Program

AMERICAN SOCIETY FOR MICROBIOLOGY VIRGINIA BRANCH 2013 ANNUAL MEETING PROGRAM

UNIVERSITY OF VIRGINIA School of Medicine Claude Moore Medical Education Building HOSTS: ALISON CRISS AND IAN GLOMSKI NOVEMBER 8-9, 2013

Welcome to the Virginia Branch ASM 2013 Annual Meeting

Virginia Branch ASM Officers 2013-2014 President – Janet Adams, Ph.D., J. Sargeant Reynolds Community College President-Elect – David Gauthier, Ph.D., Old Dominion University Secretary – Robyn Puffenbarger, Ph.D., Bridgewater College Treasurer – Lynn Lewis, Ph.D., University of Mary Washington Councilor – David Buckalew, Ph.D., Longwood University Alternate Councilor – Laura Runyen-Janecky, Ph.D., University of Richmond

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2013 VA Branch ASM

VA Branch ASM Gratefully Acknowledges our Sponsors

2013 VA Branch ASM

VIRGINIA BRANCH 2013 ANNUAL MEETING FRIDAY, NOVEMBER 8, 2013 Check-in and on-site registration throughout the afternoon All sessions will be held in the School of Medicine Claude Moore Medical Education Auditorium, Room 3110 12:00 – 5:00

Registration Desk, Third Floor

1:15 – 1:30

ASM Welcome: Janet Adams, VA Branch President and Alison Criss and Ian Glomski, UVA Hosts

Note: Names in bold print denote competitors for  “Best  Student  Presentation” Session I

Moderator: Heath Damron, University of Virginia

1:30 – 1:45

Clostridium difficile toxins and pathogen associated molecular patterns synergistically increase expression of the pathogenic cytokine IL-23 Carrie Cowardin, Sarah Kuehne, Erica Buonomo, Nigel Minton, and William A. Petri, Jr. University of Virginia, Charlottesville, VA.

1:45 – 2:00

The Inhibitory Effects of Adenylate Cyclase Toxin (ACT) on Bordetella Biofilm Formation C. Hoffman (1), M. Gray (1), G. Donato (1), Y. Reyes (1), J. Eby (1), P. Cotter (2), R. Osicka (3), P. Sebo (3), and E. Hewlett (1). (1) Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA. (2) School of Medicine, University of North Carolina, Chapel Hill, NC. (3) Academy of Sciences of the Czech Republic, Division of Cell and Molecular Microbiology, Czech Republic.

2:00 – 2:15

A Francisella FopA mutant exhibits decreased attachment to host cells and delayed phagosomal escape G. Brett Moreau, Aiping Qin, and Barbara Mann. University of Virginia, Charlottesville, VA.

2:15 – 2:30

Secretion of a nuclease by Neisseria gonorrhoeaeenhances escape from killing by neutrophil extracellular traps Richard A. Juneau and Alison K. Criss. University of Virginia, Charlottesville, VA.

2:30 – 2:45

Ethanolamine promotes expression of putative and characterized fimbriae in enterohemorrhagic Escherichia coli O157:H7 Laura A. Gonyar and Melissa M. Kendall. University of Virginia, Charlottesville, VA.

2:45 – 3:00

The Sinorhizobium meliloti chemoreceptor McpU directly binds proline and mediates chemotaxis towards host plant exudates Benjamin A. Webb, Sherry Hildreth, Richard F. Helm, and Birgit E. Scharf. Virginia Tech, Blacksburg, VA.

3:00 – 3:15

BREAK: refreshments

2013 VA Branch ASM

Session II

Moderator: Katie Crump, Virginia Commonwealth University

3:15 – 3:30

OpaR Controls a Network of Downstream Transcription Factors in Vibrio parahaemolyticus BB22 Alison Kernell-Burke, Leah T. C. Guthrie, Thero Modise, Guy Cormier, Roderick V. Jensen, Linda L. McCarter, and Ann M. Stevens. Virginia Tech, Blacksburg, VA.

3:30 – 3:45

Factors Involved in Clostridium perfringens Gliding Motility on Agar Surfaces Hualan Liu, Steve Melville, David Popham, Roderick Jensen. Virginia Tech, Blacksburg, VA.

3:45 – 4:00

Identification of Proteases in Naegleria fowleri Ishan Vyas, Melissa Jamerson, Jocilyn Budda, Francine Marciano-Cabral. Virginia Commonwealth University, Richmond, VA.

4:00 – 4:15

Toxin Inhibition by Peptides and Small Molecules Janis Doss and Dayle Daines. Old Dominion University, Norfolk, VA.

4:15 – 4:30

The role of LptA, an LOS-modifying enzyme, in gonococcal defense to human PMNs Jonathan Handing and Alison Criss. University of Virginia, Charlottesville, VA.

4:30 – 4:45

BREAK

4:45 – 6:00

Introduction of ASMBL speaker: David Gauthier

ASM BRANCH LECTURESHIP SPEAKER: Dr. Chris Marx Department of Organismic and Evolutionary Biology & FAS Center for Systems Biology, Harvard University

The Math of Metabolism: Mutations  That  Don’t  Like  Each  Other  (and  Species  That  Do) 6:00 – 6:15

Branch Photograph

6:15 – 8:30

Buffet Dinner and Poster Session

6:30 – 7:30

Poster Session 1: odd-numbered posters

7:30 – 8:30

Poster Session 2: even-numbered posters

Poster Sessions will be held in MR5, Rooms 1005 and 2005.

2013 VA Branch ASM

VIRGINIA BRANCH 2013 ANNUAL MEETING SATURDAY, NOVEMBER 9, 2013 8:00 am

Breakfast sponsored by ThermoFisher Scientific Coffee, juice, and bagels

Note:  Names  in  bold  print  denote  competitors  for  “Best  Student  Presentation”

Session III

Moderator: Dwi Susanti, Virginia Tech

8:30 – 8:45

Localization of GSLEs in Bacillus anthracis Dormant and Germinating Spores using PSICIC Sean P. Mury and David L. Popham. Virginia Tech, Blacksburg, VA.

8:45 – 9:00

Quantitative Mass Spectrometry of Bacillus subtilis Germination Proteins Yan Chen, W. Keith Ray, Richard F. Helm, Stephen B. Melville, and David L. Popham. Virginia Tech, Blacksburg, VA.

9:00 – 9:15

Correlative Algorithm for Repeat Placement Abhishek Biswas, David Gauthier, Desh Ranjan, and Mohammad Zubair. Old Dominion University, Norfolk, VA.

9:15 – 9:30

Sex hormones differentially regulate HSV-1 gene expression during the early stages of infection in vitro Thanh Kim Nguyen and Patric Lundberg. Eastern Virginia Medical School, Norfolk, VA.

9:30-9:45

Targeting Dengue Virus Dependent 3' - 5' Human Exoribonucleases Krystal Haley and Dr. Daniel Engel. University of Virginia, Charlottesville, VA.

9:45 – 10:00

BREAK: refreshments

10:00 – 11:00 Safety in the Undergraduate Teaching Laboratory - Faculty Session (and interested postdocs) Guidelines for Biosafety in Teaching Laboratories (link): http://www.asm.org/index.php/education-2/22-education/8308-new-version-availablefor-comment-guidelines-for-best-biosafety-practices-in-teaching-laboratories 10:00 – 11:00 Career Panel - Student and Postdoc Session 11:00 – 11:15 BREAK: refreshments

Session IV

Moderator: Rick Juneau, University of Virginia

11:15 – 11:30 Protein Interaction Networks in Bacteria Peter Uetz, Seesandra S Rajagopala, Roman Häuser, and Russell L. Finley. Virginia Commonwealth University, Richmond, VA. 2013 VA Branch ASM

11:30 – 11:45 The Impact of Genome Reduction on Conservation of Microbial Protein Complexes and Their Components J. Harry Caufield and Peter Uetz. Virginia Commonwealth University, Richmond, VA. 11:45 – 12:00 Complement evasion strategies of oral treponemes associated with periodontal disease Daniel P. Miller, John V. McDowell, Jessica K. Bell, J. Christopher Fenno, Richard T. Marconi. Virginia Commonwealth University, Richmond, VA. 12:00 – 12:15 The Relationship of the Lipoprotein SsaB, Manganese, and Superoxide Dismutase in Streptococcus sanguinis Virulence for Endocarditis Katie E. Crump, Brian Bainbridge, Sarah Brusko, Lauren S. Turner, Xiuchun Ge, Victoria Stone, Ping Xu, and Todd Kitten. Virginia Commonwealth University, Richmond, VA. 12:15 – 12:30 Evolution of Thioredoxin (Trx) systems and the role of Trx in Methanocaldococcus jannaschii Dwi Susantia, Joshua H. Wong, William H. Vensel, Usha Loganathan, Rebecca DeSantis, Ruth A. Schmitz, Monica Balsera, Bob B. Buchanan, and Biswarup Mukhopadhyay. Virginia Tech, Blacksburg, VA. 12:30 – 12:45 Closing Remarks 12:45 – 1:45

Lunch & Business Meeting

VA Branch ASM Business Meeting Agenda: Nov 9, 2013 I. II. III. IV. V.

VI.

VII.

VIII.

General Branch Information Treasurer’s  Report    – Lynn Lewis Secretary’s  Report  – Robyn Puffenbarger Councilor’s  Report  – Wade Bell Upcoming Branch Meetings a. 2014 – JMU b. 2015 – volunteers? Branch Officer Elections a. President-Elect: need interested candidates b. Treasurer – Lynn Lewis c. Secretary, 2015 Uses for National Funds?? a. Student Branch funds – Jan 1, funds disbursed until gone b. Region 2 funds – Nov 30, Career Panel, Student Travel Grants, etc. c. National funds – Nov 30, collaborate with a regional meeting (does not have to be another ASM Branch); increase membership New Business a. Undergraduate programming ideas b. Other new business?

2013 VA Branch ASM

Abstracts for Oral Presentations Session I Abstracts Clostridium difficile toxins and pathogen associated molecular patterns synergistically increase expression of the pathogenic cytokine IL-23 Carrie Cowardin, Sarah Kuehne, Erica Buonomo, Nigel Minton, and William A. Petri, Jr. University of Virginia, Charlottesville, VA. Clostridium difficile pathogenesis is primarily mediated by the Rho-glucosylating Toxins A and B, which activate  Nuclear  Factor  kappa  B  (NFκB)  and  the  NLRP3  inflammasome.  The  pro-inflammatory cytokine IL-23 is a pathogenic mediator in C. difficile infection, as 100% of IL-23p19-/- mice survive infection while only 16.7% of WT mice survive (p=0.005). IL-23 is induced via Pattern Recognition Receptor (PRR) and IL-1β  receptor  signaling.  We  hypothesized  that  the  ability  of  toxins  A  and  B  to  activate  NFκB   would augment signals from PRRs to increase IL-23 expression from myeloid cells in the lamina propria. To address this question, bone marrow derived dendritic cells were treated with purified toxin A and B as well as toxin-containing supernatants from isogenic toxin mutant strains. We discovered that although purified toxins were not sufficient to induce IL-23 (below detection), C. difficile toxins combined with PAMPs exerted a combinatorial effect on IL-23 expression. Supernatants from the epidemic strain R20291 induced 513 +/-123 pg/mL of IL-23 while an isogenic AB- toxin mutant strain induced only 14.8 +/-7.4 pg/mL (p value = 0.03). IL-1β  was  similarly  increased  in  cells  exposed  to  WT  supernatant  (2,737   +/-183 pg/mL) but not in supernatants without toxin (below detection) suggesting that IL-1β  may  play  a   role in the induction of IL-23. Our data demonstrate that C. difficile is capable of inducing IL-23, and that PRR signaling and IL-1β  are  likely  necessary  for  this  induction.    These  findings  implicate  toxin  and  PRR   activated signaling pathways as potential targets of therapeutic intervention to prevent pathogenic IL-23 production. The Inhibitory Effects of Adenylate Cyclase Toxin (ACT) on Bordetella Biofilm Formation C. Hoffman (1), M. Gray (1), G. Donato (1), Y. Reyes (1), J. Eby (1), P. Cotter (2), R. Osicka (3), P. Sebo (3), and E. Hewlett (1). (1) Division of Infectious Diseases and International Health, Department of Medicine, University of Virginia, Charlottesville, VA. (2) School of Medicine, University of North Carolina, Chapel Hill, NC. (3) Academy of Sciences of the Czech Republic, Division of Cell and Molecular Microbiology, Czech Republic. Bordetellae are able to form biofilm but the mechanisms are not completely understood. Biofilm formation is dependent on Bvg-regulated virulence factors; for example Filamentous Haemagglutinin (FHA) has been shown to be required for biofilm formation. In addition, a strain lacking the cyaA gene, encoding ACT, forms more biofilm than wild type (WT) bacteria, suggesting an inhibitory effect of ACT. In the present studies, the ability of Bordetellae strains to form biofilm was evaluated and quantified by the crystal violet assay, under a variety of conditions. To characterize the role of ACT, we examined the effects of exogenously added B. pertussis ACT. As hypothesized, addition of ACT to any of the strains capable of producing biofilm results in reduced biofilm accumulation. This inhibitory effect is concentration dependent and does not require the enzymatic activity of ACT (production of cAMP from ATP). We have shown previously that a physical interaction occurs between FHA and ACT. Collectively these data suggest that the dependence of biofilm formation on FHA and the inhibition of this phenomenon by ACT may reflect that interaction. To understand better the relationship between FHA and ACT, and the possible role of their interaction in biofilm formation, strains with deletions in FHA and ACT and the effects of mutations in ACT and FHA, are being characterized. To explore further this interaction we are using flow cytometry with the Amnis ImageStream to examine and quantify surfaceassociated molecules of Bordetellae, such as ACT and FHA. 2013 VA Branch ASM

