NC ASM 2012
North Carolina American Society for Microbiology
2 0 12 M e e t i n g
North Carolina State University
6 O c t o b e r 2 0 12
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NC ASM 2012
Schedule
Preliminaries
8:00
Registration
Poster and talk set-up
Coffee break
Award Committees meeting/organization
9:00
Amy Grunden
Welcome & Introductory comments
Session 1: Jim Brown, Chair
9:15
Floyd Inman III
Liquid Media Development for the Mass Production of the Entomoparasitic Nematode Heterorhabditis bacteriophora
9:30
Rinu Kooliyottil
Critical Aspects in the Mass Production of Beneficial Nematodes
9:45
Len Holmes
Mass Production of the Beneficial Nematode Heterorhabditis
bacteriophora Utilizing Fermentation Technology
10:00
Poster session 1 (Odd numbered posters should be attended)
Coffee break
Exhibitors & Vendors
Session 2: Eric Anderson, Chair
11:00
Michael I. Betteken
Oxygen Induced Resistance to Tert-butyl Hydroperoxide is Mediated by Dps in Bacteroides fragilis
The Brucella abortus Irr Protein Directly Regulates the Transcription of Genes Encoding for Iron Uptake Systems, and the
Iron Responsive Activity of Irr Is Dependent upon an Internal
Heme Binding Motif
11:15
David A. Martinson
11:30
Elhassanny Ahmed
FtrABCD is a Ferrous Iron Transporter that Is Required for Virulence in Brucella abortus 2308
11:45
Clayton C. Caswell
A Novel Small Regulatory RNA is Regulated by and Potentially
Regulates the General Stress Response in Brucella abortus 2308
12:00
Lunch
Unattended Poster Session
Exhibitors & Vendors
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Schedule
Session 3: Wrennie Edwards, Chair
1:00
Maria C. White
Equine Herpesvirus Type 1 (EHV-1) Mediated Oncolysis and Viral
Cell to Cell Spread in Human Glioblastoma Multiforme Cells Are
Enhanced by Valproic Acid
1:15
Syed Sultan
Motility but Not the Possession of Periplasmic Flagella is Crucial
for the Enzootic Life Cycle of Borrelia burgdorferi
1:30
Yajuan Lin
Estimating the Genotype Specific in situ Activity of Prochlorococcus from rRNA Content and Cell Size
NC Invitational Talk: Seán O’Connell, Chair
Manganese Is the New Black: Microbial Biogeochemistry of Mn in
Southern Appalachian Caves
1:45
Suzanna Bräuer
2:15
Poster session 2 (Even numbered posters should be attended)
Coffee break
Exhibitors & Vendors
ASM Branch Lecture: Amy Grunden, Chair
3:15
Nancy E. Freitag
From Soil to Cytosol: The Pathogenic Transition of the Environmental Bacterium Listeria monocytogenes
Postscript
4:15
Amy Grunden
Concluding remarks
Awards
4:30
Jim Brown
Business meeting
Officer elections
5:00
Adjournment
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Meeting Sponsors
The American Society
for Microbiology
North Carolina
State University
Sigma Xi
North Carolina
Academy of Science
Association of
Southeastern Biologists
Life Technologies
Advanced Microscopy
Group
Nikon
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Abstracts (talks)
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1.1) Liquid Media Development for the Mass Production of the Entomoparasitic Nematode Heterorhabditis bacteriophora
Floyd Inman III and Len Holmes
Department of Chemistry and Physics, UNC-Pembroke
Entomoparasitic nematodes (Heterorhabditis spp. and Steinernema spp.) and their entomopathogenic
bacterial symbionts are being commercialized as a safer alternative to chemical insecticides. For commercialization, these beneficial nematodes must be mass produced on a large scale to obtain sufficient
quantities. Culturing techniques can either be performed on solid media or in liquid suspension; however,
rearing on solid media is costly, labor intensive and produce insufficient numbers of nematodes. Since
rearing on solid media results in low quantities of nematodes, it is not an ideal culturing technique for
nematode mass production. Therefore, in theory, mass production in a liquid medium will result in a larger
number of nematodes if the optimal culturing conditions are provided (i.e. media conditioning with the
bacterial symbiont Photorhabdus luminescens). In this study, an enriched liquid medium was developed
to support the growth of both H. bacteriophora and its bacterial symbiont P. luminescens and compared
to a patented liquid medium. This newly developed animal based medium was able to support nematode
mass production and reduce the amount of time needed to obtain sufficient nematode quantities. Final
nematode densities obtained ranged from 1,000 to 65,000 nematodes per milliliter depending on the provided medium and the density of the nematode inoculum. Furthermore, final nematode densities obtained with the optimized were at least two-fold higher than reported densities of other utilized media. The
optimized media also reduced the culturing process by an average of one week when compared to other
reported media (4 weeks).
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1.2) Critical Aspects in the Mass Production of Beneficial Nematodes
Rinu Kooliyottil, Floyd Inman, and Len Holmes
Department of Chemistry and Physics, UNC-Pembroke
Extensive use of chemicals in agriculture is negatively impacting natural environments due to soil and
water contamination. Biological control agents (such as beneficial nematode) have shown to be an attractive organic alternative for controlling a wide range of crop insect pests. Photorhabdus and Xenorhabdus
(symbiotic bacteria of entomoparasitic nematodes of Heterorhabditis and Steinernema, respectively) are
enteric bacteria that have evolved intriguing lifestyles of nematode symbiosis and insect pathogenicity.
The bacterial symbionts are carried monoxenically in the intestine of infective juvenile stage nematodes,
where these nematodes serve as vectors for dispersing their bacterial symbionts from insect to insect.
Mass production of these beneficial nematodes is a huge challenge because of the delicate symbiosis
involved. Other complex factors that affect the mass production process include but are not limited to
nematode and bacterial physiologies, strain specificities, bacterial phase variation and nematode copulation. Successful commercialization of these biocontrol agents is only possible if high nematode densities
in submerged cultures are obtained. To date, knowledge about mass production technologies is proprietary; therefore, hindering scientific advancement and other commercialization opportunities. It is the main
goal of our laboratory to utilize fermentation technology to redevelop and optimize the nematode mass
production process.
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1.3) Mass Production of the Beneficial Nematode Heterorhabditis bacteriophora Utilizing Fermentation
Technology
Len Holmes and Floyd Inman III
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Department of Chemistry and Physics, UNC-Pembroke
Nematodes that are insect parasites are being mass produced and commercialized as biological control
agents to control populations of crop insect pests. Entomoparasitic nematodes (EPNs) provide several
advantages over chemical insecticides as they are 1) organic; 2) able to persist in the applied environment as insect hosts are available; 3) not harmful to domesticated animals or humans and 4) found globally in soil. Mass production of EPNs is cumbersome and expensive on solid media; however, several
challenges have been identified rearing EPNs in liquid media. Do to the extreme difficulty of liquid culturing; EPN producers are not willing to reveal their trade secrets of mass production and thereby hindering
scientific advancement. This study reveals how the use of principles and applications of fermentation
technology can be utilized to ease the difficulties of the mass production process in submerged culture.
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2.1) Oxygen Induced Resistance to Tert-butyl Hydroperoxide is mediated by Dps in Bacteroides fragilis
Michael I. Betteken and C.J. Smith
Department of Microbiology and Immunology, ECU
Bacteroides fragilis, an obligate anaerobe, is a member of the indigenous intestinal microflora but is the
most commonly isolated anaerobe from infections in humans. Typical infections with B. fragilis include
complications such as abdominal abscesses, perforated and gangrenous appendicitis, skin and soft tissue infections, endocarditis, and bacteremia. In order to cause infection, B. fragilis must resist oxidative
stress and express a variety of virulence factors. Increased oxygen tension at the site of infection affects
the survival of anaerobes and in B. fragilis this environmental stress is diminished by the induction of an
oxidative stress response that includes catalase, Dps, alkylhydroperoxide reductase, and the thioredoxin
TrxD. This results 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 diffusion assays with 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 oxyR deletion mutant still demonstrated
complete resistance to tert-butyl peroxide whereas an oxyR and dps double deletion mutant demonstrated extreme sensitivity even after aerobic incubation. These results were confirmed by survival assays of
cells exposed to tert-butyl hydroperoxide. The results suggest that there is a second oxygen inducible
transcriptional activator of dps present in B. fragilis which mediates this resistance in the absence of
OxyR. The development of a transposon mutagenesis screen utilizing a dps reporter gene fusion is currently underway to determine the identity of this second regulator.
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2.2) The Brucella abortus Irr protein directly regulates the transcription of genes encoding for iron uptake
systems, and the iron responsive activity of Irr is dependent upon an internal heme binding motif
Martinson, DA and Roop RM
Department of Microbiology and Immunology, 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 alpha-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 high levels of iron for their function. An isogenic B. abortus irr mutant produces significantly
less siderophore molecules when grown under iron limiting conditions compared to the wild type strain.
Intracellular iron levels are also lower in an irr mutant than that in the wild type strain. We have deterPage 6
NC ASM 2012
mined, through real-time RT PCR that numerous genes encoding for iron uptake systems are misregulated in an irr mutant. Irr binding motifs (ICE Box) are present in the promoter regions of a number of
the genes encoding for these iron uptake systems. Electrophoretic mobility shift assays are being used to
determine the ability of Irr to directly regulate these genes by binding to their promoter regions. The iron
responsive activity of Irr is unique, in that when intracellular iron levels are high, Irr is degraded and can
no longer function as a transcriptional regulator. An internal HXH heme binding motif that is highly conserved among the alpha-proteobacteria is required for iron dependent degradation of Irr in B. abortus. We
are presently exploring the mechanism behind the HXH dependent iron responsive degradation of Irr in B.
abortus in an effort to better understand how Irr coordinates the expression of iron metabolism genes.
