NC A SM 2011
North Carolina American Society for Microbiology
2 0 11 M e e t i n g
8 October 2011
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Schedule
Preliminaries
8:00
Registration
Poster and talk set-up
Coffee break
Award Committees meeting/organization
9:00
Seán O’Connell
Welcome & Introductory comments
Session 1: Seán O’Connell, Chair
9:15
9:30
9:45
10:00
10:15
Pauline Chugh
Profiling of host microRNAs following West Nile Virus infection
Allie Amick
"A Change Is Gonna Come" – HEGs and the dynamic genome of F1
mycobacteriophages
Indi Bose
Marty Roop
An RNA interference screen for virulence factors in Cryptococcus
neoformans
Identification and characterization of two small regulatory RNAs
linked to virulence in Brucella abortus 2308
Poster session I (Even numbered posters should be attended)
Coffee break
Vendors & Exhibitors
Session: 2: Betsy Wilson, Chair
11:15
David Martinson
Iron-dependent degradation controls the activity of the transcriptional regulator Irr in Brucella abortus
11:30
Neetu Kumra Taneja
Dra, a Bordetella locus homologus to the dlt loci of Gram-positive
bacteria promotes incorporation of D-alanine, resistance to antimicrobial peptides and adherence to respiratory cell lines.
11:45
Joshua Sailsbery
Fundamental characteristics and discerning sites of the eukaryotic
bHLH domain
12:00
Lunch
1:00
Poster session 2 (Odd numbered posters should be attended)
Vendors & Exhibitors
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Schedule
Session 3: Ece Karatan, Chair
What is the Serpentinite Microbiome?: Recent studies of the genomics, physiology, and ecology of microbial biofilms from deep
within the Earth
2:00
Matt Schrenk,
NC Invitational Talk
2:30
Coffee break
Vendors & Exhibitors
Awards committee meetings
Plenary session: Seán O’Connell, Chair
3:15
Jade Wang,
ASM Branch Lecture
Prevention of conflict between replication and transcription
Postscript
4:15
Seán O’Connell
Concluding remarks
Awards
4:30
Jim Brown
Business meeting
Officer election
5:00
Adjournment
Meeting Sponsors
The American Society
for Microbiology
Association of
Southeastern Biologists
(Tim Atkinson &
Marilyn Pendley)
NC Academy
of Science
(Melanie Lee-Brown)
Martin Microscope
(Terry Knorr)
Nikon
(Larry Kordon)
NC Biotechnology
Center (Jon Lawrie)
Life Technologies
(Charles Cochran)
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Abstracts (talks)
1.1) "A Change Is Gonna Come" – HEGs and the dynamic genome of F1 mycobacteriophages
K. Allie Amick, Hannah M. Berry, Iara P. Calil, Morgan E. Carter, Joshua R. Chappell, Alicia M. Hilliard,
Hannah Huntley, Anna K. Knight, William H. Kohlway IV, Nicholas A. Snow, Meredith L. Wojcik, Christine A. Zabel, Susan D. B. Carson, Eric S. Mayer, and Eric S. Miller
Department of Microbiology, NCSU
North Carolina State University is a participating institution of the HHMI Science Education Alliance, a
program that supports undergraduate research through first year Phage Hunters and Phage Genomics
courses. In the fall of 2010, NCSU students isolated 16 different bacteriophages that infect the soil bacterium Mycobacterium smegmatis, a non-pathogenic mycobacterial species often used in place of
strains that cause tuberculosis or leprosy. Of these isolated phages, one was chosen for further investigation and sequencing. Mutaforma13, the selected phage, is classified as a member of the F1 subcluster. While many mycobacteriophages within a given subcluster share sequence homology across their
entire genome, phages within the F1 subcluster show sequence homogeneity in the 5’ half of the genome, but a high degree of sequence heterogeneity in the 3’ half. The functions encoded by genes in
the conserved region are predominately for the virion structure and DNA packaging, whereas the diverse, non-conserved region contains both unique and conserved hypothetical genes as well as genes
that are typically found in bacterial genomes, not in phage genomes. Notably, the 3’ half of many F1
phage genomes contain putative homing endonuclease genes (HEGs). Mutaforma13 contains 5 putative
HEGs. Phage HEG products are able to cleave dsDNA and direct the transfer of (“mobilize”) genes from
one genome into another. We hypothesize that HEGs occur outside of the conserved genome region
where they are less likely to cause alterations to core phage genes. Rather, they predominate in the
variable region of the genome where their self-directed insertion transfers new genes that are of potential benefit to phage propagation. That is, HEGs are part and parcel / cause and effect of the dynamic, heterogeneous genome region of F1 subcluster phages.
1.2) Profiling of host microRNAs following West Nile Virus infection
Pauline E. Chugh and Dirk Dittmer
Department of Microbiology and Immunology, UNC - Chapel Hill
The innate immune response to West Nile virus (WNV) infection involves recognition of the virus through a series of RNA sensors, including TLR3 and RIG-I, leading to the secretion of cytokines and
establishment of an antiviral state. Since microRNAs (miRNAs) have been shown to play a role in viral
pathogenesis and in the immune response to infection, we investigated changes in the cellular miNA
profile upon WNV infection in several different cell types. We also performed microRNA profiling in
cells lacking the double stranded dsRNA sensor TLR3 to determine what effect TLR3 played in the microRNA response to WNV infection. Interestingly, we found that the majority of cellular miRNAs were
not influenced by the presence of TLR3 in the cell types studied suggesting that this sensor may not
play an integral role in the microRNA response to WNV infection. However, we found that WNV induced cell-type-specific miR profiles that seemed to correlate with the cell’s response to infection.
Those cell types that displayed productive infection with WNV responded with induction of different
miRNAs than those associated with solely an inflammatory cytokine response to the virus. Taken together, our in-depth, large-scale miRNA profiling combined both sequencing and Taqman-based miRNA
profiling and revealed that WNV infection induces different miRNAs in a cell-type-specific manner dependent on the cell’s response to infection. Further studies may reveal microRNAs with potential for
inhibiting WNV pathogenesis.
1.3) An RNA interference screen for virulence factors in Cryptococcus neoformans
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Indrani Bose and Tamara Doering
Department of Biology, WCU & Department of Molecular Microbiology at Washington University Medical
School
Cryptococcus neoformans is a basidiomycetous yeast found ubiquitously in nature. It is one of
a handful of fungi that can proliferate and cause systemic infections in mammalian hosts. It is an opportunistic pathogen that is the causative agent of cryptococcosis, sometimes leading to a fatal meningoencephalitis in immunocompromised patients. The ability to grow at the high body temperature of
warm-blooded hosts is critical for its ability to cause disease, and the genes responsible necessary for
its virulence. To identify genes required for growth at high temperature, we devised an RNA interference (RNAi) screen to silence genes at random. We have created an RNAi library of genomic DNA inserts, approximately 2Kb in size. These inserts are cloned in a telomeric, ADE2 marked vector, in between two GAL7 promoters present in opposite orientation. In the presence of galactose, each insert is
transcribed from the two promoters leading to the formation of double-stranded RNA (dsRNA) in the
cell. This activates the RNAi pathway, silencing genes corresponding in sequence to that of the cloned
insert. We have tested the vector for activation of the RNAi pathway by silencing known genes with
well-studied phenotypes, such as the LAC1 and URA5 genes. Silencing of the LAC1 gene renders the
strain unable to produce melanin, while silencing of the URA5 gene allows the cells to grow on 5-FOA.
Once the method was validated, this strategy was used for forward genetics to identify genes whose
function is required for viability at 37˚C. The screen has identified essential genes such as RHO1 (encoding a small GTPase) and LPC1 (required for sphingolipid biosynthesis), and non-essential genes such
as CHS5 (a chitin synthase gene) and others. This is a fast and easy way to study the phenotypes of a
wide range of genes without altering the genomic make-up of the cells.
