th Annual Wind River Conference on Prokaryotic Biologywriver.sites.unlv.edu/58th_WR_Complete_Prog_5.pdfAmerican Society for Microbiology UNLV, School of Life Sciences College of Arts

58th Annual Wind River Conference, June 4-8, 2014

58th Annual Wind River Conference on Prokaryotic Biology

Basic Microbiology - The Key to Understanding The Complexity of Life

June 4-8, 2014

Aspen Lodge

Estes Park Colorado USA

58th Annual Wind River Conference, June 4 - 8, 2014 2

WELCOME TO THE 57th ANNUAL WIND RIVER CONFERENCE ON PROKARYOTIC BIOLOGY 2014

Wind River Executive Committee: Dr. Ron Yasbin, UNLV to University of Missouri—St. Louis Dr. Uldis Streips, University of Louisville Medical School Dr. John Iandolo, University of Oklahoma Health Sciences Center Dr. Cristina Bongiorni, Danisco Dr. Diana Downs, University of Wisconsin Dr. Helen Wing, University of Nevada, Las Vegas Dr. Jorge Escalante, University of Wisconsin Dr. Charlie Moran, Emory University Dr. Rifka Rudner, Hunter College Dr. Don G. Ennis, University of Louisiana, Lafayette Dr. Mario Pedraza-Reyes, University of Guanajuato Dr. Eduardo Robleto, University of Nevada, Las Vegas Dr. Corrie Detweiler, University of Colorado Boulder Dr. R. Martin (Marty) Roop, East Caroline University Scientific program chairs: Dr. Helen Wing, University of Nevada, Las Vegas Dr. Erin Murphy, Ohio University Heritage College of Osteopathic Medicine Contributions from the following sponsors helped make this year’s conference possible and are gratefully acknowledged: National Science Foundation Monserate Biotechnology Group Sharlet Biotechnologies, Inc. American Society for Microbiology UNLV, School of Life Sciences College of Arts and Sciences, University of Missouri—St. Louis Funding provided by the National Science Foundation and the American Society for Microbiology allows us to support the travel of graduate students and post-doctoral fellows to the conference. Special acknowledgement:

Special thanks are due to Scott Miller at UNLV for expert administrative assistance.


THE WIND RIVER HALL OF FAME

PAST AND PRESENT PLENARY SPEAKERS

2014 Dr. Michael Gilmore Harvard Medical School Dr. Gisela Storz National Institutes of Health

Dr. Ferran Garcia-Pichel Arizona State University Dr. Barbara Funnell University of Toronto 2013 Dr. Nina Salama Fred Hutchinson Cancer Institute Dr. Shelley Payne University of Texas at Austin Dr. Renee Tsolis University of California Davis Dr. Ralph Isberg Tufts University 2012 Dr. David Rudner Harvard Medical School Dr. Esther Angert Cornell University Dr. Mark Gomelsky University of Wyoming Dr. Dennis A. Bazylinski University of Nevada, Las Vegas 2011 Dr. James Bardwell University of Michigan Dr. Sean Crosson University of Chicago Dr. Jo Handelsman Yale University Dr. R. Martin Roop, II East Carolina University 2010 Dr. Peter Christie University of Texas-Houston Medical School

Dr. Richard Gourse University of Wisconsin Dr. Dan Kearns University of Indiana

Dr. Rolf Thauer Max Planck Institute for Terrestrial Microbiology, Marburg 2009 Dr. Jim Imlay, University of Illinois at Urbana-Champaign Dr. Andy Camilli Tufts University, Howard Hughes Medical Institute Dr. Jade Wang Baylor College of Medicine Dr. Norman Pace University of Colorado at Boulder 2008 Dr. Roberto Kolter, Harvard Medical School Dr. Graham Hatfull, University of Pittsburgh Dr. Roberto Belas, University of Maryland

Dr. William Jacobs, Albert Einstein College of Medicine

2007 Dr. Jared Leadbetter, California Institute of Technology Dr. Tina Henkin, Ohio State University Dr. Harry Mobley, University of Michigan Medical School Dr. Lynn Zechiedrich, Baylor College of Medicine

2006 Dr. David Figurski, Columbia University Medical Center Dr. Karl E. Klose, University of Texas-San Antonio Dr. John Helmann, Cornell University Dr. Victor Dirita, University of Michigan School of Medicine

2005 Dr. Samuel Kaplan, University of Texas Houston Medical School Dr. George O’Toole, Dartmouth Medical School Dr. Dianne Newman, California Institute of Technology Dr. Tom Silhavy, Princeton University

2004 Dr. Curtis Suttle, University of British Columbia Dr. Paul Gollnick, State University of New York-Buffalo Dr. Heidi Kaplan, University of Texas Houston Medical School


Dr. Jorge Galán, Yale University School of Medicine

2003 Dr. Vincent Fischetti, Rockefeller University Dr. Robert Landick, University of Wisconsin Dr. Bonnie Bassler, Princeton University Dr. Neil Welker, Northwestern University