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2.3) The use of natural products to augment the oncolytic activity of vesicular stomatitis virus
Dylan Fehl, Kristin Johnson and Maryam Ahmed
Department of Biology, Appalachian State University
A promising cancer treatment modality is the use of oncolytic viruses to kill cancer cells. We are interested in developing the M51R M protein mutant of VSV as a candidate oncolytic agent due to its capacity to
effectively kill cancer cells and its low virulence in vivo. However, prostate cancer cell lines like PC-3 are
relatively resistant to infection with rM51R-M virus. Studies have shown that the transcription factor NF-κB
is constitutively active in PC-3, suggesting a role for NF-κB in induction of the antiviral immune response
and invasiveness in PC-3 cells. We hypothesize that the inhibition of NF-κB in cancer cells will render
them susceptible to oncolytic VSV. Our goal is to determine the ability of natural compounds known to
inhibit NF-κB (flavokavain B and curcumin) to suppress the antiviral immune response and promote VSV
induced cell killing of cancer cells. Our results show that pretreatment of PC3 prostate cancer cells with
flavokavain and curcumin inhibited the phosphorylation and activation of STAT1, a key player in the antiviral immune response. Furthermore, curcumin, but not flavokavain B, potentiated VSV-induced oncolysis after 24 hours post-infection. Neither curcumin nor flavokavain B decreased viral protein expression in
vitro, as measured by 35S-Methionine metabolic labeling. Additionally, both curcumin and flavokavain B
upregulated expression of the pro-apoptotic factor Bax in conjunction with VSV. These results suggest
that natural products have the potential to synergize with oncolytic VSV therapies by inhibiting antiviral
responses and promoting cell death. Ongoing studies will determine the mechanisms by which these
products promote anti-cancer effects and aid in oncolytic therapies.
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2.4) A novel small regulatory RNA is regulated by and potentially regulates the general stress response in
Brucella abortus 2308.
Clayton C. Caswell, Sean Crosson, and R. Martin Roop II
Department of Microbiology and Immunology, ECU
The ability of bacteria to cope with stress conditions is an essential component of their survival. The αproteobacterium Brucella abortus, which infects both animals and humans, must sense and react to a
variety of harsh conditions, including oxidative and low pH stresses. These bacteria have evolved an intricate genetic circuitry that responds to general stress. This system activates the transcription of genes
whose products function to protect the bacterium from these harsh conditions. The extracytoplasmic
function sigma factor RpoE1 is a central regulator of the general stress response in B. abortus, and we
have recently identified a small regulatory RNA (sRNA) whose expression is absolutely dependent upon
RpoE1. In a B. abortus strain containing an in-frame deletion in rpoE1, the expression of this sRNA,
called Bsr6, is abolished. Moreover, the promoter of bsr6 contains a consensus RpoE1 activation motif,
providing evidence for a direct link between bsr6 transcription and RpoE1. Current efforts are aimed at
determining the role of Bsr6 in 1) the ability of B. abortus to withstand oxidative and acid stress and 2) the
virulence of B. abortus in macrophages and mice. The regulatory targets of Bsr6 are being defined using
microarray and quantitative proteomic analyses. Overall, these studies will characterize the role of Bsr6
in the B. abortus general stress response, and will also define the role of this sRNA in the biology and
virulence of Brucella.
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3.1) Equine herpesvirus type 1 (EHV-1) mediated oncolysis and viral cell to cell spread in human glioblastoma multiforme cells are enhanced by valproic acid
Maria C. White, Michael J. Courchesne, and Arthur R. Frampton, Jr.
Department of Biology and Marine Biology, UNC-Wilmington
Glioblastoma multiforme is the most common form of primary brain tumor in humans. This aggressive
cancer is highly refractory to conventional modes of treatment, including radiotherapy, chemotherapy, and
surgical resection. Viral oncolytic therapy offers an alternative means to combat malignancies that are
unresponsive to these standard treatment options. Our group has previously shown that the cytolytic animal virus equine herpesvirus type 1 (EHV-1) could effectively infect and kill a panel of human glioma cell
lines to a varying degree. Antibody blocking assays showed that the virus uses human major histocompatibility complex class 1 (MHC-I) to enter these cells, and the degree of glioma infectivity was positively
correlated with MHC-I expression. This current study aimed at enhancing the oncolytic potential of EHV-1
on two glioma cell lines that were moderately infected and killed by the virus. We demonstrate that the
histone deacetylase inhibitor valproic acid (VPA) synergizes with EHV-1 and leads to significant augmentation in viral entry, replication, cell to cell spread, and cell lysis. Expression of the MHC-I receptor was
also increased after VPA treatment, suggesting that this may be one mechanism by which VPA enhances
EHV-1 mediated cell killing. A library of fractions derived from marine microorganisms was then screened
in an effort to identify novel compounds that could synergize with EHV-1 and increase viral mediated oncolysis. Currently, the hits obtained from this initial screen are being further investigated.
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3.2) Borrelia burgdorferi requires cyclic-di-GMP to switch from glucose to glycerol metabolism for survival
in the tick phase of the enzootic cycle
Elizabeth A. Novak, Syed Z. Sultan, Michael R. Miller, Melissa J. Caimano, and M.D. Motaleb
Department of Microbiology and Immunology, ECU
Borrelia burgdorferi cycles between disparate environments of the Ixodes scapularis tick vector and
mammalian host. During transmission between hosts, B. burgdorferi detects changes in its environment,
such as pH, CO2, nutrient availability, and temperature and responds appropriately by modulating its
gene expression. Cyclic-di-GMP is primarily instrumental in orchestrating the adaptation of B. burgdorferi
to the tick environment. B. burgdorferi possesses only one set of cyclic-di-GMP-metabolizing genes (one
diguanylate cyclase and two distinct phosphodiesterases) and one cyclic-di-GMP-binding PilZ-domain
protein designated as PlzA. PilZ proteins were found to alter the expression of several genes, including
glycerol uptake and utilization (gap) genes, and have also been shown to act as a transcriptional or translational regulator in other bacteria. Our lab has previously demonstrated that while PlzA-deficient spirochetes exhibited a modest defect in the mouse host, they were markedly deficient in surviving in fed ticks.
We show by qRT-PCR and iTRAQ that expression of gap genes (bb0240-bb0243) and other proteins
were differently expressed in ΔplzA. Interestingly, Pappas et al. demonstrated the essential requirement
of glycerol for fitness of B. burgdorferi in the tick host. Thus, it is possible that the decreased survivability
of ΔplzA in its tick host may be due, in part, to the down-regulation of genes utilized for glycerol metabolism at a time when they are essential because glucose, the preferred carbohydrate, is limited. Currently,
we are investigating if constitutive expression of gap genes in ΔplzA will restore the survivability of the
mutant spirochetes in the tick vector. We are also determining if PlzA directly regulates the expression of
gap as well as other potential genes that may be important in the viability of B. burgdorferi in the arthropod. These studies may lead to the identification of mechanisms critical to the transmission and virulence
of B. burgdorferi during the enzootic cycle. 88
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3.3) Estimating the genotype specific in situ activity of Prochlorococcus from rRNA content and cell size
Yajuan Lin, Erik R. Zinser, Veronica P. Lance, and Zackary I. Johnson
Division of Marine Sciences and Conservation, 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. While much is known
about the abundance and genetic diversity of this genus, little is known about the activity of the different
clades in their natural environment. To address this gap, here we use the Prochlorococcus clade eHL-II
(eMIT9312), the dominant clade in most temperate and tropical open ocean waters, as a model organism
to develop and apply a tool to examine the in situ activity using rRNA content and cell size as metrics of
cellular physiology. For two representative isolates (MIT9215 and MIT9312) grown over a range of lightregulated growth conditions, rRNA cell-1 increases linearly with specific growth rate whereas cell size
indicated by side scatter (SSC) is anticorrelated. Although each strain has a unique relationship between
cellular rRNA (or cell size) and growth rate, both strains have the same strong positive correlation between rRNA cell-1SSC-1 and growth rate suggesting a conserved relationship for members of this clade
and possibly other marine microbes. Using this approach, we investigated the in situ activity of this dominant Prochlorococcus clade eHL-II in the field including two vertical profiles (0-200m) in the Pacific Ocean
and a horizontal transect from Honolulu to San Diego. We observed distinct activity patterns across different environmental gradients such as light, nutrients and temperature, and these patterns are dynamic
and uncoupled with the biomass distribution. This tool provides unique insight into the ecology of Prochlorococcus and could potentially be expanded to include other types of Prochlorococcus (or other microbes) to explore the biogeographic patterns of activity and help to understand the mechanisms driving
these patterns.
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The North Carolina Invitational speaker is Dr. Suzanna
Bräuer, Assistant Professor in the Department of Biology at Appalachian State University. I am broadly interested in the biogeochemistry, ecology and diversity of microorganisms in various environments, including rivers,
coastal margins, wetlands/estuaries, deep-sea hydrothermal vents, and caves; and I am interested in studying the
impact of these microorganisms on the biogeochemical cycling of Fe, Mn and C in the environment.
Suzanna Bräuer, Department of Biology, Appalachian State University
Manganese is the new black: Microbial biogeochemistry of Mn in southern Appalachian caves
Bräuer SL, Carmichael MJ, Carmichael SK, Johnson K, Strom A, Santelli C, Roble L and B Zorn
The upper Tennessee River Basin contains the highest density of our nation’s caves; yet, little is known
regarding speleogenesis or Fe and Mn biomineralization in these predominantly epigenic systems.
Mn:Fe ratios at some of the sites approached 1.0 representing an order of magnitude increase above
the bulk bedrock ratio and suggesting that biomineralization processes play an important role in the
formation of these cave ferromanganese deposits. Estimates of total bacterial SSU rRNA genes in fer7
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romanganese biofilms and crusts measured approximately 7x10 - 9x10 cells/g wet weight sample. In
2008, one of the Mn oxide-rich seeps bloomed in a massive Mn(II) oxidizing biofilm, anecdotally related
to a release of sewage in a nearby sinkhole. A SSU-rRNA based molecular survey of this biofilm material revealed that 21% of the 34 dominant OTUs were closely related to known metal-oxidizing bacteria
or clones isolated from oxidized metal deposits. Several different isolates that promote the oxidation of
-8
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Mn(II) compounds were obtained from this site, some from high dilutions (10 -10 ) of cave biofilm material. In contrast to studies of caves in other regions, SSU rRNA sequences of Mn-oxidizing bacterial
isolates in this study most closely matched those of Pseudomonas, Leptothrix, Flavobacterium, and
Janthinobacterium, suggesting that this site harbors a unique consortium of microorganisms, perhaps
due to exogenous nutrient inputs. Molecular methods (16S rRNA) confirmed the presence of human fecal indicators Bacteroides-Prevotella, and most probable number (MPN) assays and ion chromatography of the associated seep water confirmed nutrient enrichment at this site. From 2009-2011, the
seep exhibited a dramatic visual reduction in Mn(IV) oxide production, which was hypothesized to correlate with a decrease in fecal nutrient input. Taken together, analyses suggest that Mn(II) oxidation at
the seep was correlated with heterotrophic activity (most likely due to reactive oxygen species production), in this case due to point source nutrient loading. Preliminary phylogenetic analysis (using SSU,
LSU, and ITS rRNA) to identify Mn(II) oxidizing fungi associated with cave litter revealed the presence
of Plectosphaerellaceae sp. DCIF (growing on discarded battery), and two Genus incertae sedis (Fungal sp. YECT1, and Fungal sp. YECT 3, growing on discarded electrical tape) that are not closely related to any known Mn oxidizing species.