1.4) Identification and characterization of two small regulatory RNAs linked to virulence in Brucella
abortus 2308
Clayton C. Caswell, Jennifer M. Gaines, Pawel Ciborowski, Derek Smith, Christoph Borchers, Christelle
Roux, Paul Dunman and R. Martin Roop II
Department of Microbiology and Immunology, Brody School of Medicine, ECU
Hfq is an RNA-binding protein that functions in post-transcriptional gene regulation by mediating interactions between messenger RNAs and small, regulatory RNAs (sRNA). Mutation of hfq in B. abortus 2308
leads to reduced survival and replication in macrophages and attenuation in animal models of infection. While the role of Hfq in the wild-type virulence of B. abortus 2308 has been firmly established,
the identities of the sRNAs working with Hfq to regulate gene expression have remained elusive. Recently, two proteins were shown to be highly over-produced in the hfq mutant compared to both the
parent strain, and these proteins were identified as the products of the genes designated BAB2_0612
and BAB1_1794. Orthologs of BAB1_1794 and BAB2_0612 in Agrobacterium tumefaciens are regulated
by two sRNAs, called AbcR1 and AbcR2. Because Brucella and Agrobacterium are close phylogenetic
relatives, we hypothesized that these sRNAs could also be present in B. abortus and, moreover, that
they might regulate the expression of BAB1_1794 and BAB2_0612. Northern blot analyses confirmed
that sRNAs homologous to the A. tumefaciens AbcR1 and AbcR2 are expressed by B. abortus 2308, and
these sRNAs were similarly named AbcR1 and AbcR2 in B. abortus 2038. The abcR1 and abcR2 single
mutant strains do not demonstrate any deficiency in their ability to survive in murine macrophages, but
the abcR1 abcR2 double mutant exhibits significant attenuation in macrophages when compared to the
parental strain 2308. Additionally, the abcR1 abcR2 double mutant is significantly attenuated in a
mouse model of infection compared to strain 2308. Recent experiments using quantitative proteomics
and microarray analysis have determined that the AbcR sRNAs are regulating genes putatively involved
in amino acid transport and metabolism, and ongoing work in our laboratory is aimed at elucidating the
molecular mechanisms of AbcR-mediated gene regulation.
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2.1) Iron-dependent degradation controls the activity of the transcriptional regulator Irr in Brucella
abortus
David Allen Martinson and Marty Roop
Department of Microbiology and Immunology, Brody School of Medicine, ECU
Members of the genus Brucella belong to the alpha-proteobacteria group of Gram negative bacteria. As an intracellular pathogen, B. abortus must overcome iron sequestration in the host cell by
utilizing highly efficient iron transport systems. These systems must be regulated, however, as excess
intracellular iron is toxic to the bacterial cells. Most of the α–proteobacteria rely on a transcriptional
regulator known as the iron response regulator (Irr) to control the expression of their iron metabolism
genes. Irr serves as an activator of genes involved in iron acquisition and represses the transcription of
genes encoding products that require a lot of iron for their function (e.g. heme biosynthesis genes).
Two mechanisms for controlling Irr activity in response to cellular iron levels have been described in
the α–proteobacteria. In Bradyrhizobium japonicum, Irr is degraded in response to intracellular iron
levels, and this degradation is dependent upon a direct interaction between the heme biosynthesis enzyme ferrochelatase and an N-terminal heme regulatory motif (HRM) on Irr. When intracellular iron
levels are high, Irr is degraded and can no longer function as a transcriptional regulator. In contrast,
the Rhizobium leguminosarum Irr lacks the N-terminal HRM. When cellular iron levels exceed a certain threshold, heme binds to the R. leguminosarum Irr and prevents its binding to DNA, but the Irr
protein is not degraded. Like its R. leguminosarum counterpart, the Brucella Irr lacks the N-terminal
HRM, but studies in our laboratory have shown that this protein is degraded in a dose-responsive manner in response to increasing intracellular levels of iron in B. abortus 2308. We are presently exploring
the mechanism behind the HRM-independent, iron-responsive degradation of Irr in this strain in an effort to better understand how this regulator coordinates the expression of its iron metabolism genes.
2.2) Dra, a Bordetella locus homologus to the dlt loci of Gram-positive bacteria promotes incorporation
of D-alanine, resistance to anti-microbial peptides and adherence to respiratory cell lines
Neetu Kumra Taneja and Rajendar Deora
Department of Microbiology and Immunology, Wake Forest School of Medicine
The Gram positive dlt loci encode proteins that alanylate teichoic acids resulting in resistance
to host immune components. Recently, the dlt operon was detected in Gram-negative bacterial genera,
including the three classical Bordetella species. Bordetella colonize the mammalian respiratory tracts
and cause a variety of respiratory diseases in humans, animal and birds. Nothing is known about the
functional role of the putative dlt loci in Bordetella or in any other Gram-negative bacteria. We show
that the dlt locus is present in laboratory and clinical strains of B. pertussis and B. bronchiseptica and
is transcribed as an operon. While there were no differences in the growth rates between the WT and
the ∆dlt strains, the mutant strain of B. bronchiseptica appeared longer than the WT strain, when visualized under phase contrast microscope. Examination of the autolysis rates in the presence of TritonX100 showed that the rate of autolysis was more rapid for the mutant than the WT strain. The ∆dlt mutant had a statistically significant higher negative surface charge and displayed enhanced killing by
CAMPs such as polymyxin B and LL-37. The ∆dlt mutant was also defective in attachment to human lung
and laryngeal epithelial cell lines A549 and Hep2. Finally, total membrane preparations of the WT
strain had 2.4 fold higher content of 14C D-alanine compared to the ∆dlt mutant. These studies
strongly suggest that the Bordetella Dra locus is involved in the incorporation of D-alanine, imparting
resistance from CAMPs and in promoting adhesion.
2.3) Fundamental characteristics and discerning sites of the eukaryotic bHLH domain
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Joshua K. Sailsbery, William Atchley, and Ralph A. Dean
Department of Genetics, NCSU
The highly conserved bHLH domain, found in many transcription factors, has been well characterized separately in Plants, Animals, and Fungi. Accordingly, we investigated the fundamental architecture of the bHLH domain across all Eukarya. We identified five essential amino acid sites that are
highly characteristic of each Kingdom, and developed classification methods that accurately determined the origin of a bHLH sequence. Expertly aligned bHLH domains were used to generate Hidden
Markov Models (HMM), which were also highly accurate for identifying bHLH domains. The HMMs and
classification methods were tested against a well known environmental sample to determine the Kingdom of previously unknown bHLH domains. Last, we have created an online tool that can align, extract, and classify bHLH sequences.
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Matt Schrenk is an assistant professor in the Department of Biology at East Carolina University and an adjunct in the Department of Geology. He earned his B.Sc. in Geology at the University of Wisconsin-Madison and M.Sc. and Ph.D in the School of
Oceanography at the University of Washington. Matt completed
a post-doc at Carnegie Institution of Washington in the Department of Terrestrial Magnetism and Geophysical Laboratory. His
work is broadly defined as ecological and he brings a unique perspective to environmental microbiology. His research takes him
to exotic and extreme ecosystems across the globe and he is
keenly interested in questions related to life elsewhere in the
universe. He studies the feedbacks between microorganisms and
their environments using molecular, microscopic, and chemical
analyses. In particularly, he is interested in microbial growth on
and within rocks in polymer-encased structures known as
biofilms. His work is focused upon biofilms in a number of extreme habitats such as highly acidic environments associated
with acid mine drainage, high pH environments associated with
old oceanic crust, and high temperature, high pressure environments associated with deep-sea hydrothermal vents.