A Francisella FopA mutant exhibits decreased attachment to host cells and delayed phagosomal escape G. Brett Moreau, Aiping Qin, and Barbara Mann. University of Virginia, Charlottesville, VA. Francisella tularensis is the causative agent of the potentially life threatening disease tularemia. It is also a significant biosecurity threat and is classified as a Tier 1 select agent because it is easily aerosolized and has a low infectious dose. As an intracellular pathogen, attachment and internalization into host cells, as well as rapid escape from the phagosome, are critical steps in Francisella virulence. However, the bacterial factors involved in attachment and internalization and their downstream affects on intracellular growth have not been well characterized. An in vitro competition assay between wildtype Francisella novicida and individual Francisella novicida transposon mutants determined that a transposon mutant in the fopA gene, which encodes an abundant outer membrane protein, was deficient in the ability to competitively attach and internalize into a lung epithelial cell line. Further characterization of Tn:fopA revealed that it was not attenuated in internalization into either epithelial or macrophage cell lines or intracellular growth at 24 hours post-infection, but was deficient in attachment. These results were recapitulated within the highly virulent Francisella tularensis ssp. tularensis Schu S4 strain. Interestingly, a  Schu  S4  ∆fopA  mutant  exhibited  a  decrease  in  intracellular  growth  within  both  epithelial  and   macrophage cell lines at 6 hours post-infection. Immunofluorescence analysis determined  that  ∆fopA   more readily colocalized with the phagosomal marker LAMP1 at 2 hours post-infection than wildtype, indicating that there is likely a delay in phagosomal escape. These data suggest that FopA could play a key role in Francisella virulence by enhancing phagosomal escape. Secretion of a nuclease by Neisseria gonorrhoeaeenhances escape from killing by neutrophil extracellular traps Richard A. Juneau and Alison K. Criss. University of Virginia, Charlottesville, VA. Symptomatic infection by Neisseria gonorrhoeae, the gonococcus (Gc), results in a highly inflammatory environment caused by a potent infiltration of neutrophils, which possess within their antimicrobial arsenal both oxidative and non-oxidative components. These components can be directed to the phagolysosome, or released by exocytosis, to kill microbes. Another approach used by neutrophils to trap and kill microbes is the release of neutrophil extracellular traps (NETs). NETs are web-like structures comprised of chromatin, decorated with antimicrobial proteins, and are released from neutrophils undergoing a unique form of cell death. In spite of these robust activities, viable bacteria are recovered from gonorrheal disease exudates. We seek to define the bacterial virulence factors that contribute to gonococcal survival after exposure to neutrophils. The NG0969 open reading frame encodes a thermonuclease  (nuc)  that  contributes  to  gonococcal  biofilm  architecture  and  remodeling†.  To  examine   the potential role of nuc in Gc resistance to neutrophils and in NET association, we generated a nuc insertion-deletion  mutant  in  strain  FA1090.    While  neutrophils  infected  with  both  parent  and  ∆nuc  mutant   produce  NETs,  the  overall  area  of  extracellular  DNA  was  significantly  higher  in  ∆nuc  mutant compared to parent Gc infection. Additionally, purified Nuc protein cleaved human neutrophil DNA. When exposed  to  neutrophils  induced  to  undergo  NET  formation,  the  ∆nuc  mutant  survived  less  well  than  the   parent, even in the presence of the phagocytosis-inhibiting chemical cytochalasin D. DNAse I addition prior  to  introduction  of  the  bacteria  partially  rescued  ∆nuc  mutant  survival.    These  studies  suggest  a   model in which Nuc enables Gc escape of NET trapping and killing during symptomatic infection. †Steichen,  et  al.    “The  Neisseria gonorrhoeaeBiofilm Matrix Contains DNA, and an Endogenous Nuclease  Controls  Its  Incorporation.”  Infection  and  Immunity.    1504-11. 79(4), April 2011. Ethanolamine promotes expression of putative and characterized fimbriae in enterohemorrhagic Escherichia coli O157:H7 Laura A. Gonyar and Melissa M. Kendall. University of Virginia, Charlottesville, VA. 2013 VA Branch ASM

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is a foodborne pathogen that causes severe bloody diarrhea and hemolytic uremic syndrome. In order to colonize the human gastrointestinal (GI) tract and cause disease, EHEC must be able to sense the host environment and promote expression of virulence genes essential for adherence. Ethanolamine (EA) is a major component of both mammalian and bacterial membranes, and due to the turnover of enterocytes, EA is present in the GI tract at concentrations that support growth of EHEC. EA is not only an important metabolite for EHEC but also serves as a signal of the host environment. We have determined that EA influences EHEC adherence to epithelial and bacterial cells, and we hypothesized that these EA-dependent adhesion phenotypes were mediated through fimbrial expression. EHEC encodes 16 fimbrial operons which are thought to be important for adhesion and early interactions with host tissue; however, very little is known about the conditions that promote expression of these fimbriae or their roles in host colonization. Using quantitative RT-PCR, we found that EA promoted transcription of genes encoded in characterized and putative fimbrial operons. Moreover, putative fimbrial structures were produced by EHEC cells grown with EA. These findings describe transcription of several putative fimbrial loci in EHEC for the first time. Importantly, the biologically relevant molecule EA, which is abundant in the GI tract, promoted expression of EHEC fimbriae. The Sinorhizobium meliloti chemoreceptor McpU directly binds proline and mediates chemotaxis towards host plant exudates Benjamin A. Webb, Sherry Hildreth, Richard F. Helm, and Birgit E. Scharf. Virginia Tech, Blacksburg, VA. The soil bacterium Sinorhizobium meliloti and the agriculturally important legume, alfalfa can form an agriculturally  important  mutualism  inside  the  plant’s  roots  that  leads  to  drastic  increase  in  plant  yield.  A   prelude to this symbiosis is chemotaxis of S. meliloti through the rhizosphere toward the host plant. This behavior is mediated by seed and root exudation of attractant compounds into the soil. Amongst the multitude of compounds in the exudate, we identified the strong attractant, proline. Here we show that methyl accepting chemotaxis protein U (McpU) of S. meliloti is important for chemotaxis toward proline and that McpU binds proline directly. By testing single residue substitution mutants in capillary assays, we found conserved aspartate residues are important for chemotaxis toward proline. These tests were followed up with in vitro binding assays between proline and variant proteins of McpU. The results indicate that proline binds in a conserved Cache domain of the periplasmic region of McpU and affects chemotaxis to allow for cell movement up a gradient of proline. In all, chemotaxis to proline of a germinating seed is likely a key step in the early establishment of the mutualism. Further studies on the interaction between attractants and their chemoreceptors may aid in propagating a greater efficiency in symbiosis. Session II Abstracts OpaR Controls a Network of Downstream Transcription Factors in Vibrio parahaemolyticus BB22 Alison Kernell-Burke, Leah T. C. Guthrie, Thero Modise, Guy Cormier, Roderick V. Jensen, Linda L. McCarter, and Ann M. Stevens. Virginia Tech, Blacksburg, VA. Vibrio parahaemolyticus is an emerging pathogen that is associated with food-borne gastroenteritis when raw or undercooked seafood is consumed. The pathogenesis of this organism is controlled by the phenomenon known as quorum sensing and the master regulator of quorum sensing in V. parahaemolyticus is OpaR. OpaR controls the virulence of V. parahaemolyticus, as well as the colony and cellular morphology associated with growth on a surface and biofilm formation. RNA-Seq, whole transcriptome Next Generation sequencing, was utilized to determine the direct and indirect targets controlled by OpaR in V. parahaemolyticus BB22. This work confirms but expands upon a previous microarray analysis done with a V. parahaemolyticus RIMD chip. There was excellent correlation 2013 VA Branch ASM

between the microarray and RNA-Seq data. Eleven transcription factors under OpaR control have been identified and further studied using qRT-PCR. A previously published position specific weighted matrix (PSWM) was utilized to assist in identification of possible OpaR binding sites. Eight direct targets were ascertained via in vitro electrophoretic mobility shift assays (EMSA) with purified hexahistidine tagged OpaR. Discovery of the direct and indirect targets of OpaR, including sRNAs, will enable a network map of regulatory interactions important for the switch between the nonpathogenic and pathogenic state to be modeled. Factors Involved in Clostridium perfringens Gliding Motility on Agar Surfaces Hualan Liu, Steve Melville, David Popham,Roderick Jensen. Virginia Tech, Blacksburg, VA. C. perfringens is a Gram-positive, anaerobic, spore-forming bacterium, which can cause several diseases in humans, from mild food poisoning to life-threatening gas gangrene. C. perfringens possesses gliding motility, which helps the bacteria to move across an agar surface. We isolated two natural mutants from C. perfringens strain SM101, which is the causative pathogen for C. perfringens foodborne illness. Compared to the wild type, those spontaneous mutants are hypermotile and form much larger and thinner spreading colonies. Peptidoglycan structure analysis indicated those mutants have thinner peptidoglycan layers. Video microscopy showed the mutants form long cells in chains during growth and have defects in cell division. Whole genome sequencing found several mutations across the genomes of two hypermotile isolates, one in minE and the other strain in ftsI. To determine whether they are the actual cause for hypermotility, we are complementing each strain with a wild-type copy of the cell division gene they are defective in. We also developed a random mariner transposon mutagenesis system in C. perfringens strain 13 and identified a peptidoglycan hydrolase homologue, SagA, as being required for gliding motility. Since many pathogenic bacteria adopt motility mechanisms for host colonization, it would be very valuable to understand the mechanism for forming those hypermotile strains, and also to evaluate the role of hypermotility in the pathogenesis of C. perfringens. Identification of Proteases in Naegleria fowleri Ishan Vyas, Melissa Jamerson, Jocilyn Budda, and Francine Marciano-Cabral. Virginia Commonwealth University, Richmond, VA. Naegleria fowleri (N. fowleri), a free-living amoeboflagellate, is the etiologic agent of Primary Amoebic Meningoencephalitis, a rapidly fatal disease of the central nervous system. Proteases have been shown to be essential for invasion of host tissue by protozoan parasites and may be a major factor contributing to the pathogenicity of N. fowleri. The purpose of the present study was to detect protease activity using zymography. Whole cell lysates were used to detect protease activity in amoebae, while conditioned medium was used to characterize secreted protease activity. In addition, invasion assays were performed using matrigel as an ECM substrate to characterize invasion patterns and examine protease secretion over time from virulent mouse-passaged versus weakly-pathogenic N. fowleri. A differential pattern of protease activity was observed in the culture medium of highly-pathogenic versus weakly-pathogenic amoebae. These results suggest that select proteases play a key role in amoebic invasion of the host ECM and host invasion leading to damage of brain tissue. Studies are in progress to characterize these proteases and to establish their functional relevance in the neuropathogenesis process. Toxin Inhibition by Peptides and Small Molecules Janis Doss and Dayle Daines. Old Dominion University, Norfolk, VA. Nontypeable Haemophilus influenzae (NTHi) is a common cause of otitis media and infections of the respiratory tract in humans. Like other prokaryotes, NTHi contains several type II toxin-antitoxin (TA) systems, in which an endoribonuclease toxin is bound to a labile antitoxin that inhibits the activity of the toxin. The antitoxin is degraded during conditions of stress, allowing the toxin to inhibit the growth of 2013 VA Branch ASM