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The Keynote address will be given by Dr. Nancy E. Freitag,
Professor of Biology, in the Department of Microbiology and
Immunology at the College of Medicine at the University of
Illinois at Chicago. Dr. Freitag has been continuously funded
for her research on different aspects of Listeria monocytogenes pathogenesis for over 15 years. She is internationally
recognized for her work on microbe-host interactions, and she
has published more than 50 papers and book chapters while
also serving as a member of journal editorial boards that include Infection and Immunity and Molecular Microbiology. Dr.
Freitag has been a member or ad hoc member on numerous
NIH study section panels, and she has served as an ad hoc reviewer for international foundations such as the Wellcome
Trust. Dr. Freitag has been an invited speaker at more than 80
conferences, seminars, and colloquia, and has participated in
a number of Science Career forums organized by various foundations, community colleges, and universities. She maintains
an active interest in science education, and has been actively
engaged in high school teacher and student science research
mentoring programs, as well as serving for five years as an
ASM judge for the Intel International Science and Engineering
Fair.
Dr. Freitag’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.
From soil to cytosol: the pathogenic transition of the environmental bacterium Listeria monocytogenes
Nancy E. Freitag, University of Illinois at Chicago
Many organisms inhabit soil and water, yet a relatively small percentage of these organisms have developed the capacity to cause human disease. L. monocytogenes, long used as a model for understanding innate and adaptive immune responses and various aspects of cell biology, also serves as a
model for deciphering how a bacterium transitions from life in the soil to life within the cytosol. The
Freitag Lab has been working to define how a key transcriptional regulator known as PrfA functions as
the molecular switch that balances L. monocytogenes life as a saprophyte with life as an intracellular
parasite.
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Poster presentations
1 Anthony D. Angotti
Vibriobactin Mediated Biofilm Formation in Vibrio cholerae
2 Matt K. Bowen
Microbial Kinetics of Glucose Utilization in Photorhabdus luminescens
3 Steven R. Cockerell
Norspermidine Responsive Biofilm Development in Vibrio cholerae
4 Bridget E. Conley
Generation of Campylobacter rectus CiaB mutants
5 Katy M. Davis
Predictable Cycles and Pulse Disturbances: What Drives Microbial
Abundances in a Shallow North Carolina Estuary?
6 Maria de Oca
Temperature and Nutrient Concentrations Determine Prochlorococcus Genetic Diversity in the Surface Pacific Ocean
7 Kristen N. Delaney
Campylobacter jejuni Gene Cj0372 (Ortholog of E. coli gspS), is Temperature Regulated and Plays a Role in Cell Size and Resistance to
Oxidative Stress
8 Amy A. Devlin
Characterization of novel, secreted Borrelia burgdorferi Type I IFNstimulatory ligand(s)
9 Robert. M. Dodder
Iron Uptake as a Target for Antimicrobial Development in Pseudomonas aeruginosa
10 Keely Dulmage
Uncovering the Regulatory Relationship between Archaeal Chromosomes and Histone Proteins
11 Dylan Fehl
The Use of Natural Products to Augment the Oncolytic Activity of Vesicular Stomatitis Virus
12 Rabin Gyawali
Survival and Changes in Cell Morphology of Escherichia coli O157:H7
in the Presence of Caffeine
13 Haylea Hannah
The Role of Polysaccharide Degrading Enzymes in the Interaction of
Agrobacterium tumefaciens with Plants
14 Ashley N. Hawkins
Methanogenesis in Acidic Bogs of the Southern Appalachian Region of
North Carolina and Investigation of a Surprising Abundance of
Crenarchaeota
15 Saeed A. Hayek
Use of Sweet Potato (Ipomoea batatas) to Develop a Medium for Cultivation of Lactic Acid Bacteria
16 Salam A. Ibrahim
Development of Food Protection and Defense Educational Materials
and Workshops for Middle/High School and Undergraduate Students
17
Rushyannah KillensCade
18 Priya Jayaraman
Characterization of a recombinant Metallosphaera sedula lipase for
use in algal-based biofuel production
The Role(s) of RNA Sensing TLRs and Adapter Molecules in the Host
Defense to Borrelia burgdorferi and in B. burgdorferi RNA-mediated
Type I IFN Induction
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19 Rebecca L. Kitchener
Improved Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of Organophosphorus Nerve Agents
20 Alyse Larkin
Influence of Ocean Temperature on Prochlorococcus Genetic Diversity Gradients in the North Pacific
21 Alicia Li
Calcineurin Governs Dimorphism and Virulence of Mucor circinelloides
22 Akarsh Manne
Study of BB0367, an Rrp1-regulon, and its effect on the enzootic life
cycle of Borrelia burgdorferi
23 Stephanie L. Mathews
The Retention of Pathogenic E. coli O157 by Salad Vegetables
24 Ki Hwan Moon
Cyclic-adenosine Monophosphate (cAMP): a Potential Second Messenger in Borrelia burgdorferi
25 Tia Morgan
MtrR Regulates Two Major Lytic Transglycosylases Responsible for
Peptidoglycan-Derived Cytotoxin Release and Autolysis in Neisseria
gonorrhoeae
26 Ivan Ndamukong
The Alternative Sigma Factor and its Repressor, EcfO/Reo, Have a
Defined Regulon and Protect Bacteroides fragilis Against Prolonged
Oxidative Stress
27 Elizabeth A. Novak
Borrelia burgdorferi Requires Cyclic-di-GMP to Switch from Glucose to
Glycerol Metabolism for Survival in the Tick Phase of the Enzootic
Cycle
28 Chirayu Patel
Response of Cervical Cancer Cells to Infection with Vesicular Stomatitis VIrus
29 Steven Schreck
Characterization of a Halophilic Lipase from Chromohalobacter salexigens for Use in an Algal-based Biofuel System
30 Horia Todor
The Dynamic Regulation Of Metabolism In Response Nutrients In a
Halophilic Archaeon
31 Christopher S. Ward
DNA extraction method influences apparent diversity of marine bacterioplankton community
32 Aaron Yerke
Hunting for the collar protein seen in Borrelia burgdorferi periplasmic flagella
33 Cheuk Man Yung
Seasonal partitioning of Vibrio populations in coastal seawater
34 Bryan Zorn
Mn(II)-oxidizing fungal populations in caves of the southern Appalachian region
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Abstracts (posters)
1414
1. Vibriobactin Mediated Biofilm Formation in Vibrio cholerae
Anthony D. Angotti and Ece Karatan
Department of Biology, Appalachian State University
Vibrio cholerae transitions between a free-swimming planktonic lifestyle and a multicellular community
called a biofilm, which is used as protection from environmental hazards. Biofilm formation is influenced
by a number of factors and can be up-regulated by the polyamine norspermidine. Norspermidine also
forms the backbone of the siderophore vibriobactin, which is secreted in response to iron deficient conditions. The dual role of norspermidine as a biofilm signaling molecule and a key component of vibriobactin
suggests that there is a link between norspermidine synthesis, biofilm formation, and environmental iron
concentrations. To analyze the role of vibriobactin in biofilm formation we used confocal microscopy and
biofilm assays. These analyses utilized mutants defective in vibriobactin transport (viuA::tetR) or synthesis (∆vibF) and were assayed in iron-replete (LB) and iron-deplete (100 µM EDDA) conditions. Our results
show that biofilm cell density is not significantly different between wild-type V. cholerae, viuA::tetR, and
∆vibF over a period of 1-4 days in 100 µM EDDA. Although overall biofilm cell density does not seem to
be affected by iron availability, confocal microscopy experiments using a Live-Dead staining kit show that
biofilm architecture is altered substantially in both viuA::tetR and ∆vibF mutants compared to wild-type
under iron-deplete conditions over a period of 1-4 days. These results show a link between vibriobactin
and biofilm development in V. cholerae.
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2. Microbial Kinetics of Glucose Utilization in Photorhabdus luminescens
Matt K. Bowen, Floyd Inman III, and Len Holmes
Chemistry and Physics, UNC-Pembroke
Photorhabdus luminescens is a Gram-negative enteric bacterium that is symbiotically associated with the
insect parasitic nematode Heterorhabditis bacteriophora. Upon entrance into the insect hemocoel, the
nematodes release P. luminescens into the insect hemolymph where the bacteria proliferates causing
insect mortality within 24-48 hours. Bacterial proliferation occurs rapidly due to the available glucose concentration (190 mg L-1) within the insect hemolymph. Microbial kinetics was employed to determine glucose utilization of P. luminescens to understand bacterial proliferation within the insect as a function of
glucose. In this study, defined media containing various concentrations of glucose (1.8 – 198 mg L-1) was
used to determine specific growth rates of P. luminescens for respective glucose concentrations. Monod
substrate kinetics was utilized to determine the maximum specific growth rate (0.96 h-1) and the substrate saturation coefficient (2.2 mg L-1) for glucose utilization in P. luminescens. Furthermore, the results
of this study suggest that P. luminescens may be proliferating at its maximum specific growth rate within
the insect based upon hemolymph glucose concentrations. Additional research is needed to determine if
the maximum specific growth rates for readily available preferred substrates can be correlated to virulence or pathogenicity.
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3. Norspermidine responsive biofilm development in Vibrio cholerae
Steven R. Cockerell, Samuel S. Pendergraft and Ece Karatan
Biology Department, Appalachian State University
Polyamines are small organic molecules containing two or more amine groups. The polyamine norspermidine is a positive regulator of biofilm formation in the aquatic pathogen Vibrio cholerae. The regulatory
effect of norspermidine is thought to be exerted by altering intracellular pools of the bacterial second
messenger cyclic diguanosine monophosphate (c-di-GMP). Alteration of c-di-GMP concentration is putatively affected by a signaling complex composed of NspS, a periplasmic binding protein, and MbaA, an
integral membrane protein in the GGDEF/EAL family. When MbaA is removed from the cell, biofilm inPage 14
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creases dramatically whereas when NspS is removed there was very little biofilm formation. In addition,
presence of both of these proteins is required for the effect of norspermidine on biofilms. These results
support the hypothesis that NspS binds norspermidine and then interacts with MbaA to downregulate its
phosphodiesterase activity. To test this hypothesis, Scanning Fluorimetry was used to determine if NspS
was able to bind norspermidine. Addition of norspermidine to purified NspS caused a large shift in the
thermal stability of NspS indicating a binding event. Next, co-Immunoprecipitation experiments were performed which showed that MbaA and NspS do interact in the cell. Our work presents evidence for the first
norspermidine-responsive signaling system in bacteria. As a future direction, enzymatic assays using purified MbaA will demonstrate the ability or inability of MbaA to act as a phosphodiesterase.