What is the Serpentinite Microbiome?: Recent studies of the genomics, physiology, and ecology of microbial biofilms from deep within the Earth
Matthew O. Schrenk
Department of Biology, ECU
Microbial habitats hosted in ultramafic rocks constitute substantial, globally-distributed portions of the subsurface biosphere, occurring both on the continents and beneath the seafloor. The
aqueous alteration of ultramafics, in a process known as serpentinization, creates energy rich, high pH
conditions, with low concentrations of inorganic carbon which place fundamental constraints upon microbial metabolism and physiology. Despite their importance, very few studies have attempted to directly access and quantify microbial activities and distributions in the serpentinite subsurface microbiome. We have initiated microbiological studies of subsurface seeps and rocks at three separate continental sites of serpentinization in Newfoundland, Italy, and California and compared these results to
previous analyses of the Lost City field, near the Mid-Atlantic Ridge. In all cases, microbial cell densities in seep fluids are extremely low, ranging from approximately 100,000 to less than 1,000 cells per
milliliter. Culture-independent analyses of 16S rRNA genes revealed low-diversity microbial communities related to Gram-positive Firmicutes and hydrogen-oxidizing bacteria. Interestingly, unlike Lost
City, there has been little evidence for significant archaeal populations in the continental subsurface
to date. Culturing studies at the sites yielded numerous alkaliphilic isolates on nutrient-rich agar and
putative iron-reducing bacteria in anaerobic incubations, many of which are related to known alkaliphilic and subsurface isolates. Finally, metagenomic data reinforce the culturing results, indicating
the presence of genes associated with organotrophy, hydrogen oxidation, and iron reduction in seep
fluid samples. Our data provide insight into the lifestyles of serpentinite subsurface microbial populations and targets for future quantitative exploration using both biochemical and geochemical approaches.
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The Keynote address will be given by Jade Wang, Associate
Professor of Biology at Baylor College of Medicine.
Dr. Jue D. ("Jade") Wang is an Associate Professor in the Departments of Molecular and Human Genetics, Biochemistry and
Molecular Biology, and Molecular Virology and Microbiology at
Baylor College of Medicine, and a Co-Director of the Interdepartmental Graduate Program in Cellular and Molecular Biology. She graduated with a B.Sc degree in physics from McGill
University in Canada. Her Ph.D. in biochemistry, which focused on the mechanisms of action of molecular chaperones,
was in Jonathan Weissman's laboratory at the University of
California, San Francisco (UCSF) in collaboration with Carol
Gross. She became a microbiologist and genomicist during her
postdoc with Alan Grossman at the Massachusetts Institute of
Technology (MIT). Dr. Wang's lab currently focuses on control
of bacterial DNA replication by nutritional availability, the
conflicts between replication and transcription and the
mechanism by which they are resolved, and genome organization and replication stress in B. subtilis and E. coli. She is a
recipient of an NIH Director's New Innovator Award (20082013) and a winner of the 2010 Rosalind Franklin Young Investigator Award by the Genetics Society of America and the Peter and Patricia Gruber Foundation.
Prevention of conflict between replication and transcription
Jue D. (“Jade”) Wang
Baylor College of Medicine
There is a genome-wide conflict between replication and transcription, with important consequences
to cellular fitness and genome integrity. Bacteria have developed diverse mechanisms to deal with
this conflict. The conflict between replication and transcription can be prevented by a genome-wide
strand bias to encode genes in the leading strand. In addition, a functional analog of the eukaryotic
transcription factor TFIIS is crucial for prevention of this conflict. Our discoveries laid the foundation
for addressing the following questions: How do transcription factors prevent the conflict between
transcription and replication? What is the physical nature of the transcription barrier and how it is
formed in response to stress? What are the evolutionary consequences? We are combining biochemical, genetic and genomic approaches to answer these questions.
Dr. Wang’s plenary lecture is supported by the ASM Branch Lectureship Program. The ASMBL program, formerly known as the
Waksman Foundation for Microbiology Lectures Program, allows
ASM branches to secure outstanding lecturers for their scientific meetings. The program has been operating for over 40
years, and lecturers continue to enhance scientific meetings at
the local level.
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Poster presentations
1
2
3
4
Cameron B. Adams
Scanning electron microscopy of the entomopathogenic bacterium,
Photorhabdus luminescens and it symbiotic nematode partner, Heterorhabditis bacteriophora
John E. Baumgartner
Characterization of the organic hydroperoxide resistance system of
Brucella abortus 2308
Michael Betteken
Oxygen induced resistance to tert-butyl hydroperoxide is mediated by
DPS in Bacteroides fragilis
Sarah Chowdhury
Interesting enrichment cultures from sub-surface serpentinite ecosystems.
5 Bridget E. Conley
6
7
8
9
10
Michael J. Courchesne
Equine herpesvirus type 1 (EHV-1) mediated oncolysis of human
glioblastoma multiforme cells
Shurrita S. Davis
Prevalence of Escherichia coli O157:H7 isolates from fresh produce
Kristen N. Delaney
Campylobacter jejuni gene Cj0372 is temperature regulated and
serves as a functional glutathionylspermidine synthetase
Amy A. Devlin
Ahmed E. Elhassanny
11 Kendall L. Fuller
12
13
Type I IFN-stimulatory ligands present within Borrelia burgdorferi
culture supernatants and sonicate are proteins, but are not lipoproteins
Characterization of a putative high affinity Fe2+ transporter in Brucella abortus 2308 that is required for virulence in the mouse model
Comparison of bacterial communities in the rhizosphere of living and
dead Eastern Hemlock (Tsuga canadensis)
John B. Goforth
Effects of spermidine on Vibrio cholerae virulence properties
Morgan J Gregg
Methicillin-resistant Staphylococcus aureus (MRSA) Prevalence in
Pregnant women and transmission risk to newborns
14 Ashley N. Hawkins
15
Generation of a murine polyclonal antibody specific for the Campylobacter jejuni protein Cj0371
Floyd L. Inman, III
Methanogenesis in bogs and fens of the Southern Appalachian region
of North Carolina and characterization of the associated microbial
community
Microbial growth kinetics of Photorhabdus luminescens in glucose
batch culture
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16 Sarah N. Justice
17
18
Ten years of microbe hunting in Great Smoky Mountains National
Park: Where is Pseudomonas aeruginosa?
Rachel Krasich
Determining the fate of transcription at DNA-protein crosslinks
Yajuan Lin
Estimating the growth rate and biomass production of genetically diverse Prochlorococcus using rRNA/rDNA ratios
Tanner Love, Savannah
Reducing surface bacteria using UV-C radiation
19 Morgan, and Daniel
Morrow
20
Jenifer Ojeda
21 Michael A. Reott
22
Alex Rutkovsky
23 Martin C. Wilson
The bhuTUV and bhuO gene products play vital roles in the ability of
Brucella abortus to use heme as an iron source
Investigations into the regulation and activity of B. fragilis thioredoxins during oxidative stress
Characterization of the PotD1 protein in Vibrio cholerae
Isolation and identification of antimicrobial-producing microbes from
soil associated with an Eastern Hemlock in the Great Smoky Mountains
National Park
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Abstracts (posters)
1. Scanning electron microscopy of the entomopathogenic bacterium, Photorhabdus
luminescens and it symbiotic nematode partner, Heterorhabditis bacteriophora
Cameron B. Adams, Floyd L. Inman, III, and Leonard Holmes
Department of Chemistry and Physics, UNC Pembroke
Photorhabdus luminescens is a bioluminescent entomopathogenic bacterium that is readily
found living in symbiosis with its nematode partner, Heterorhabditis bacteriophora. Scanning electron
microscopy was used in this study to obtain topographical images of P. luminescens cultured in nutrient
broth supplemented with 1.0% trehalose and infective juveniles of H. bacteriophora obtained from a
distributor. The detailed images from this study show that P. luminescens is approximately 5 microns
long and 1 micron in width and is surrounded by a “protective layer” of trehalose. This “protective
layer” was also seen surrounding nematodes that were also treated with a 1.0% trehalose solution.