2002 Dr. Stephen Farrand, University of Illinois-Urbana Dr. Barry Wanner, Purdue University Dr. Sanford A. Lacks, Brookhaven National Laboratory Dr. Janet Yother, University of Alabama-Birmingham Dr. Malcolm Winkler, Lilly Research Laboratories

2001 Dr. Kim Lewis, Tufts University Dr. Austin Newton, Princeton University Dr. Julie Theriot, Stanford University Dr. Dale Kaiser, Stanford University

2000 Dr. Thomas A. Trautner, Max-Planck Institute for Molecular Genetics Dr. Ryland F. Young, Texas A&M University Dr. Abigail A. Salyers, University of Illinois Dr. John W. Costerton, Montana State University Dr. Tony Romeo, University of North Texas Health Science Center

1999 Dr. Frederick Neidhardt, University of Michigan Dr. Olaf Schneewind, University of California at Los Angeles Dr. Roger W. Hendrix, University of Pittsburgh Bacteriophage Institute Dr. Eugene Nester, University of Washington

1998 Dr. Scott Hultgren, Washington University School of Medicine Dr. Susan M. Rosenberg, Baylor College of Medicine Dr. Mary Lou Guerinot, Dartmouth College Dr. Peter Greenberg, University of Iowa

1997 Dr. Malcolm Winkler, University of Texas Houston Medical School Dr. John Baross, University of Washington Dr. Philip Harriman, National Science Foundation Dr. Susan Golden, Texas A&M University


Wednesday, June 4, 2014

3:00-6:00 PM Registration 4:00-6:30 PM Welcome Reception (Lobby of main lodge) 6:30-8:00 PM Dinner & Opening remarks 8:00-9:00 PM

Evolution of Enterococci into Leading Hospital Pathogens

in the Antibiotic Era

Michael S. Gilmore

Harvard Medical School Departments of Ophthalmology and Microbiology, Boston, MA, USA

9:00PM- Happy Hour at the lounge and bar

Thursday, June 5, 2014

7:00-8:00 AM Breakfast (Great Room Dining Room) 8:10–8:30 AM 8:35-8:50 AM 8:55-9:10 AM 9:15-9:35 AM 9:40-9:55 AM

Role of the PrrF and PrrH Small RNAs in Virulence of Pseudomonas aeruginosa Amanda G. Oglesby-Sherrouse University of Maryland, Baltimore, MD, USA Role of the PrrF and PrrH Small RNAs in virulence of Pseudomonas aeruginosa Alexandria Reinhart University of Maryland, Baltimore, MD, USA Twin sRNAs RyfA1 and RyfA2 of Shigella dysenteriae William H. Broach Ohio University, Athens, OH, USA

A cyclic-di-GMP riboswitch regulates stability of a small RNA in Vibrio cholerae Benjamin R. Pursley Michigan State University, East Lansing, MI, USA Regulation of Shigella dysenteriae Virulence Factors by RNA Thermometers Andrew B. Kouse Ohio University, Athens, OH, USA

10:00-10:20 AM Coffee Break


10:20-10:35 AM 10:40-10:55 AM 11:00-11:20 AM 11:25-11:40 AM 11:45-12:05 PM 12:10-12:30 12:30-2:00 PM 2:00-2:20 PM 2:25-2:50 PM 2:55-3:15PM

Characterization of Brucella abortus hypothetical proteins and their role in virulence James A. Budnick Virginia Tech, Blacksburg, VA, USA A LysR-type transcriptional regulator activates sRNA-encoded genes and plays a critical role in virulence of Brucella abortus Lauren M. Sheehan Virginia Tech, Blacksburg, VA, USA The alternative sigma factor σB is a global regulator of small RNAs (sRNAs) in Staphylococcus aureus Ronan Carroll

University of South Florida, Tampa, FL, USA The δ subunit of RNA polymerase guides promoter selectivity and virulence in Staphylococcus aureus Andy Weiss University of South Florida, Tampa, FL, USA Mechanisms of AmrZ-mediated activation and repression of Pseudomonas aeruginosa virulence genes Binjie Xu The Ohio State University, Columbus, OH, USA Characterization of the forgotten SlyA in Shigella flexneri Natasha Weatherspoon-Griffin University of Nevada Las Vegas, Las Vegas, NV, USA Lunch (Great Room Dining Room) Thiol biochemistry in methane-producing Archaea Nicole R. Buan University of Nebraska-Lincoln, Lincoln, NE, USA Use of bioinspired topographies to reduce bacterial surface contamination and medical device related infections Ethan E. Mann Sharklet Technologies, Inc, Aurora, CO, USA Functional Characterization of Genes Required for Lignocellulose Degradation in Cellvibrio japonicas Jeffery G. Gardner University of Maryland-Baltimore County