NTHi, which helps the organism evade antibiotics and the host immune system. Developing a more stable molecule that could bind to and inhibit the toxin would prevent NTHi from entering this dormant state. Our model for this study was the VapBC-1 TA system, in which VapC-1 is the toxin and VapB-1 is the antitoxin. Initially, we used a phage display procedure to identify peptides from a commercial 7-mer phage library that could bind to the VapC-1 toxin and performed fluorescence-quench RNase interference assays to determine which bound peptide could interfere with the activity of the VapC-1 toxin. We also tested a set of seven synthetic 15-mer overlapping peptides based upon the VapB-1 antitoxin sequence. We then expanded our investigation to include the discovery of small molecules that could inhibit the toxin. We are currently working with the National Center for Advancing Translational Sciences (NCATS) to adapt our assay for high-throughput screening of their small molecule libraries to identify possible chemical inhibitors of VapC-1. The role of LptA, an LOS-modifying enzyme, in gonococcal defense to human PMNs Jonathan Handing and Alison Criss. University of Virginia, Charlottesville, VA. Infection with Neisseria gonorrhoeae(Gc) is marked by a rapid influx of polymorphonuclear leukocytes (neutrophils or PMNs) to the site of infection. Despite a robust PMN immune response, viable Gc can be recovered from PMN-rich gonorrheal secretions and from PMNs infected with Gc in vitro. We hypothesize that Gc has defensive measures that help confer resistance to PMNs. Lipooligosaccharide (LOS), an essential component of the bacterial outer membrane, has been implicated in gonococcal defense but its role in interactions with human PMNs are underexplored. Of particular importance, Gc can enzymatically modify the lipid A portion of LOS by the addition of phosphoethanolamine (PEA) moieties to  phosphate  groups  at  the  1’  and  4’  positions.  Loss  of  LptA,  the  enzyme  catalyzing  this  reaction,  leads  to   an increased sensitivity of the bacteria to killing by human complement1 and cationic antimicrobial peptides (CAMPs)2. We have demonstrated an lptA mutant in the FA1090 background shows a significant survival defect in PMNs, compared to WT and an lptA complement (lptA+). Further examination via Baclight viability dyes has demonstrated that the lptA mutant exhibits increased susceptibility to PMN killing both intra- and extracellularly relative to WT and lptA+, however we observed no difference in bacterial internalization. The observed survival defect could be due to altered PMN responses to the lptA mutant and/or intrinsic sensitivity to PMN components. We have demonstrated the lptA mutant is more sensitive to a purified PMN granule extract and select cationic components indicating that the mutant survival defect is in part due to intrinsic sensitivity to components of PMN arsenal. 1. L. Lewis et al. Infection and Immunity. 2013 Jan;81(1):33-42. 2. L. Lewis et al. Infection and Immunity. 2009 Mar;77(3):1112-20 Session III Abstracts Localization of GSLEs in Bacillus anthracis Dormant and Germinating Spores using PSICIC Sean P. Mury and David L. Popham. Virginia Tech, Blacksburg, VA. During germination of Bacillus anthracis spores, the cortex peptidoglycan is degraded by germinationspecific lytic enzymes (GSLEs). Without the action of these critical enzymes, the germination process will not be completed, and the spore will be unable to resume metabolism as a vegetative cell. The limit of differentiation of two signals using light microscopy is approximately 250nm. Our project involves the localization of GSLEs in the spore that may be separated by considerably less distance. GSLE localization is made possible through the use of fluorescent proteins created by fusing mcherry to genes encoding GSLEs and protein markers. The mCherry signal is analyzed and compared to that of Mitotracker Green, a lipophilic membrane stain. The distance between these two signals will be analyzed 2013 VA Branch ASM

with the program Projected System of Internal Coordinates using Interpolated Contours (PSICIC). Statistical analysis will elucidate the spatial relationship of GSLEs and marker proteins in relation to the membrane. By arranging the relative locations of these signals, a distance weighted map will be created in the dormant spore and changes will be tracked during cortex degradation in the germinating spore. Knowledge of the localization of GSLEs can provide information that will be indispensible in defining GSLE accessibility and function for intentional germination protocols. Compared with spores, vegetative Bacillus anthracis cells are much more susceptible to heat, antibiotics, radiation, and chemical treatment. Deliberate germination of spores by activating GSLEs will be crucial in designing cheaper, more effective, decontamination protocols to counteract the use of Bacillus anthracis spores as a bioweapon. Quantitative Mass Spectrometry of Bacillus subtilis Germination Proteins Yan Chen, W. Keith Ray, Richard F. Helm, Stephen B. Melville, and David L. Popham*. Virginia Tech, Blacksburg, VA. The majority of Bacillus subtilis dormant spores will germinate rapidly in response to nutrient germinants with dramatic changes in the physical properties of the cells. However, a small subpopulation termed superdormant spores are resistant to germination. The germinant valine was used to prepare rapidlygerminating and superdormant spore populations. The purified superdormant spores (~1% of spore population) germinated poorly with the germinant used for isolation. However, they germinated as well, if not better, than the initial dormant spores in response to a germinant that targets a different germinant receptor. A targeted LC-MS/MS method was developed using multiple reaction monitoring to quantify 11 membrane-associated germination-related proteins. Membrane-associated YpeB, GerD, PrkC, GerAC and GerKC decreased 6.8, 3.5, 3.8, 1.9 and 2.4-fold, respectively, during spore germination. In contrast, SpoVAD was 1.7-fold more abundant in germinated spore membranes than in those of dormant spores. GerAC, GerKC, and GerD were the only proteins that were significantly less abundant in superdormant spores than in the initial dormant spores, showing 3.4, 1.9, and 1.8-fold decreases, respectively. Similarities in the protein-abundance differences between dormant spores and superdormant or germinated spores suggest that the superdormant spore isolation procedure may have resulted in certain early, non-committal germination steps in the superdormant spores, but that they were blocked at a particular stage in the process. Our results suggest that, in addition to low levels of germinant receptor proteins, a deficiency in the GerD lipoprotein can contribute to heterogeneity of spore germination. Correlative Algorithm for Repeat Placement Abhishek Biswas, David Gauthier, Desh Ranjan, and Mohammad Zubair. Old Dominion University, Norfolk, VA. De-novo genome assembly from DNA fragments is primarily based on sequence overlap information. In addition mate-pair reads or paired-end reads provide linking information for joining gaps and bridging repeat regions. Genome assemblers in general assemble long contiguous sequences (contigs) using both overlapping reads and linked reads until the assembly runs into an ambiguous repeat region. These contigs are further bridged into scaffolds using linked read information. However, errors can be made in both phases of assembly due to high error threshold of overlap acceptance and linking based on too few mate reads. Identical as well as similar repeat regions can often cause errors in overlap and mate-pair evidence. Also, the problem of setting the correct threshold to minimize errors and optimize assembly of reads is not trivial and often requires a time consuming trial and error process. Therefore, we propose a novel scaffolding tool, Correlative Algorithm for Repeat Placement (CARP), capable of joining low error contigs using mate pair reads, resolved repeat structures and verification of joins based on synteny with one or more reference organisms. The CARP tool requires a set of long repeat sequences such as insertion sequences that can be manually determined or found computationally. The tool is designed to match very low error contigs with strong overlap using the ambiguous partial repeat sequence at the ends of the 2013 VA Branch ASM

contig. These matches are verified by synteny with reference to one or more related organisms. We show that the CARP tool can be used to verify low mate pair evidence regions, independently find new joins and significantly reduce the number of scaffolds. Sex hormones differentially regulate HSV-1 gene expression during the early stages of infection in vitro Thanh Kim Nguyen and Patric Lundberg. Eastern Virginia Medical School, Norfolk, VA. The sexual dimorphism of Herpes Simplex Virus-1 (HSV-1) pathogenesis has been demonstrated where mortality and morbidity are more severe in male mice compared to female mice suggesting that gender and/or sex hormones have a role in viral pathogenesis. We propose that sex hormones can alter HSV-1 gene expression thereby changing the pathogen life cycle and consequently pathogenesis. By bioinformatic search, putative HREs were identified throughout the HSV-1 genome. Custom HSV-1 microarrays were performed to examine HSV-1 gene expression changes in the presence of E2 (bestradiol) or DHT (dihydroxytestosterone) in both sex hormone receptor neutral (CV-1) and positive (MCF7) cell lines. In MCF7 cells, global HSV-1 gene expression is upregulated in the presence of DHT and unchanged in the presence of E2 compared to the unstimulated control; no differences in viral gene expression were noted in CV-1 cells. Additionally, qRT-PCR demonstrated that a key HSV-1 regulatory gene ICP0 was upregulated more than 3-fold in the presence of DHT while no difference occurred in the presence of E2. ChIP analysis confirmed the location of viral HREs within the ICP0 promoter. qPCR demonstrated a functional change in viral replication when HSV copy number was drastically increased in the presence DHT compared to E2 and the unstimulated control. We show that androgens provide an environment where viral gene expression is enhanced and viral replication increased thus showing the direct effect of sex hormones on HSV-1. To our knowledge, this is the first study to demonstrate sexual dimorphism at the level of the pathogen. Targeting Dengue Virus Dependent 3' - 5' Human Exoribonucleases Krystal Haley and Dr. Daniel Engel. University of Virginia, Charlottesville, VA. The World Health Organization reports that dengue fever is the fastest spreading mosquito-transmitted disease in the world, with a 30-fold increase in incidence over the last 50 years. Correlatively, dengueassociated morbidity and mortality are on the rise, causing a significant public health burden. Since mosquito population control is currently the only form of disease prevention, therapeutic discovery is critical. To date, dengue virus has eluded virus-targeting therapies, such as vaccines and antivirals, due in part to serotype differences. An alternative treatment approach is targeting host cell factors, which are required  for  each  stage  of  the  dengue  virus  life  cycle.  Recent  studies  have  demonstrated  that  human  3’  –> 5’  exoribonucleases  – EXD2, ERI3, and DIS3L2 – are necessary for dengue virus replication in cell culture. Therefore, we aim to identify novel compounds that inhibit both virus replication and cellular exoribonuclease activity. We expressed each exoribonuclease in S. cerevisiae, and each induced a slow growth phenotype. We then used a high-throughput, toxicity suppression screen and identified small molecule compounds that improved transformed S. cerevisiae growth. Verified compounds will be selected for future experiments based on their low cellular toxicity and potent antiviral properties. We will also investigate the role of each exoribonuclease during dengue virus replication. Lastly, we plan to evaluate the potential use of these inhibitors in the treatment or prevention of dengue virus infection in humans.

2013 VA Branch ASM

Session IV Abstracts Protein Interaction Networks in Bacteria Peter Uetz, Seesandra S Rajagopala, Roman Häuser, and Russell L. Finley. Virginia Commonwealth University, Richmond, VA. Protein-protein interactions (PPIs) are a powerful way to elucidate the functions of proteins, including those of unknown function. However, PPIs have been neglected in the study of enzymes and enzymatic activities, at least in large-scale studies. We have systematically mapped PPIs in bacteria using a novel Yeast Two-Hybrid (Y2H) system that uses multiple bait and prey vectors [1]. We have conducted systematic Y2H screens in Treponema pallidum [2], Escherichia coli, Streptococcus pneumoniae, and Helicobacter pylori that resulted in 3649, 2234, 2057, and 2089 PPIs, respectively. Given the limitations of the Y2H we estimate that these numbers represent no more than a third of all interactions. When combined with data from protein complex purifications and mass spectrometry data, we were able to map another third of all interactions in protein complexes of E. coli, including multiprotein enzyme complexes. The E. coli genome encodes an estimated ~1,500 enzymes (i.e. proteins having an EC number in Uniprot). Among the 2,234 binary PPIs that we found in E. coli 1,276 involve at least one enzyme. Similarly, of the 1,400 proteins found to be in complexes [3] 655 are enzymes. We conclude that PPIs can affect both regulatory and metabolic networks. [1] Stellberger etc 2010, Proteome Sci. 8: 8 [2] Titz etc 2008, Plos ONE 3: e2292 [3] Hu etc 2009, Plos Biol. 7: e1000096 The Impact of Genome Reduction on Conservation of Microbial Protein Complexes and Their Components J. Harry Caufield and Peter Uetz. Virginia Commonwealth University, Richmond, VA. Protein-protein interactions and protein complexes have been systematically studied for only a few bacterial species. For instance, about 300 and 116 heteromeric complexes have been isolated from E. coli and Mycoplasma pneumoniae, respectively. Although there are currently more than 20,000 binary protein-protein interactions published for several species, they rarely capture the full extent to which proteins participate in complexes. We have coupled the results of published mass spectrometrycharacterized protein complexes with databases of gene orthology, conservation and essentiality to better characterize protein interactions. We used complexes isolated from E. coli to identify proteins that are either conserved or lost in other species, shedding light on the essentiality of these proteins and complexes. Complexes isolated from Mycoplasma pneumoniae, a model genome-reduced species, were used as a minimal set of protein complexes for most bacterial species. Microbial protein complexes will help us to understand the biological role of uncharacterized proteins and complexes, especially in lessstudied bacterial species. Complement evasion strategies of oral treponemes associated with periodontal disease Daniel P. Miller, John V. McDowell, Jessica K. Bell, J. Christopher Fenno, and Richard T. Marconi. Virginia Commonwealth University, Richmond, VA. Periodontal disease, the most prevalent disease of microbial etiology in humans, is a chronic inflammatory disease that initiates with a shift in the bacterial flora of the subgingival crevice. Oral spirochetes, which typically represent less than 1% of the total bacterial population in the healthy subgingival crevice, account for 50% of the bacterial population in periodontal pockets. We have 2013 VA Branch ASM