Through the use of these images, the length of H. bacteriophora was determined to be about 500 microns long with an oral cavity width of approximately 5 microns, mid-length width of about 20 microns
and the tail tip, 1 micron.
2. Characterization of the organic hydroperoxide resistance system of Brucella abortus 2308
John E. Baumgartner, Clayton C. Caswell, Daniel W. Martin, and R. Martin Roop II
Department of Microbiology and Immunology, Brody School of Medicine, ECU
The organic hydroperoxide resistance protein Ohr has been identified in numerous bacteria
where it functions in the detoxification of and resistance to organic hydroperoxides, and expression of
ohr is often regulated by a MarR-type transcriptional regulator called OhrR. Brucella abortus, a pathogenic α-proteobacterium, encodes a potential ohr-ohrR system, but whether or not these genes contribute to organic hydroperoxide resistance in B. abortus has not been established. Using isogenic ohr
and ohrR mutants and lacZ promoter fusions, it was determined that the ohr-ohrR system contributes
to resistance to organic hydroperoxide, but not hydrogen peroxide, in B. abortus 2308, and OhrR is a
transcriptional repressor of both ohr and ohrR. Electrophoretic mobility shift assays and DNase I footprinting revealed that OhrR binds directly to a specific region termed an “OhrR box” in the ohr-ohrR
promoter region, and, interestingly, the B. abortus OhrR box is highly similar to OhrR boxes described
in other bacteria. While the ohr-ohrR system plays a prominent role in the protection of B. abortus
from organic hydroperoxide stress, this system is not required for wild-type survival of B. abortus in
primary murine macrophages. Similarly, the ohr mutant exhibits wild-type levels of spleen colonization
in a mouse model of Brucella infection; however, the ohrR mutant is significantly attenuated in the
mouse model of infection. Ongoing experiments are aimed at elucidating the OhrR regulon in hopes of
illuminating the molecular basis of attenuation of the ohrR mutant.
3. Oxygen induced resistance to tert-butyl hydroperoxide is mediated by DPS in Bacteroides fragilis
Michael Betteken, E. Rocha, M. Reott, and C.J. Smith
Department of Microbiology and Immunology, Brody School of Medicine, ECU
Bacteroides fragilis, an obligate anaerobe, is one of the most commonly isolated organisms
from anaerobic infections in humans. Infections from B. fragilis can result in complications such as abdominal abscesses, perforated and gangrenous appendicitis, skin and soft tissue infections, endocarditis
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and bacteremia. In order to cause infection, B. fragilis must adhere, resist increased oxygen tension,
and express a variety of virulence factors. The increased oxygen tension presents a large challenge for
the survival of the anaerobic B. fragilis. This challenge is addressed through the coordination of several
oxidative stress response genes such as OxyR, DPS, AhpC, and an extensive thioredoxin system resulting
in increased aerotolerance and resistance to peroxides such as hydrogen peroxide, cumen hydroperoxide, and tert-butyl hydroperoxide. B. fragilis resistance to tert-butyl hydroperoxide was characterized
in this report. Under anaerobic conditions, disk inhibition assays utilizing tert-butyl hydroperoxide
(55mM) demonstrated 55mm zones of inhibition. However, assays with plates incubated in air for three
hours prior to anaerobic incubation were completely resistant to tert-butyl hydroperoxide. Studies
showed that DPS mediated this increased resistance to tert-butyl hydroperoxide after aerobic incubation. OxyR is a known activator of DPS transcription; however, an ΔoxyR mutant still demonstrated
complete resistance to tert-butyl peroxide whereas an ΔoxyRΔdps double mutant demonstrated zones
of inhibition of 60mm even after aerobic incubation. These results suggest that an oxygen induced alternative transcriptional activator of dps is present in B. fragilis which mediates this resistance.
4. Interesting enrichment cultures from sub-surface serpentinite ecosystems.
Sarah Chowdhury, Bridget Nelson, Billy Brazelton, and Matt Schrenk
Department of Biology, ECU
The aqueous alteration of ultramafic minerals is a process known as serpentinization. This occurs at spreading ridges on the ocean floor and on continents where the mantle layer has become uplifted as a result of tectonic activity. Serpentinization generates energy for chemolithotrophy in microorganisms located in the deep subsurface biosphere. Our lab engages in exploration of the serpentinite hosted subsurface biosphere at the Northern Apennine ophiolite in the Ligurian region of Italy,
the Tablelands ophiolite at Gros Morne National Park, Canada and the Coast Range ophiolite at
McLaughlin Natural Reserve, California. Ultrabasic (pH 11) waters from McLaughlin and the Tablelands
were cultured in an anaerobic enrichment experiment using a block design of carbon and energy
sources and electron acceptors. Cultures were incubated for several weeks and community differences
were observed through TRFLP. The isolation of an iron reducing Firmicute from samples enriched with
complex organic compounds along with ferric citrate is of interest because it has close relatives with
hydrogen generating capabilities. We continue to generate data on the physiology of the subsurface
serpentinite associated microbial communities and isolates derived from them. These data along with
chemical and genetic data from parallel samples will provide insight into the environmental constraints
facing organisms in an important component of Earth’s subsurface.
5. Generation of a murine polyclonal antibody specific for the Campylobacter jejuni protein Cj0371
Bridget E. Conley, Kristen N. Delaney, and Deborah S. Threadgill
Department of Microbiology, NCSU
Campylobacter jejuni can colonize chickens without causing overt pathology, but when a human becomes infected with C. jejuni the result is gastroenteritis which can be quite severe. We are
seeking genes which may be involved in determining C. jejuni tropism and pathogenicity. One of the
main differences between the human and chicken gut is temperature; the chicken body temperature is
42°C as opposed to 37°C for humans. Microarray analysis revealed a number of proteins that are differentially regulated at the two temperatures. One gene which was upregulated at 42°C was the hypothetical protein Cj0371. In order to study the function of this gene product we generated a Cj0371specific antibody. The ectodomain of Cj0371 (base pairs 103-606) was amplified from C. jejuni 11168.
This PCR product was cloned into the multiple cloning site of pET21a at the Nde1 and Xho1 sites. The
resulting plasmid pET21a-371 was transformed into NovaBlue and BL-21 chemically competent cells for
DNA analysis and protein expression, respectively. Recombinant Cj0371 was induced in liquid culture
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by IPTG and purified on a nickel column. Recombinant protein was dialyzed into PBS and filter sterilized prior to immunization. BALB/c mice were immunized three times i.p. with 10µg of recombinant
Cj0371 adsorbed on alum gel (adjuvant). Ten days after the immunization, plasma samples were collected and evaluated for antibody production by ELISA. All immunized mice developed α-Cj0371 antibodies.
6. Equine herpesvirus type 1 (EHV-1) mediated oncolysis of human glioblastoma multiforme cells
Michael J. Courchesne, Maria C. White and Arthur R. Frampton Jr
Department of Biology and Marine Biology, UNCW
Viral oncolytic therapy offers a means to combat malignancies that are unresponsive to other
treatments. Over the past 20 years, many viruses have been evaluated for their oncolytic potential
against various cancers including the highly aggressive brain cancer, glioblastoma multiforme (GBM). In
this study, the cytolytic animal virus equine herpesvirus type 1 (EHV-1) was evaluated for its oncolytic
potential against five human glioblastoma cell lines, A-172, Hs 683, LN-18, SNB19, and U251. EHV-1
productively infected four of these cell lines and the degree of infection was positively correlated with
glioma cell death. Previous work from our group showed that equine major histocompatibility complex
class 1 (MHC-I) is an entry receptor for EHV-1. This finding, coupled with the ability of EHV-1 to infect
cells from myriad species, led us to investigate the level of MHC-I expression on the panel of glioma
cells and to examine whether this expression correlated with EHV-1 infection. RT-PCR assays showed
that four of the five glioma cell lines expressed human MHC-I, but no MHC-I transcript was detected on
the EHV-1 resistant Hs 683 cells. However, Hs 683 cells were rendered susceptible to EHV-1 infection
after an equine MHC-I receptor was expressed in these cells, indicating that a major block to infection
of these cells is the lack of a suitable EHV-1 receptor.
