North Carolina American Society for Microbiology
2014
Meeting
Appalachian State
University
September 27 , 2 0 1 4
NC ASM 2014
Schedule
Preliminaries
8:00
9:00
Registration
Poster and talk set-up
Breakfast (Sponsored by Sartorius)
Awards committee meeting/organization
Vendors/Sponsors
Ece Karatan
Welcome & Introductory comments
Session 1 : Ece Karatan, Chair
9:15
Melissa Ramirez
9:30
Emily Snavely
9:45
Ahmed Elhassanny
10:00
Shaun Steele
10:15
Victoria Carpenter
10:30
Cell cycle regulation and adaptations to stress in Mycobacterium tuberculosis
Identifying type II secreted proteins during Chlamydia trachomatis infection
Regulation of the acid-responsive expression of ftrABCD, the genes
encoding a ferrous iron specific transporter, in Brucella abortus 2308
Trogocytosis-Associated Intercellular Transfer of Intracellular Pathogens
The Type III Secretion Effector, TepP induces type 1 interferon gene
activation in Chlamydia trachomatis infected cells
Poster session 1 (Even numbered posters should be attended by presenters)
Coffee Break (Sponsored by ASB-Association of Southeastern Biologists)
Vendors/Sponsors
Session 2 : Suzanna Bräuer, Chair
11:15
Walter Patterson
Comparison of Growth Kinetics Models Using Pseudomonas fluorescens
11:30
Layton Ashmore
Detection and enumeration of spoilage microbes by Real Time q-PCR
11:45
Christopher Ward
12:00
Stephanie Mathews
12:15
12:30
Melanie Lee-Brown
Episodic disturbances modify seasonal patterns of microbial community
composition in a dynamic coastal environment
Biodegradation and Bioconversion of Pulping Waste by Paenibacillus
glucanolyticus
Where is your PULSE? National Movement to Transform Life Science
Departments
Lunch (Sponsored by North Carolina Biotechnology Institute and Appalachian University College
of Arts and Sciences)
Session 3 : Maryam Ahmed, Chair
1:30
Audrey Brown
The effect of Moringa oleifera on the oncolytic activity of vesicular stomatitis
virus in cervical cancer cell lines
1:45
Jerome McKay
PD-1 suppresses protective immunity to Streptococcus pneumoniae through
a B cell-intrinsic mechanism
2
NC ASM 2014
2:00
Stephanie Johnstone
2:15
Eric Miller
2:30
3:15
3:45
Infection of equine endothelial cells with non-neurovirulent equine
herpesvirus-1 strain KyA elicits greater down regulation of inflammatory
cytokines CCL3, IL8, TGF-2β and CXCL3 compared to neurologic strain
OHIO 2003
The HHMI SEA-PHAGES program at NC State University – how your campus
can reinvigorate undergraduate STEM education using bacteriophages
Poster session 2 (Odd numbered posters should be attended by presenters)
Coffee Break (Sponsored by Alfa Aesar)
Vendors/Sponsors
Rita Tamayo
NC Invitational Talk
Regulation of Clostridium difficile colonization factors by cyclic
diguanylate
Intermission
Coffee break
Vendors/Sponsors
Awards committee meeting
Plenary session
4:00
4:15
Awards
Michael Federle
ASM Branch Lecture
Microbial Wikileaks: Bacteria that conspire to harm, and small molecules
that may protect
Postscript
5:15
Ece Karatan
5:30
Jim Brown
Concluding remarks
Business meeting
Officer election
6:00
Reception (Sponsored by WorldWide, Kapa Biosystems, ASU Graduate School, Exela)
8:00
Adjournment
We thank Eastern Carolina University Department of Biology and Department of Microbiology and
Immunology for sponsoring printing of the Conference Booklet.
3
NC ASM 2014
Sponsors & Vendors:
Cratis D. Williams
Graduate School
4
NC ASM 2014
Abstracts (talks)
1.1. Cell cycle regulation and adaptations to stress in Mycobacterium tuberculosis
Melissa V. Ramirez*, Clinton C. Dawson, Rebecca Crew**, Kathleen England***, and Richard A. Slayden
"Mycobacteria Research Laboratories, Department of Microbiology, Immunology, and Pathology, Colorado
State University
*Current affiliation: Department of Biological Sciences, NC State University
**Current affiliation: Pulmonary Division, Department of Medicine, National Jewish Health
***Current affiliation: Department of Infectious Diseases, Stanford University School of Medicine"
Despite intense research efforts, mechanisms employed by Mycobacterium tuberculosis (Mtb) for the
establishment of non-replicating persistence (NRP) are poorly understood. In order to investigate mechanisms
associated with NRP, we asked how cell cycle regulation is coordinated with induction of adaptive responses.
Based on historical work and our recent studies, we reason that cells are halted in the cell cycle at the point
of septum formation in order to establish NRP. We employed a bioinformatic approach to identify cell cycle
regulatory elements and assessed the ability of the putative regulator to halt cell cycle progression and elicit
adaptive responses. We show that rv1708 encodes an alternative Soj protein, referred to as SojMtb, and
coordinates a regulatory mechanism involved cell cycle control at the point of septum formation and
adaptive responses associated with NRP. Overexpression of sojMtb induces the MazF6 toxin component of the
MazEF6 toxin:antitoxin (TA) pair, a system that has been implicated in stress responses in bacteria. We
demonstrate that MazF6 induces bacteriostasis and is inhibited by interaction with its cognate antitoxin,
MazE6, and show a physical interaction between the two proteins in vivo. Further, we demonstrate that this
overexpression of MazF6 is capable of inducing tolerance to ethambutol, an important antibiotic for the
treatment of tuberculosis. Finally, sojMtb, mazEF6 TA pair are expressed in bacteria obtained from infected
tissues, further supporting our hypothesis that MazF6 has a role in facilitating adaptations to the alternative
environments encountered during infection. Importantly, the data provide evidence of the first coupled
regulatory system linking the MazF6 toxin with cessation of cell division via SojMtb and demonstrates that
this TA system is part of a global response involved in the adaptations leading to the establishment of and
possibly enhancing the non-replicating persistent state of Mtb.
1.2. Identifying type II secreted proteins during Chlamydia trachomatis infection
EA Snavely, BD Nguyen, and RH Valdivia.
Department of Molecular Genetics and Microbiology, Center for Microbial Pathogenesis, Duke University
Medical Center
Chlamydia trachomatis is the most common bacterial cause of sexually transmitted diseases and the leading
cause of preventable blindness worldwide. As an obligate intracellular pathogen, C. trachomatis secretes
proteins such as degradative enzymes and toxins to acquire nutrients and manipulate host cell processes via
multiple secretion mechanisms, including the type III secretion (T3S) and the type V secretion (T5S) systems.
Work in our laboratory suggests C. trachomatis also has a functional type II secretion system (T2SS). We
recently identified a C. trachomatis strain with a mutation in GspE, a conserved ATPase required for T2S.
gspE Chlamydia mutants display a five- to seven-fold reduction in the production of infectious progeny, fail
to secrete the protease CPAF into the host cytoplasm, accumulate large glycogen granules in the lumen of
the pathogenic vacuole (“inclusion”), presumably as a result of the failure to secrete a glycogen hydrolase,
and accumulate proteins in the periplasm. We hypothesized a subset of the periplasmic accumulating
proteins are T2S substrates that are unable to be secreted in this strain. Using label-free quantitative liquid
chromatography-tandem mass spectrometry (LC-MS/MS) we established a comprehensive analysis of proteins
in WT and gspE mutant bacteria. We identified numerous candidate type II secreted proteins that can
translocate to the periplasm and are preferentially accumulated in gspE mutant bacteria. Overall, these
findings indicate that Chlamydia has an active T2SS that facilitates the export of factors that contribute to
optimal bacterial growth in the intracellular environment.