3:00-5:00 PM 5:00-6:30 PM 6:30-7:30 PM 8:00-9:00 PM 9:00 -

Free Time & Poster Set-up Poster & Happy Hour Dinner (Great Room Dining Room)

Intricate regulation provided by small RNAs and small proteins

Gisela Storz Eunice Kennedy Shriver National Institute of Child Health and Human Development

Cell Biology and Metabolism Program Bethesda, MD, USA

Happy Hour at the lounge and bar

Friday, June 6, 2014

7:00-8:00 AM

Breakfast (Great Room Dining Room)

8:10–8:30 AM 8:35-8:55 AM 9:00-9:15 AM 9:20-9:35 AM

Oral transmission of Mycobacteria: Investigations of M. marinum Association with and breaching the gut epithelia of fish Don G. Ennis University of Louisiana, Lafayette, LA, USA Fatty acid alterations protect Enterococcus faecalis from membrane damaging agents including the antibiotic daptomycin Elizabeth M. Fozo University of Tennessee, Knoxville, TN, USA Deletion of an ABC transporter renders Enterococcus faecalis susceptible to lysozyme Sriram Varahan

University of Kansas, Lawrence, KS, USA Regulation of a dual diguanylate cyclase-phosphodiesterase (GGDEF/EAL) protein in A. tumefaciens Nathan Feirer Indiana University, Bloomington, IN, USA


9:40-9:55 AM Sick and twisted: A structure-function analysis of the Helicobacter pylori stomach colonization factor Csd4 Kris M. Blair University of Washington, Seattle, WA, USA


10:20-10:40 AM 10:45-11:05 AM 11:10-11:30 AM 11:35 - 11:50 AM 11:55-12:10 PM 12:15-12:30 PM 12:35-12:45PM 12:45-2:00 PM

Biogenesis of BamA (Omp85), a core protein of the β-barrel assembly machine of Gram negative bacteria, mitochondria and chloroplasts Rajeev Misra Arizona State University, Tempe, AZ, USA Regulation of agglutination in Staphylococcus aureus Heidi A. Crosby University of Iowa, Iowa City, IA, USA Triclosan induces Staphylococcus aureus nasal colonization Adnan K. Syed University of Michigan, Ann Arbor, MI, USA Peptidoglycan fragments as a potential signal for biofilm dispersal David E. Payne University of Michigan, Ann Arbor, MI, USA Role and regulation of extracellular proteases in Staphylococcus epidermidis Alexandra E. Paharik University of Iowa, Iowa City, IA, USA The disulfide bonding system suppresses an alternate cellulose activation system David A. Hufnagel University of Michigan, Ann Arbor, MI, USA Group Picture Lunch (Great Room Dining Room)


2:00-4:00 PM 4:00-6:30 PM 5:00-6:30 PM 6:30-7:30 PM 8:00-9:00 PM 9:00 -

Free Time Posters Session I Happy Hour Dinner (Great Room Dining Room)

THE NEIL WELKER MEMORIAL PLENARY ADDRESS Introduction by Uldis StreIps

Microbe vs. microbe at the OK corral; Global change and the biogeography of soil microbes

Ferran Garcia-Pichel

Arizona State University, School of Life Sciences Tempe, AZ, USA


Saturday, June 7, 2014

7:00-8:00 AM Breakfast (Great Room Dining Room)

8:10–8:30 AM 8:35-8:50 AM 8:55-9:10 AM 9:15-9:35 AM 9:40-9:55 AM

Galleria mellonella as an infection model of Group A Streptococcus Scott V. Nguyen University of Oklahoma Health Sciences Center, Oklahoma City, OK, USA A proposed model for the regulation of IncA/C plasmid conjugation transfer Kevin S. Lang

University of Minnesota, Saint Paul, MN, USA

Regulation of the alternative sigma factor σX, during transformation in Streptococcus pneumoniae Yanina Tovpeko University of Illinois-Chicago, Chicago, IL, USA Development of competence leads to mutagenesis in stressed Bacillus subtilis cells Amanda A. Prisbrey University of Nevada, Las Vegas, Las Vegas, NV, USA Non-B DNA promotes genetic diversity in B. subtilis stationary phase cells Carmen Vallin University of Nevada, Las Vegas, Las Vegas, NV, USA



10:20-10:40 AM 10:45-11:05 AM 11:10-11:25 AM 11:30-11:50 AM 11:55-12:10 PM 12:15-12:30 PM 12:30-2:00 PM

An iron transporter limits Salmonella replication in single-infection but is required for mixed-infection of mice and macrophages Toni A. Nagy University of Colorado-Boulder, Boulder, CO, USA Ironing it out: the role of the Yersinia type III secretion system in the gut Leah Schwiesow University of California-Santa Cruz, Santa Cruz, CA, USA Nutritional stress activates the Salmonella pathogenicity island 2 type III secretion system intracellular virulence program Timothy Tapscott