7. Prevalence of Escherichia coli O157:H7 isolates from fresh produce
Shurrita S. Davis, Janak R. Khatiwada, and Leonard L. Williams
The Center for Excellence in Post-Harvest Technologies, NC A&T
The Center of Disease Control and Prevention (CDC) estimates that 48 million illnesses, 128,000
hospitalizations, and 3,000 deaths are traced to foodborne pathogens. Multiple foodborne illness outbreaks have been source-linked to the consumption of fresh produce contaminated with Escherichia
coli O157:H7. This study investigated the prevalence of E. coli O157:H7 and detected the presence of
Shiga-toxin type-1 and type-2 (stx1 and stx2). The samples were isolates from 455 fresh vegetable samples consisting of green-onion, lettuce, cilantro, and spinach. The samples were purchased from local
and non-local farms in North Carolina. Samples were streak onto selective media, Sorbitol MacConkey
agar for isolation and identification according to the Bacteriological Analytical Manual (BAM). The DNA
from the positive isolates was extracted for pathogenic testing. Recognition of virulence factors was
done using PCR. Thirty-two of the 455 samples (7%) tested were shown to be positive for E. coli
O157:H7 and 21 of the 32 samples (65.5%) samples that tested positive was shown to contain stx1 or
stx2 or both. The findings of this study indicate the prevalence of E. coli O157:H7 in fresh produce is
low but a majority of strains the E. coli found had the potential to be pathogenic.
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8. Campylobacter jejuni gene Cj0372 is temperature regulated and serves as a functional glutathionylspermidine synthetase
Kristen N. Delaney, Jason M. Andrus, Angelika Jährig, Jinzhi Wang, Alain Stintzi, and Deborah S.
Threadgill
Department of Microbiology, NCSU
C. jejuni was cultured in hydrogen-containing microaerophillic conditions to resemble the environment of the human (37°C) or chicken (42°C) intestinal tract. Microarray analysis revealed that
cj0372 was upregulated when bacteria were cultured at 42°C. Since C. jejuni colonizes the GI tract of
chickens without causing overt pathology we hypothesized that this gene may be involved in persistence. Cj0372 is annotated as a hypothetical glutathionylspermidine synthetase (GspS). In E. coli GspS
catalyzes the fusion of glutathione and spermidine while consuming ATP. Using an in vitro GSP synthesis assay, recombinant Cj0372 released inorganic phosphate from ATP when provided with precursors
for this reaction. These data are consistent with the hypothesis that Cj0372 is a GSP synthetase. We
generated a chromosomal knockout of Cj0372 in C. jejuni (DST372) by interrupting the gene with a
chloramphenicol resistance cassette. The DST372 strain demonstrates accelerated growth. DST372
also shows a loss of motility at 37°C and 42°C. The loss of motility may contribute to enhanced proliferation in the mutant. In addition to accelerated growth, DST372 reached higher optical densities at
stationary phase. One possible explanation for this is that DST372 was less sensitive to oxidative stress
in an H2O2 disc diffusion assay. We also observed decreased cell size in DST372 as determined by
scanning electron microscopy. This may be related to the increased rate of cell division.
9. Type I IFN-stimulatory ligands present within Borrelia burgdorferi culture supernatants and sonicate
are proteins, but are not lipoproteins
Amy A. Devlin, H. Maylor-Hagen, and J. C. Miller
Department of Microbiology, NCSU
Borrelia burgdorferi infection of C3H mice causes severe Lyme arthritis, whereas infected C57BL/6
mice only develop mild arthritis. Previously, we identified a novel and unappreciated role for Type I
IFN induction in the development of severe Lyme arthritis within genetically susceptible C3H mice.
Utilizing mouse bone marrow-derived macrophages (BMDMs) as model cells, we identified multiple Borrelia burgdorferi (Bb) associated Type I IFN-stimulatory ligands, which included products released by
the bacteria into the culture supernatant and cell-associated sonicate ligands. Bb RNA, itself an IFNstimulatory ligand, was not responsible for the IFN stimulatory activities mediated by Bb culture supernatant or sonicate ligands. Bb sonicate analyzed by 16S rRNA quantitative RT-PCR was shown to be
RNA-free, and RNase-treated or untreated Bb culture supernatants induced IFN-stimulated genes (ISGs)
to comparable levels in stimulated mouse BMDMs. High speed ultracentrifugation of Bb culture supernatants performed to remove any possible bacterial membrane blebs, did not alter its macrophage IFNstimulatory activity. The IFN-stimulatory capacity of Bb culture supernatant and sonicate ligands were
greatly reduced following heat inactivation and proteinase K treatments, suggesting that these Bbderived ligands are proteins. ISG induction was unaltered in Bb culture supernatant- or sonicatestimulated TLR-2-/- BMDMs and TLR-2 antibody-blocked RAW264.7 macrophage-like cells, compared
with stimulated wild-type BMDMs or non-antibody treated RAW cells. These data indicate that the IFNstimulatory activities of Bb culture supernatants and sonicate ligands are non-lipoprotein mediated.
Current efforts are focused on HPLC-mediated fractionation of Bb culture supernatants and sonicate.
HPLC fractions will be applied to RAW cells and screened by RT-PCR for IFN-stimulatory activity. Bb
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proteins present within IFN-stimulatory HPLC-fractions will be identified by LC-MS² and MASCOT analyses. Identification of these IFN-stimulatory proteins will broaden our knowledge of bacterially elicited
Type I IFN signaling pathways and may lead to improved therapeutics for individuals suffering from
Lyme arthritis.
10. Characterization of a putative high affinity Fe2+ transporter in Brucella abortus 2308 that is required for virulence in the mouse model
Ahmed E. Elhassanny, Eric S. Anderson, and R. M. Roop II
Department of Microbiology and immunology, Brody School of Medicine, ECU
Like most bacteria, Brucella strains require iron as an essential micronutrient. However, because iron is tightly sequestered in mammalian hosts these bacteria have adapted a variety of strategies to acquire enough iron from this limiting environment. Siderophore-dependent ferric iron-specific
and heme acquisition systems have been identified in Brucella as important means of acquiring this
essential metal; however, no ferrous iron (Fe2+)-specific transport system has been characterized. The
genes designated BAB2_0837-0840 in the B. abortus 2308 genome sequence are predicted to encode
Fe2+ transporter belonging to the recently described Cup-II-type family of prokaryotic Fe2+ transporters.
These Brucella genes have been given the provisional designations bfeA-D; bfeA (BAB2_0840) is predicted to encode a 19 kDa periplasmic iron binding protein, bfeB (BAB2_0839) a CupII-type ferroxidase,
bfeC (BAB2_0838) an Ftr1 homolog high affinity iron permease and bfeD (BAB2_0837) a polyferredoxin
that is thought to maintain the redox balance of the transporter. Compared to the parental 2308
strain, an isogenic bfeA mutant displays a decreased capacity to use Fe2+ as an iron source in vitro and
displays severe attenuation in cultured murine macrophages and experimentally infected mice. Transcriptional analyses indicate that bfeA expression is elevated in B. abortus 2308 in response to iron
deprivation, and mutational studies indicate that this pattern of expression is mediated by the ironresponsive regulator Irr. Correspondingly, a predicted Irr binding site is located 55 nt upstream of a
transcriptional start identified for bfeA by primer extension, and a direct interaction between a recombinant version of the Brucella Irr and the bfeA promoter region has been observed in an electrophoretic mobility shift assay. Our working hypothesis is that the BfeABCD complex allows Brucella
strains to successfully scavenge Fe2+ during their residence in acidified vacuoles, where ferrous iron
Fe2+ should be readily available, in the early stages of their interactions with host macrophages.