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4. Generation of Campylobacter rectus CiaB mutants
Bridget E. Conley, Erin Harrell, Kristen N. Delaney, Deborah S. Threadgill
Department of Microbiology, NCSU
Campylobacter rectus is a Gram negative anaerobic oral pathogen involved with periodonitits which has
been linked to preterm delivery and low birth weight in pregnant women. Campylobacter invasion antigen
B (CiaB) is a conserved hypothetical protein sharing homology with Campylobacter jejuni CiaB which aids
in invasion to allow pathogenesis. In order to better understand the activity of CiaB in C. rectus and its
role in periodonitis, a mutant was created by disrupting the CiaB gene. Invitrogen’s Gateway cloning system was used to create a vector containing three fragments, an upstream pre-CiaB fragment, a spectomycin resistance cassette and a downstream post-CiaB fragment. Electroporation of C. rectus ATCC
33238 was performed to transform the plasmid which allowed a homologous recombination event to occur. The disruption of the endogenous CiaB gene with spectomycin resistance was confirmed by sequencing.
1515
5. Predictable cycles and pulse disturbances: what drives microbial abundances in a shallow North Carolina estuary?
Katy M. Davis, T. Phillips, C.S. Ward, C.M. Yung, Z.I. Johnson and D.E. Hunt
Division of Marine Science & Conservation, Duke University
The composition and temporal dynamics of the heterotrophic and autotrophic micro-plankton assemblage
is important in determining nutrient and carbon fluxes in estuarine systems. Using flow cytometric analysis we investigated the community dynamics of heterotrophic bacteria and three categories of phytoplankton in the Newport river estuary over the course of a year. Fixed-point sampling of the microbial community and environmental parameters at hourly, daily, and weekly intervals allowed us to capture much of
the variability in the shallow, tidally-flushed estuary. Our sampling also captured pulse disturbances such
as storm events that potentially introduce large amounts runoff from agricultural and urban developments
at the head of the estuary. We observed two to 100-fold changes in phytoplankton and bacterial abundance within a two-week period when sampling at the daily scale. Seasonally, microbial abundance exhibited a positive correlation with temperature and was lowest during the winter months of December
through February. Interestingly, Hurricane Irene (August 2011) did not exert strong influence on the seasonal microbial dynamics in spite of increased nutrient levels following the storm. Instead, we hypothesize that tidal advection and changes in water residence time shape the bacterioplankton and phytoplankton community. Identifying the drivers of estuarine microbial community dynamics is essential in predicting how organisms and biogeochemical cycles in the Newport River estuary will respond to disturbances
and seasonal changes.
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6. Temperature and nutrient concentrations determine Prochlorococcus genetic diversity in the surface
Pacific Ocean
Maria de Oca, Yajuan Lin, and Zackary Johnson
Division of Marine Science & Conservation, Duke University
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Prochlorococcus, the most abundant photosynthetic microbe in the open ocean, is comprised of multiple
genetic clades, but the breadth of this diversity and the environmental variables that regulate it are poorly
understood. We investigated the genetic diversity of Prochlorococcus in surface waters of the Equatorial
Pacific Ocean during a cruise from August to September 2006 covering a longitudinal transect from
140°W to 140°E and between 2°N and 2°S (EUCFe) representing multiple environmental gradients to
uncover relationships between genetic diversity and the environment. DNA was extracted from samples
of 19 stations and 16S-23S ribosomal DNA Internal Transcribed Spacer sequences were amplified and
aligned with a custom Prochlorococcus ecotype database. Multivariate analysis was used to infer the
patterns of association between ecotype abundance and environmental variables. High light clade II ecotypes (HLII) were numerically dominant throughout the transect, and the type strain found in the highest
numbers was MIT9301. Stations were clustered in two major groups according to their similarity in ecotype abundance. This classification was found to be significantly correlated with the temperature and nutrient concentrations of the water masses encountered: the Pacific Warm Pool and the Pacific Cold
Tongue. The latter region was found to support a more genetically diverse Prochlorococcus population,
composed of low and high light ecotypes in high relative abundances. These data indicate that, when light
conditions are constant, temperature and nutrients determine ecotype distribution in the ocean surface
waters.
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7. Campylobacter jejuni gene Cj0372 (ortholog of E. coli gspS), is temperature regulated and plays a role
in cell size and resistance to oxidative stress.
Kristen N. Delaney, Jason M. Andrus, Angelika Jährig, Jinzhi Wang, Alain Stintzi, 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. 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 and 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. 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. 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. Glutathione regulates the oxidative state of the cellular environment by reducing reactive oxygen species. When Cj0372 is disrupted the resulting mutant is more sensitive to oxidative stress
than the parental strain. In addition to the original mutant strain, we recently generated the same mutation in an additional C. jejuni strain and have been confirming the phenotypes for loss of the putative
GspS in this genetic background. Additionally we have investigated a single mutant of E. coli gspS as well
as an E. coli triple mutant defective for both gspS and two gspS-like genes to determine if the pleiotropic
effects seen in C. jejuni gspS mutants are replicated in the E. coli mutants.
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8. Characterization of novel, secreted Borrelia burgdorferi Type I IFN-stimulatory ligand(s)
Amy A. Devlin, Jennifer C. Miller
Department of Microbiology, NCSU
Borrelia burgdorferi (Bb) causes Lyme disease, the most prevalent arthropod-borne disease in the US.
60% of untreated patients develop arthritis that is associated with the presence of bacteria within the joint.
We recently found that Bb-elicited Type I IFN production enhanced murine Lyme arthritis severity, without
impacting host defense. We recently identified multiple Bb-associated macrophage Type I IFN-stimulatory
ligands, including products released by Bb into the culture supernatant. High speed ultracentrifugation of
Bb culture supernatants was performed to remove potential bacterial membrane blebs. Bb RNA, itself an
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IFN-stimulatory ligand, did not govern the IFN-stimulatory activities mediated by ultracentrifuged Bb supernatant. Ultracentrifuged Bb supernatant did not contain the dominant lipoprotein Osp A or other Borrelial membrane proteins. Ultracentrifugation of Bb supernatant did not alter its IFN-stimulatory capacity, as
IFN-responsive genes were still induced in mouse bone marrow-derived macrophages (BMDMs). These
data indicate that IFN-stimulatory activity of ultracentrifuged Bb supernatant is not attributable to Bb genomic RNA, or to lipoprotein-rich membrane blebs released by lysed Bb. Cyclic-di-GMP, a bacterial second messenger, has been shown to up-regulate Type I IFN production in multiple cell types. Stimulation
of BMDMs with Bb cyclic-di-GMP pathway members indicated that cyclic-di-GMP is dispensable in the
Type I IFN response. The IFN-stimulatory capacity of Bb culture supernatant ligand(s) was reduced following heat inactivation/proteinase K treatments, suggesting that these Bb-derived ligand(s) are proteins.
The IFN-stimulatory capacity of ultracentrifuged Bb supernatant was retained following serum-depletion
treatments aimed at reducing the biological complexity of the serum-rich culture medium. Current efforts
are focused on anion-exchange chromatography, reverse-phase C18 liquid chromatography, and 2D-gel
electrophoresis to further fractionate ultracentrifuged Bb supernatant, prior to LC-MS²-based identification
of these proteins. Uncovering these new IFN-stimulatory ligand(s) will prompt new therapeutic strategies
that could lessen the symptoms of arthritis patients without interfering with Lyme disease antibiotic treatment regimens.
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9. Iron Uptake as a Target for Antimicrobial Development in Pseudomonas aeruginosa
Robert. M. Dodder, Ryshonda P. Lewis, Alvin B. Lu, Eladio Camarillo Jr., Scotti D. Tootle, Brittany A.
Rogers and Eric S. Anderson
Department of Biology, ECU
Approximately 30,000 children and adults are affected by cystic fibrosis in the United States. Pseudomonas aeruginosa is an opportunistic pathogen that establishes persistent biofilms in the lungs of these patients resulting in severe and often fatal respiratory infections. Like most bacteria, Pseudomonas requires iron and captures it from the environment using small secreted compounds called siderophores.
Biosynthesis and uptake of one of these siderophores, pyochelin, is controlled using the AraC-like regulator, PchR. In this pathway, the siderophore serves as a co-activator for its own biosynthesis when bound
to iron. Metabolically, this represents a very efficient system. When iron levels are low, pyochelin is produced at a basal level. If ferric iron is present, pyochelin captures the metal and returns it, signaling the
presence of a viable iron source. This increases production of that specific uptake pathway. If pyochelin
returns without iron, basal production continues and other acquisition systems are utilized to acquire sufficient levels of this essential metal. This pathway presents an attractive target for antimicrobial development. These antimicrobials would not only be pathogen specific, but would also stimulate their own uptake, thus increasing their efficacy even further. The net result would be an antimicrobial that is highly
effective at very low concentrations. Our work focuses on the ability of gallium-complexed pyochelin (GPc) to target this pathway. Gallium (Ga3+) is a metal with an ionic radius similar to ferric iron (Fe3+) and
can be incorporated into iron binding sites in the bacterium. However, gallium is toxic, as it is not able to
be reduced, a critical property for iron to act as a biological cofactor. Preliminary experiments indicate
that G-Pc is 1000x more effective than gallium alone at killing liquid cultures of P. aeruginosa. We are
currently examining the effect of G-Pc on both initial biofilm formation and established biofilms.
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10. Uncovering the regulatory relationship between archaeal chromosomes and histone proteins.
Keely Dulmage, Claudia Ofori-Marfoh, and Amy Schmid
University Program in Genetics and Genomics, Duke University
In all three domains of life, organisms employ non-specific DNA-binding proteins to compact and organize
the genome as well as to regulate transcription. Our current understanding of genome organization in
archaea is shallow and often contradictory. Although most methanogens encode histone proteins, only
class 1 methanogenic archaea compact the chromosome into nucleosomes. On the other hand, class 2
methanogens primarily employ the archaeal nucleoid protein MC1. We ask if halophilic archaea require
histone proteins for genome compaction and whether this plays a part in regulating genome-wide tranPage 17
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scription. This is significant because the halophilic archaea employ a unique “salt-in” strategy in order to
adapt to high salt conditions, resulting in an intracellular environment approaching 5M potassium. Therefore we theorize that DNA, a highly charged species, is likely under unusual constraints. In order to better
understand genome organization in the halophiles, we knocked-out HpyA, the sole putative histone protein encoded in the genome of Halobacterium salinarum. Preliminary mass spectrometry analysis of the
DNA/protein complexes suggests that it may not constitute the major portion of DNA-bound protein. However, light microscopy, sucrose gradient ultracentrifugation, and growth curve studies indicate that HpyA
is important for many cellular functions and is possibly a global regulator of transcription.