University of Colorado School of Medicine, Aurora, CO, USA Multiple flagellins in Agrobacterium tumefaciens: function and coordination Bitan Mohari

Indiana University Bloomington, Bloomington, IN, USA

FlgM is secreted in Bacillus subtilis Rebecca A. Calvo Indiana University, Bloomington, IN, USA Characterization of swrD function in Bacillus subtilis swarming motility Ashley N. Roarty Indiana University, Bloomington, IN, USA Lunch (Great Room Dining Room)

2:00-4:00 PM 4:00-6:30 PM 5:00-6:30 PM 6:30-7:30 PM 8:00-9:00 PM

Free Time Poster Session II Happy Hour Dinner (Great Room Dining Room)

Surfing on the bacterial chromosome: the dynamics of plasmid partition promoted by ParA ATPases

Barbara Funnell


9:00 -

University of Toronto Department of Molecular Genetics

Toronto, Ontario, CANADA


7:00-8:30 Sunday, June 9, 2013 Breakfast (Great Room Dining Room) Departure


Abstracts for Talks, in Oder of Presentation Evolution of Enterococci into leading Hospital Pathogens in the Antibiotic Era Michael S. Gilmore,1,2 Francois Lebreton,1,2 Daria Van Tyne,1,2 and Ashlee M. Earl2

1Harvard Medical School Departments of Ophthalmology and Microbiology Boston, MA, 02114 2Broad Institute of MIT and Harvard Cambridge, MA, 02142 The enterococci are believed to be members of an ancient gastrointestinal tract consortium that assembled as long as 500 Million years ago. The evidence for this is their ubiquitous presence in the intestines of nearly all forms of terrestrial life, from insects to man, implying their occurrence in the last common ancestor. However, in the antibiotic era, they emerged among the vanguard of multidrug resistant hospital pathogens, where they infect the bloodstream, urinary tract and surgical wounds. Most multidrug resistant hospital infections caused by enterococci are caused by specific clades of the species E. faecalis and E. faecium. Comparative genomics has proven to be a powerful tool for determining how these specific clades evolved following the introduction of antibiotics. E. faecalis occurs as an ancient branch of the genus, and much of the divergence of the most common hospital lineages can be accounted for by the entry of mobile elements, including pathogenicity islands, fitness modules, and phages – possibly as the result of the loss of CRISPR protection of the genome. Hospital strains of E. faecalis often possess genomes 25% larger than commensal strains, with all of the difference being accounted for by mobile elements. The leading hospital pathogenic lineage of E. faecium appears to have emerged from a commensal lineage found in the intestines of animals, in which the carbohydrate utilization pathways have been systematically replaced. Additionally, mobile elements have also played a prominent role in forming the highly hospital adapted clade. This lineage appears to be highly mutable, and the E. faecium clade causing most VRE infections has arguably now become a new species.


Role of the PrrF and PrrH Small RNAs in Virulence of Pseudomonas aeruginosa Angela Nguyen1, Maura O’Neill1, Daniel Powell1,2, Robert Ernst2,3, Angela Wilks1, Amanda Oglesby-Sherrouse1,2 1University of Maryland School of Pharmacy, Department of Pharmaceutical Sciences 2University of Maryland School of Medicine, Department of Microbiology & Immunology 3University of Maryland School of Dentistry, Department of Microbial Pathogenesis Baltimore, MD 21201 Pseudomonas aeruginosa is an opportunistic bacterial pathogen that requires iron for growth and virulence. To balance its requirement for iron with the potential for iron toxicity, P. aeruginosa employs a hierarchy of regulatory systems to maintain iron homeostasis. The iron-responsive PrrF1 and PrrF2 small RNAs (sRNAs) play a central role in this regulatory network (Wilderman, et al, 2004). The PrrF sRNAs are produced when iron is limiting and block the production of iron-containing proteins, thus “sparing” iron when this nutrient is scarce. Additionally, Oglesby, et al, previously showed that PrrF regulation spares a metabolite - anthranilate - for production of a bacterial pheromone referred to as Pseudomonas quinolone signal (PQS). PQS induces expression of virulence genes, indicating a regulatory mechanism by which PrrF may contribute to P. aeruginosa pathogenesis. While all Pseudomonads encode for the PrrF sRNAs, P. aeruginosa is unique in its tandem arrangement of the prrF1 and prrF2 genes on the chromosome. This arrangement allows for the expression of the PrrH sRNA that is responsive to heme (Oglesby-Sherrouse and Vasil, 2010), an abundant source of iron in the human host. Recent work in our lab shows that PrrH regulates genes for heme metabolism, heme acquisition, and virulence, indicating a role for this unique sRNA in pathogenesis. Here, we show by mass spectrometry that deletion of the genes encoding the PrrF and PrrH sRNAs (∆prrF1,2 mutant) affects heme homeostasis by P. aeruginosa. Additionally, we show that the ∆prrF1,2 mutant is completely attenuated for virulence in a murine model of lung infection. Thus, the sRNAs encoded by the P. aeruginosa prrF locus are predicted to be essential mediators of iron and heme homeostasis during infection. As such, we propose that the PrrF and PrrH sRNAs are potential targets for the development of novel antimicrobials.