demonstrated that T. denticola binds FH via its FhbB protein and exploits FH activity to downregulate complement activation on the bacterial cell surface. We further demonstrate that T. denticola cleaves bound FH using the dentilisin protease. It is our hypothesis that this activity results in the local depletion of FH with subsequent damaging immune dysregulation. To better understand the FH-FhbB interaction we have determined the molecular structure of FhbB (1.7A resolution), mapped FH-FhbB contact points, and through gene deletion analyses, demonstrate the essential role of FhbB in serum resistance. To date FhbB-FH studies have focused on T. denticola strain 35405. In this study we seek to define the potential of other oral treponeme species and diverse T. denticola isolates to influence complement regulation. FH binding, dentilisin activity and FH cleavage by each isolate was assessed and the influence of FhbB sequence variation on these processes determined. Lastly, we conducted the first comprehensive analysis of the serum sensitivity of oral treponemes. The data obtained from this study enhances our understanding of complement manipulation by T. denticola and other oral treponemes and will facilitate efforts to develop therapeutic and preventive strategies for this significant human health concern. The Relationship of the Lipoprotein SsaB, Manganese, and Superoxide Dismutase in Streptococcus sanguinis Virulence for Endocarditis Katie E. Crump, Brian Bainbridge, Sarah Brusko, Lauren S. Turner, Xiuchun Ge, Victoria Stone, Ping Xu, and Todd Kitten. Virginia Commonwealth University, Richmond, VA. Streptococcus sanguinis is a normal inhabitant of the mouth and an important cause of infective endocarditis. SsaB, a protein belonging to a family of orthologous metal-transport proteins termed Lipoprotein Receptor Antigen I (LraI), is critical for endocarditis virulence in S. sanguinis. While LraI proteins have been shown to transport manganese and additional metals, we are interested in the role of metal homeostasis in oxygen tolerance. Using isogenic mutants lacking SsaB, the manganese-dependent superoxide dismutase, SodA, or both, we addressed the specific role of manganese in oxygen tolerance and virulence in S. sanguinis. The role of SsaB as a metal transporter was confirmed by reduction of cellular manganese and iron levels in the ssaB mutant. In an animal model of endocarditis, all three mutants exhibited reduced virulence, with the double mutant exhibiting the greatest defect and the sodA mutant, the least. This reduction of virulence was not due to diminished resistance to phagocytic killing or heart valve colonization in either single mutant. Furthermore, the relative virulence of wild-type and mutant strains was reproduced by a growth assay employing only normal rabbit serum and physiological levels of oxygen. Using these physiological conditions, SodA activity was decreased but not absent in the ssaB mutant. Additionally, antioxidant studies suggested that increased damage by superoxide was necessary  for  the  ssaB  mutant’s  reduced-growth phenotype, but not sufficient, with additional contributions by hydroxyl radicals likely. Together, these studies indicate that SsaB-mediated manganese uptake enables virulence by providing resistance to oxidative stress through SodA-dependent and independent mechanisms. Evolution of Thioredoxin (Trx) systems and the role of Trx in Methanocaldococcus jannaschii Dwi Susantia, Joshua H. Wong, William H. Vensel, Usha Loganathan, Rebecca DeSantis, Ruth A. Schmitz, Monica Balsera, Bob B. Buchanan, and Biswarup Mukhopadhyay. Virginia Tech, Blacksburg, VA. Thioredoxin (Trx), a small redox protein, controls a spectrum of processes in Eukaryotes and Bacteria by changing the thiol redox status (SH/S-S) of selected proteins. Some the notable examples of Trxcontrolled systems are photosynthesis in plant and cell death and aging in human. Although the Trx is present in the Archaea, the role of this redox protein in the Archaea remained unknown. We are studying the development of the Trx systems in a broad range of microorganisms and their distribution and roles in methanogens. For the last item we focused on Methanocaldococcus jannaschii, a deeply-rooted hyperthermophilic methanogen inhabiting a deep-sea hydrothermal vents, as the model. 2013 VA Branch ASM

The organism performs only hydrogenotrophic methanogenesis, one most ancient respiratory metabolism of Earth. A bioinformatics analysis suggested that the two known Trx systems, one (NTR) being dependent on a flavin containing NADPH-Trx reductase (NTR) and other involving an iron-sulfur cluster containing ferredoxin-thioredoxin reductase (FTR) arose independently. FTR was likely invented by the ancient Bacteria and NTR system was likely developed by the Archaea beginning with an ancestral form (XTR) that was not dependent on nicotinamides. Their original roles in the control of carbon dioxide fixation and defense against oxidative damage apparently have been preserved through their evolutionary development. Trx was found to be nearly universal in methanogens. Deeply-rooted hydrogenotrophic methanogens, which are predominantly thermophiles, carried two Trx homologs on average, whereas their more evolved, nutritionally versatile counterparts carry 4-9 Trx homologs. Methanocaldococcus jannaschii carries two Trx homologs: Trx1, a canonical Trx, reduces insulin and accepts electrons from E. coli NADP-thioredoxin reductase. Trx2 is atypical. Proteomic analyses identified 154 potential Trx1 targets in air-oxidized M. jannaschii cell extracts. These targets represented a broad range of cellular processes, including methanogenesis, biosynthesis, transcription, translation, and oxidative response. In enzyme assays, Trx1 activated partially deactivated forms of two of these targets, F420-dependent methylenetetrahydromethanopterin dehydrogenase, a methanogenesis enzyme, and sulfite detoxifying F420-dependent sulfite reductase, validating proteomics observations. These results suggest that Trx assist methanogens in combating oxidative stress and synchronizing metabolic activities with the availability of reductants such as H2, thereby influencing global carbon cycle and methane emission.

2013 VA Branch ASM

Abstracts for Poster Presentations Session A 1 Suppression of Dendritic Cell Maturation, Activation, and T Cell Stimulatory Capacity by Melanoma-derived Factors Johnathon Drake Bishop and Kristian Michael Hargadon. Hampden-Sydney College, Farmville, VA. We have previously shown that melanoma-derived factors suppress the maturation/activation of the murine dendritic cell (DC) line DC2.4. Importantly, the extent of this suppression is dependent upon the tumorigenicity of the melanoma under study, with the highly aggressive B16-F1 melanoma exhibiting greater suppressive effects than D5.1G4 melanoma, a mutated variant of B16 whose growth and metastasis is immunologically controlled in a large percentage of hosts. We wished to extend our previous studies with the in vitro DC2.4 cell culture system by exploring whether melanoma-derived factors also suppress the maturation and activation of bona fide immature DC isolated from the murine spleen. Here, we show in an ex vivo system that melanoma-derived factors also differentially suppress the maturation and activation of DC, which in turn regulates the T cell stimulatory capacity of these cells. Finally, we identify partial roles for both melanoma-derived TGFbeta1 and VEGF-A in the suppression of DC function. 2 Antimicrobial Analysis of the Xylylene Series of Novel Amphiphiles Tara Gallagher, Nicholas Minahan, Kyle Bonifer, Jade LaDow, Kevin Caran, Kevin Minbiole, and Kyle Seifert. James Madison University, Harrisonburg, VA. Antibiotic resistance is advancing ubiquitously via multiple modes of horizontal gene transfer (HGT), and has given rise to highly resistance life-threatening human pathogens such as Mycobacterium tuberculosis and methicillinresistance Staphylococcus aureus (MRSA). To date, our collective aim has been to develop a new class of potent, broad-spectrum antimicrobials, based on novel series of amphiphiles that will be difficult for organisms to counteract or to resist via mutation. In order to determine the biological activity our amphiphiles, minimum inhibitory concentration (MIC) assays and minimum bactericidal concentration (MBC) assays were performed for each compound to determine the inhibitory efficacy of each compound against known bacterial pathogens. Notably, our amphiphiles have shown greater inhibitory and bactericidal activity than control compounds DTAB, CTAB and benzalkonium chloride (Lysol), falling within the low micromolar range. Although our xylylene series (oX, mX, pX) amphiphiles do not match the inhibitory or bactericidal activity of our mesitylene series (M) or paraquat series (PQ) amphiphiles, they achieve striking synergy in combination against both Gram-positive and Gram-negative organisms. Synergy of our amphiphiles is determined by the  fractional  inhibitory  concentration  (FIC)  index,  where  an  FIC  index  of  ≤0.5   indicates synergy. For example, amphiphiles mX,14,14/mX-1,14 of the xylylene series have an FIC index of 0.19 for Gram-negative Escherichia coli and 0.50 for Gram-positive Staphylococcus aureus. All  the  while,  our  xylylene  series  amphiphiles  maintain  MIC  and  MBC  values  as  low  as  4μM  for   Staphylococcus aureus and  8μM  for  Escherichia coli. 3 Phylogenetic Analyses of Streptococcus parauberis from Fish and Cattle Keaira Thornton and Ashley Haines. Norfolk State University, Norfolk, VA. Streptococcus parauberis is a gram-positive lactic acid bacterium that infects cattle and fish, which can negatively impact dairy and aquaculture fisheries. Recently it has become an emerging pathogen in these industries. In this work, nucleic and amino acid sequences of multiple housekeeping genes from fish and cattle isolates were analyzed. These gene sequences were used to construct phylogenetic trees using publicly available software. We hypothesized that analysis of multiple genes 2013 VA Branch ASM

will better predict the closest relative to S. parauberis than the previous single gene analyses. In addition, amino acid sequences may further clarify our proposed phylogenies. Our data suggest that S. parauberis may be more closely related to Streptococcus iniae (a fish pathogen) than to Streptococcus uberis (a cattle pathogen), the two model species that are most closely related to S. parauberis. These phylogenetic analyses improve our understanding of the evolutionary relationships between related bacterial species from different animal hosts. Focusing on the molecular epidemiology of S. parauberis will help explain how different host species are infected and may help prevent future outbreaks. 4 Neisseria gonorrhoeae phagosomes delay fusion with primary granules to enhance bacterial survival inside neutrophils M. Brittany Johnson and Alison K. Criss. University of Virginia, Charlottesville, VA. Gonorrhea is a global health problem with 106 million cases each year worldwide. Symptomatic infection with Neisseria gonorrhoeae(Gc) promotes inflammation featuring influx of polymorphonuclear leukocytes (PMNs), yet some Gc survive PMN exposure during infection. We demonstrate a novel mechanism of Gc resistance to PMNs: Gc phagosomes avoid maturation into phagolysosomes by delayed fusion with primary granules, which contain antimicrobial components including serine proteases. Reduced phagosome-primary granule fusion was observed via immunofluorescence in gonorrheal exudates and human PMNs infected ex vivo. Using bacterial viability dyes along with antibodies to primary granules revealed that Gc survival in PMNs correlated with early residence in primary granule-negative phagosomes. However, when Gc was killed prior to PMN exposure, dead bacteria were also found in primary granule-negative phagosomes. These results suggest that Gc surface characteristics, rather than active bacterial processes, influence phagosome maturation and that Gc death inside PMNs occurs after phagosome-granule fusion. In support of this hypothesis, we observed that delayed phagosome-granule fusion could be overcome by opsonizing Gc with immunoglobulin or by Gc surface expression of opacity-associated proteins (Opa). The residence of Gc in mature phagosomes due to opsonization or Opa expression resulted in decreased Gc viability, which was attributed in part to serine protease activity. We conclude that one method for Gc to avoid PMN clearance in acute gonorrhea is by delaying primary granulephagosome fusion via Gc surface composition, thus preventing formation of a phagolysosome. 5 Stabilization of Vaccines against Heat – I Samson S. Stratton, Jeffrey D. Raiford, Anthony W. Thomas, and Carl W. Vermeulen. Paul D. Camp Community College, Franklin, VA. As almost all vaccines require refrigeration, nearly 20% of the world (1 billion people and even more livestock) has little or no access to their protections. In this Phase I, various enzyme models for vaccines were straight-jacketted in pure dried starch and continuously subjected to temperatures up to 70c for many weeks with little if any loss of activity. Furthermore, dried starch is inherently its own delivery device as thin "noodles" of it have the strength to penetrate intramuscularly through skin, and the body's amylases degrade it within minutes releasing its vaccine content. 6 Identification of Fungi Associated with Rhizome Rot in the Medicinal Plant, Actaea racemosa (Black Cohosh) Michelle Donahoe, Lindi Hutchinson, Jasmine L. Vaughn, Christine Small, and Georgia A. Hammond. Radford University, Radford, VA. Black Cohosh (Actaea racemosa) is a widely known and used medicinal plant that is endemic to the southeast region of the United States, more specifically, the Appalachian mountains of Virginia, West Virginia, Tennessee, North Carolina, and Kentucky. Black Cohosh is in high demand because it is 2013 VA Branch ASM