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11. The use of natural products to augment the oncolytic activity of vesicular stomatitis virus
Dylan Fehl, Kristin Johnson and Maryam Ahmed
Department of Biology, Appalachian State University
A promising cancer treatment modality is the use of oncolytic viruses to kill cancer cells. We are interested in developing the M51R M protein mutant of VSV as a candidate oncolytic agent due to its capacity to
effectively kill cancer cells and its low virulence in vivo. However, prostate cancer cell lines like PC-3 are
relatively resistant to infection with rM51R-M virus. Studies have shown that the transcription factor NF-κB
is constitutively active in PC-3, suggesting a role for NF-κB in induction of the antiviral immune response
and invasiveness in PC-3 cells. We hypothesize that the inhibition of NF-κB in cancer cells will render
them susceptible to oncolytic VSV. Our goal is to determine the ability of natural compounds known to
inhibit NF-κB (flavokavain B and curcumin) to suppress the antiviral immune response and promote VSV
induced cell killing of cancer cells. Our results show that pretreatment of PC3 prostate cancer cells with
flavokavain and curcumin inhibited the phosphorylation and activation of STAT1, a key player in the antiviral immune response. Furthermore, curcumin, but not flavokavain B, potentiated VSV-induced oncolysis after 24 hours post-infection. Neither curcumin nor flavokavain B decreased viral protein expression in
vitro, as measured by 35S-Methionine metabolic labeling. Additionally, both curcumin and flavokavain B
upregulated expression of the pro-apoptotic factor Bax in conjunction with VSV. These results suggest
that natural products have the potential to synergize with oncolytic VSV therapies by inhibiting antiviral
responses and promoting cell death. Ongoing studies will determine the mechanisms by which these
products promote anti-cancer effects and aid in oncolytic therapies.
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12. Survival and changes in cell morphology of Escherichia coli O157:H7 in the presence of caffeine
Rabin Gyawali, Alani Adkins, Radiah Minor, and Salam A. Ibrahim
Family and Consumer Sciences, NCA&T SU
Illnesses resulting from consumption of food contaminated with pathogenic bacteria such as Escherichia
coli O157:H7 has been of serious concern to public health. There is a growing consumer demand for natural methods to control such foodborne pathogens. Caffeine can be used in food products as natural preservatives. The objective of this study was to investigate the survival and changes in cell morphology of
E. coli O157:H7 in the presence of caffeine in a laboratory medium and in skim milk. In addition, flow cytometry was applied to detect changes in the shape of E. coli O157:H7 in the presence of caffeine. The
inhibitory effect of caffeine at different concentrations was determined by inoculating E. coli O157:H7 in
tryptic soy broth (TSB) broth and skim milk samples. Samples were incubated at 37ºC for 48 h, and survival and growth of E. coli O157:H7 population was enumerated by plating onto trypticase soy agar. Our
results showed that caffeine significantly (P < 0.05) inhibited the growth of E. coli O157:H7 in laboratory
media (TSB) and milk samples. A greater than 3.0 log cfu/mL inhibition was observed in milk containing
0.5% caffeine within 12 h of incubation. Moreover, using flow cytometry, marked changes in the morphology of E. coli O157:H7 also were observed. Caffeine has potential as an antimicrobial agent and could be
used as an effective natural preservative to improve the safety of food products.
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13. The Role of Polysaccharide Degrading Enzymes in the Interaction of Agrobacterium tumefaciens with
Plants
Haylea Hannah, Stephanie L. Mathews, Ann G. Matthysse
Department of Microbiology, UNC-CH
Agrobacterium tumefaciens causes crown gall tumors in plants. These tumors result from the expression
of a bacterial DNA fragment (T DNA) transferred to the plant cell. The bacterium transfers its T DNA using a type IV secretion system to cross the bacterial membranes, cell wall, and the plant plasma membrane. The type IV pilus must also cross the plant cell wall but little is known about the mechanism by
which this occurs. Bacterial enzymes, produced by A. tumefaciens, which degrade plant cell wall components may aid in this process. To explore this possibility, mutations in genes predicted to encode glycosyl
hydrolases were constructed and characterized for virulence, binding to various sites on tomato roots,
growth on various polysaccharides, and enzymatic activity. Mutations in the genes Atu3104, Atu3129,
Atu3312, Atu4560, and Atu4561 were all avirulent on Bryophyllum diagremontiana (B. diagremontiana).
Of these genes, only a mutation in Atu3312 was virulent on tomato stems. Additionally, a mutation in
Atu4560 resulted in reduced virulence on carrots while mutations in Atu3129 and Atu4561 were virulent.
The protein products of these genes were tested for enzymatic activity. Atu3104 appears to encode an
arabinosylfuranosidase. Atu3129, the protein encoded by the gene Atu3129, was able to degrade β-1-4
linkages, while Atu4560 was not. A mutation in Atu3312 was only able to colonize the lower root, and
showed little to no growth at other sites on or near the root. Atu4560 did not grow as well as the wild type
in the soil surrounding the root. Based on the findings presented above, each of these genes plays a distinct role in interaction with plant surfaces and some may be involved in determining host range.
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14. Methanogenesis in Acidic Bogs of the Southern Appalachian Region of North Carolina and Investigation of a Surprising Abundance of Crenarchaeota
Ashley N. Hawkins, and Suzanna L. Bräuer
Cell and Molecular Biology, Appalachian State
Methanogenesis from wetland archaea is a major source of methane emissions and, as a result, a major
influence on levels of this potent greenhouse gas in the atmosphere. The process of methanogenesis,
when performed biologically, involves specialized archaea of the Euryarchaeal phylum termed methanogens. Methanogens are only able to utilize a narrow range of substrates in the biochemical pathway leading to methane production and as such, occupy specialized ecological niches of which wetlands are a
major component. In order to fully understand the underlying processes governing the production and
release of methane from these understudied ecosystems, this study focuses on the effects that several
different variables, present in most wetlands, have on the archaeal community composition present in the
environment and their ability to produce methane. Gas chromatography analyses are being utilized to
quantify the amount of methane being produced from each of three separate sample sites over the
course of a three week period during each of the four seasons. During the course of the seasonal study,
archaeal clone libraries were built as well in order to characterize the community composition of each site
as well as the changes it might experience seasonally. Preliminary results from this analysis reveal a surprisingly high number of Crenarchaeota present in the bogs. This over-abundance is currently being investigated with quantitative PCR (qPCR). In addition to seasonal variations, an influence of vegetation
type is being revealed through gas chromatography as well as qPCR studies. A fourth component of the
research currently being conducted is the detection of ARMAN-like (Archaeal Richmond Mine Acidophilic
Nanoorganisms) sequences present in one study site upon initial screening. Such organisms are also
members of the Euryarchaea and were initially discovered in acidic mine drainage in 2006 along with
subsequent sequence identification in acidic bog sites of Southern Finland.
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15. Use of Sweet potato (Ipomoea batatas) to develop a medium for cultivation of lactic acid bacteria
Saeed A. Hayek, Abolghasem Shahbazi, and Salam A. Ibrahim
Energy and Environmental Sciences, NCA&T SU
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Sweet potatoes (Ipomoea batatas) are rich with many nutrients including carbohydrates, vitamins, and
minerals that could support the growth of lactic acid bacteria (LAB). The objective of this study was to develop a medium for LAB using sweet potato as a basic component. Fresh sweet potatoes were baked at
400°C for 1 h, cooled, peeled, cut, and blended with 50% (w/v) distilled water, and centrifuged then supernatant was collected to form the sweet potato medium (SPM). A group of components including: sodium acetate (5g), potassium monophosphate (buffer) (2g), ammonium citrate (2g), Tween 80 (1ml), magnesium sulfate (0.2g), and manganese sulfate (0.05g) were added to 1 L of SPM then divided into two
portions of 500 ml for NSPM and PSPM. PSPM was enhanced with beef extract 1%, yeast extract 0.5%,
and peptone 1%. MRS was prepared then SPMs and MRS were autoclaved for 15 min at 121°C. Nine
strains of LAB were individually inoculated at an average of 2.27±0.44 log CFU/ml into batches of MRS,
PSPM, and NSPM. Samples were incubated for 48 h at 37°C and bacterial growth was monitored using
turbidity reading, pH value, and bacterial population count CFU/ml. Our results showed no significant (P >
0.05) differences between LAB growth rates in MRS and PSPM whereas NSPM showed slightly slower
growth rate. After 48 h of incubation, LAB populations reached 11.34±0.45 log CFU/ml in MRS,
11.74±0.36 log CFU/ml in PSPM, and 10.88±0.31 log CFU/ml in NSPM. These finding indicated that
sweet potatoes can support the growth of LAB and could be used as a low cost medium for laboratorial
and industrial applications.
2020
16. Development of food protection and defense educational materials and workshops for middle/high
school and undergraduate students
Salam A. Ibrahim, Marcella Cheek-Crook, Rabin Gyawali, Saeed A. Hayek, and Mehradad Tajkarimi
Family and consumer Sciences, NCA&T SU
In order to protect American food supplies, there is a need to educate our middle/high school and undergraduate college students in food and agriculture-related fields about food protection and defense. The
food and nutritional sciences at North Carolina A&T State University has received several educational
grants to develop instructional materials for middle/high and undergraduate students and to start a new
food protection and defense curriculum. Three food defense and protection courses have been approved
for teaching as part of the food science curriculum. A new educational manual was also developed and
used in these educational activities. Our educational activities demonstrated the public’s interest in learning about the importance of food protection. Most of the students participating in these activities showed
interest in taking more formal training in this area and would recommend these educational activities to
others. The food and nutritional science at North Carolina A&T State University plan to expand these activities and include graduate students.
2020
17. Characterization of a recombinant Metallosphaera sedula lipase for use in algal-based biofuel production
Rushyannah Killens-Cade, Rachel Turner, Christine MacInnes, and Amy Grunden
Department of Microbiology, NCSU
Lipid-producing microalgae are emerging as the leading platform for producing alternative biofuels in response to diminishing petroleum reserves. Optimization of fatty acid production is required for efficient
conversion of microalgal fatty acids into usable transportation fuels. Microbial lipases can be used to enhance fatty acid production because of their efficacy in catalyzing hydrolysis of esters into alcohols and
fatty acids while minimizing the potential poisoning of catalysts needed in the biofuel production process.