5
NC ASM 2014
1.3. Regulation of the acid-responsive expression of ftrABCD, the genes encoding a ferrous iron specific
transporter, in Brucella abortus 2308
Ahmed E. Elhassanny and R. Martin Roop II
Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University
FtrABCD is a ferrous iron (Fe2+)-specific transporter that is essential for the wild-type virulence of B. abortus
2308 in experimentally infected mice. As expected, our studies have found that the expression of ftrABCD is
responsive to iron deprivation. This response is mediated by the iron response regulator (Irr), the
predominant iron-responsive regulator in Brucella and the other alpha-proteobacteria. These genes are also
induced by exposure to acidic pH in both B. abortus 2308 and an isogenic irr mutant, indicating that the ironand pH-responsive regulation of these genes are independent processes. This acid-responsive expression of
the ftr locus is important because it potentially allows the brucellae to fine-tune the expression of their iron
acquisition genes to adapt to the acidic environment they encounter in the endolysosomal Brucellacontaining vesicles, where Fe2+ is thought to be a biological relevant iron source. Phenotypic analysis of
Brucella regulatory mutants has identified two potential candidates for the acid-responsive regulator of
ftrABCD expression. The first is the two-component regulator FeuPQ, which is homologous to the BqsRS
system in Pseudomonas. BqsRS senses extracellular Fe2+. Since acidic pH favors the stability and solubility of
Fe2+, it is possible that the expression of the Brucella ftrABCD is induced in response to extracellular Fe2+
instead of acidic pH per se. The second potential acid-responsive regulator of the Brucella ftrABCD operon is
MucR, a zinc finger protein that coordinates the transcription of numerous genes in the α-proteobacteria
required for the successful endosymbiotic and pathogenic interactions of these bacteria with their animal
and plant hosts. Current studies are aimed at defining the relative roles of FeuPQ and MucR in regulating the
acid-responsive expression of the ftrABCD operon in B. abortus 2308 and determining if these regulatory links
are direct or indirect.
1.4. Trogocytosis-Associated Intercellular Transfer of Intracellular Pathogens
S. Steele (presenter), J. Brunton, S. Taft-Benz and T. Kawula
Department of Microbiology and Immunology, University of North Carolina-Chapel Hill
Franciella tularensis is a highly infectious, facultative intracellular bacterium that infects a wide range of
cell types. F. tularensis primarily replicates within host cells during infection. Yet during murine infections,
the number of cells infected rapidly exceeds the initial inoculum. Despite these data and extremely low F.
tularensis induced cell cytotoxicity, the field has assumed that F. tularensis egress from infected cells
requires cell lysis. However, we found that intracellular F. tularensis can transfer directly from cell to cell
via a novel mechanism where both the donor and recipient cell remain viable. Intercellular F. tularensis
transfer correlates with an intercellular process called trogocytosis. Trogocytosis is a process that both
eukaryotic and prokaryotic cells use to exchange functional surface exposed proteins between cells.
Furthermore, F. tularensis transfer is cell type specific, contact dependent, and cell death independent.
These data indicate that extracellular bacterial transfer is not the primary mechanism F. tularensis uses to
transfer from an infected to uninfected cell. Importantly, we observed a similar mechanism in both
Salmonella typhimurium and with magnetic beads. Thus, intercellular transfer mechanism during
trogocytosis may be exploited by many different pathogens to migrate between cells without entering the
extracellular space.
6
NC ASM 2014
1.5. The Type III Secretion Effector, TepP induces type 1 interferon gene activation in Chlamydia trachomatis
infected cells.
Victoria Carpenter, Yi-Shan Chen, and Raphael H. Valdivia
Molecular Genetics and Microbiology, Duke University
The obligate intracellular bacterial pathogen Chlamydia trachomatis is the leading cause of preventable
blindness worldwide and the most prevalent bacterial cause of sexually transmitted infections in the western
world. Despite the socioeconomic impact relatively little is known about the biology of C. trachomatis and
the way in which these bacteria modify the host cell to facilitate their intracellular survival and replication.
The manipulation of host cell processes by C. trachomatis is accomplished by bacterial proteins, termed
effectors, which are secreted directly into the host cell via the chlamydial type 3 secretion (T3S) system. The
recently described C. trachomatis effector protein Translocated early phosphoprotein (TepP) is delivered
into the host cell early in infection and is phosphorylated at multiple sites. We have previously shown that
phosphorylated TepP interacts with CT10 regulator of kinase like protein (Crk), thereby recruiting it to the
bacterial replicative vacuole during infection. Additionally preliminary data has shown that TepP activates
type 1 interferon (IFN) induced gene transcription and that TepP induces differential phosphorylation
patterns in the host cell during infection. Recently we have identified that TepP induced type 1 IFN gene
transcription occurs in a STING and cyclic-di-nucleotide dependent manner. Furthermore we have shown that
this change is dependent on the presence of Crk in the host cells. .Finally, TepP induces host cell morphology
changes which lead to the dispersion of cells in a confluent monolayer. Altogether this has led us to propose
a model in which TepP mediates cytoskeletal rearrangements through Crk, leading to the leakage of inclusion
components, including the bacterial second messenger cyclic-di-AMP, which activates type 1 IFN gene
transcription.
2.1. Comparison of Growth Kinetics Models Using Pseudomonas fluorescens
Walter Patterson, Devang Upadhyay and Leonard Holmes
Sartorius Stedim Biotechnology Laboratory, Biotechnology Research and Training Center, The University of
North Carolina at Pembroke, Pembroke, NC 28372 USA.
Pseudomonas fluorescens a soil dwelling gram negative bacillus species, was exposed to minimal media
containing limiting carbon substrates in shake flasks. The specific growth rates of the exponential phase of
the cultures were determined by means of the natural logarithm of the optical density from the given
cultures. The resulting specific growth rates were graphically compared to the concentration of the limiting
carbon substrate. The plotted data was then fitted to mathematical models to determine the half-velocity
constant (ks) for the given substrates as well as a calculated maximum specific growth rate. The models
resulting from the non-linear fits were compared. The substrates used in this study were glycerol, fructose
and glucose at varying concentrations The models used in this study were the Monod, Moser and Tessier.
7
NC ASM 2014
2.2. Detection and enumeration of spoilage microbes by Real Time q-PCR
Layton Ashmore, Dr. Seth Cohen
Department of Fermentation Science, Appalachian State University
With the rise in popularity of fermented foods and beverages, quick and reliable tools for the detection of
spoilage microbes are critical for quality control and product safety. The objective of this project is to
validate methods that allow for quick and accurate detection of spoilage microbes from various food and
beverage matrices. We compared several methods using real time PCR (RT-PCR) and real time quantitative
PCR (RT-qPCR) to determine efficacies and relative efficiencies for several common spoilage organisms in
fermented beverages. For the detection of metabolically active organisms RNA extraction and subsequent
cDNA synthesis was used for testing with genus and species specific primers. For detecting and determining
the relative abundance of organisms (active and biofact) two methods of DNA extraction were used with
genus and species specific DNA primer. A simple method involving sonication of bulk cell material (via
centrifugation) followed by DNA extraction and cleanup was compared to a method involving thermal
disruption (freezing and subsequent heating of samples) to induce lysis and direct RT-PCR analysis of cell
lysate. Both methods yielded comparable results, though the second method required less time for sample
preparation. The results of both methods were used to test primer efficiency and accuracy using samples
containing known microbes.