internationally used as an alternative treatment for menopausal symptoms. A number of studies have been devoted to transplantation of black cohosh rhizomes because there is severe danger of overharvest and extinction of the plant. During transplantation, Black Cohosh has been found to be susceptible to fungal infections that are loosely referred to as rhizome rot. Rhizome rot severely limits the success of transplantations. Our research focuses on identifying the fungi causing rhizome rot in transplantations that are ongoing in southwestern Virginia, using ITSrRNA sequencing. The ITS region is found in all fungi and it varies in term of sequence and there for can be used for identification. We have isolated fungal DNA from infected rhizomes, as well as from soils from preand post-transplantation sites. Our goal is to identify the various fungi that are associated with the rhizome rot, to identify any fungi that could potentially be in the soil and to use these data to design strategies to improve transplantation of this valuable native plant. 7 Identifying bacteria that live in high concentrations of arsenic Jessica O'Grady, Lindi Hutchinson, and Dr. Georgia Hammond. Radford University, Radford, VA. Microorganisms are exceedingly abundant and diverse in the soil, making it difficult for scientists to characterize them. Some bacteria can thrive in extreme environmental conditions due to their genetic makeup. Our focus is on bacteria capable of metabolizing arsenic, a heavy metal. We obtain soil samples from a headwater stream at an abandoned arsenic mine in Floyd County, Virginia. Erosion at this site has caused arsenic to leach out of the soil and into a headwater stream nearby, potentially contaminating major river systems in Virginia. According to our previous research, bacteria isolated at this site are able to live in arsenic rich environments because they contain arsenic resistance genes, and these genes are expressed at high levels when pure lab cultures of bacteria from the site are grown in arsenic. Our goal is to characterize these bacteria down to the genus and if possible, species level using the 16s rRNA gene. This gene sequence is highly conserved among bacteria; however, it contains variable regions with point mutations that can be used for identification purposes. To accomplish this, we isolated DNA from pure cultures of the bacteria, amplified the 16s rRNA genes with a PCR reaction, purified the PCR product, and sequenced the DNA. We then put the obtained sequences into databases to compare our sequenced data with known ribosomal subunit sequences of identified bacteria. Our results contribute to knowledge regarding bacteria capable of surviving in environments containing the heavy metal, arsenic. 8 The quorum sensing regulator ExpR directly regulates symbiotically important Flp pili in Sinorhizobium meliloti Hardik Zatakia, Cassandra Nelson, Anjali Sharma, and Birgit Scharf. Virginia Tech, Blacksburg, VA. Type IVb pili (Tfpb) are mostly studied in enteropathogens and have diverse functions in bacterial aggregation, attachment, and microcolony formation. The Sinorhizobium meliloti genome contains two pil gene clusters; one on the chromosome (pil1) and the other on the pSymA megaplasmid (pil2), both of which code for the genes required for Tfpb synthesis. pilA1 in the pil1 gene cluster encodes the putative pilin subunit belonging to the flp (fimbrial low-molecular-weight protein) family of pilins. To establish the role of Tfpb in the symbiotic interaction of S. meliloti and its host Medicago sativa, we conducted competitive nodulation assays. The S. meliloti pilA1 deletion strain was about 30% deficient in nodulation as compared to the wild type. Absence of pili in the pilA1 deletion strain was confirmed using transmission electron microscopy. Transcriptional reporter gene assays illustrated that the expression of pilA1 peaks at early stationary phase and is repressed by ExpR. ExpR is a LuxR-type transcriptional regulator and part of the quorum sensing system in S. meliloti. It regulates exopolysaccharides production and motility in S. meliloti. Direct binding of AHL-activated ExpR to the pilA1 promoter region was confirmed with electrophoretic mobility shift assays. A 28-bp protected region on the pilA1 promoter was defined using DNase I footprinting analyses. This protected region comprised a 17-bp sequence that matches the consensus sequence for ExpR binding. 2013 VA Branch ASM

Thus, our studies show that S. meliloti coordinates temporal expression of Type IVb pili with other cellular processes namely exopolysaccharide synthesis and motility for optimal interaction with its host. 9 Integration of a new chemotaxis protein CheT in the chemosensory signaling chain of Sinorhizobium meliloti Umair Syed and Birgit E. Scharf. Virginia Tech, Blacksburg, VA. Sensory signal transduction in bacterial chemotaxis is generally mediated through a two-component regulatory system consisting of a histidine autokinase, CheA, and a response regulator, CheY. The symbiotic alpha-proteobacterium Sinorhizobium meliloti employs an indirect phosphate sink mechanism rather than direct dephosphorylation to inactivate the motor-binding response regulator, phosphorylated CheY2. In this mechanism, phosphoryl groups are shuttled from CheY2-P back to CheA, which in turn phosphorylates CheY1. While reported for the first time for S. meliloti, the presence of more than one response regulator in the chemotactic signaling chain in bacterial species outside the enterobacterial group suggests that a phosphate sink is a widespread mechanism for signal termination. In addition to the canonical chemotaxis genes, the che1 operon contains two open reading frames coding for novel proteins, CheS and CheT. We have recently shown that CheS reduces CheY2-P levels by enhancing the binding of CheY1 to the kinase CheA. Behavioral assays of DcheT cell populations showed that CheT has the opposite effect in promoting an increase in CheY2P levels by a yet unknown mechanism. Bacterial chemoreceptors prototypically assemble into polar clusters where they form large sensory complexes that recruit cytoplasmic components of the signaling pathway. The current study analyzes the localization of CheT fused to the fluorescent proteins eGFP and mCHERRY by fluorescence microscopy. Furthermore, the interaction of CheT with all eight chemotaxis proteins will be determined using individual deletion strains. Results of this study will give insight into the placement of CheT in the signaling transduction chain of S. meliloti. 10 Towards an Understanding of the Role of Motility and Chemotaxis in Antitumor Efficacy of Salmonella typhimurium VNP 20009 Katie Broadway and Dr. Birgit Scharf. Virginia Tech, Blacksburg, VA. Several bacterial species have been found to be attracted to tissues ridden with cancerous cells. This leads to colonization of the tissue, effective tumor shrinkage and host survival. Salmonella, a facultative anaerobic, enteric, motile bacterial species, is tumor targeting. These bacteria preferentially penetrate and replicate within tumors, suppressing their growth and metastasis. The selected bacterial strain used in this project is VNP 20009. This is a mutagenized derivative of Salmonella enterica serovar Typhimurium 14028, a species commonly studied in virulence and immunology. The long term goal is, in a collaborative effort joining the fields of engineering and microbiology, to assess the effect of motile VNP 20009 bacteria loaded with anticancer drug carrying nanoparticles on tumor eradication. The aim of this project is to characterize and maximize the positive chemotactic response VNP20009 possesses towards tumor cells. In previous work, it has been shown that motility is critical for this strain to accumulate in tumor tissue. Various mutant strains of the VNP 20009 were constructed by lambda-red genetic engineering, and their behavior compared to the parental 14028 strain. In addition, the genome of VNP 20009 will be annotated and the genes responsible for the tumor seeking characteristic will be investigated. With this knowledge, it is anticipated that the bacteria can then be genetically modified to increase their affinity to cancerous cells in host tissue. Through understanding and analyses of this strain's ability to colonize tumors by the bacterial cell-drug delivery complex, cancerous cell death can be maximized. 11 Designing Tricistronic and Bicistronic Lenti-Viral Vectors using Scar-less DNA Assembly Methods and Web-based Software to Study GRK4 2013 VA Branch ASM

Christophe Langouet-Astrie, Louise Temple, Dora Wang, John Gildea, and Robin Felder. James Madison University, Harrisonburg, VA. In this study, the goal is to develop a diagnostic tool for salt sensitivity that involves detection of specific protein, in this case G protein-coupled  receptor  kinase  4  (GRK4).    To  measure  this  protein’s   activity and production, two issues arise: constructing a delivery system to introduce the DNA in a timely and cost-effective manner and quantifying the amount of protein produced in live cells. For the former issue, novel gene cloning techniques known as scar-less multipart DNA assembly methods have been created that have been developed to optimize cost and time effectiveness. Due to the difficulty in optimized protocols for these methods, a web-based software known as j5 contains programs that aid in creating a protocol. For the latter issue, these assembly techniques and the webbased software are used to construct lenti-viral delivery system containing bicistronic and tricistronic vectors. Bicistronic vectors are commonly used for measuring protein synthesis my indirect measurement of a fluorescence protein. Development of tricistronic vectors allow for introduction of one protein with two modes of measurement i.e. microscopy and antibiotic selection. Two bicistronic vectors were constructed containing GRK4 and Zeocin resistance in one vector and an AT1 receptor and a CYPHR protein in the other. In addition, a tricistronic vector is being constructed containing GRK4, a Zeocin resistance, and a fluorescence protein with self-cleaving peptide sequences and internal ribosomal entry sites. These tools will help measure GRK4 production and activity in order to help develop an assay for detecting salt sensitivity in humans. 12 Identifying Environmental Bacillus Vincent A. Gentilcore, Dylan S. McKnight, and Georgia A. Hammond. Radford University, Radford, VA. The Bacillus genus is one of a large group of bacteria found in nature. Many environmental Bacillus species  can’t  be  identified  using  the  Small  Subunit  rDNA  sequence.    Our  study  focuses  on  Small   Acid-Soluble proteins (SASPs), which are unique to endospore-forming bacteria including Bacillus. SASP proteins in Bacillus species are unique relative to SASP proteins found in other genera of endospore formers such as Clostridium. Using DNA sequences for two major SASP proteins (SASPA and SASP-B) from Bacillus subtilis, we have developed PCR primers to use in PCR reactions that examine the DNA of bacteria that we collect from the environment. Our objective is to use these primers in a PCR reaction that uses a gradient of annealing temperatures to detect major SASP genes in unknown species. Detection of a PCR product would be very good evidence that the unknown bacterium was a member of the genus Bacillus. 13 Antibiotic resistance and putative virulence factors of clinical and environmental strains of Vibrio vulnificus James Conrad, Pradeep Vasudevan, and Joanna Mott. James Madison University, Harrisonburg, VA. Antibiotic resistance in the pathogen, Vibrio vulnificus, is a major concern for the medical community as well as aquaculture and saltwater aquaria hobbyists. Infections progress rapidly and effective treatment is crucial. V. vulnificus is found in warm brackish coastal waters, sediments and associated with organisms such as oysters and fish. Several virulence factors have been correlated with clinical isolates, including mannitol fermentation, salicin metabolism, and presence of pilF. Previously 253 V. vulnificus isolates were collected from infected patients, oysters, and water samples from several states. Each isolate was characterized based on its pilF gene and D-mannitol fermentation. In this study, a subsample of these isolates was subjected to antibiotic resistance analysis (ARA) to determine any correlations between antibiotic resistance and clinical or environmental origin. Additional isolates were collected from residential and commercial saltwater aquaria. Each isolate was confirmed as V. vulnificus by amplification of the vvhA gene and then subjected to ARA. A drug 2013 VA Branch ASM