Although studies have extensively focused on lipases produced by mesophilic organisms, our understanding of lipases produced by thermophilic, acidic tolerant microbes, such as Metallosphaera sedula, is
limited. It is likely that enzymes produced by M. sedula will exhibit high specific activities at high temperatures (>70°C) and acidic pHs (<5). These characteristics will be important for the conversion of microalgal
fatty acids/lipids into transportation fuel because of temperature and pH conditions used for fuel conversion. The primary goal of this study was to recombinantly express the M. sedula gene Msed_1072, which
encodes a thermoactive lipase, in Escherichia coli to enable its biochemical characterization. It is hypothesized that this lipase will be more stable at elevated temperatures and acidic pHs compared to homologous mesophilic lipases, which has important implication for its use in the lipid to fuel conversion process.
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The gene encoding Msed_1072 was cloned into the pET-28a and pET21-b expression plasmids for fusion with an N-terminal (Msed_1072Nt) and C-terminal (Msed_1072Ct) histidine-tag, respectively. Recombinant protein was expressed in E. coli strain BL-21, purified using affinity chromatography, and biochemically characterized using a variety of substrates over a range of temperatures and pH and in the
presence of metal ions, organic solvents, and detergents. Msed_1072Nt and Msed_1072Ct demonstrated
enzymatic activity over a range of temperatures and pH as well as tolerance of different metal ions, organic solvents, and detergents.
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18. The role(s) of RNA sensing TLRs and adapter molecules in the host defense to Borrelia
burgdorferi and in B. burgdorferi RNA-mediated Type I IFN induction
Priya Jayaraman, Amy A. Devlin, and Jennifer C. Miller
Department of Microbiology, NCSU
The causative agent of Lyme disease, Borrelia burgdorferi, causes a multitude of symptoms, including
arthritis. Lyme arthritis develops in 60% of untreated patients, and is correlated with the presence of bacteria within a large joint. The critical roles of TLR2 and MyD88 in host defense to Borrelia burgdorferi have
been well documented. TLR2-/- mice contain hundreds additional B. burgdorferi, whereas MyD88-/- mice
harbor thousands of spirochetes within their ankle joints, compared with the burden present within wildtype joints. This implies that other TLR(s) may participate in the host defense to B. burgdorferi. We recently discovered that B. burgdorferi RNA is a potent Type I IFN-stimulatory ligand for mouse bone marrow-derived macrophages (BMDMs). We examined whether TLR7, a MyD88-dependent receptor for single-stranded RNA, was required for clearance of B. burgdorferi from mouse ankle joints. Equivalent numbers of B. burgdorferi were detected by quantitative real-time PCR within the ankle joints of wildtype and
TLR7-/- C57BL/6 (B6) mice sacrificed at 4 weeks post-infection. Type I IFN-stimulated gene transcript
(ISG) levels were also assessed within B6 and TLR7-/- mouse joints at 1-week post-B. burgdorferi infection. While B6 ankle joint cells did not induce the Type I ISG Oasl2, its transcription was markedly upregulated within TLR7-/- ankle joints. Type I ISG transcript levels were induced, to similar levels in wildtype B6
and TLR7-/- BMDMs following stimulation with either live B. burgdorferi or B. burgdorferi RNA. Taken together, our data indicate that TLR7 is not required for Borrelia burgdorferi clearance from mouse ankle
joints or Type I IFN-responsive gene induction. Current studies are focused on the role(s) of the TLR
adapter molecules MyD88 and TRIF in the Bb RNA-mediated BMDM Type I IFN response. These results
will lead to the development of improved therapeutic agents for the treatment of Lyme disease.
2121
19. Improved Catalytic Activity of Hyperthermophilic Pyrococcus horikoshii Prolidase for Detoxification of
Organophosphorus Nerve Agents
Rebecca L. Kitchener, Casey M. Theriot, Saumil S. Shah, and Amy M. Grunden
Department of Microbiology, NCSU
Prolidase is a multifunctional enzyme that hydrolyzes the unique bond found in X-Pro dipeptides and can
also degrade organophosphorus (OP) compounds rendering them harmless by cleaving their P-F or P-O
bonds. Initial characterization of Pyrococcus horikoshii prolidase homolog 1, or Ph1prol, showed that this
enzyme is more catalytically active over a broader temperature and pH range than both P. horikoshii
prolidase (Phprol) and Pyrococcus furiosus prolidase (Pfprol). Ph1prol is also more thermostable and
has a higher affinity for metal than its previously studied counterparts. In an effort to further optimize
Ph1prol performance against OP compounds such as sarin and soman, randomly mutated enzymes were
prepared, of which four were selected for characterization based on their increased activity at lower temperatures. Biochemical characterization of Ph1prol and the four variants (A195T/G306S-, Y301CK342N-,
E127G/E252D- and E36V-Ph1prol) has been completed, and Ph1prol and its variants have superior enzymatic attributes when compared to Phprol and Pfprol for the purpose of OP compound degradation.
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20. Influence of Ocean Temperature on Prochlorococcus Genetic Diversity Gradients in the North Pacific
A. Larkin, Y. Lin, and Z.I. Johnson
Division of Marine Science and Conservation, Duke University
In the oligotrophic regions of the world’s oceans Prochlorococcus, a unicellular cyanobacteria, comprises
21-43% of the photosynthetic biomass and accounts for 13-48% of the net primary production, thus contributing substantially to marine biogeochemistry. However, increasing temperatures due to climate
change could result in major shifts in the geographic distribution and genetic structure of Prochlorococcus, potentially affecting open ocean ecology. To better understand the relationship between environmental temperature and Prochlorococcus community structure, we collected DNA from surface samples
on two ~4000 km ocean transects crossing major sea surface temperature gradients in the North Pacific
Ocean. The first transect ran from Seattle, WA to Honolulu, HI in October 2011 and the second ran from
Honolulu, HI to San Diego, CA in March 2012. The internal transcribed spacer (ITS) region between 16S
and 23S, a highly variable region previously shown to distinguish ecologically distinct clades of marine
cyanobacteria, was amplified and the products were pyrosequenced (271,168 total reads, 328 bp mean
length). The two transects showed a significant difference (p<0.01) in operational taxonomic unit (OTU)
abundance. However, both transects had similar trends in the distribution of Prochlorococcus strains
across the North Pacific. For the HI to CA cruise, the transition between taxonomic zones as well as the
peak in OTU abundance occurred at approximately 18-19°C. Additionally, OTU abundance was signifi2
cantly correlated with temperature (R =0.9143). These data correspond with laboratory experiments
showing 18-19°C to be the transition point between the two dominant clades of Prochlorococcus, eHLII
(eMIT9312) and eHLI (eMED4). These data suggest that changing ocean temperatures may have a substantial effect on the population structure and genetic diversity of Prochlorococcus in the North Pacific
Ocean and ultimately its role in ocean ecology and biogeochemistry.
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21. Calcineurin Governs Dimorphism and Virulence of Mucor circinelloides
Alicia Li, Soo Chan Lee, Joseph Heitman
Molecular Genetics and Microbiology, Duke University Medical Center
Fungal dimorphism is evolutionarily conserved in many lineages in the fungal kingdom. The dimorphic
transition is associated with pathogenicity of pathogenic fungi. However, in human pathogenic zygomycetes, our understanding of dimorphism and virulence is limited. Mucor circinelloides is a human pathogenic zygomycete that exhibits dimorphism. Under certain conditions, such as high levels of oxygen, Mucor grows as a filamentous fungus. Under other conditions, such as high levels of carbon dioxide, Mucor
2+
exhibits yeast growth. Interestingly, we found that the Ca calmodulin-dependent serine/threoninespecific protein phosphatase calcineurin controls this morphogenic transition. In the presence of FK506,
a specific calcineurin inhibitor, Mucor exhibits only multi-budded yeast growth, and thus phenotypically
mirrors the yeast growth under microaerobic and high CO2 conditions. Mucor has three calcineurin catalytic A subunits, CnaA, CnaB, and CnaC, and one calcineurin regulatory B subunit, CnbR. Our studies
focused on the underlying genetics in the yeast-hyphal transition by gene disruption of cnbR, which is
essential for calcineurin activity. Through transformation and homologous recombination, we obtained
two independent cnbR mutants. The cnbR mutants grow exclusively as yeast. This observation provides
compelling evidence that calcineurin regulates the dimorphic transition in Mucor. In virulence tests using
Galleria larvae as a heterologous host we found that cnbR yeast cells are significantly less virulent than
the wild type, indicating that the hyphal growth phase of Mucor is more virulent than the yeast form. Our
findings demonstrate that calcineurin governs the dimorphic transition and virulence in Mucor.
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22. Study of BB0367, an Rrp1-regulon, and its effect on the enzootic life cycle of Borrelia burgdorferi
Akarsh Manne and MD. Motaleb
Department of Microbiology and Immunology, ECU
Lyme disease, the most common arthropod-borne infection in the United States, is caused by the spirochete Borrelia burgdorferi. In nature, B. burgdorferi has an obligate enzootic cycle involving a mammalian
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host reservoir and an Ixodes tick vector. There is abundant evidence that B. burgdorferi adapts to these
disparate milieus by altering its transcriptional, antigenic, and metabolic profiles in response to host- and
tick-specific signals. The complex changes associated with “host adaptation” are orchestrated, in part, by
the spirochete’s 2 two-component systems—one of which is Hpk1-rrp1 that operates through the signaling molecule c-di-GMP. Rrp1 is the response regulator that synthesizes c-di-GMP when phosphorylated,
presumably by Hpk1. Others and our laboratory have demonstrated that c-di-GMP is crucial for the tickphase of the spirochete’s life cycle. The Rrp1-regulon includes genes that are responsible in metabolic,
chemotaxis, and transport pathways. Rrp1-deficient spirochetes are unable to survive in ticks, a required
function in order to transmit infection to a mammal. We postulate that Δrrp1 cells are unable to survive in
the tick environment as it modulates the expression of genes required for alternative carbon sources.
bb0367, a gene considered to function as a putative phosphotransferase for carbohydrate metabolism
was significantly down-regulated in Δrrp1. To demonstrate if bb0367 can restore the survivability of Δrrp1
spirochetes in ticks, it was constitutively expressed using the flgB-promoter in Δrrp1. Ticks containing wild
type, ∆rrp1, or ∆rrp1/PflgB-bb0367 were assayed to determine the spirochetes load per tick over a sevenday period. However, ∆rrp1/PflgB-bb0367 cells were unable to restore the viability of the mutants in ticks.
The Rrp1-regulon consists of 10% of the genome required for survivability in ticks; therefore, the phenotype of ∆rrp1/PflgB-bb0367 cells could have been similar to the ∆rrp1 cells. Our laboratory is continuing
to study the function of bb0367 in the enzootic life cycle of B. burgdorferi.