2.3. Episodic disturbances modify seasonal patterns of microbial community composition in a dynamic coastal
environment
Christopher S. Ward, Katherine M. Davis, Cheuk-Man Yung, Sara K. Blinebry, Zackary I. Johnson, Dana E. Hunt
Marine Science and Conservation, Nicholas School of the Environment, Duke University
Microbes are essential players in marine food webs and biogeochemical cycling. However, the field of
microbial ecology has only a basic understanding of the factors that influence microbial community
dynamics, including the relative importance of seasonal changes and episodic events. Here, we examine the
microbial community at a dynamic, coastal site (Beaufort Inlet, Beaufort NC USA) weekly over a 2.5-year
period (Jan 2011 – Aug 2013). Using 16S rRNA gene library sequencing, we observe recurring seasonal
variation in microbial community composition, largely driven by changes in the relative abundance of
dominant groups Pelagibacter, Synechococcus and Acidimicrobiales. Despite the strong seasonal pattern,
episodic disturbances to the microbial community resulted in deviations from the community trajectory. Most
notably, in Aug-Sept 2011 the composition of the microbial community changed significantly for
approximately 8 weeks following a Category 1 hurricane (Irene), which dramatically altered water chemistry
at our site. During this period, the community composition shifted towards higher evenness in its rank
abundance distribution. This is in contrast to the two subsequent years when summer and autumn
communities were less even than winter and spring communities, suggesting that this disturbance altered
normal seasonal characteristics in 2011. To further examine the impacts of episodic events, we assessed
disturbances to the microbial community by identifying short-term fluctuations in community diversity, as
measured by Bray-Curtis dissimilarity. This approach detected numerous events that caused significantly
increased rates of change in community composition, including several rainfall events of <1.5 cm that were
associated with only minor changes in measured environmental variables. These findings demonstrate that
episodic disturbances in dynamic coastal systems impact microbial communities at moderate frequency.
These disturbances modify seasonal patterns and result in more complicated and less predictable community
dynamics over both weekly and annual time scales.
8
NC ASM 2014
2.4. Biodegradation and Bioconversion of Pulping Waste by Paenibacillus glucanolyticus
Stephanie L. Mathews, Joel Pawlak and Amy M. Grunden
Department of Plant and Microbial Biology and Department of Forest Biomaterials, North Carolina State
University
The pulping material black liquor is generated by the kraft process, and this underutilized waste stream has
potential for downstream bioconversion. A microorganism was isolated from a black liquor sample collected
from the Department of Forest Biomaterials at North Carolina State University. The organism was identified
as Paenibacillus glucanolyticus using 16S rDNA sequence analysis and was shown to be capable of growth on
black liquor as the sole carbon source based on minimal media growth studies. Minimal media growth curves
demonstrated that this facultative anaerobic microorganism can degrade black liquor as well as cellulose,
hemicellulose, and lignin. High performance liquid chromatography (HPLC) and gas chromatography-mass
spectrometry (GC-MS) were used to identify the products generated by P. glucanolyticus when grown
anaerobically on black liquor. Fermentation products which could be converted into high-value chemicals
such as succinic, propanoic, lactic, and malonic acids were detected. Vanillic and gallic acids were also
produced which suggest that P. glucanolyticus can degrade lignin. GC-MS analysis of P. glucanolyticus culture
supernatant when grown on cellulose, hemicellulose, and lignin as the sole carbon source correlated the
production of fermentation products with the components of black liquor. These results indicate that P.
glucanolyticus can grow on black liquor by degrading the carbon sources that make up this pulping byproduct
and in the process produce high-value chemicals.
2.5. Where is your PULSE? National Movement to Transform Life Science Departments
Melanie J. Lee-Brown
Department of BIology and Office of Undergraduate Research & Creative Endeavors, Guilford College and
PULSE Leadership Fellow
The Partnership for Undergraduate Life Science Education (PULSE) was conceived and organized by NSF,
NIH/NIGMS and HHMI in 2012 to stimulate systemic, transformative change of undergraduate life science
education. This transformation includes all types of post-secondary educational institutions from Research 1
to Community Colleges and is based upon the 2011 report Vision and Change in Undergraduate Biology
Education: A Call to Action. Cultural and institutional change requires targeting such things as reward
structures, enhanced student learning, faculty development, teaching tools and strategies to develop and
transfer skills between colleagues. In this session you will access resources and learn about the latest
initiatives of PULSE in year two of their work. You will also find out how to get involved and raise your PULSE
at any stage in your career.
9
NC ASM 2014
3.1. The effect of Moringa oleifera on the oncolytic activity of vesicular stomatitis virus in cervical cancer
cell lines
A. Brown, N. Mowa, J. Emrani, and M. Ahmed
Department of Biology, Appalachian State University and Department of Chemistry, North Carolina A & T
State University
Extracts obtained from the plant Moringa oleifera (MO) have demonstrated anticancer and anti-inflammatory
qualities and are currently being assessed for their use in combination therapy. We hypothesize that MO will
promote killing of aggressive cancer cells by oncolytic viruses such as vesicular stomatitis virus (VSV) due to
their ability to inhibit signaling pathways in cancer cells that promote cell survival and regulate antiviral
immunity, such as the NF-κB pathway. To test this hypothesis, we determined the effect of various extracts
of MO (aqueous, butanolic, methanolic, ethanolic and hydroethanolic) on proliferation and killing of cervical
cancer cells (C4-II, SiHa and HeLa) by VSV and on stimulation of immune cells. Our results indicated that the
ethanolic extract of MO inhibited C4-II and HeLa cell proliferation and promoted the ability of a wild-type
strain of VSV (rwt virus) to kill cells. The decrease in cell viability in HeLa cells correlated with an
enhancement of virus replication in infected cells and a decrease in activation of the transcription factor,
NF-κB. The ethanolic extract of MO also stimulated immunity in infected peripheral blood mononuclear cells
by inducing maturation of plasmacytoid and myeloid dendritic cell (DC) subsets and inducing the expression
of the inflammatory cytokines, IL-6 and TNF
.
contains compounds with anticancer and immunostimulatory functions. Future studies will elucidate
upstream components of the NF-κB pathway that are influenced by MO and determine if MO impacts the
antiviral response in infected cells.
3.2. PD-1 suppresses protective immunity to Streptococcus pneumoniae through a B cell-intrinsic mechanism.
Jerome T. McKay, Ryan P. Egan, Rama D. Yammani, and Karen M. Haas
Department of Microbiology and Immunology, Wake Forest School of Medicine
PD-1 is an inducible inhibitory receptor expressed on activated B and T cells. Despite the emergence of the
PD-1:PD-1 ligand (PDL) regulatory axis as a promising target for treating multiple human diseases, remarkably
little is known about how this pathway regulates responses to extracellular bacterial infections. We found
that PD-1-/- mice, as well as wild type mice treated with a PD-1 blocking antibody, exhibited significantly
increased survival against lethal Streptococcus pneumoniae infection following either priming with a lowdose pneumococcal respiratory infection or immunization with pneumococcal capsular polysaccharide (PPS).
Enhanced survival in mice with disrupted PD-1:PDL interactions was explained by significantly increased PPSspecific B cell proliferation, isotype switching, and anti-capsular IgG production. Importantly, PD-1 was
expressed on PPS-specific B cells and suppressed IgG production by a B cell-intrinsic mechanism. PD-1
ligands, B7-H1 and B7-DC, were both necessary for suppression. Overall, our results highlight a central role
for B cell-expressed PD-1 in regulating protection against encapsulated bacterial infections.
10
The se
NC ASM 2014
3.3. Infection of equine endothelial cells with non-neurovirulent equine herpesvirus-1 strain KyA elicits
greater down regulation of inflammatory cytokines CCL3, IL8, TGF-2β and CXCL3 compared to neurologic
strain OHIO 2003
Stephanie E. Johnstone and Arthur R. Frampton Jr.
Department of Biology and Marine Biology, University of North Carolina Wilmington.
Equine herpesvirus-1 (EHV-1) infection of endothelial cells within the central nervous system (CNS) has been
shown to contribute to equine herpesvirus myeloencephalopathy (EHM), a severe neurologic disease. Due to
the recent increase in the incidence of EHM cases, and lack of an effective vaccine or therapy, it is critical
that the underlying molecular mechanisms by which EHV-1 causes EHM are investigated so appropriate
preventative or therapeutic interventions can be developed. Previous research has shown that different EHV1 strains elicit different inflammatory immune responses in peripheral blood mononuclear cells and increased
levels of some of these mediators may be correlated with an increased risk of EHM. Using a multiplex PCR
array expression assay, transcript levels of host innate and adaptive inflammatory immune response genes
were measured in equine endothelial (EE) cells after infection with different EHV-1 strains.