Role of the PrrF and PrrH Small RNAs in Virulence of Pseudomonas aeruginosa Alexandria A. Reinhart1, Daniel A. Powell1, Robert K. Ernst1,2, and Amanda G. Oglesby-Sherrouse1,3 1Department of Microbiology and Immunology, School of Medicine, 2Department of Microbial Pathogenesis, School of Dentistry, 3Department of Pharmaceutical Sciences, School of Pharmacy, University of Maryland, Baltimore, MD 21201 Pseudomonas aeruginosa is an opportunistic pathogen that causes pulmonary infections in compromised individuals. Iron is required for P. aeruginosa virulence and is obtained through siderophore, heme, and ferrous iron uptake systems. Excessive iron induces oxidative stress, and iron uptake is therefore regulated by intracellular iron concentrations. In high iron conditions, iron binds to the Fur protein, which in turn represses expression of genes encoding iron and heme uptake systems. Fur also blocks expression of the PrrF small non-coding RNAs (sRNAs) (Wilderman et. al. 2004). The PrrF sRNAs contribute to iron homeostasis by inducing the degradation of mRNAs encoding iron-containing proteins. Regulation by the PrrF sRNAs also spares a metabolite that is required for the production of the Pseudomonas Quinolone Signal (PQS), an important signaling factor in P. aeruginosa virulence (Oglesby et.al. 2008). Thus, the PrrF sRNAs are involved in regulating cellular communication necessary for P. aeruginosa virulence. The genes encoding the PrrF sRNAs are arranged in tandem on the P. aeruginosa genome, allowing for expression of a third, heme-regulated, sRNA named PrrH (Oglesby-Sherrouse and Vasil, 2010). Transcription of PrrH initiates at the prrF1 promoter, reads through the prrF1 terminator, and terminates at the prrF2 terminator. Thus, the PrrH sRNA possesses sequence derived from the prrF1-prrF2 intergenic region that is unique from the PrrF sRNAs. Here, we identify the vreR mRNA as a putative PrrH target by in silico analysis of the PrrH unique sequence. VreR is part of the Vre cell surface signaling system, responsible for virulence gene expression in P. aeruginosa (Llamas et. al. 2008). We show that heme regulates the stability of the vreR mRNA in a prrF locus-dependent manner, putatively by PrrH binding over the Shine-Delgarno sequence. Unexpectedly, this putative interaction stabilizes the mRNA as opposed to targeting it for degradation. Thus, both the PrrF and PrrH sRNAs appear to regulate genes required for P. aeruginosa virulence. Additionally, we found that the ∆prrF1,2 mutant, which lacks production of all three sRNAs, was avirulent in an acute lung infection mouse model. Furthermore, the ∆prrF1,2 mutant vaccinated mice against a subsequent challenge with wild type P. aeruginosa. Thus, the sRNAs encoded by the prrF locus are required for P. aeruginosa virulence. Current work aims to determine the mechanism of PrrH regulation of vreR and how this regulation contributes to virulence in P. aeruginosa.


Twin sRNAs RyfA1 and RyfA2 of Shigella dysenteriae William H. Broach1, Sarah E. Klim 2, Erin R. Murphy, PhD1 1Ohio University Heritage College of Osteopathic Medicine Department of Biomedical Sciences Athens, OH 45701 2Ohio University Biological Sciences Athens, OH 45701 Shigella species are the causative agents of Shigellosis, a severe diarrheal disease characterized by bloody, mucoid diarrhea as well as fever, nausea and abdominal cramping. Small regulatory RNA molecules (sRNAs) are a class of trans-encoded ribo-regulators that modulate target gene expression using a variety of molecular mechanisms. The sRNA RyfA was originally found to be produced by Escherichia coli during the mid-logarithmic phase of grown and, based on nucleic acid conservation, was predicted to be conserved in Shigella flexneri 2A. Analysis of the S. dysenteriae SD197 genome on the NCBI database revealed the presence of two genes with 90% and 91% identity to S. flexneri ryfA; genes dubbed ryfA1 and ryfA2 respectively. Initial in silico analysis predicted 95% nucleotide sequence identity between RyfA1 and RyfA2 with 5 of the 15 nt that vary between the two molecules occurring consecutively in what has been termed the variable region. Mfold analysis suggests that the sequences contained within each variable region corresponds to an open loop within an otherwise conserved structure shared by RyfA1 and RyfA2. While the ryfA1 and ryfA2 gene sequences are quite similar, the promoter regions are quite different, suggesting a difference in the expression profile and/or level of the two genes. To test this prediction, transcriptional reporter plasmids were constructed and the activity of the ryfA1 and ryfA2 promoters tested under host-like and non-host-like environmental conditions. Both the ryfA1 and ryfA2 promoters are more active at 30°C, a non-host condition, as compared to their activity at 37°C, human body temperature. Additionally, activity of the ryfA1 promoter, but not that of the ryfA2 promoter, is affected by iron availability such that it is more active in an iron rich environment, another non-host-like condition. The ryfA1 and ryfA2 genes were cloned under the control of an IPTG inducible plasmid promoter to determine the affects of over-expression of each on the growth and virulence of S. dysenteriae. Growth curve analysis shows two strikingly different growth patterns with over-production of RyfA1 and RyfA2. Tissue culture based assays demonstrated that while both RyfA1 and RyfA2 influence virulence-associated processes, they do so uniquely. Together these data demonstrate that while the twin sRNA molecules RyfA1 and RyfA2 are nearly identical in sequence and predicted structure, their functions are not redundant as each uniquely influences virulence-associated processes in S. dysenteriae.