panel was selected based on CLSI M45-A2 recommendations as well as antibiotics frequently used to test V. vulnificus. At least one antibiotic from each class in CLSI M45-A2 was included in the drug panel. Isolates were then grouped based on their ARA, pilF, and D-mannitol fermentation profiles to determine any correlations between putative virulence factors, ARA patterns, and origin. This data will provide information on the current levels of antibiotic resistance in V. vulnificus from different sources. 14 Antimicrobial Analysis of the Meta-Pyridine series of Novel Ampiphiles Brandi Volkers and Kyle Seifert. James Madison University, Harrisonburg, VA. In the US, approximately 2 million patients acquire nosocomial infection each year during their stay in health care facilities, resulting in over 100,000 deaths. Multi-drug resistant organisms initiate many of these infections, making the development of novel effective antimicrobials paramount in hospital settings. As a continuation of our previous research on bicephalic amphiphiles, which correlated antibacterial activity with head group arrangement and chain length, the antimicrobial activity of three distinct series of amphiphiles were investigated. The first series, gemini, is characterized by a bi-substituted arene core with identical head groups each with hydrocarbon tails of the same length (14 carbons). The second series, bicephalic, is also characterized by a bi-substituted arene core with a pyridine group in place of a head group and hydrocarbon tail. The final series, mesitylene, contains an arene core substituted with two identical head groups each with hydrocarbon tails of the same length (10-16 carbons) and a non-substituted pyridine head group. The series of amphiphiles were assayed against Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Bacillus subtilis, Streptococcus agalactiae and Escherichia coli, by performing standard minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) assays. MIC and MBC values for these amphiphiles were in the low micromolar range. Specifically, the compound M-P,12,12 from the  mesitylene  series,  “meta-pyridine,12  carbon  chain,  12  carbon  chain”,    showed  greater  inhibition   and bactericidal activity than control compounds, DTAB, CTAB and Lysol. Synergy assays performed against E. coli and S. aureus and biofilm disruption assays against P. aeruginosaalso yielded positive results, bringing biofilm levels down significantly in some cases. 15 Mutational Analysis of Disulfide Bond Oxidoreductase DsbA2 Function in Legionella pneumophila Zegbeh Z. Kpadeh, Shandra R. Day, and Paul S. Hoffman. Division of Infectious Diseases and International Health, Department of Medicine, Department of Microbiology, Immunology and Cancer Biology, University of Virginia School of Medicine, Charlottesville, VA. Background: Legionella pneumophila (Lpn) is a Gram negative, intracellular parasite of free-living protozoa  that  when  aerosolized  causes  Legionnaires’  disease,  an  occasionally  fatal  pneumonia.  The   Dot/Icm type IVb secretion system (T4SS) is the main virulence mechanism and is dependent on correct disulfide bond (DSB) formation catalyzed by a novel and essential DSB oxidoreductase DsbA2 and not by DsbA1, a nonessential DSB oxidoreductase. DsbA2 is a bifunctional enzyme containing a highly conserved N-terminal dimerization domain enabling the protein to form a homodimer. Deletion of the dimerization domain produced the monomer (DsbA2N), which no longer exhibits protein disulfide isomerase activity, but complements DsbA activity in an E. coli dsbA mutant. Methods: A cis-proline mutant of DsbA2N (P198T) was expressed in Lpn AA100 strain to assess the effect on virulence. Intracellular growth in Acanthamoeba castellanii, attachment and invasion of HeLa cells and T4SS contact-dependent hemolysis of erythrocytes was compared to empty vector control. The interacting DSB partners of DsbA2N were identified in an E. coli background using a soft agar motility assay. 2013 VA Branch ASM

Results: A measurable defect in intracellular growth by expressing DsbA2NP198T in A. castellanii was seen at 48 and 72 hours, and attachment and invasion of HeLa cells was significantly reduced (p=0.005); suggesting DsbA2 is important for pathogenesis. Erythrocyte hemolysis was significantly attenuated by expression of DsbA2NP198T (p1.00in) and the fecal indicator bacteria Escherichia coli and Enterococcus spp. were enumerated through standard membrane filtration methods. Quantification of fecal indicator bacteria levels after rainfall revealed an increase in both E.coli and Enterococcus levels at all of the sampling sites, both entering and leaving the ponds, with no discernible trend between sites. The increase in fecal indicator bacteria levels following rainfall indicates the run-off from the Arboretum may be a source of fecal contamination in Newman Lake. As such, this poses a potential problem for water quality  downstream  through  Black’s  Run  and  into   the city of Harrisonburg. 28 Effect of Lipomannan from M. smegmatis on RAW 264.7 Cells Stephanie DeRonde and David Freier. Lynchburg College, Lynchburg, VA. The dose response activation of RAW 264.7 murine macrophages by M.smegmatis lipomannan (LM), a TLR2 ligand, and lipopolysaccharide from E. coli (055:B5), a TLR4 ligand, will be compared. Concentrations ranging from 10 ng/mL to 3 ug/mL are used. Activation measures include nitric oxide (NO) production as measured by the Greiss reaction, western blot analysis of MAPK p38, and ELISA for TNF-α  production  in  culture  supernatants.  Initial  experiments  examining  10ng/ml  and  3µg/ml  LM   concentrations in comparison to 100 ng/ml of LPS (from E.coli O55:B5) demonstrated that the 3µg/ml concentration of LM produced a response of 60 µM nitrite, while the 100 ng/ml concentration of LPS induced a response of 108 µM of nitrite. This effect was seen in cells grown in DMEM, but not ones grown in RPMI 1640. This is an unexpected and potentially novel observation. The broad purpose of this research is to elucidate mechanisms by which Mycobacterium activate innate immune / inflammatory responses in order to provide a better understanding of Mycobacterium infection through comparison and analysis of the role of the TLR-2 pathway relative to the bioactive lipid and signaling molecule sphingosine-1-phosphate (S1P). 29 Characterizing the Invasion Domain of Anaplasma phagocytophilum Outer Membrane Protein A Kathryn S. Hebert*, David Seidman, and Jason A. Carlyon. Department of Microbiology and Immunology, Virginia Commonwealth University, Richmond, VA. Obligate intracellular bacteria use outer surface proteins called invasins to enter eukaryotic host cells. Infection by these organisms can be prevented or cured simply by blocking internalization. Thus, it is desirable to characterize invasins of obligate intracellular bacteria, specifically the domains of these proteins that are critical for entry into host cells. Anaplasma phagocytophilum is a tick-transmitted obligate intracellular rickettsial pathogen that causes the emerging disease, human granulocytic anaplasmosis (HGA). Our lab previously determined that the A. phagocytophilum outer membrane protein A (OmpA) is critical for invasion of mammalian host cells and its invasion domain lies within its N-terminus. Here, we generated a series of OmpA N-terminal peptide-specific antisera and assessed their efficacies at blocking A. phagocytophilum infection. Antiserum targeting OmpA amino 2013 VA Branch ASM

acids 59 to 74 (OmpA59-74)  significantly  inhibited  bacterial  binding  to  the  OmpA  receptor,  α2,3sialylated PSGL-1 and infection of host cells. Recombinant OmpA proteins carrying insertions of 5amino acids in the same region as that targeted by the blocking antiserum, but not any other region, were unable to bind host cells or competitively inhibit A. phagocytophilum infection. Using alanine substitutions, we pinpointed glycine 61 and lysine 64 as being critical for OmpA cellular adherence. These studies represent the most detailed characterization of any rickettsial invasin to date. 30 Orientia tsutsugamushi ankyrin repeat-containing proteins, Ank8 and Ank15, traffic to the lumen of host cell endoplasmic reticulum Sean Evans, Andrea R. Beyer, Lauren VieBrock, Smita Singh, and Jason A. Carlyon. Virginia Commonwealth University, Richmond, VA. Orientia tsutsugamushi, an obligate intracellular bacterium, is the causative agent of scrub typhus, a potentially deadly disease that afflicts one million persons annually in the Asia-Pacific region. The virulence factors that this understudied pathogen uses to take over eukaryotic host cells are poorly defined. An emerging theme among intracellular bacterial pathogens is the delivery of eukaryotic-like ankyrin-repeat containing effectors (Anks) into host cells. Ankyrin repeats are 33-amino acid tandem motifs that mediate protein-protein interactions. Prokaryotic Anks interact with host cell proteins to mimic, alter, or disrupt eukaryotic cellular functions and thereby promote pathogen survival. We previously confirmed that O. tsutsugamushi expresses 21 different anks during infection. As a first step in functionally characterizing the Anks, we monitored the subcellular localization of ectopically expressed Anks in host cells. Several were observed to traffic to distinct locales within the endoplasmic reticulum (ER). Two such ER-tropic Anks are Ank8 and Ank15. Using confocal miscroscopy, we observed that both Ank proteins colocalize with the ER lumenal marker, protein disulfide isomerase but not with ER membrane markers. Ank8, but not Ank15, also carries a second motif that mimics the F-box, which functions in eukaryotes to nucleate polyubiquitination machinery. Using a GST-pulldown assay, we demonstrated that Ank8 interacts with polyubiquitination machinery in an F-box dependent manner. These data suggest that Ank8 facilitates proteasomal degradation of an ER protein component and that both Ank8 and Ank15 could hijack the host cell secretory pathway to promote O. tsutsugamushi survival inside the host cell. 31 Acute  Dose  Response  Effects  of  γ-HCH (lindane) on Measures of Innate and Adaptive Immune Function in Female Swiss Mice Roslyn Alexander and David Freier. Lynchburg College, Lynchburg, VA. The primary functions of macrophages are defense against bacterial pathogens. To assess macrophage function, levels of nitric oxide production can be observed following the stimulation by bacterial lipopolysaccharides. In vivo, peritoneal exudate cells (PEC) can serve as a source of macrophages. Elicitation of PEC by 4% TG (thioglycolate) will be examined in comparison to the antibody forming (AFC)  cell  response,  following  treatment  with  γ  Hexachlorocyclohexane  (lindane).  Five  groups  of   female Swiss mice, four in each group will be treated with: Saline Vehicle (gavage), 10 mg/kg Lindane (gavage), 50 mg/kg Lindane (gavage), 100 mg/kg lindane (gavage) for 10 days, or 200 mg/kg Cyclophosphamide (IP) given 4 days prior to assay. Two mice per treatment will be used for elicitation of PEC by IP injection of sterile 4% TG. For the AFC response, each mouse receives an IP injection of 1x108 sheep erythrocytes 4 days prior to assay. The AFC response and PEC response to bacterial LPS are done in a separate series of experiments. Mice are euthanized by CO2 inhalation at day 0, and either peritoneal exudate cells or a single cell suspension of splenocytes will be prepared for evaluation of immune responses and the acute immunotoxicity of lindane. Previous literature has suggested a biphasic effect on T-dependent antibody responses, but no effect on macrophage phagocytic activity. 2013 VA Branch ASM