11. Comparison of bacterial communities in the rhizosphere of living and dead Eastern Hemlock (Tsuga
canadensis)
Kendall L. Fuller and Sean O'Connell
Department of Biology, WCU
Eastern Hemlock (Tsuga canadensis) is an imperiled tree species in Eastern North America due to the
actions of an exotic insect, Hemlock Woolly Adelgid (Adelges tsugae). Large tracts of forest dominated
by hemlock have been decimated by this pest. Little is known about the microbial partners of hemlock,
especially in the rhizosphere, the critical interface of the plant and soil. Samples were collected from
rhizosphere soil of living and dead hemlock trees (n=3 each) at Albright Grove, Great Smoky Mountains
National Park. DNA was extracted from the soil and DNA extracts were subjected to PCR targeting the
16S rDNA using primers 27F and 1492R. Molecular cloning was used to generate 181 DNA sequences for
a portion of the 16S rRNA gene. Results indicated that seven phyla were represented in the clone libraries with Acidobacteria (68.5%, 63.3%) and Proteobacteria (17.4%, 26.0%) being the dominant phyla
for dead and living trees, respectively. For Acidobacteria, Group 1 and Group 2 were most commonly
observed with Group 1 more common in the rhizosphere of living trees and Group 2 more numerous in
the dead trees. The only occurrence of Betaproteobacteria was observed for living trees. Two phyla
each appeared only in the living or dead hemlock, while the Planctomyces appeared in both clone liPag e 16
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braries. The differences observed in the bacteria associated with living and dead hemlock may represent a shift in the soil chemistry associated with photosynthate versus organic chemicals resulting from
decomposition. Ongoing work is also assessing seasonal trends in microbial diversity of hemlock and
species diversity within the clone libraries. Identification of bacterial partners linked to hemlock health
may help preserve existing trees or repopulate stands of trees lost to the adelgid.
12. Effects of spermidine on Vibrio cholerae virulence properties
John B. Goforth and Ece Karatan
Department of Biology, ASU
Vibrio cholerae is the causative agent of the severe diarrheal disease Cholera. The disease occurs when V. cholerae are ingested and successfully colonize the small intestine. Successful colonization of the small intestine by V. cholerae is dependent upon the ability to synthesize the type 4b pilus
toxin-coregulated pilus (TCP). This colonization factor is composed of the repeating homopolymer
TcpA. TCP allows cells to autoagglutinate and form microcolonies, which are thought to aid in the
colonization of the small intestine.
Many environmental signals have been shown to regulate TCP syntheses including pH, amino acids, bile
salts, temperature and osmolarity. V. cholerae will likely encounter a plethora of signals in the small
intestine, many of which are unknown. The objective of this work was to see if one class of small
molecules called polyamines, which are abundant in the small intestine have an effect on V. cholerae
virulence. Polyamines are small, organic polycationics that are synthesized by almost all organisms and
are essential for normal cell growth. Due to its abundance in the small intestine the polyamine spermidine, synthesized by the human host as well as the intestinal microbiota, was tested for its effect on
autoagglutination. Agglutination was measured by assaying the rate of sedimentation of V. cholerae
cultured under optimal agglutinating conditions with increasing spermidine concentrations. Spermidine
concentrations ≥5 mM significantly reduced agglutination. Fluorescent microscopy also revealed a reduction in microcolony size in cultures containing spermidine concentrations ≥5 mM. Interestingly,
western blot analysis using an anti-TcpA antibody showed spermidine had no effect on TcpA levels in
cultures exhibiting attenuation of agglutination. In conclusion, this data indicates spermidine has an
inhibitory effect on microcolony formation by a mechanism that is independent of an effect on TcpA
levels.
13. Methicillin-resistant Staphylococcus aureus (MRSA) Prevalence in Pregnant women and transmission
risk to newborns
Morgan J Gregg, Robin L LaCroix, and Yuko J Miyamoto
Department of Biology, Elon University
MRSA is emerging as a major pathogen in pediatric and adult populations alike. However,
minimal information is available describing the prevalence of MRSA colonization in pregnant women or
the risk of transmission to the infant. Through the review of hundreds of medical records in this retrospective study, prevalence and incidence of MRSA infection or colonization in peripartum mothers and
their newborns MRSA colonization was determined using real time automated polymerase chain reaction. Of 102 evaluable mother/baby pairs examined in this study, 8 were positive for MRSA in both
mother and baby, providing an overall MRSA transmission rate of 7.84%
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14. Methanogenesis in bogs and fens of the Southern Appalachian region of North Carolina and characterization of the associated microbial community
Ashley N. Hawkins and Suzanna L. Bräuer
Department of Biology, ASU
Biological methane production by archaea in wetlands is a major source of methane emissions
resulting in an influence on levels of greenhouse gases in the atmosphere. To fully understand the
processes governing the production and release of methane from these understudied and threatened
ecosystems, this study focuses on the effects that several variables have on the archaeal community
and their ability to produce methane. Gas chromatography analyses are being utilized to quantify
methane being produced from each of three sample sites over the course of a three week period during
each of the four seasons. Preliminary results from the summer season have suggested an influence of
pH and the average position of the water table. The average, site-wide pH from each sample area was
as follows: Pineola, NC (pH 4.96), Sugar Mountain, NC (pH 4.67) and Tater Hill, NC (pH 5.73) taking into
account a range of pHs within each site. Initial summer measurements indicate an increase in methane
production from sites with a shallow water level as observed from the final methane percentage of
6.0560% from the Sugar Mountain sample, (average water table depth of ~6.35cm), compared to a final
methane percentage of 3.513% and 5.310% from samples within the Tater Hill and Pineola sites respectively (average water table depth of ~11.43cm). A general trend of increased methane production in
more neutral areas within sample sites was also indicated with a final methane percentage of ~5.310%
at pH 5.2 compared with ~1.463% at pH 4.81 from the Pineola sample area after three weeks of incubation. Archaeal clone libraries have also been built and are currently being analyzed. These analyses
may reveal distinct differences in community composition across the sites. Additionally, we are collecting DNA samples for qPCR analyses in order to characterize any seasonal changes in community composition across sites and within sites. A fourth component of the current research is the examination of
ARMAN-like organisms detected in clone libraries of one study site. These organisms were initially discovered in acidic mine drainage in 2006 along with subsequent sequence identification in acidic bog
sites of Southern Finland. They are currently being investigated as some of the smallest free-living archaeal organisms.
15. Microbial growth kinetics of Photorhabdus luminescens in glucose batch culture
Floyd L. Inman, III and Leonard Holmes
Department of Chemistry and Physics, UNC Pembroke
The entomopathogenic bacterium, Photorhabdus luminescens, is a species of bioluminescent
enteric bacteria that is symbiotically associated with the beneficial nematode, Heterorhabditis bacteriophora. It has been hypothesized that studying the growth kinetics of P. luminescens will allow for
the optimization and formulation of a liquid medium for the mass production of H. bacteriophora. Microbial growth kinetics of P. luminescens was studied in a 10 mM glucose batch culture utilizing a 2 liter Sartorius Stedim Biostat A plus fermentation system. Constant fermentation conditions include: airflow rate (0.65 vvm), temperature (28°C) and agitation (200 rpm). Measurements of luminosity, turbidity, and biomass were recorded and graphed as a function of time. The specific growth rate (µ) was
determined by utilizing the natural log of the biomass (0.54 h-1) and the natural log of the absorbance
(0.40 h-1). According to biomass data, luminosity greatly increased once the bacteria entered into the
deceleration phase of growth.