EE cells were infected for 10 hours at a multiplicity of infection of 3 with either the non-neurovirulent
Kentucky A (KyA) strain or the neurovirulent Ohio 2003 strain of EHV-1. Data collected from three
independent experiments showed that cells infected with KyA exhibited a significant decrease in numerous
pro-inflammatory genes including, CCL3, IL8, TGF-β2, and MIP-2β. In contrast, only a modest reduction in the
same set of pro-inflammatory cytokines was observed in cells infected with Ohio 2003. The KyA-induced
down-regulation of CCL3, IL8, TGF-β2, and MIP-2β was validated in triplicate using a single gene qPCR assay,
which yielded results similar to the PCR array. Data obtained from this study suggest that the nonneurovirulent KyA strain elicits a greater overall down regulation of inflammatory immune response
compared to Ohio 2003. These data suggest that the differential host inflammatory response to infection
with neurovirulent versus non-neurovirulent strains of EHV-1 may impact severity of disease outcome."
3.4 The HHMI SEA-PHAGES program at NC State University – how your campus can reinvigorate undergraduate
STEM education using bacteriophages
Eric S. Miller
Department of Plant & Microbial Biology, North Carolina State University
Bacteriophages are the most abundant biological entities on earth, infecting bacteria throughout the
phylogenetic tree. In 2009 NC State University joined the Howard Hughes Medical Institute’s Science
Education Alliance to advance STEM education through experiential undergraduate courses. The SEA-PHAGES
program uses Mycobacterium smegmatis as a host for the isolation of new bacteriophages from local soil
samples. Instructors receive training on working with bacteriophages, followed by training in bioinformatics
to lead characterization of the sequenced genomes from student-isolated phages. For three years,
participating campuses receive training, lab manuals, supplies, genome sequencing services, bioinformatics
support, a buddy-system of experience instructors, and participation in a capstone symposium at the HHMI
Janelia Research Campus. Early in their scientific careers, students receive a research experience that
conveys the excitement of scientific discovery, while establishing fundamental skills for successful biological
research. The SEA description can be found at http://www.hhmi.org/programs/science-education-alliance
and student mycobacteriophage research outcomes are detailed at http://phagesdb.org/. First year NC
State University undergraduates have isolated over 50 new bacteriophages in Phage Hunters, most infecting
M. smegmatis. Every student has obtained an electron micrograph of their named phage and initiated the
characterization of the DNA genomes, and then they vote to sequence a phage genome isolated during the
course. Following an introduction to NGS, assembly and annotation, student teams use the second semester,
Phage Genomics, to annotate and explore specific gene functions revealed in the genome. NCSU
mycobacteriophages Microwolf, Mutaforma13, Astraea and others have provided avenues of study into host
range, lysogeny, tmRNA, programmed frameshifting and other advanced topics. Recently, NCSU Phage
Hunters study bacteriophages of Paenibacillus larvae, a honeybee pathogen associated with American
11
NC ASM 2014
Foulbrood disease. Newly isolated P. larvae phages Diva, Lily, Rani, Redbud and others have provided new
opportunities for research, hypothesis-driven science education, and outreach that instill students with
enthusiasm for science discovery. Our positive experience with the HHMI SEA program recommends that
other North Carolina colleges and universities apply (October 31, 2014 deadline) to join the 70+ institutions
engaged in this empowering STEM education program.
12
NC ASM 2014
NC Invited Lecture
Rita Tamayo
UNC-Chapel Hill
Regulation of Clostridium difficile colonization factors by cyclic diguanylate
C. difficile is a Gram-positive, spore-forming, obligate anaerobe that causes a spectrum of intestinal
diseases. C. difficile diseases are among the most commonly acquired nosocomial infections in the developed
world. Treatment of C. difficile infections costs more than $3 billion in the U.S. alone. Yet little is known
about how this organism senses its entry into the host and adapts to the intestinal environment. We are using
C. difficile as a model organism to explore the roles of c-di-GMP in Gram-positive bacteria, in which c-di-GMP
has been poorly studied. We have developed tools for manipulating c-di-GMP levels in C. difficile and have
identified numerous pathways regulated by c-di-GMP, including motility and cell-cell adhesion. We are
currently investigating the molecular basis of how c-di-GMP controls these various pathways.
ASM Branch Lecture
Michael Federle
University of Illinois at Chicago
Microbial Wikileaks: Bacteria that conspire to harm, and small molecules that may protect
We are focused on understanding how bacteria coordinate gene expression across a population using cell-tocell communication. Many important behaviors and activities of bacteria, including the ability to become virulent,
to form biofilms, or to enter the competent state, are controlled through intercellular communication. This
process, referred to as quorum sensing, facilitates inter-bacterial communication and the ability to communicate
between bacteria and host, thus potentially affecting the health of the host. Intercellular communication relies on
secreted signaling molecules (which we refer to as pheromones) that are detected by various types of receptor
proteins in recipient cells. Pheromone detection potentiates differential gene expression.
We have identified and characterized new quorum sensing pathways in Streptococcal species. The protein
family known as Rgg is central to these signaling pathways, dually acting as cytoplasmic receptors of peptide
pheromones and transcriptional regulators. Rgg proteins contribute to a complex quorum-sensing network in
Streptococcus pyogenes (Group A Streptococcus, or GAS). GAS is responsible for a wide variety of diseases
that range in severity from mild cases of impetigo and pharyngitis (strep throat), to life-threatening necrotizing
fasciitis, myonecrosis, and toxic shock. Acute rheumatic fever and rheumatic heart disease are a leading cause
of death from streptococcal infections in developing countries. We are currently investigating the molecular
nature of the streptococcal quorum sensing network, and its role in controlling virulence factors that contribute
to disease. Our studies will lead to the development of new methodologies that disrupt infectious diseases by
interfering with bacteria's ability to coordinate their assault on the human body.
Dr. Federle’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.
13
NC ASM 2014
Poster Presentations
1
Xavier Atkinson
Using Microbiology as a Tool for Engaging Future Scientists: An ECU /
Beddingfield High School Community Science Initiative
2
Joe Blackburn
Characterization of Microbes Isolated from Serpentinizing Soils
3
Will Brennan
Regulation of Vibrio cholerae biofilm formation and norspermidine production
by iron
4
Chequita Brooks
Do Microbial Assemblages Differ by Brand and Flavor of Kombucha?
5
Justine Celli
Asymptomatic chlamydia and gonorrhea infections in UNCW students visiting
the health center for HIV testing
6
Yi-Shan Chen
A Type III Secretion Effector Mediates Recruitment of Phosphoinositide 3kinase to the Pathogenic Vacuole of Chlamydia trachomatis
7
Mara Cloutier
Illumina sequencing of bacterial and fungal populations associated with
manganese oxide deposits among caves in the southern Appalachian Mountains
8
Algenis De Jesus
Potential DNA Repair Mechanisms of the Oral Bacteria Campylobacter rectus
9
Susan Gardner
The role of copper homeostasis in oxidative stress resistance and host
colonization of Campylobacter jejuni
10
Austin Harbison
Novel anaerobic bacteria cultured from Pineola Bog, NC
11
Joshua Martin
Investigation of an ExbB/ExbD proton channel and its role in gliding motility in
Flavobacterium johnsoniae
12
Brittany Miller
A novel component of SecA2 protein export in Mycobacterium tuberculosis
13
Blake Sanders
Characterization of the PotABCD2D1 polyamine transport system in Vibrio
cholerae
14
Robert Schilke
MRSA Carriage in UNCW Nursing Students
15
Caroline Smith
Development of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to
Improve Carbon Fixation in the Seed Oil Crop Camelina sativa
16
Richard Sobe
Regulation of polyamine-mediated biofilm formation by NspS and MbaA in
Vibrio cholerae
17
Maliha Talib
Isolation and identification of bacteria from yogurt samples capable of
producing antimicrobials
18
Jerome McKay
PD-1 suppresses protective immunity to Streptococcus pneumoniae through a B
cell-intrinsic mechanism
Abstracts (posters)
14
NC ASM 2014
1. Using Microbiology as a Tool for Engaging Future Scientists: An ECU / Beddingfield High School
Community Science Initiative
Xavier J. Atkinson1, Justin B. Perry1, Emily Cayton2, Mike Carter2 and Eric S Anderson1
1East Carolina University, Greenville, NC 27858. 2BeddingField High School, Wilson, NC 27893.