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23. The Retention of Pathogenic E. coli O157 by Salad Vegetables
S. L. Mathews, R. B. Smith and A. G. Matthysse
Department of Microbiology, NCSU (Presenting work from UNC-CH)
This study examines the importance of plant species and plant organ or part in the retention of pathogenic E. coli O157 by salad vegetables by measuring the number of viable bacteria retained by washed plant
parts. E. coli was rapidly retained by the cut edges of leaves and fruits. Retention by the intact epidermis
was slower than by cut edges in all cases and depended more on the organ examined than the plant
species. E. coli was retained by the cut leaf edge more rapidly and in greater numbers than Agrobacterium tumefaciens or Sinorhizobium meliloti; suggesting that the rate of retention and number of bacteria
retained depended on the bacterial species tested. Both E. coli O157:H7 and K12 were retained by cut
leaves and fruits of a variety of plant species. Wounding increased the number of bacteria retained by
leaves and fruits but not sprouts. Although the characteristics of bacterial retention by fruits and leaves
were similar for the species examined, the effects of mutations in exopolysaccharide synthesis differed
between fruits and leaves suggesting that there may be differences in the mechanisms involved. Understanding the role of plant species and organ in the retention of E. coli O157:H7 by plants will aid in the
development of methods to reduce contamination of salad vegetables with these bacteria.
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24. Cyclic-adenosine monophosphate (cAMP): a potential second messenger in Borrelia burgdorferi
Ki Hwan Moon and MD. Motaleb
Department of Microbiology and Immunology, ECU
Bacteria alter their gene expression by using signal transduction pathways which are mediated by a variety of second messengers. Borrelia burgdorferi, the causative agent of Lyme disease, is exposed to disparate host environments comprised of an Ixodes tick vector and a vertebrate host. This organism regulates its gene expression profile by utilizing c-di-GMP, the only second messenger signaling system currently known in this pathogen. B. burgdorferi lacks classical adenylate cyclases in its genome; however, it
does encode for an unrelated adenylate cyclase, BB_0723 (a putative cyaB), which is a class IV adenylate cyclase. In order to verify the existence of a functional cAMP signaling system in B. burgdorferi, a
ΔcyaB mutant strain was constructed to measure intracellular cAMP concentration. Furthermore, the adenylate cyclase activity of CyaB will be analyzed by performing a cAMP enzyme assay on recombinant B.
burgdorferi CyaB expressed and purified from Escherichia coli. If the B. burgdorferi CyaB is found to be
functional, other components of the cAMP signaling system, such as a cAMP-specific phosphodiesterase
and cAMP-receptor protein (CRP) will be investigated. cAMP is known to have a central role in regulating
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motility and virulence in several other bacterial species. Therefore, we will perform swarm plate assays to
determine if cAMP plays a role in motility. Additionally, we will test to determine not only if the ΔcyaB
cells are able to infect C3H/HeN mice by needle inoculation, but also the requirement of cAMP for survival in the tick vector and transmission from the arthropod to the mammalian host. Together, these results
will reveal the existence of a functional cAMP signaling system and, its contribution to motility and the
enzootic life cycle of B. burgdorferi.
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25. MtrR Regulates Two Major Lytic Transglycosylases Responsible for Peptidoglycan-Derived Cytotoxin
Release and Autolysis in Neisseria gonorrhoeae
Tia Morgan, Ronald R. McMillan, Daniel Williams
Department of Biology, NCCU
The multiple-transferable resistance protein (MtrR) is a transcriptional repressor of the mtrCDE-encoded
drug efflux system and Type IV pilus biosynthesis genes and activator of PBP1 expression in Neisseria
gonorrhoeae. More recently, published microarray data showed that MtrR is an activator of ltgA expression in gonococci. LtgA is a lytic transglycosylase responsible for recycling approximately half of the cell
wall in GC and releasing peptidoglycan-derived cytotoxins, which cause epithelial damage and elicit specific inflammatory cytokine responses. Based on the previous microarray data, we decided to further define MtrR’s capacity to regulate ltgA and ltgD. Although ltgD was not detected by the microarray, we decided to determine whether it is MtrR regulated since ltgD is responsible for the remaining portion of recycled cell wall. To this end, we examined ltgA and ltgD mRNA-transcription and LtgA protein levels. We
determined that MtrR directly increases ltgA expression, resulting in increased LtgA levels and increased
ltgD expression in gonococci. Disruption of mtrR reduced PG monomer release and cell lysis, which suggests that MtrR regulation of ltgA and ltgD may impact gonococci’s ability to recycle and release peptidoglycan and modulate autolysis during growth. Thus, the study provides insight as to how lytic transglycosylases are regulated to ensure cell growth without inducing lysis and release of peptidoglycan-derived
cytotoxins.
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26. The Alternative Sigma Factor and its Repressor, EcfO/Reo, Have a Defined Regulon and Protect
Bacteroides fragilis Against Prolonged Oxidative Stress
Ivan Ndamukong, Jason Gee, Anita Parker, Samantha Palethorpe, Jeffrey Smith
Department of Microbiology and Immunology, ECU
Bacteroides fragilis is an important component of the human intestinal microbiota. It thrives predominantly
in the colon, which provides a favorable anaerobic environment and a rich source of complex carbohydrate substrates. A complex symbiotic relationship with the host is maintained under these conditions.
When Bacteroides escape the gut’s anaerobic environment into more aerated sites such as the peritoneal
space, significant pathological states such as abscess formation and bacteremia develop. Aerotolerance
has been linked to a robust oxidative stress response which in turn is necessary for maximal virulence in
a mouse intra-abdominal abscess model. During oxidative stress there is a dynamic change in gene expression that encompasses a third of the genome but there is a paucity of information on factors that control this response. A large number of transcription regulators including a set of about 15 structurally diverse extracytoplasmic function (ECF) sigma factors are induced by oxidative stress and one of these,
EcfO, was used as a model of ECF sigma factor activity during this stress. Genetic and biochemical experiments show that EcfO was located in an operon with a structurally unique antisigma factor, Reo. Cells
deleted for EcfO were impaired during exposure to oxygen or other forms of oxidative stress whereas reo
mutants were more resistant to the stress. Protein-protein interaction experiments demonstrated that Reo
directly interacts with and regulates the activity of EcfO. Expression microarray and chromatin affinity precipitation assays were used to identify target genes regulated by EcfO and an EcfO recognition sequence
was identified. The results revealed EcfO controls a regulon of novel lipoproteins whose distribution in
nature is restricted to members of the Bacteroidetes phylum. The role of these genes in the context of
oxidative stress resistance and the pathogenesis of abscess formation is under investigation.
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27. Borrelia burgdorferi requires cyclic-di-GMP to switch from glucose to glycerol metabolism for survival
in the tick phase of the enzootic cycle
Elizabeth A. Novak, Syed Z. Sultan, Michael R. Miller, Melissa J. Caimano, and M.D. Motaleb
Department of Microbiology and Immunology, ECU
Borrelia burgdorferi cycles between disparate environments of the Ixodes scapularis tick vector and
mammalian host. During transmission between hosts, B. burgdorferi detects changes in its environment,
such as pH, CO2, nutrient availability, and temperature and responds appropriately by modulating its gene
expression. Cyclic-di-GMP is primarily instrumental in orchestrating the adaptation of B. burgdorferi to the
tick environment. B. burgdorferi possesses only one set of cyclic-di-GMP-metabolizing genes (one
diguanylate cyclase and two distinct phosphodiesterases) and one cyclic-di-GMP-binding PilZ-domain
protein designated as PlzA. PilZ proteins were found to alter the expression of several genes, including
glycerol uptake and utilization (gap) genes, and have also been shown to act as a transcriptional or translational regulator in other bacteria. Our lab has previously demonstrated that while PlzA-deficient spirochetes exhibited a modest defect in the mouse host, they were markedly deficient in surviving in fed ticks.
We show by qRT-PCR and iTRAQ that expression of gap genes (bb0240-bb0243) and other proteins
were differently expressed in ΔplzA. Interestingly, Pappas et al demonstrated the essential requirement of
glycerol for fitness of B. burgdorferi in the tick host. Thus, it is possible that the decreased survivability of
ΔplzA in its tick host may be due, in part, to the down-regulation of genes utilized for glycerol metabolism
at a time when they are essential because glucose, the preferred carbohydrate, is limited. Currently, we
are investigating if constitutive expression of gap genes in ΔplzA will restore the survivability of the mutant
spirochetes in the tick vector. We are also determining if PlzA directly regulates the expression of gap as
well as other potential genes that may be important in the viability of B. burgdorferi in the arthropod. These studies may lead to the identification of mechanisms critical to the transmission and virulence of B.
burgdorferi during the enzootic cycle.
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28. Response of cervical cancer cells to infection with vesicular stomatitis virus
Chirayu Patel and Maryam Ahmed
Department of Biology, Appalachian State University
Vesicular stomatitis virus (VSV) is currently being studied as a candidate oncolytic agent due to its ability
to induce apoptosis in a variety of cancer cells. Previous studies have shown that matrix (M) protein mutants of VSV, such as rM51R-M virus, act as selective anti-cancer agents by targeting cancer cells while
sparing normal cells. Our goal is to promote the use of VSV for the treatment of cervical cancers. The
cervical cancer cell line Siha has been shown in previous studies to be permissive to infection and killing
by VSV. We hypothesized that cervical cancer lines are sensitized to VSV due to blockage of the type-1
interferon (IFN) response by human papillomavirus (HPV) oncoproteins. However, our results indicated
that Siha cells retained their ability to respond to type I IFN. Furthermore, in contrast to previous studies,
we observed that Siha cells were sensitive to killing by both wild-type (wt) and M protein mutant VSV
(rM51R-M virus) only when infected at high multiplicities of infection. In addition, cells were more sensitive to killing by rM51R-M virus than wt VSV. In conclusion, our results show that Siha cells are resistant
to VSV infection, perhaps due to the maintenance of intact antiviral responses. We are currently investigating this effect in different cervical cancer cell lines and developing combination therapeutic strategies
with natural compounds to augment VSV-induced oncolysis of cervical cancers.