16. Ten years of microbe hunting in Great Smoky Mountains National Park: Where is Pseudomonas aeruginosa?
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Sarah N. Justice, Chris Beyer, Kendall Fuller, and Sean O'Connell
Department of Biology, WCU
Every fall, two classes from Western Carolina University sample soil and water to isolate bacteria from Great Smoky Mountains National Park (GSMNP). This is the tenth year of this project and many
patterns about bacterial distribution in natural environments are emerging. Each student isolates a
single bacterial culture and characterizes it via microscopy, culture conditions, and biochemical and
metabolic properties. The final data generated is a sequence of a portion of the 16S rRNA gene. The
data presented here reflect the results of the 16S rDNA survey for all ten years, including 267 bacterial
sequences. In addition, a focused effort has been undertaken to isolate Pseudomonas aeruginosa from
these and other samples in GSMNP. Five phyla, 12 classes, 15 orders, 32 families, and 66 genera have
been detected while another 24 sequences have been found to have no close relatives to described
bacteria. Water samples have produced 27 genera not observed from soil while soil diversity has shown
25 unique genera. Seventeen genera have occurred in both soil and water. Common water genera
included Janthinobacterium and Rhodoferax while soil has been dominated by Streptomyces and Burkholderia. Bacillus and Flavobacterium have been commonly found in both soil and water. One surprising result is that out of 17 Pseudomonas isolates (and hundreds of clones from other samples), none
were identified as P. aeruginosa, and furthermore, directed selective culturing also has not worked
thus far for finding this species. Novel bacterial species have been recovered from GSMNP, including
some that may represent new families or higher taxonomic levels. These microbial species may include
some with unusual chemical capabilities and metabolic byproducts of interest to science and industry
and further work is warranted.
17. Determining the fate of transcription at DNA-protein crosslinks
Rachel Krasich and Kenneth N. Kreuzer
Department of Biochemistry, Duke University
DNA-protein crosslinks (DPCs) are a form of DNA damage where a protein becomes covalently
bound to DNA. In bacteria, they have been shown to block replication, transcription, and protein synthesis, and eventually lead to cell death(1-3). Compared to other types of DNA damage, DPCs remain
amongst the most poorly understood due to their complex nature; the agents that cause DPCs, their
method of crosslinking, the proteins that are affected, and the locations of DPCs all vary. Our lab is
particularly interested in the fate of transcription elongation complexes stalled by DPCs. Using E. coli
as our model system, we found that cells deficient in SsrA, a specialized tmRNA molecule that releases
ribosomes stalled on a truncated peptide and tags the peptide for degradation, were hypersensitive to
DPC formation. Furthermore, this hypersensitivity was dependent solely on tmRNA’s ability to release
stalled ribosomes. In order to isolate the effect on transcription blockage, we used the transcription
elongation inhibitor Streptolydigin (Stl) which inhibits elongating RNA polymerase (RNAP). We found
that cells deficient in SsrA were also hypersensitive to Streptolydigin. To identify the protein(s) responsible for releasing the stalled RNAP, we tested a number of knockouts of transcription elongation
and termination proteins, including proteins known to dislodge stalled or paused RNAP. Previous work
showed that Streptolydigin-inhibited polymerases are released from the DNA transcript in vivo4. Surprisingly, we found that mutants deficient in the transcription-coupled repair factor, Mfd, were slightly
resistant to Stl treatment. Conversely, overexpressing Mfd led to hypersensitivity to Stl treatment.
However, cells deficient in both Mfd and SsrA activity were no longer hypersensitive, indicating Mfd
activity creates substrates for SsrA activity. Cells overexpressing Mfd also had elevated levels of SsrA
tagging, indicating high levels of prematurely terminated translation. Taken together, this data implies a unique situation where Mfd activity is leading to premature termination and growth inhibition,
both when transcription is inhibited by Stl and when Mfd is overexpressed.
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18. Estimating the growth rate and biomass production of genetically diverse Prochlorococcus using
rRNA/rDNA ratios
Yajuan Lin and Zackary I. Johnson
Duke Marine Lab, Duke University
The marine cyanobacteria Prochlorococcus is the most abundant phytoplankton in tropical and
subtropical open oceans and accounts for 5-25% of global marine primary production (Chisholm et al.,
1988; Partensky et al., 1999; Vaulot et al., 1995). While much is known about the abundance (Johnson
et al., 2006; Zwirglmaier et al., 2007) and genetic diversity (Kettler et al., 2007; Rocap et al., 2002) of
this genus, little is known about the activity of the different clades in their natural environment. Here
we show that in several Prochlorococcus cultured strains the rRNA/rDNA ratio is strongly correlated
with their specific growth rate over a range of light-regulated growth conditions. Moreover, a field
study reveals that the rRNA depth profile of the dominant clade eMIT9312 is consistent with the total
Prochlorococcus primary production. This evidence suggests that rRNA/rDNA ratio may be used to estimate the in situ biomass production of coexisting Prochlorococcus clades and reveal their differential
contribution to carbon uptake and ecology. Using this approach, we designed primer sets specific for
two major Prochlorococcus clades, eMIT9312 (high-light adapted) and eMIT9313 (low-light adapted),
and measured their rRNA content in three representative stations in the open ocean. The growth rate
or biomass production inferred from their rRNA/rDNA ratio is uncoupled with their abundance in the
water column, and is significantly influenced by light and nutrient availability. Further exploring the
biogeography patterns of production in combination with environmental variable data will greatly help
unravel the ecological and biogeochemical contribution that these genetically distinct, but morphologically similar marine microbes have in a changing ocean environment.
19. Reducing surface bacteria using UV-C radiation
Tanner Love, Savannah Morgan, Daniel Morrow, and Barbara Morrow
Providence Day School
Many schools in North Carolina (and throughout the country) have implemented antibacterial
strategies (mainly through the use of hand sanitizers and urging students to wash hands frequently) to
avoid the spread of pathogenic bacteria. However convenient these methods may be, they are not as
effective in reducing the amount of pathogens present as other potential methods. Ultra-violet-C radiation has a unique effect on microorganisms. While the high concentration of alcohol in hand sanitizers is able to kill a bacterium by damaging its membrane and denaturing the proteins within its cell,
this process is not as efficient in killing the bacterium as the process it undergoes when exposed to UVC radiation. Several experiments were conducted to determine an “optimal exposure time and distance” from a UV-C light source at which bacterial cultures (grown in nutrient broth) were most effectively reduced. Nutrient broth was inoculated from bacteria on the surface of five randomly chosen
keyboards in Providence Day School’s technology lab and a heterogeneous “slurry” of unknown bacteria
was cultured. Spectrophotometry was implemented to determine the amount of light transmitted by
each sample at varied exposure times and distances from the UV-C source. After eleven trials, (exposure times of 15 min- 10 hours and distance ranges of 0.25 m-2.0 m), it was calculated that an optimal
exposure time is 240 minutes and the optimal distance was 0.25 m. The growth of the cultures, when
irradiated at this optimal distance and time, was reduced by 19.7%. The optimal time and distance factored not only the amount of light transmitted by the bacteria in the cultures, but also the operational
cost of implementing UV-C lights into a typical classroom setting.