32 The Bacterial Hyaluronan Synthase Caitlin Hubbard and Jochen Zimmer. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA. Hyaluronan (HA) is an extracellular, linear polysaccharide consisting of alternating residues of glucuronic acid and N-acetyl-glucosamine. It is synthesized by the membrane integrated Hyaluronan Synthase (HAS), an enzyme that is highly conserved and ubiquitously expressed among vertebrates, where it forms a major component of the extracellular matrix. In addition to performing many physiological and structural roles in the body, HA is implicated in cancer, rheumatoid arthritis and wound healing, as well as the virulence of some bacterial species. Only a limited number of pathogenic prokaryotes produce HA. In the group A Streptococcal bacterium Streptococcus equisimilis, and Streptococcus pyrogenes, HA is extruded and surrounds the bacterial cell with a thick capsule of non-immunogenic HA to avoid an immune response by its host. Additionally, acquisition of HAS by Bacillus cereus allows the bacterium to cause an anthrax-like disease. While HA is synthesized inside the cell, it performs its biological function on the outside of the cell. Our aim is to understanding how the HA polymer is translocated across biological membranes. We established an in vitro assay for HA translocation from purified components to address whether HAS is sufficient for HA synthesis and membrane translocation. To this end, we purify HAS in a detergent solubilized system and reconstitute the protein into proteoliposomes that are then used for in vitro activity and translocation assays. Polymer translocation across the vesicle membrane can be assayed with the help of hyaluronidases, which leave translocated HA intact. This analysis demonstrates that HAS is necessary and sufficient for HA synthesis and translocation. 33 Analysis of plasmids from Streptococcus parauberis Armonee Avent and Ian Copeland. Norfolk State University, Norfolk, VA. Streptococcus parauberis is a gram-positive lactic acid bacterium that can be found in fish and dairy cows. This bacterium infects the kidneys and brain causing inflammation and necrosis of tissue, which can lead to death in certain fish species. It has been found in aquacultured fish in Spain and Korea and recently in striped bass in the Chesapeake Bay. In these fish strains of S. parauberis, a plasmid was identified recently. A plasmid is a genetic structure that can replicate independent of the chromosomes. As part of this project, the genes of this plasmid are being sequenced. This will assist in the identification of antibiotic resistance genes, virulence genes, and host-specificity genes. Another goal includes the characterization of this plasmid using a recently proposed plasmid classification system. Understanding the types of plasmids as well as any antibiotic resistance genes found in these pathogenic strains should facilitate the effective treatment of infected aquaculture fishes. These data may also contribute towards future development of a vaccine for this bacterium. Together these efforts should improve the profits of fishing industries and limit the emergence of this pathogen in wild populations, such as here in the Chesapeake Bay. 34 Protein interaction networks, complexes, and domains in bacteria J. Harry Caufield, Christopher Wimble, Marco Abreu, Norman Goodacre, and Peter Uetz. Virginia Commonwealth University, Richmond, VA. Conservation of protein binary interaction networks. We have recently produced binary protein interaction data for several species of bacteria, including Escherichia coli, Helicobacter pylori, Treponema pallidum, and Streptococcus pneumoniae. We have analyzed the conservation and evolution of protein-protein interactions across these and other species. This data will also assist in the identification of protein function for those previously uncharacterized (around 30% of all proteins in each proteome is currently of unknown function). Our preliminary analysis suggests that only few interactions are conserved, which is not surprising, given that only a fraction of all proteins are 2013 VA Branch ASM

conserved. Conservation of protein complexes across bacteria. Large-scale screens for microbial protein-protein interactions rarely capture the extent to which proteins participate in complexes. We have coupled the results of published mass spectrometry-characterized protein complexes with databases of gene orthology and essentiality to better characterize the extent to which proteins interact, the complexes they form, and how this is related to physiological traits. Comparative analysis of protein interaction domains in bacteria. Protein domains are conserved structural and functional elements of proteins, suggesting that domain-domain interactions will be preserved across species. We are analyzing protein domains and their interactions in multiple bacterial species (including E. coli, Treponema pallidum, and others) to find out to what extent there interactions and thus functions are conserved. This should lead to a better understanding of domains of unknown function (DUFs), uncharacterized proteins, and previously studied proteins. 35 Protein interaction networks in microbes and ecosystems Neha Sakhawalkar, Jitender Mehla, Andrey V. Matveyev, and Peter Uetz. Virginia Commonwealth University, Richmond, VA. Various methods and techniques are used to study protein-protein interactions, including the Yeast Two Hybrid system (Y2H) and affinity purification combined with mass spectrometry (AP/MS). However, none of them is truly a high-throughput method that allows to capture all interactions in a cell or organism at reasonable cost in a short time. We are developing a new methodology that allows us to map whole interactomes in a single experiment using a modified Y2H and next-generation sequencing. The technology could also be applied to meta-interactomes in an ecosystem, e.g. to map all interactions among the proteins in a microbiome, including interactions among species (e.g. virus-human, phage-bacteria etc.). In parallel, we are working to adapt a bacterial two-hybrid system (B2H) to next-gen sequencing. The Bacterial Adenylate Cyclase Two Hybrid (BACTH) system has been developed to study PPIs between both cytoplasmic and as well as membrane proteins. Furthermore, the existence of Gateway vectors and cloning should enable high throughput screening of interactions, in order to describe microbial interactomes as well as meta-interactomes (e.g. between viruses and their hosts). 36 Comparison of the Nitric Oxide Response of RAW 264.7 Murine Macrophages Grown in RPMI 1640 and DMEM Diana Spangler and David Freier. Lynchburg College, Lynchburg, VA. Research literature in which RAW 264.7 murine macrophages are used to study inflammatory activity vary significantly in factors such as cell number, duration of stimulus, and basic culture conditions. This study is designed to compare the two most common culture media; RPMI 1640 and DMEM. Both of these are supplemented with 10% fetal bovine serum, sodium bicarbonate, 100 IU Penicillin Streptomycin 100mg, and 25mM HEPES. The most significant difference is that DMEM contains 4.5g/l glucose where RPMI has 2 g/l. This comparison examines RAW 264.7 stimulation by a dose response of bacterial LPS and the production of nitric oxide. RAW 264.7 cells were incubated for  24  hours  in  a  5%CO2/  37˚C  environment  to  lipopolysaccharide  from  E. coli (serotype O55:B5) at concentrations from 1-100 ng/mL. Cells are kept in continuous culture with passage every 4 days and are at a limit of 25 passages or 3 months from thaw. Experiments are conducted on a 24-well plate 500ul volume at 4x105 cells/well. After stimulation, supernatants are collected to evaluate nitric oxide response by the Greiss reaction. Experiments suggest that cells grown in DMEM have a nitric oxide response that is 4-5 times greater than that of cells grown inRPMI1640. Both DMEM and RPMI grown cells show a dose response to LPS, but at a concentration of 100 ng/mL DMEM cells 2013 VA Branch ASM

produce a 100uM concentration of nitrite, while RPMI grown cells produce only 17uM. This difference in responsiveness of RAW 264.7 cells could significantly change interpretation of current and past primary scientific literature. 37 Utilizing Coding Sequences to Increase Phylogenetic Resolution of Pilobolaceae Heaven Cerritos and Dale Beach. Longwood University, Farmville, VA. The family of Pilobolaceae is a collection of fairly ubiquitous fungal species associated with the Mucorales,  and  best  known  as  “Shotgun  Fungi”  capable  of  shooting  sporangia  great  distances.  The   Pilobolaceae typically are found in the feces of grazing animals such as horses, cows, deer, and antelope. Animals eat the fungal spores that can be found attached to grass shoots. Once the spores are re-deposited in the field, small shoots grow and provide a launching platform to disperse the spores away from the dung. Continued study of these organisms is complicated since they are difficult to maintain long term cultures in the lab, and traditional methods of identifying samples to the level of species relies on difficult and ambiguous measurements of spore morphology. Such phenotypic characteristics of spores alone are difficult due to overlapping characteristics across genera. Recent application of phylogenetic methods has focused on ribosomal RNA sequences to successfully differentiate genera within the family of Pilobolaceae. We are extending this work by developing select protein coding sequences to classify isolates to species level. Using stored genomic DNA samples from type strains as well as novel isolates we are determining the Tubulin gene sequence to serve  as  a  molecular  “barcode”  to  differentiate  samples  and  evaluate  phylogenetic   relationships. In addition, we have begun the full-genome sequencing and assembly of a novel Pilobolus sp. isolate from Prince Edward County, Va. 38 Quantification of Enterococcus spp. in litter from an organic turkey farm Ben Holland, Steven McBride, Pradeep Vasudevan, and Joanna Mott. James Madison University, Harrisonburg, VA. Enterococcus, a Gram-positive gut commensal bacterial group, routinely contaminate waterways through runoff from agriculture, and can present health risks to humans and animals who come in contact with them. Their importance as indicators of fecal contamination has increased in recent decades because of their growing antimicrobial resistance, high levels within fecal wastes, and ability to survive in a wide range of conditions. Assessment of fecal contamination is an important step towards keeping humans and animals safe from infections in environmental and clinical settings. In this study, Enterococcus levels in turkey litter were assessed and species prevalence was analyzed. Over one hundred presumptive Enterococcus isolates were collected from the litter of a single flock of  turkey  poults  during  their  first  six  weeks  of  rearing  at  an  organic  “No  Antibiotic  Use,”  turkey  farm   in Shenandoah Co., VA. These isolates were first isolated using membrane-Enterococcus Indoxyl-βD-Glucoside agar, and then speciated using the Biolog MicrologTM system. The results of this study will provide information on levels of specific species of Enterococcus from agricultural settings that could potentially become sources of environmental and waterway contamination. 39 Identification of the Staphylococcal Cassette Chromosome mec (SCCmec) element in an Environmental Bacteriophage Population Amber Brooke Sauder, Emily Pelto, and Dr. Louise Temple. James Madison University, Harrisonburg, VA. Methicillin-resistant Staphylococcus aureus has routinely been viewed as the responsible pathogen for many nosocomial infections, but in the past decade, more disease has occurred outside hospitals, indicating the possible spread of methicillin resistance primarily in agricultural settings. The genetic element that carries the methicillin-resistance gene, mecA, is the SCCmec element. Because SCCmec 2013 VA Branch ASM

is a mobile genetic element, it could be transferred in three ways - conjugation, transformation, or transduction. The method of interest to this study is transduction. In order to determine if bacteriophages are transferring the SCCmec element between bacteria, a protocol was developed to isolate the phage population and use PCR and sequencing technologies to examine the genetic information contained within the bacteriophages. The developed protocol consists of sample filtration and sterilization, phage precipitation, DNA extraction, PCR, agarose-gel electrophoresis, and sequencing. This protocol was developed around positive controls, spiking samples, and general troubleshooting. The protocol was altered several times to extract as much phage DNA as possible, prevent the degradation of DNA, and deal with possible PCR inhibitors. The current protocol has produced one sample that showed a PCR product of the right size for a gene in the SCCmec element and was confirmed positive by sequencing. The sequence shows that the element is type I.2. Future goals of this experiment include: collecting and processing more samples, sequencing more PCR products, whole sample sequencing using PacBio technology, and possible chromosome walking to determine the extent of the SCCmec element carried by the bacteriophages. 40 Temperature Study of Mycobacterial Infections in Striped Bass Smith, D.E., Vogelbein, W.K., Korngiebel-Rosique, M., and Gauthier, D.T. Old Dominion University, Norfolk, VA. Striped bass (Morone saxatilis) are the most popular recreational sportfish in Chesapeake Bay. Mycobacterial infections within these fish are present at very high levels and are an area of concern within the fishing community. Mycobacterium shottsii and Mycobacterium pseudoshottsii appear to be the major agents associated with disease in this system. With increased eutrophication, the deeper waters of Chesapeake Bay have become increasingly hypoxic and it is speculated that striped bass are subject  to  a  “thermal  oxygen  squeeze”;;  hypoxia  in  the  deeper  waters  pushes  the  fish  into  shallower,   warmer waters, potentially leading to increased susceptibility to infection. In this study, we examined the effect of temperature on infection with M. shottsii and M. pseudoshottsii in striped bass. Striped bass were experimentally infected with 10⁶ colony-forming units of M. shottsii or M. pseudoshottsii, then  held  at  20˚C.  Quantitative  PCR  and  bacteriology  were  used  to  determine  splenic  bacterial   densities at 4 weeks post-infection. Water  temperatures  were  then  changed  to  25˚  or  30˚C,  or  kept   constant  at  20˚C  for  an  additional  4  wk.    Prior  to  the  temperature  change,  there  were  no  significant   differences in M. pseudoshottsii or M. shottsii densities between systems. Four weeks after the temperature change, there were significant differences in density for both bacteria across temperatures.    The  highest  densities  for  both  bacteria  were  observed  at  20˚C,  and  the  lowest  densities   were  observed  at  30˚C.    Our  data  therefore  reveal  an  inverse relationship between temperature and infection; which is inconsistent with the thermal oxygen squeeze hypothesis. 41 Genomic and Phenotypic Analysis of Bacillus pumilus Bacteriophage Lysis Cassette Carly Starke, Nathaniel Burkholder, Dr. Ron Raab, and Dr. Louise Temple. James Madison University, Harrisonburg, VA. A novel lysis cassette in bacteriophage Pappano, isolated from Bacillus pumilus strain BL8, was identified from whole genome sequence annotation. The lysis cassette consists of genes encoding hydrolase and holin-like products predicted to be capable of lysing B. pumilus cells. Another gene upstream of the hydrolase and holin genes encoding a peptidase was also speculated to play a role in cell lysis. Various lysis cassette combinations were PCR amplified, ligated into broad host range vector pTYB2 downstream of an IPTG inducible promoter, and transformed into E. coli. Cultures containing the desired constructs were grown to mid-log phase and induced with IPTG. Approximately one hour post induction of the entire cassette, a steep lysis curve was observed. It was observed that the holin when expressed alone was capable of inducing cell lysis at a slightly lower rate compared to the full cassette, but cells expressing the hydrolase alone did not undergo lysis. 2013 VA Branch ASM