A cyclic-di-GMP riboswitch regulates stability of a small RNA in Vibrio cholerae Benjamin R. Pursley1 and Christopher M. Waters1 1Michigan State University Department of Microbiology and Molecular Genetics East Lansing, MI, 48824 Cyclic di-guanosine-monophosphate (c-di-GMP) is an important second messenger signaling molecule that is utilized by a wide variety of organisms throughout the bacterial domain. It has been shown to play a central role in many diverse phenotypes including biofilm formation, motility, cell cycle regulation, and virulence gene expression. In Vibrio cholerae, c-di-GMP is clinically relevant because it is understood to mediate the lifestyle transition from a free-living aquatic organism to a human pathogen, although the genetic regulatory changes that underlie this transition are not yet well understood. One of the most important genetic inputs for c-di-GMP regulation in V. cholerae is the Vc2 riboswitch. This non-coding RNA element specifically recognizes and binds to c-di-GMP, altering is secondary structure in the process. Traditional riboswitches use structural changes to elicit regulatory control over downstream genes by altering the transcriptional or translational efficiency of messenger RNA. However, we have determined that the Vc2 riboswitch is actually expressed and regulated independently of its nearest gene, tfoY. Unlike other riboswitches, the Vc2 riboswitch uses a novel mechanism to regulate the abundance of an upstream small RNA by altering post-transcriptional stability of the RNA strand from the 3’-end. This small RNA is abundant at high intracellular levels of c-di-GMP and scarce at low levels of c-di-GMP. Current work in our lab is focused on using transcriptomic, proteomic, and metabolic analyses to identify the regulatory targets of this small RNA, and to elucidate its role in global c-di-GMP regulation of V. cholerae.


Regulation of Shigella dysenteriae Virulence Factors by RNA Thermometers

Andrew B. Kouse1, Kevin F. Gross1, Amanda E. Dunson1, Erin R. Murphy Ph.D.2 1Ohio University Biological Sciences Department Athens, OH, 45701 2Ohio University Heritage College of Osteopathic Medicine Athens, OH, 45701 Shigella dysenteriae is a gram-negative enteroinvasive pathogen and a causative agent of shigellosis, a severe form of dysentery which afflicts over 90 million people annually. The ability of S. dysenteriae to survive and cause disease is dependent upon the ability of the pathogen to sense and adapt to the varied environments encountered throughout its infectious cycle. Among the environmental cues sensed by Shigella is temperature, a condition that varies between the host and non-host environments, and one that has been shown to influence the expression of approximately 10% of the genes present in these pathogens. The most well defined thermoregulatory mechanism utilized by S. dysenteriae is the temperature-dependent modulation of target gene expression by the transcriptional repressor H-NS. Recent findings, however, suggest that RNA thermometers also play a significant role in the thermoregulation of Shigella gene expression. An RNA thermometer is a cis-acting RNA element located within the 5’ untranslated region (utr) of the target gene that, under non-permissive temperatures, forms an inhibitory hairpin that occludes the Shine-Delgarno sequence and thus inhibits ribosomal binding and translation. This study identifies and characterizes several S. dysenteriae RNA thermometers that function to control a variety of virulence-associated genes. In silico analysis of the Shigella genome predicts the presence of RNA thermometers within genes responsible for virulence (ompA), nutrient-acquisition (shuA and shuT) and heat-shock (htrA). Our studies to date indicate that the expression of several genes predicted to harbor an RNA thermometer is subject to temperature-dependent post-transcriptional regulation, a pattern that is consistent with regulation via this class of ribo-regulators. Mutagenic analyses of the putative RNA thermometers within the 5’ untranslated regions of ompA and shuA confirmed the functionality of these predicted regulatory elements. Studies are ongoing to investigate the functionality of the RNA thermometers predicted within shuT, htrA and other S. dysenteriae genes. These data were the first to experimentally demonstrate that RNA thermometer regulated gene expression in and Shigella species and suggest that this regulatory method is widespread within these important pathogens.