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20. The bhuTUV and bhuO gene products play vital roles in the ability of Brucella abortus to use heme
as an iron source
Jenifer Ojeda, James T. Paulley, and R.M. Roop II
Department of Microbiology and Immunology, ECU
Cattle are a natural host for Brucella abortus, and brucellosis is also a zoonotic disease affecting humans worldwide, particularly in endemic countries. Brucellosis causes a chronic flu-like illness characterized by an undulant fever in humans, and the current strategy employs prevention of the illness
through herd vaccinations. Iron is an essential nutrient for most organisms, and bacteria employ various strategies to overcome the strict iron limitation imposed by the host. Many pathogenic bacteria
utilize heme as an iron source in order to sustain an infection within their host. Brucella are intracellular pathogens, and reside within a vacuole that interacts with the endoplasmic reticulum (ER) of the
host macrophage, making heme an attractive iron source since the macrophage recycles senescent red
blood cells to the ER. B. abortus requires a functional heme uptake system in order to maintain infection within its host. The heme utilization system of Brucella includes a TonB-dependent outer membrane transporter (BhuA), a heme-binding periplasmic protein (BhuT), an ATP-dependent cytoplasmic
membrane permease (BhuUV), and a heme oxygenase (BhuO) to remove the iron center from the heme.
While BhuA has been previously characterized (Paulley, et al 2007), our data have determined that the
bhuTUV locus is required for the utilization of heme as an iron source in vitro. Further, BhuO, a heme
oxygenase of Brucella identified by Puri, et al in 2006, plays a role in iron acquisition from heme in
vitro, and is required for maintenance of infection in the mouse model. These data support the hypothesis that heme serves as an essential iron source for B. abortus in the mouse model of chronic infection, and that the bhuTUV and bhuO gene products play vital roles in the ability of Brucella to obtain iron from heme.
21. Investigations into the regulation and activity of B. fragilis thioredoxins during oxidative stress
Michael A. Reott, Jr., A. C. Parker, E. R. Rocha, and C. J. Smith
Department of Microbiology & Immunology, Brody School of Medicine, ECU
Bacteroides fragilis is a gram-negative, obligate anaerobe native to the intestinal tract. Upon
injury, B. fragilis can migrate out to the intraperitoneal space, where it contributes to the formation
of life-threatening intra-abdominal abscesses. The ability to colonize this space is mediated in part by
a highly effective oxidative stress response (OSR) that allows B. fragilis to survive and persist in aerobic
regions. The OSR is a coordinated regulation of several sets of genes which react to an oxidative stress
condition. Previous studies have shown the importance of the thioredoxin (Trx) system in the ability of
B. fragilis to manage oxidative stress. Experiments utilizing mutant strains lacking the different trx
genes found that these six trx genes had some ability to compensate for each other in growth and survival during the OSR. However, the Trx proteins may also have individual roles in B. fragilis. TrxD was
shown to be important during disulfide stress and thus the current study investigated the roles and
regulation of this Trx. Further investigations showed that TrxD appeared to be important for the reduction of specific proteins, potentially during the OSR. Recently, competition studies and microarray
analysis utilizing an artificial abscess model in rats have revealed that while the Trx system does not
appear to be necessary for survival within the abscess, TrxD was shown to be the highest induced Trx
within the B. fragilis genome during the course of the infection. These data may indicate a significant,
while not necessary, role for TrxD during survival within an abscess environment. Taken together,
these studies demonstrate that the B. fragilis Trx system is an important component of the OSR and
that particular portions of this system may be playing prominent roles for persistence in an abscess.
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22. Characterization of the PotD1 protein in Vibrio cholerae
Alex Rutkovsky and Ece Karatan
Department of Biology, ASU
Vibrio cholerae is an intestinal pathogen that thrives in aquatic environments by forming
biofilms, which allow the bacterium to survive otherwise harmful conditions. Biofilm formation is regulated by numerous environmental signals including polyamines. Polyamines are small cationic molecules that promote normal cell growth in most living organisms. The polyamine spermidine enters V.
cholerae through an ABC-type polyamine transporter causing a decrease in biofilm formation. The purpose of this project was to characterize the interaction of spermidine with PotD1, the ligand-binding
protein of this transporter. To study PotD1-spermidine interaction, three amino acid residues within
the putative spermidine binding cleft of PotD1, which are conserved in PotD1 orthologs, were altered
by site-directed mutagenesis. The point mutants were introduced into a ∆potd1 strain and cellular
levels of spermidine were determined using high performance liquid chromatography. Because V.
cholerae cannot synthesize spermidine under these conditions, levels of cellular spermidine is indicative of uptake of spermidine. The W252L and D254N mutants did not support spermidine uptake indicating these residues are required for PotD1 function, whereas, E168A had reduced spermidine uptake
indicating this residue plays a lesser role. In addition, W252L and E168A supported a biofilm phenotype
similar to wild-type, whereas, D254 was required for the inhibitory effect of PotD1 on biofilms. Our
results suggest that PotD1 may regulate biofilm formation through distinct mechanisms one of which is
independent of spermidine uptake.
23. Isolation and identification of antimicrobial-producing microbes from soil associated with an Eastern Hemlock in the Great Smoky Mountains National Park
Martin C. Wilson and Betsy Wilson
Department of Biology, UNCA
Microbes were isolated from soil associated with an eastern hemlock (Tsuga canadensis) in
Great Smoky Mountains National Park by cultivation on oligotrophic media containing polymeric carbon
compounds. Antimicrobial-producing microbes were identified by measuring their inhibition of the
growth of antimicrobial-sensitive organisms on agar plates.
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Presentation Awards
The Mary Poston Award was established to recognize the best paper
given by a student at meetings of the NC Branch of the ASM. Mary Poston was a longtime employee of Duke University who contributed much
to the NC Branch and she was held in high esteem both by her colleagues and by medical students. She contributed much to the NC
Branch, including service as Branch Secretary-Treasurer from 1950 until
her death in 1961. Many letters of appreciation have been written over
the years by student recipients of the Mary Poston Award, commenting
on the confidence the award gave them and on the importance of the
competition for the award as part of their graduate training.
Last year’s winner: Gökhan Tolun
University of North Carolina Chapel Hill
More than the sum of its parts: Physical and mechanistic coupling in the
viral two-component recombinases
The Thoyd Melton Award was established to recognize an outstanding
oral presentation by a graduate student. At the time of his premature
death on Nov. 22, 2000, Thoyd Melton was Associate Vice Chancellor for
Academic Affairs and Dean of graduate studies at N.C. A&T State University. Prior to this position, Dr. Melton was a member of NC State University's Department of Microbiology and an Associate Dean of the
Graduate School. Dr. Melton was very active in research and particularly
in graduate education. In 1999, he received the William A. Hinton Research Training Award from ASM. This award honors an individual who
has made significant contributions toward fostering the research training of underrepresented minorities in microbiology.
Last year’s winner: Caitlin Mattos Briggs
Wake Forest University School of Medicine
Stimulation of primary human macrophages with Gram positive bacteria enhances susceptibility to Paramyxovirus infection
The Best Poster award is open to anyone presenting a poster at the NC
ASM meeting.
Last year’s winner: Eric Anderson
East Carolina University
Identification of the tpd-ftr1 locus, encoding a putative high affinity
ferrous iron (Fe++) transport system necessary for virulence in Brucella
abortus 2308.
The Paul Phibbs Award is awarded for the best presentation by an undergraduate student at NC ASM Branch meetings.
Last year’s winner: Stephanie Lambeth
University of North Carolina Chapel Hill
Polysaccharide degrading enzymes in Agrobacterium tumefaciens
A check for $100 will be given for each of these awards at the conclusion of the meeting.
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Meeting Organization Committee / NC ASM Officers
Seán O’Connell
President-elect
Western Carolina University
Marty Roop
President
East Carolina University
Melanie Lee-Brown
Past-president
Guilford College
Jim Brown
Secretary
NC State University
Geraldine Luginbuhl
Treasurer
NC State University
Ece Karatan
Councilor
Appalachian State University
Ed Swords
Alternate Councilor
Wake Forest University
Joe Wolf
Historian
Peace College
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