There did not appear to be a difference in the rate of lysis in cultures with or without the peptidase gene. Lysis of BL8 infected with Pappano at a multiplicity of infection (MOI) of 3 was observed within five minutes post inoculation. Even though the lysis cassette exists in the genome of BL8 with its hydrolase and holin genes in opposite orientation than typically found in other phages, we have shown that it is a functional, novel lysis cassette. We have also shown that this cassette is capable of lysing gram-negative cells that have a different cell wall composition. 42 Francisella tularensis interspecies communication Scott N. Dean, Collette Marchesseault, and Monique van Hoek. George Mason University, Fairfax, VA. The goal of this study is to determine the interspecies communication of Francisella tularensis in the context of the natural polymicrobial environment in the soil. These results will enhance our knowledge of the persistence of this organism in its natural habitat. In this study we focused on Francisella and its interaction with Gram-negatives, Pseudomonas aeruginosa and Burkholderia sp., known pathogens present in the soil. P. aeruginosa and produces quinolines (such as HQNO) and monounsaturated fatty acids signals that cause significant effects on the metabolism, growth, and biofilm formation of F. novicida. We hypothesize that small soluble factors produced by Pseudomonas and Burkholderia may have specific effects on F. tularensis lifestyle through dysregulation  of  Francisella  gene  expression.  In  our  studies,  we  have  shown  the  effect  of  100  μg/mL   HQNO and P. aeruginosa conditioned media on static F. tularensis novicida biofilm, and that these small soluble signals significantly decrease biofilm mass (p < 0.01) as quantified by COMSTAT (without affecting bacterial growth). The metabolism and growth of Francisella was also studied in response to P. aeruginosa signaling molecules. We have shown that P. aeruginosa produces signals that significantly alter F. novicida metabolism, growth, and biofilm. In future work, we will assess the effect of HQNO and other small signaling molecules on Francisella virulence and gene expression. These studies may lead to a greater knowledge of F. tularensis persistence in the natural polymicrobial environment. This work was supported by HDTRA1-12-1-030 Francisella interspecies interaction with Pseudomonas, a soil microbe. 43 Birds as Vectors for Rickettsia parkeri Jessica Vincent, Chelsea Wright, Erin Heller, Dr. David Gauthier, Dr. Eric Walters, and Dr. Holly Gaff. Old Dominion University, Norfolk, VA. Rickettsia parkeri is a bacterial pathogen carried by Amblyomma maculatum, the Gulf Coast tick. In 2004, R. parkeri was identified as a cause of disease in humans, which was called Tidewater spotted fever because of its initial diagnosis in the Tidewater area of Virginia and similarity to Rocky Mountain spotted fever. Increasing diagnoses of Tidewater spotted fever in the eastern United States have raised concerns in endemic areas. Gulf Coast ticks are concomitantly spreading north in a rapid and punctuated pattern. This dispersal is atypical of terrestrial animal vectors, and implicates birds as likely dispersal agents. In order to help determine whether birds have played a role in the spread of A. maculatum and, in turn, Tidewater spotted fever, we are attempting to detect Rickettsia parkeri in wild avian blood samples. We use a TaqMan assay designed for Rickettsia spp., and intend to test positive samples for species-level identification. Currently, out of the 250+ samples tested, none have been positive. If the pathogen is detected in avian blood, this will be a transformative finding in our understanding of tick ecology. If not, this may indicate that the pathogen is not detectable in the bird blood or that birds do not play a vector role in the transmission of R. parkeri. To differentiate between the two possibilities, we plan on testing A. maculatum collected from birds for Rickettsia parkeri .

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44 Genome-Wide Association Study of Mycobacteriophage Host Preference Hayley Norian and Steven G. Cresawn. James Madison University, Harrisonburg, VA. Mycobacteriophages are bacteriophages that infect the genus of bacteria Mycobacterium, which includes pathogens such as Mycobacterium tuberculosis and Mycobacterium ulcerans. Full genome sequences of 461 mycobacteriophages are currently available and have been grouped into clusters of related genome sequences. Host range information, or whether or not the phages infect or do not infect a particular host, is available. In a recent paper, some 220 phages were isolated on Mycobacterium smegmatis strain mc2155, sequenced and placed into 42 groups of related genome sequences. When testing host range, three different levels of infectivity were observed: phages that were incapable of infecting the host, phages that were capable of infecting the host, and some that were at a plating efficiency less than one relative to mc2155. The phages that are capable of infecting a particular host are of particular interest. Using software to compare genome sequences, we can observe subtle differences in the genomes that might play a role in whether or not the phage can infect a particular host. Identifying crucial genes in phages to infecting bacterial hosts could be potentially useful in the future in terms of therapeutics and treatments. 45 Identification and Characterization of Staphylococcus Spp. In Stream Water and Sediment Michael Partin, Kevin Libuit, and Dr. Jim Herrick. James Madison University, Harrisonburg, VA. Staphylococcus is comprised of 41 known species, of which 18 can colonize humans. Despite the prevalence of infectious Staphylococcus within hospital settings and agriculture, there are few reports of Staphylococcus in natural bodies of water. A recent study by the US FDA found substantial contamination of poultry and other meats with Staphylococcus. We hypothesized that intensive farming of poultry adjacent to streams would result in contaminated runoff, resulting in at least transient occurrence of Staphylococcus spp. in streams waters and sediments. This study intends to determine whether Staphylococcus occurs and persists within agriculturally impacted streams in the central Shenandoah Valley. In preliminary work, large numbers of Staphylococcus spp. were detected in water from Muddy Creek, which runs adjacent to a poultry processing plant. Mannitol Salt Agar (MSA) was used to isolate 11Staphylococcus spp. from the Muddy Creek site. As the occurrence of Staphylococcus in Muddy Creek seems to be transient and highly variable, perhaps depending upon runoff and season, continuing efforts are underway to determine the temporal and spatial variability in its occurrence in both water and sediment. Future studies will include determining the susceptibility of Staphylococcus isolates to a range of relevant antibiotics – including oxacillin, which would identify methicillin resistance – and whether resistance is transmissible and/or plasmid borne. Overall, this study will provide insight into the occurrence of staphylococci in freshwater and a possible link between the microflora of agricultural animals and that of native streams. 46 Brain-specific angiogenesis inhibitor-1 (BAI1) regulates macrophage bactericidal activity and cytokine responses to Gram-negative bacteria Emily A. Billings, Soumita Das, Chang Sup Lee, Kodi S. Ravichandran, and James E. Casanova. University of Virginia, Charlottesville, VA. The detection of a pathogen and initiation of an immune response occurs through a limited set of germ-line encoded receptors called pattern recognition receptors (PRR). The coordinated actions of these receptors result in microbicidal activity, antigen presentation, cytokine and chemokine production, and loss of these receptors can have devastating consequences on immunity. Brainspecific angiogenesis inhibitor-1 (BAI1) has recently been characterized as a phagocytic receptor that drives uptake of Gram-negative bacteria. Here we investigate the role of BAI1 in the cellular response to Gram-negative bacteria, including crosstalk with other PRRs. We find that BAI1deficient macrophages exhibit both impaired uptake of and microbicidal activity against a model 2013 VA Branch ASM

Gram-negative species, E. coli K-12. BAI1 ligation stimulates the production of reactive oxygen species (ROS), suggesting that BAI1-mediated microbicidal activity depends on the ROS burst. ROS induction may be enhanced through BAI1-driven activation of Rac2. We have shown that BAI1 binds lipopolysaccharide (LPS), however TLR4 is the main immunostimulatory receptor during LPS recognition. Interestingly, we find that TLR4 associates with BAI1, suggesting the formation of a signaling complex, and in fact BAI1-deficient macrophages exhibit attenuated inflammatory signaling. While activation of NFkB appears normal in BAI1-deficient cells, activation of IRF3 and induction of TRIF-dependent cytokines and chemokines is significantly impaired. Collectively, these results suggest that BAI1 may regulate the clearance of microbes through enhanced phagocytosis and microbicidal activity and may also drive the immune response by influencing the cytokine milieu during infection. Future studies will investigate the importance of BAI1 in-vivo using a peritoneal infection model. 47 Manipulation of host signaling by the Salmonella translocon/effector protein SipC Carissa B. Meyer, Adam Greene, and James E. Casanova. University of Virginia, Charlottesville, VA. Salmonella enterica are a genus of Gram-negative bacteria that cause a range of human diseases and present a significant global health burden. To invade cells and evade host defenses, Salmonella injects  ‘effector’  proteins  into  the  host  cell  through  a  ‘molecular  syringe’  called  a  Type  III  secretion   system (T3SS). Bacterial effectors hijack numerous host signaling pathways to stimulate bacterial uptake and create an intracellular niche for replication, the Salmonella containing vacuole (SCV). SipC is an essential component of the invasion-associated T3SS that also functions as an effector. Our previous work identified eukaryotic SipC-interacting proteins through a yeast-two-hybrid screen using the SipC C-terminal tail as bait, including proteins involved in modulating actin, membrane trafficking  and  signaling.  Here  we  investigate  the  function  of  several  ‘hits’  during  Salmonella   infection of non-phagocytic cells. We have verified that filamin A, MAP4K4, PICALM, and Bax interact with SipC-C in HeLa cell extracts. MAP4K4 is a mitogen-activated serine/threonine kinase that regulates cortical actin architecture and JNK activity through multiple targets. Depletion of MAP4K4 enhanced Salmonella invasion, suggesting that SipC co-opts MAP4K4 to limit bacterial uptake. Bax is a pro-apoptotic Bcl2 family protein whose activity is inhibited by Akt. We find that SipC-C also interacts with Akt in HeLa cell extracts, suggesting that SipC may influence apoptosis through the Akt/Bax signaling axis. Finally, we show that SipC localizes to the SCV and persists on many SCVs for up to four hours post-invasion. Collectively, our data suggests that SipC influences bacterial invasion and intracellular survival by hijacking diverse cellular pathways. 48 Integrated Phylogenomic Approaches Toward Pinpointing the Origin of Mitochondria Zhang Wang and Martin Wu. Department of Biology, University of Virginia, Charlottesville, VA. "Overwhelming evidence supports the endosymbiosis theory that mitochondria originated once from within  α-proteobacteria. However, exactly when it happened remains highly debated. In this study, we took advantage of integrated phylogenomic approaches to pinpoint the origin of mitochondria. In particular,  we  1)  filled  the  gaps  in  the  mitochondrial  tree  of  life  by  sequencing  genomes  of  19  αproteobacteria that represent a broad phylogenetic diversity, 2) identified a number of mitochondrialderived nuclear genes  as  “well-behaved”  phylogenetic  markers  with  lower  evolutionary  rates  and  less   compositional bias, and 3) applied more realistic phylogenetic models that better account for LBA and sequence compositional bias. With cutting-edge phylogenetic methods and more and better data, our integrated phylogenomic approaches revealed an interesting tree topology. On the one hand, the mitochondria were grouped with Rickettsiales, which is consistent with the well-accepted topology. On the other hand, Pelagibacter, the smallest free-living bacteria previously claimed to be the sister clade of mitochondria, were found to be distantly related to the mitochondria. In addition, 2013 VA Branch ASM

reconstruction of genome content of mitochondrial ancestor suggested it lacked a hydrogenproducing machinery but likely possessed a complete aerobic pathway. Our results therefore indicated  a  lack  of  evidence  for  the  “hydrogen  hypothesis”  as  opposed  to  the  alternative  “oxygen   scavenger  hypothesis”  for  the  origin of mitochondria. 49 Highly Dynamic Gut Micriobiota of Wild Baboon Populations Tiantian Ren1, Laura Grieneisen2, Elizabeth Archie2, and Martin Wu1. 1Department of Biology, University of Virginia, Charlottesville, VA. 2Department of Biological Sciences, University of Notre Dame, South Bend, IN. Gut microbiota has been shown to play essential roles in host biology. However, it is less clear what forces are important in shaping the gut microbiota and how the microflora changes over a host’s   lifetime. To investigate environmental factors and host traits that influence gut microbiota over time, we determined the bacterial composition of 132 fecal samples collected from 32 wild baboons in the Amboseli National Park over 17 years. Our 16S rRNA survey showed that baboon gut microbiota is typical of an omnivorous primate. However, it is highly dynamic between and within individuals. Strikingly, samples collected from the same individual one month apart can be as different as samples collected over 10 years apart. Our study showed that bacterial richness increases with age (from 2 months to 20 years). Canonical correspondence analysis (CCA) revealed that age and rainfall are the only factors associated with the bacterial composition, suggesting that microbial exposure and seasonal diet shifting might play important roles in determining gut microbial structure. Our findings support  the  “rare  biosphere”  theory, that rare microbes can serve as a dormant seed bank and can resuscitate under favorable environmental conditions.

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