45.0 KD from SDS-PAGE. PLM-28 from fraction number 74 was
exhibited higher anti-angiogenic effect as 82.8%, 65.9%, 30.2%, and 22.3%
at the concentration of 18.5ug/mL, 7.4 ug/mL, 3.7 ug/mL and 0.74 ug/mL,
respectively, this fact finally would be based on blocking the pathway from
VEGFR2, VE-cadherin, PI3K and beta-catenin to NF-κB by PLM-28 from
western blot. PLM-28 also suppressed activated U937 monocytic cells
adhesion to HUVECs, and non-activated U937 to IL-1β activated
HUVECs, and the adhesion of U937 cells to IL-1 β-stimulated HUVECs
was predominantly decreased when both cell types were pretreated with
PLM-28. New and plenty of anti- ngiogenic materials exhibited higher
antioxidative capacity and non-toxicity can be expected from these results.
Keywords: Angiogenesis, Antioxidative capacity, Fusiform fish, PLM-28,
VEGFR2, VE-cadherin, PI3K, beta-catenin
I028
Preparation of Enzymatic Bioreactor in Mesoporous
Media
1
2
1
Jin Hyung LEE , Jungbae KIM , Kyoung-Woong KIM , Chul
1
3
Han SONG , and Man Bock GU *
1
2
Department Environmental Science and Engineering, GIST, Pacific
3
Northwestern National Laboratory(PNNL), USA, College of Life Science and
Biotechnology, Korea University
*Corresponding author:mbgu@korea.ac.kr
Enzymatic reactor was constructed in mesocellular mesoporous silica particles by
immobilizing lipase in the media.Mesocellular Mesoporous Silica (MMS) are
small particles (200-500nm) with many meso-size pores having 37nm diameter
and connected by mesoporous channels (13nm). Enzyme was immobilized in
meso-size pores by 1) only adsorption and 2) adsorption followed by
cross-linkage using 0.5% glutaraldehyde (GA) treatment to construct nanoscale
enzyme reactors and their efficiencies were compared through the measurement
of activity and stability. The GA treated reactor showed higher activity than
GA-untreated one and there is no significant difference in the amount of enzyme
loading with or without GA treatment. In our experiment, GA treated reactor
showed 1.6 times hi