Antibiotic resistance by disease-causing bacteria is a major medical concern, and the discovery of new
antibiotic compounds has not kept up with the current rates of resistance. Many of the antibiotics currently
in use were isolated from soil microorganisms. Given the vast number of soil microbes, and the natural
selective pressure on these organisms to produce new and unique antibiotics, it is likely that the soil beneath
our feet represents an untapped resource for identifying new antibiotic compounds.
East Carolina University and Beddingfield High School in Wilson, NC have teamed to examine this potential
resource with a community science outreach program. In this program, Beddingfield High School biology
students, under the supervision of their instructor and graduate students from ECU, collect and characterize
soil samples, isolate potential antibiotic producers and perform initial screening of the candidate organisms.
The techniques these students use will be incorporated into their regular classroom activities to provide
reinforcement of the material they are expected to learn for the biology End of Curriculum (EOC) exam. In
this way, the program supports the teaching goals of the educators, invests the students in a legitimate,
“hands-on” scientific project and paves the way for the discovery of new antibiotics that could potentially be
used to treat infections that are resistant to currently available therapies.
2. Characterization of Microbes Isolated from Serpentinizing Soils
Joe W. Blackburn1, Matthew O. Schrenk2 and Eric S. Anderson1
1East Carolina University, Greenville, NC 27858. 2Michigan State University, East Lansing, MI 48824
Serpentinization occurs on a global scale and has been observed in marine and terrestrial environments; this
metamorphic process occurs when ultramafic rocks, characteristic of the Earth’s lower crust and upper
mantle, are uplifted by tectonic activity and interact with water. This interaction creates an environment
rich in electron donors such as hydrogen and methane; short-chain hydrocarbons and small organic acids are
also produced to a lesser extent. Serpentinization leads to alkaline conditions in excess of pH 10, limited
access to dissolved inorganic carbon and terminal electron acceptors, and low biological diversity. These
factors make serpentinizing environments unique and challenging for inhabiting microbes.
In 2011, Dr. Matt Schrenk and colleagues from East Carolina University began studies on serpentinizing
environments within the Coast Range Ophiolite Microbial Observatory (CROMO) in California. CROMO was
established to monitor the geochemical and microbial activity in a terrestrial serpentinizing environment.
Soil samples were collected directly from CROMO via drill cores to observe the microbial communities in the
serpentinite subsurface. This study focuses on the microbes isolated from those drill core samples.
Thus far, 26 distinct microbes have been cultured from these serpentinizing soils and identified based on 16S
ribosomal RNA sequence. Of those identified, 5 were discovered to be previously uncultured microorganisms.
Currently, we are performing basic metabolic characterizations of these organisms, and examining their
growth kinetics under a range of pH conditions to determine whether these organisms have biologically
adapted to thrive in the extreme conditions of a serpentinizing environment.
15
NC ASM 2014
3. Regulation of Vibrio cholerae biofilm formation and norspermidine production by iron
Will Brennan and Ece Karatan
Department of Biology, Appalachian State University
Vibrio cholerae is a pathogenic bacterium and the causative agent of the severe diarrheal disease cholera.
V. cholerae can be in either a motile, or planktonic state, or in a multicellular community known as a
biofilm. The switch between these two states is regulated by environmental signals; one such signal
norspermidine, a rare polyamine, has been shown to upregulate biofilm formation. Another regulatory input
is iron; V. cholerae has an absolute requirement for iron and it has been shown that under iron-deplete
conditions biofilm formation is inhibited. V. cholerae has multiple pathways for iron acquisition; one such
pathway involves the synthesis of vibriobactin, a molecule with high affinity for ferric iron known as a
siderophore. Interestingly, vibriobactin contains a norspermidine backbone. The use of norspermidine in
both iron-acquisition and biofilm formation indicates that these two processes may be coordinately
regulated. The objective of this study was to determine if siderophore production has an impact on biofilm
formation through the use of mutants defective in vibriobactin synthesis (ΔvibF) and vibriobactin uptake
(viuA::tetR). Our results to date support previous research showing that iron limitation inhibits biofilm
formation and furthermore we have shown that the inability to synthesize vibriobactin leads to an increase in
biofilm formation under iron-replete conditions and leads to a change in biofilm morphology when visualized
via SEM. We hypothesize that biofilm formation is enhanced via one of two potential pathways. First,
inhibition of vibriobactin results in accumulation of norspermidine in the cell, leading to an increase in
biofilm formation, and second, initial accumulation of norspermidine levels leads to a decrease in the
transcription of nspC encoding the enzyme responsible for norspermidine synthesis, which indirectly
enhances biofilm formation. Currently, we are measuring norspermidine levels in the cells via HPLC and
quantifying transcription of nspC via RT-PCR in order to test these hypotheses.
4. Do Microbial Assemblages Differ by Brand and Flavor of Kombucha?
Chequita Brooks, Emily Ashe, and Sean O'Connell
Department of Biology, Western Carolina University
Over the last 10 years kombucha consumption has experienced a dramatic rise in popularity from individuals
looking for a healthy alternative to soft drinks. Kombucha is the product of fermenting sweetened black tea
using a consortium of bacteria and yeasts. Due to the presence of these microorganisms, including
Saccharomyces spp. and lactobacilli, it has been suggested that kombucha is probiotic in nature. The
purpose of this study was to characterize the microbial assemblages in differing brands and flavor varieties of
kombucha. Microscopic, Biolog phenotypic profiling, most probable number of acetic and lactic acid bacteria
(MPN), and rDNA sequencing tests were employed to characterize the microorganisms from four brands of
kombucha: Buchi, Celestial Seasonings, GT’s, and Reed’s. The MPN results indicated greater numbers of both
acetic and lactic acid microbes in Buchi and GT’s and that acetic acid numbers were higher than lactic acid
producing microorganisms. Isolates from the MPNs yielded mostly yeasts in the genus Brettanomyces from
both media. Gluconobacter spp. from acetic acid MPNs were most common. Results from carbon source
utilization patterns in Biolog YT plates showed distinct patterns for Buchi cultures versus the other three
brands. Preliminary findings also show that flavor and/or color additives to Buchi products impact the
response of the microbes in Biolog plates, but not necessarily in MPN assays. The Buchi blend containing
cayenne pepper had the lowest response for both Biolog and MPN tests. Generally, MPN counts ranged from
106 to 108 for acetic acid microbes and 106 to 107 for lactic acid producers. Brand and flavor additions of
kombucha clearly have an effect on the microbial makeup of these beverages. Further testing of the
probiotic characteristics of kombucha (e.g., organic acids, key microorganisms) is warranted, especially
considering the potential for this beverage containing a high degree of species diversity.
16
NC ASM 2014
5. Asymptomatic chlamydia and gonorrhea infections in UNCW students visiting the health center for HIV
testing
Justine M. Celli, K.L. McKenna, C. Passerella, K.B. Kiser
Department of Biology, University of North Carolina Wilmington
The purpose of this study was to develop a urine-based PCR assay to test college students for asymptomatic
chlamydia and gonorrhea infections. This research is critical to understanding the dangers associated with
contracting Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) and how to minimize the risk of
transmission. UNCW students visiting the Abrons Student Health Center (SHC) for free HIV testing will be
recruited to provide urine samples for CT/NG while waiting for their HIV swab results. Because these
students are already visiting the SHC for HIV testing, they are most likely sexually active and amongst a highrisk group for sexually transmitted infections (STIs). The student will read and sign a consent form as well as
a brief questionnaire then collect a first-void urine sample. The consent form, questionnaire, and collection
container will all be labeled with a unique code so professionals at the SHC will be able to contact students
with results while keeping identities of participants confidential from researchers. These samples will later
be transported to the lab then stored at 4ºC until testing analysis is conducted. DNA isolation, Polymerase
Chain Reaction (PCR), and gel electrophoresis will be used to identify the presence of CT and NG DNA in the
urine sample. The lower limits for reaction volume, cycling parameters, and DNA concentrations were
determined as cost and time saving measures. Along with providing free testing for individuals, this study
will increase the awareness of the importance of safe sex practices and regular testing for STIs. The data
collected will also be useful for observing STI trends in this high-risk population.