Characterization of Brucella abortus Hypothetical Proteins and Their Role in Virulence James A. Budnick1 and Clayton C. Caswell1

1Virginia Tech Department of Biomedical and Veterinary Sciences, Virginia-Maryland Regional College of Veterinary Medicine Blacksburg, VA. 24060

Brucella spp. are the leading cause of zoonotic infection worldwide with over 500,000 human cases per year. These bacteria are Gram-negative intracellular pathogens belonging to the α2 subclass of proteobacteria and can infect both domestic and wild animal species. Brucella spp. cause a chronic infection leading to spontaneous abortions and infertility in domestic animals, and chronic fevers in humans. Small regulatory RNAs (sRNA) are important regulators of protein function and mRNA expression in bacteria. AbcR1 and AbcR2 are two sRNAs in Brucella abortus that are crucial for the survival and replication of the bacteria within macrophages and the host. Mutations in both abcR1 and abcR2 caused attenuation of Brucella survival in mice.

Recently, we have identified a LysR-type transcriptional regulator that controls the expression of AbcR2. The B. abortus lysR mutant shows no transcription of the AbcR2 sRNA. The lysR mutant had attenuated survival in macrophages and mice spleens. The experiments also revealed the transcriptional regulation of three genes putatively encoding hypothetical proteins in Brucella abortus. Genetic and amino acid analysis reveals that two of these hypothetical proteins share over 90% protein similarity signifying that they may be serving redundant functions. Interestingly, there are over 100 orthologs of these hypothetical proteins found in other organisms. Why are these hypothetical proteins so well conserved among bacteria? This is one question that our lab is trying to answer by characterizing these proteins in Brucella abortus.

The goal of this project is to 1) identify whether these hypothetical proteins are encoding proteins or sRNA, 2) determine the molecular function of each hypothetical gene, and 3) assess the role of these hypothetical genes in regards to Brucella’s ability to survive and colonization in the host.


A LysR-type transcriptional regulator activates sRNA-encoding genes and plays a critical

role in virulence of Brucella abortus Lauren M. Sheehan1, Clayton C. Caswell1 Virginia Polytechnic Institute and State University Department of Biomedical and Veterinary Sciences, Virginia-Maryland Regional College of Veterinary Medicine Blacksburg, VA, 24060 Small regulatory RNAs (sRNAs) play critical roles in both bacterial virulence and gene regulation. In Brucella abortus 2308, the highly similar sRNAs AbcR1 and AbcR2 are essential for the survival and replication of the bacteria within macrophages, as well as the ability of the bacterium to chronically colonize mice. Recently, we have examined how abcR2 expression is controlled at the transcriptional level by a LysR-type regulator we named VtlR (Virulence-associated Transcriptional LysR-family Regulator). VtlR is found flanking abcR2 on chromosome 1 and activates the transcription of abcR2, not abcR1. Importantly, B. abortus vtlR mutant exhibits a reduced ability to survive and replicate in murine macrophages and is also unsuccessful at causing a wild-type, chronic infection in mice. In terms of regulation, electrophoretic mobility shift assays (EMSAs) revealed that VtlR binds directly to the abcR2 promoter, but not to the abcR1 promoter, further clarifying is specificity. To gain further insight into the relationship between VtlR and Brucella gene regulation, microarray analysis was performed using RNA from the parental strain 2308 and the vtlR mutant strain. We discovered that in addition to regulating transcriptional expression of abcR2, VltR is also the activator of three small hypothetical protein-encoding genes (bab1_0914, bab1_0574, bab1_0512). Current research is looking into characterizing the role and function of these genes and determining their individual roles in B. abortus virulence. Interestingly, orthologs of VtlR are found amongst a wide range of other alphaproteobacteria including Sinorhizobium, Bartonella and Agrobacterium. So why is this one LysR transcriptional regulator so conserved among a variety of host-associated bacteria? The goals of this project are to: 1) determine the mechanism and function of VtlR in Brucella abortus 2308 and, 2) understand the conservation and importance of VtlR orthologs in other bacteria.