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29. Characterization of a Halophilic Lipase from Chromohalobacter salexigens for Use in an Algal-based
Biofuel System
Steven Schreck, Rushyannah Killens, Amy M. Grunden
Department of Microbiology, NCSU
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In recent years, environmental, geo-political, and economic forces have brought into sharp focus problems intrinsic to conventional, petroleum based energy sources. To combat these problems, countries
have increasingly turned to alternative energy sources, including ethanol and bio-diesel. While promising, these "first generation" alternative energy sources are unable to scale to meet the heavy consumer
demand for transportation fuel and often carry environmental detriments of their own. Thus, stepwise
improvements in production efficiency, cost and scale are needed to ensure that significant cost and
environmental savings may be realized. Towards this end, researchers are investigating ways to leverage biotechnology to engineer fuel producing microorganisms such as oil-producing microalgae. One
method to increase algal oil production involves the use of lipases which are able to increase oil production efficiency by cleaving algal lipids into free fatty acids, which can be used directly in existing
fuel conversion processes. The research project described here focused on the recombinant expression
of the Chromohalobacter salexigens gene Csal_26202 (TesA homolog – CsTesA), which encodes for a
halophilic lipase in an effort to biochemically characterize the lipase for its application in marine algal
biofuel production. CsTesA was cloned into both the pET-21b and pQE1 plasmids resulting in Cterminal or N-Terminal histidine tagged proteins, respectively. An untagged enzyme was generated by
post purification removal of the N-terminal tag. Recombinant proteins were expressed in Escherichia
coli strain BL-21 or M15[pRep4], purified using affinity chromatography, and were biochemically characterized using a variety of substrates over a range of temperatures, pH and salinity. Both tagged and
untagged enzymes showed enzymatic activity over a range of temperatures, pH, and salt concentrations, with the untagged enzyme providing higher specific activity than either of its tagged counterparts.
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30. The dynamic regulation of metabolism in response nutrients in a halophilic archaeon
Horia Todor, Kriti Sharma, Adrianne Pittman, Olga Ilkayeva, Chris Newgard, Amy Schmid
Department of Biology, Duke
Halobacterium salinarum is a hypersaline archaeon found in the Great Salt Lake. In its environment, H.
salinarum is constantly exposed to seasonal and daily fluctuations in nutrients, UV radiation, oxygen, and
salinity. Surviving these varied and extreme environments requires unique adaptations such as dynamic
restructuring of the gene regulatory network (GRN) to integrate varied stimuli into a coherent response. A
central regulatory node in the GRN of H. salinarum is the TrmB transcription factor, which has been
shown to regulate 113 genes in central carbon metabolism and biosynthesis of purines, cobalamin, and
thiamine. The TrmB-DNA interaction is regulated by nutrient availability. We hypothesize that glucose
acts as an integrator of diverse metabolic pathways, working through TrmB to regulate metabolism dynamically in response to nutrient fluctuations in the high salt environment. To test this hypothesis, we integrated mRNA levels of 100 genes with ChIP-qPCR measurements of TrmB binding site occupancy and
metabolite dynamics in response to a glucose stimulus. Using an ODE model of TrmB regulation, we
were able to establish the importance of TrmB to the regulation of specific genes, as well as establish the
presence or absence of secondary regulators. We found that TrmB mediates varied dynamic responses
to nutrient fluctuations. When overlaid on a metabolic map, these dynamic responses form clusters of
biological significance, confirming the importance of TrmB to metabolic balance.
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31. DNA extraction method influences apparent diversity of marine bacterioplankton community
Christopher S. Ward, Zackary I. Johnson, and Dana E. Hunt
Division of Marine Science and Conservation, Duke University
Over the past two decades, microbial ecologists have increasingly relied on culture-independent methods
to characterize complex environmental communities. Molecular analysis of bacterial diversity is possible
via sequencing of 16S rRNA or other universally conserved genes. However, non-quantitative extraction
of populations within the sample leads to underestimation of bacterial diversity. In order to ensure the reliability of diversity estimates, we evaluated several extraction methods for retrieving bacterioplankton
DNA from seawater filters. Using Ion Torrent 16S rRNA gene amplicon pyrosequencing, we found that
there was compositional dissimilarity between the three extraction methods, as determined by betaPage 26
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diversity metrics. Sucrose buffer lysis followed by phenol-chloroform extraction yielded substantially higher percentages of marine cyanobacteria Synechococcaceae and chloroplast sequences from unicellular
eukaryotes Stramenopila and Chlorophyta, suggesting better extraction efficiencies of difficult-to-lyse algal cells. Since the other methods resulted in lower cyanobacterial contributions to the total extracted
DNA pool, less prevalent taxa were over-represented. These findings illustrate the effect of differential
extraction efficiencies on community diversity analysis and indicate the need for determining the appropriate DNA isolation method for a particular sample type.
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32. Hunting for the collar protein seen in Borrelia burgdorferi periplasmic flagella
Aaron Yerke, Xiaowei Zhao, Jun Liu and Md. A. Motaleb
Department of Microbiology & Immunology, ECU
The most prevalent vector-borne disease in the United States is Lyme disease, and is caused by the spirochete, Borrelia burgdorferi. Carried by Ixodes ticks, infection with B. burgdorferi can cause numerous
symptoms such as erythema migrans, cardiac anomalies, Lyme arthritis, and nervous system disorders.
Motility is crucial for B. burgdorferi’s virulence –its ability to disseminate from the bite site in the skin to the
colonization sites (joints, heart, and nervous system), and migration within ticks. Our goal is to further our
understanding of the mechanism of B. burgdorferi’s motility by examining the periplasmic flagellar motor
rotation or assembly. Several of the main proteins in this apparatus are still unidentified. An example of
this is the collar, a structure unique to spirochetes. In Cryo-electron tomography (Cryo-ET), B. burgdorferi’s periplasmic flagellar apparatus, the structure appears as a large collar-shaped organelle encompassing p-ring. The p-ring is a bushing that houses the flagellar rod in the peptidoglycan.
B. burgdorferi’s fliZ is a gene of unknown function with no significant homology to other bacterial species.
It is located in an operon consisting of genes encoding predominantly periplasmic flagellar biosynthesis
proteins. Given its location, plus the fact that it is exclusively found in spirochetes, fliZ had a high likelihood of being a flagellar protein and possibly the collar, so, it was inactivated by homologous recombination. Once inactivated and confirmed, the mutant bacteria were observed under a dark-field microscope
and their flagellar apparatus was examined using Cryo-ET. The mutant cells were motile and had a flatwave morphology and possessed the collar structure seen in the wild-type cells. No observable differences between the fliZ-knockout and wild-type bacteria were detected. These results indicate that fliZ
does not encode the collar protein. We are investigating this mutant to identify if it exhibits phenotype related to periplasmic flagellar assembly or motility.
27
33. Seasonal partitioning of Vibrio populations in coastal seawater
CM Yung, KD Davis, MK Vereen, A Herbert, A Kantorowska, DE Hunt
Division of Marine Science and Conservation, Duke University
Temperature is suggested to be one of the most important factors in structuring bacterioplankton communities and populations. This study uses culturable vibrio as a model system to study the seasonal microbial population dynamics in coastal sea surface water. The temporal population dynamics among vibrio
strains was studied by using biweekly time series seawater sampling taken from East Pivers Island,
Beaufort, NC from Sept 2011 to Jan 2012 (water temp range 9.6°C to 27.3°C). A partial hsp60 gene
maximum likelihood tree reveals that there are distinct population distributions varying by season, yet
there are several clades of closely related strains isolated from different temperature. To further examine
the relationship between isolation temperature and temperature of maximal growth, we have measured
growth rates over a temperature range for six closely related strains with identical hsp60 alleles, isolated
at three time points, where the seawater temperature was 10°C, 15.6°C, or 27.3°C. We measured growth
rates for these strains in glucose minimal media at a range of temperature (8 temperatures spanning 836°C). We were able to identify differences in growth rate and thermal niche width. In general, strains isolated from warmer temperature grow faster than hose from colder temperature when the incubation temperature is above 16°C, but warmer strains have a narrower growth range. This suggests vibrio may
demonstrate an adaptive trade-offs in thermal performance.
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34. Mn(II)-oxidizing fungal populations in caves of the southern Appalachian region
Zorn B, Carmichael SK, Santelli C, Roble L, Carmichael MJ and Bräuer SL
Department of Biology, Appalachian State University
The cycling of manganese (Mn) in the environment remains relatively poorly characterized when compared with other metals such as iron (Fe). Fungi of the Ascomycota have been observed under scanning
electron microscopy (SEM) to collect Mn (predominantly Mn(IV) oxides) on the periphery of vegetative
hyphae as well as at sites of cellular differentiation upon sporocarp formation. Recent studies indicate that
at least some oxides may form by reaction of Mn(II) with reactive oxygen species (ROS). Ultimately the
oxides may provide a protective barrier or assist in the capture of other miscellaneous trace metals, although the exact evolutionary functions are unclear. In this study, three southern Appalachian epigenictype caves are being examined for fungal diversity using 18S ribosomal RNA (rRNA) clone libraries as
well as culturing analyses of isolates exhibiting Mn oxidation potential. qPCR data of Mn-oxide rich crusts,
clay and biofilm samples indicate that fungi are present in low abundance (<1%) in all locations sampled
within the cave. However, field and culturing data suggest that fungi readily colonize anthropogenic litter;
a hypothesis which is currently being investigated in situ. Culturing efforts have thus far yielded members
of the orders Glomerales and Pleosporales as well as two Genus incertae sedis (Fungal sp. YECT1, and
Fungal sp. YECT3, growing on discarded electrical tape) that do not appear to be closely related to any
other known Mn oxidizing species. Preliminary metagenomic analyses of clone library data indicate that
over 88% of amplified DNA is representative of the phylum Basidiomycota (predominantly Agaricomycetes), 2.74% of Ascomycota, 2.28% of Blastocladiomycota and Chytridiomycota, 0.46% of Zygomycota,
and 3.65% of Eukarya or Fungi incertae sedis.
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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:
Rachel Krasich, Duke University, “Determining the fate of transcription
at DNA-protein crosslinks”
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:
David Martinson, East Carolina University, “Iron-dependent degradation
controls the activity of the transcriptional regulator Irr in Brucella abortus”
The Best Poster award is open to anyone presenting a poster at the NC
ASM meeting.
Last year’s winner:
Jenifer Ojeda, East Carolina University, “The bhuTUV and bhuO gene
products play vital roles in the ability of Brucella abortus to use heme
as an iron source”
The Paul Phibbs Award is awarded for the best presentation by an undergraduate student at NC ASM Branch meetings.
Last year’s winner:
Allie Amick, North Carolina State University, “"A Change Is Gonna Come"
– HEGs and the dynamic genome of F1 mycobacteriophages”
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
Amy Grunden
President-elect
NC State University
Seán O’Connell
President
Western Carolina University
Marty Roop
Past-president
East Carolina University
Jim Brown
Secretary
NC State University
Wrennie Edwards
Treasurer
Novartis
Ece Karatan
Councilor
Appalachian State University
Ed Swords
Alternate Councilor
Wake Forest University
Eric Anderson
Historian
East Carolina University
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