6. A Type III Secretion Effector Mediates Recruitment of Phosphoinositide 3-kinase to the Pathogenic
Vacuole of Chlamydia trachomatis
Yi-Shan Chen and Raphael H. Valdivia
Department of Molecular Genetics and Microbiology, Duke University
Chlamydia trachomatis, the causative agent of trachoma and sexually transmitted infections, employs a type
III secretion (T3S) system to deliver effector proteins into host epithelial cells to establish a replicative
pathogenic vacuole. Aside from the phosphoprotein TARP, a Chlamydia effector that promotes actin rearrangements, very few T3S effectors mediating bacterial entry and early inclusion establishment have been
identified or characterized. Like many T3S effectors, TARP requires a chaperone (Slc1) for efficient
translocation into host cells. Our lab previously defined proteins that associate with Slc1 in invasive
Chlamydia elementary bodies (EB) by immunoprecipitation coupled with liquid chromatography-tandem mass
spectrometry (LC-MS/MS). In this manner, we identified a previously uncharacterized protein, which we
renamed TepP (Translocated early phosphoprotein), as a new T3S effector. Immunoprecipitation of
endogenous TepP during Chlamydia infection coupled to LC-MS/MS revealed an association of TepP with
multiple host proteins, including the catalytic subunit and regulatory subunit of Phosphoinositide 3-kinase
(PI3K) and CrkL, a SH2 and SH3 domain-containing host scaffolding protein, implying this protein acts as a
“scaffolder” that recruits different proteins to hijack host signaling. Indeed, the level of phospho-Akt
decreased in cells infected with Chlamydia expressing TepP compared to Chlamydia lacking TepP, indicating
the activity of PI3K is prohibited in the presence of TepP. We propose a model wherein TepP sequesters PI3K
to Chlamydia pathogenic vacuole, leading to the dampening of downstream signaling events which may be
important for establishing a replicative niche for Chlamydia.
17
NC ASM 2014
7. Illumina sequencing of bacterial and fungal populations associated with manganese oxide deposits
among caves in the southern Appalachian Mountains
Cloutier, M.L, Zorn, B.T., Carmichael, S.K., Braϋer, S.L.
Department of Biology, Appalachian State University
Manganese (MnII)-oxidizing microorganisms are abundant in nature and have been shown to oxidize Mn(II) up
to 100,000 times faster than abiotic reactions. The environmental significance of Mn(III/IV) oxides is still
ambiguous, but potential impacts include; the formation of protective barriers around microbes, the
adsorption and storage of heavy metal ions, and scavenging of reactive oxygen species. The microbial ecology
of Mn(III/IV) oxide deposits are not well distinguished and even less is understood concerning factors that
stimulate Mn(II)-oxidizing microbes in situ. This study will assess what bacterial and fungal groups may be
enriched in response to nutrients such as casamino acids, arabinose, succinic acid, malt extract and glucose
and may yield insights into which Mn(II)-oxidizing microorganisms may be important in the cave environment.
Four caves systems were investigated, including two relatively pristine caves, and two environmentally
impacted caves. Leucoberbelin blue (LBB) tests were used to select incubation sites with minimal Mn(II)
oxidation. DNA samples were taken from each site at the beginning of the experiment and agar plates
containing a variety of media were deployed in order to stimulate bacterial and fungal Mn(II) oxidation.
Plates with media aimed at enriching bacterial Mn(II) oxidizers were incubated in situ for approximately 10
weeks, at which point LBB tests were performed and DNA samples were collected from plates that tested
positive for Mn(III/IV) oxides. DNA samples will also be collected from plates that test positive for Mn(III/IV)
oxides after a 16 week incubation period. Afterward, DNA will be extracted from the samples and the SSU
rRNA bacterial gene as well as the ITS1 fungal region will be PCR amplified in preparation for Illumina
amplicon sequencing. Results from a previous study indicated that sites where Mn(II) oxidation was
stimulated by glucose plates demonstrated a shift in fungal populations away from Basidiomycota and
Ascomycota, and towards Zygomycota, a phylum of fungi unknown to oxidize Mn(II). Microorganisms play
important roles in maintaining ecosystems through bottom-up effects, and identifying how exogenous carbon
sources affect microbial communities will aid in understanding the effects of anthropogenic impacts on
biogeochemical cycling within those ecosystems.
8. Potential DNA Repair Mechanisms of the Oral Bacteria Campylobacter rectus
Algenis De Jesus, Kristen Delaney, Ph.D., Jessica Vickery
Department of Microbiology, Fayetteville State University, Fayetteville North Carolina 28301
Campylobacter rectus is a gram negative bacteria found in the oral microbiota of most periodontitis patients.
Little is known about the potential for pathogenesis in C. rectus. In this study we examine a gene encoding a
protein of unknown function in C. rectus that has a high level of homology (80%) to a known DNA repair
protein (UmuC). The ability to repair DNA damage confers a survival advantage to an organism - especially if
it lives in a harsh competitive environment like the human oral cavity. Preliminary data with an Escherichia
coli mutant lacking umuC supports that UmuC plays a role in survival after exposure to ultraviolet light. If
the gene in C. rectus is involved in DNA repair mechanisms, then a genomic knockout will show an increased
rate of death when exposed to UV light. A mutagenesis vector will be generated containing a large span of
DNA homologous to the regions flanking the gene of interest. When this vector is transformed into C. rectus
33238 via electroporation the genome will undergo a homologous recombination event that will result in the
excision of the target gene. Future studies will compare the mutant to the parental strain in a variety of
assays to characterize growth, morphology, and the ability to repair multiple types of DNA damage.
Acknowledgements: This work was supported by the Ronald E. McNair Scholars Program, Integrated STEM
Academic Success, and Fayetteville State University Research Initiative for Scientific Enhancement (FSU-RISE)
Program
18
NC ASM 2014
9. The role of copper homeostasis in oxidative stress resistance and host colonization of Campylobacter
jejuni
Susan P. Gardner, Jonathan W. Olson
Department of Biological Sciences, North Carolina State University, Raleigh, NC
Campylobacter jejuni is considered a commensal in poultry, and is found in very high numbers in the cecal
crypts of broilers and turkeys. The slaughtering process leads to contaminated carcasses and, consequently,
pathogenesis in humans who consume this poultry. Genes have been identified with predicted roles in copper
homeostasis in the Campylobacter jejuni genome, however their role in C. jejuni survival and host
colonization is unknown. We have isolated strains with mutations in two of these genes: a multicopper
oxidase (cueO:CM), and a Cu(I) translocating P-type ATPase (copA:KAN). In vitro, these mutants were less
capable of handling the addition of copper and more susceptible to induced oxidative stress. When these
strains were inoculated into one-week-old chickens, their cecal contents contained fewer C. jejuni when
assayed three weeks post inoculation. The deficiency was exacerbated by the addition of copper to the feed.
Further studies will focus on the multicopper oxidase as a potential target for small molecule inhibitors that
can be administered prophylactically to domestic poultry.
10. Novel anaerobic bacteria cultured from Pineola Bog, NC
Austin Harbison and Suzanna L Bräuer
Department of Biology, Appalachian State University
Peat-forming wetlands are one of the largest natural sources of atmospheric methane and as such are
important contributors to global climate change. In these anaerobic environments, plant material is slowly
decomposed by bacteria who then release H2, CO2, acetate and other methane precursors. Little is known
about the microbes involved in these processes, and a large proportion of the organisms are novel and
uncultured. The aim of this project was to culture and identify anaerobic decomposers in local peat bogs and
characterize their physiological properties. Samples were taken from bogs located in Pineola, NC and Panther
Town, NC, and were anaerobically cultured in an N2 or N2/CO2-rich headspace with varied carbon sources
such as glycerol, sucrose, xylose, lactose, maltose or casamino acids for fermentive bacteria. Two cultures of
interest, CS4 and PC4 exhibited marked growth on glycerol and sucrose and were diluted to extinction in
order to obtain pure cultures. The cultures were also grown both aerobically and anaerobically on agar plates
supplemented with yeast and peptone in an attempt to isolate and ensure purity. The PC4 culture was
observed microscopically and found to contain long squared-ended rods approximately 0.5 μm by 4 μm
occurring singly or in short chains. Phylogenetic analysis revealed high identity (99%) to several Closdridium
species including C. puniceum, C. corinoforum, C. favososporum, and C. saccharoperbutylacetonicum. The
CS4 culture was observed microscopically and found to contain small curved rods approximately 0.3 μm by
0.5 μm occurring in clumps. Phylogenetic analysis revealed low identity (~94%) to cultured strains including
Rhizomicrobium palustre and Rhizomicrobium electricum described in 2010 and 2011, respectively. This
organism represents a novel species, and most likely, a novel genus within the Hyphomicrobiaceae family of
the Alphaproteobacterial class.
19
NC ASM 2014
11. Investigation of an ExbB/ExbD proton channel and its role in gliding motility in Flavobacterium
johnsoniae
Joshua T. Martin and Ryan G. Rhodes
University of North Carolina Wilmington, Department of Biology and Marine Biology
ExbB and ExbD are polypeptides that form a proton channel that uses energy from the proton motive force to
drive active transport by high-affinity outer membrane transporters. In Myxococcus xanthus the ExbB and
ExbD homologs, AglR and AglQ/S, form a proton channel that provides the energy necessary for gliding
motility. Inspection of the Flavobacterium johnsoniae genome revealed three loci that may encode ExbB and
ExbD-like proton channels that could provide the energy needed for gliding motility. In this study we used an
allelic exchange system to delete the exbB/exbD homologs encoded by fjoh_0459 and fjoh_0460,
respectively. Primers were designed with engineered restriction sites, and the region flanking each gene was
amplified by PCR and cloned into the suicide vector pRR51. Cloning of each fragment was confirmed by
colony PCR and restriction digest. The deletion construct (pJTM02) was introduced into F. johnsoniae by triparental conjugation, and incorporation of the suicide vector was selected for by plating cells on medium
containing erythromycin. Second recombinants were selected for by plating cells on medium containing
streptomycin. Deletion of the genes of interest was identified by performing colony PCR, and confirmed by
DNA sequencing. Mutant cells cultured on PY2 agar exhibited spreading colonies similar to wild-type cells,
indicating that fjoh_0459 and fjoh_0460 are not required for gliding motility.
12. A novel component of SecA2 protein export in Mycobacterium tuberculosis
Brittany K. Miller, Lauren S. Ligon, Nathan W. Rigel, and Miriam Braunstein
Department of Microbiology & Immunology, University of North Carolina, Chapel Hill, NC, USA
Proteins are transported from the cytoplasm to extracellular spaces of bacteria through a variety of general
and specialized export systems. One specialized pathway, denoted as the SecA2-dependent export pathway,
secretes proteins important for virulence in several pathogens. In Mycobacterium tuberculosis, ∆secA2
mutants are attenuated in macrophages and mice. SecA2 is an ATPase that provides energy for exporting a
small subset of proteins through the canonical Sec translocase across the cytoplasmic membrane. In the nonpathogenic model Mycobacterium smegmatis, a mutation in the ATP binding region, referred to as SecA2KR,
renders SecA2 nonfunctional and dominant negative. Mutations arise spontaneously that suppress secA2KR
phenotypes. By sequencing extragenic suppressor mutations, our goal is to identify proteins that interact
with SecA2. We have sequenced the genomes of six independent, extragenic suppressors of secA2KR that
have mutations in the gene encoding a conserved, hypothetical protein denoted Msmeg1684. This suppression
indicates that Msmeg1684 may be a novel component of SecA2 protein export.
Our preliminary work confirms that an M. smegmatis ∆msmeg1684 mutant expressing SecA2KR suppresses the
SecA2KR phenotype. Additionally, we’ve shown that we can complement an ∆msmeg1684 mutant expressing
SecA2KR with a plasmid expressing Msmeg1684. Msmeg1684 has a homolog in M. tuberculosis denoted Rv3311
(64% identical, 78% similar). Like secA2, rv3311 is predicted to be an essential gene for survival in
macrophages. We have shown that rv3311 can complement an ∆msmeg1684 mutant, indicating that these
proteins have homologous functions in M. tuberculosis and M. smegmatis respectively. Our future work will
focus on elucidating the function of Rv3311/Msmeg1684 and their role in SecA2-dependent export. Because
Rv3311 homologs and SecA2 homologs are conserved together in Actinobacteria, we anticipate that the role
of Rv3311 in SecA2-dependent export is not limited to M. tuberculosis and M. smegmatis.
20
NC ASM 2014
13. Characterization of the PotABCD2D1 polyamine transport system in Vibrio cholerae
Blake Sanders, Libby Villa, and Ece Karatan
Department of Biology, Appalachian State University
Polyamines are a ubiquitous class of molecules that are involved in cell growth and regulate a wide variety of
biological functions. Bacteria contain multiple polyamine transport systems, generally ABC transporters
composed of a substrate binding protein in the periplasm, two channel forming proteins, and a membrane
associated ATPase involved in energy supply. Vibrio cholerae has a putative ABC transporter, PotABCD2D1.
Previous research in our lab has investigated the roles of the substrate binding proteins of this system, PotD1
and PotD2. It was shown that PotD1 is responsible for transport of the polyamines spermidine and
norspermidine into V. cholerae, while PotD2 did not appear to play a role in the import of these polyamines.
Moreover, PotD1 mutants displayed an increased biofilm phenotype when compared with wild type. The
roles of the other components of the polyamine transport system still need further investigation. The
objectives of this study were to more thoroughly characterize the norspermidine/spermidine uptake system
PotABCD2D1, and determine whether imported polyamines or the components of the transporter system
affect biofilm formation. Also, due to the capability of PotD1 to facilitate transport of both norspermidine
and spermidine, the preference of this protein for one polyamine over the other was investigated. Our
results to date indicate that PotB, one of the transmembrane channel forming proteins, is involved in
spermidine uptake. PotB and PotD mutants both display increased biofilm growth compared to wild type,
suggesting that the components of the transport system do not seem to play a role in biofilm formation, but
rather spermidine import into the cell mediates this phenotype. In addition, PotD1 does not appear to have
a preference for norspermidine over spermidine. The roles of PotA and PotC of the uptake system are still
under investigation.
14. MRSA Carriage in UNCW Nursing Students
Robert M. Schilke, M. Brandon Ludlum, Meghan L. Dalziel, Haleigh R.J. Nelson, Garrett R. Hutchings, Samuel
J. Gmuca, and Kevin B. Kiser
University of North Carolina Wilmington, Department of Biology
Methicillin-Resistant Staphylococcus aureus (MRSA) is an opportunistic pathogen that negatively impacts the
healthcare community. MRSA causes numerous infections in hospital patients. Nursing students likely come in
contact with this pathogen during their clinical rotations, which may increase their risk of becoming MRSA
carriers. To understand the relationship between exposure and MRSA carriage, we took nose and throat swab
samples from 117 UNCW nursing students. These individuals were asked to participate in the study by filling
out consent forms, as well as conveying background information on possible past exposure to MRSA. Swab
samples were plated on CHROMagar plates to identify S. aureus. These isolates were then tested for
coagulase activity. Disk diffusion was used to test antibiotic susceptibility of the bacteria. MRSA was
characterized by cefoxitin resistance. Of the 117 individuals, 44% of them were S. aureus carriers. 18
individuals had S. aureus in both their nose and throat. There were 12 students that had it solely in their
nose and 21 students had it solely in their throat. There were no incidences of MRSA carriage in the spring
2014 cohort.
21
NC ASM 2014
15. Development of a Microbe-Derived, Synthetic Condensed Reverse TCA Cycle to Improve Carbon
Fixation in the Seed Oil Crop Camelina sativa
Caroline Smith, Denise Aslett, Mikyoung Ji, Kai Li, Xuili Lin, Hannah Wapshott, Benjamin Bobay, De-yu Xie,
Heike Sederoff, Amy M. Grunden
Department of Plant and Microbial Biology, North Carolina State University
With the rising cost and diminishing quantities of fossil fuels, the generation of sustainable, cost-effective,
high quality biofuels is vital. While many plant based biofuel methods are capable of producing sustainable,
high quality fuels, they are either not cost effective, lack proper infrastructure for implementation, or utilize
food crops. Camelina sativa is an ideal candidate for biofuel production as it is not a food crop, can be grown
on marginal land, and has a high seed oil content. However, producing biofuel from Camelina alone is
unlikely to be sufficiently cost effective to compete with currents fuels. One of the issues limiting cost
effectiveness is the amount of usable biomass the plant is capable of producing that can be converted into
fuel. To overcome this limitation, a synthetic carbon fixation cycle (SynCycle) is being generated for use in
Camelina sativa. The SynCycle is composed of five microbial enzymes which form the shortest, energetically
feasible reverse TCA cycle known. This SynCycle will scavenge carbon dioxide and bicarbonate from the
chloroplast and generate glyoxylate. The glyoxylate will feed into an engineered photorespiratory bypass and
be incorporated into the Calvin-Benson cycle. By increasing the amount of fixed carbon available to the
plant, an increase in overall plant biomass and seed oil production will be possible. In order to ensure that
the SynCycle will function in Camelina the enzymes were selected, cloned, expressed, purified, and tested
for activity individually and in combination using a variety of spectrophotometric, NMR, and LC-MS
techniques.
16. Regulation of polyamine-mediated biofilm formation by NspS and MbaA in Vibrio cholerae
Richard Sobe and Ece Karatan
Department of Biology, Appalachian State University
Biofilms are surface-attached communities believed to play major roles in the pathogenesis of many diseasecausing bacteria including the intestinal pathogen Vibrio cholerae. Transition to the biofilm-associated state
and loss of motility is mediated by increased concentrations of the second messenger, c-di-GMP. c-di-GMP is
synthesized by diguanylate cyclases (DGC) and is degraded by phosphodiesterases (PDE). Environmental
signals stimulate signal transduction pathways leading to modulation of intracellular c-di-GMP levels.
Specifically, the triamines norspermidine and spermidine have been shown to enhance and inhibit biofilm
formation in V. cholerae, respectively. These signals are believed to influence c-di-GMP levels by signaling
through the periplasmic binding protein, NspS, and the integral membrane protein and phosphodiesterase,
MbaA. NspS deletion mutants are inhibited in biofilm formation while MbaA deletion mutants produced robust
biofilms. Furthermore, the effects of norspermidine and spermidine are abolished in mutants of NspS or
MbaA. Therefore, we hypothesize that norspermidine and spermidine exert their effects on biofilm formation
through a signaling pathway mediated by NspS and MbaA.
In our model, norspermidine binding to NspS enhances the interaction between NspS and MbaA resulting in
decreased PDE activity while spermidine hinders this interaction. Previous studies in our lab have shown that
NspS interacts with norspermidine and spermidine and that MbaA is a c-di-GMP-specific PDE; however, the
interaction between NspS and MbaA has not been conclusively demonstrated. The purpose of this work was to
probe the hypothesized NspS-MbaA interaction and determine the effects of norspermidine and spermidine
on this interaction using a multipronged approach. Plasmid and chromosomal-based expression systems are
currently being used to investigate this interaction via co-immunoprecipitation and bacterial two-hybrid
assays.
22
NC ASM 2014
17. Isolation and identification of bacteria from yogurt samples capable of producing antimicrobials
Maliha Talib Presenter, Dr. Melissa Ramirez, and Dr. Alice Lee
"Maliha Talib, Department of Biochemistry, NC University
Dr. Melissa Ramirez and Dr. Alice Lee, Department of Microbiology, NC University
The Centers for Disease and Control (CDC) estimates approximately 2 million people acquire bacterial
infections that are resistant to one or more antibiotics and at least 23,000 die as a result of these infections
(1). The improper use of antibiotics is the most important factor leading to antibiotic resistance.
Concurrently, there has been a rapid decline in the discovery and development of new classes of antibiotics
in the last 40 years (2). As a result, there is a critical need for the discovery and development of new
antimicrobials. One group of antimicrobial compounds being studied are bacteriocins, which are small,
peptide products from bacteria. Bacteriocins exhibit some valuable properties as an antimicrobial, they
may have a broad or narrow spectrum of activity, exhibit low toxicity, high potency, activity against
antibiotic-resistant strains, and amenable to gene-based peptide engineering (3). Many bacteriocins are
produced by lactic acid bacteria (LAB).
Nisin is the first (and only) bacteriocin to be approved for
widespread use to control food spoilage pathogens. Bacteriocins hold great promise as viable alternatives to
antibiotics. The aim of this preliminary study is to isolate and identify bacteriocin producing LAB from
fermented dairy products. The lactobacilli selective medium, deMan, Rogosa and Sharpe (MRS) was used to
isolate LAB from both commercial and homemade yogurt. These candidate LAB strains were screened against
the nonpathogenic indicator strains Escherichia coli and Bacillus subtilis. Three isolates exhibited zones of
inhibition when plated against the indicator strains. Microscopic analysis of these isolates indicated that
they were Gram-negative cocci. Biochemical tests indicated that these strains were able to ferment glucose
and were catalase negative. All isolates were facultative aerobes. Next steps will be to determine if the
antimicrobial activity of these LAB isolates are due to bacteriocin production. Culture supernatant fluids of
these isolates will be tested for antimicrobial activity against selected indicator strains. The potential
identification of a novel bacteriocin from isolated LAB strains would serve as a promising candidate for
further characterization, with potential applications in the field of medicine and could contribute to the
control of the emergence of antibiotic resistance.
18. PD-1 suppresses protective immunity to Streptococcus pneumoniae through a B cell-intrinsic
mechanism
Jerome T. McKay, Ryan P. Egan, Rama D. Yammani, and Karen M. Haas
Wake Forest School of Medicine
PD-1 is an inducible inhibitory receptor expressed on activated B and T cells. Despite the emergence of the
PD-1:PD-1 ligand (PDL) regulatory axis as a promising target for treating multiple human diseases, remarkably
little is known about how this pathway regulates responses to extracellular bacterial infections. We found
that PD-1-/- mice, as well as wild type mice treated with a PD-1 blocking antibody, exhibited significantly
increased survival against lethal Streptococcus pneumoniae infection following either priming with a lowdose pneumococcal respiratory infection or immunization with pneumococcal capsular polysaccharide (PPS).
Enhanced survival in mice with disrupted PD-1:PDL interactions was explained by significantly increased PPSspecific B cell proliferation, isotype switching, and anti-capsular IgG production. Importantly, PD-1 was
expressed on PPS-specific B cells and suppressed IgG production by a B cell-intrinsic mechanism. PD-1
ligands, B7-H1 and B7-DC, were both necessary for suppression. Overall, our results highlight a central role
for B cell-expressed PD-1 in regulating protection against encapsulated bacterial infections.
Meeting Organization Committee / NC ASM Officers
23
NC ASM 2014
Ece Karatan
President-elect
Appalachian State University
Eric Anderson
President
East Carolina University
James W. Brown
Secretary
NC State University
Wrennie Edwards
Treasurer
Novartis
Ece Karatan
Councilor
Appalachian State University
24
NC ASM 2014
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.
The Thoyd Melton Award was established to recognize an outstanding oral
presentation by a Grad 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.
The Best Poster award is open to anyone presenting a poster at the NC ASM
meeting.
The Paul Phibbs Award is awarded for the best presentation by an
Undergradate at NC ASM Branch meetings.
.
A check for $100 will be given for each of these awards at the conclusion of the meeting.
25