The alternative sigma factor σB is a global regulator of small RNAs

(sRNAs) in Staphylococcus aureus Ronan K. Carroll, Andy Weiss and Lindsey N. Shaw University of South Florida Department of Cell Biology, Microbiology and Molecular Biology Tampa, FL, 33620 Staphylococcus aureus is a versatile human pathogen capable of infecting a variety of niches throughout the human body. Regulation in S. aureus is complex with over 130 transcription factors encoded within the genome. In recent years the number of small regulatory RNAs (sRNAs) identified and characterized in S. aureus has rapidly expanded, revealing a previously underappreciated level of post-transcriptional regulation by these non-coding RNA species. Current estimates now suggest that the number of sRNAs encoded in the S. aureus genome exceeds that of proteinaceous transcription regulators. Despite this recent acceleration in discovery and investigation, the comprehensive global identification and analysis of sRNAs remains challenging, and very little is known about the factors that control their expression. Previous work has shown that the alternative sigma factor, σB, is involved in the regulation of specific sRNAs in S. aureus; however, the extent of sRNA regulation by this element is currently unknown. In this work we perform a screen to identify σB regulated sRNAs using total RNA sequencing (RNA-seq). The identification and analysis of sRNAs by RNA-seq is dependent on a fully sequenced, annotated reference genome. Therefore, to facilitate the global analysis of previously identified sRNAs, and to search for novel σB-dependent sRNAs, we constructed a genbank file for strain NCTC8325, containing 110 previously characterized S. aureus sRNAs. RNA-seq data analysis using this modified file facilitated the simultaneous identification of all previously identified σB-regulated sRNAs in S. aureus, and the identification of a number of novel sRNAs under the control of this regulator. This work demonstrates for the first time the importance of accurate genome annotation in the identification and analysis of sRNAs by RNA-seq.


The δ subunit of RNA polymerase guides promoter selectivity and virulence in

Staphylococcus aureus

Andy Weiss1, Ronan K. Carroll1, J. Antonio Ibarra1 and Lindsey N. Shaw1

1University of South Florida

Department of Cell Biology, Microbiology and Molecular Biology,

Tampa, FL 33620

Abstract:

In Gram-positive bacteria, and particularly Firmicutes, the DNA-dependent RNA polymerase (RNAP) complex contains an additional subunit, termed the δ factor, or RpoE. This enigmatic protein has been studied for over 30 years in various organisms, but its function is still not well understood. In this study we investigate its role in the major human pathogen Staphylococcus aureus. We show conservation of important structural regions of RpoE in S. aureus and other species, and demonstrate binding to core-RNAP that is mediate by the β and/or β’ subunits. To identify the impact of the δ subunit on transcription we performed RNA-seq analysis, and observed 191 differentially expressed genes in the rpoE mutant. Ontological analysis revealed, quite strikingly, that many of the downregulated genes were known virulence factors, whilst several mobile genetic elements (SaPI5 and prophage ΦSA3usa) were strongly upregulated. Phenotypically, the rpoE mutant had decreased accumulation and/or activity of a number of key virulence factors, including α-toxin, secreted proteases and PVL. We further observed significantly decreased survival of the mutant in whole human blood, increased phagocytosis by human leukocytes, and impaired virulence in a murine model of infection. Collectively, our results demonstrate that the δ subunit of RNAP is a critical component of the S. aureus transcription machinery and plays an important role during infection.


Mechanisms of AmrZ-mediated activation and repression of

Pseudomonas aeruginosa virulence genes Binjie Xu1,3, Christopher Jones2,3,4, Daniel Wozniak1,2,3

Departments of Microbiology1, Department of Microbial Infection and Immunity2, Center for Microbial Interface Biology3, The Ohio State University, Columbus, OH, 43214 Department of Microbiology and Environmental Toxicology4, University of California Santa Cruz, Santa Cruz, CA, 95064 The Gram negative bacterial pathogen Pseudomonas aeruginosa (Pa) causes a number of infections, such as lung infections in cystic fibrosis (CF) patients. Non-mucoid Pa is the colonizer in CF lungs; however, in later infections, Pa is often seen to adapt the mucoid phenotype. Mucoidy is characterized by overproduction of the polysaccharide named alginate, and disappearance of flagella. Alginate-dominant biofilms acts as a thick protective layer against phagocytic cells and antibiotics, while the loss of flagella mediates immune evasion. However, the question remains how Pa mediates this coordination. The transcription factor AmrZ activates alginate overproduction through activating transcription of the algD operon, and inhibits flagella expression through repressing transcription of the flagella master regulator fleQ. Nevertheless, the molecular mechanism is unclear how AmrZ achieves activation and repression of different targets. Thus, we sought to identify what factors determine the outcome of AmrZ-mediated regulation. Our ChIP-seq experiments characterized AmrZ binding sites (ZBSs) throughout the Pa genome, and identified precise positions of ZBSs in PalgD and PfleQ. Both promoters contain two ZBSs, which have been verified by in vitro DNA binding assays. Nevertheless, their positions relative to the respective transcription start site differ dramatically in these two promoters. In PalgD, transcriptional fusion assays showed that ZBS1 contributed significantly to PalgD activation, while the effect of ZBS2 scrambling was relatively mild but significant. In addition, we saw an additive effect of these two ZBSs when both were scrambled. Current progress is focused on elucidating roles of individual ZBSs of PfleQ during AmrZ-mediated repression, and determining whether AmrZ binding to a target with two ZBSs leads to DNA bending or DNA looping. Overall, this study has its potential to reveal delicate regulatory mechanisms of different virulence factors in Pa, and to provide targets for control measures against Pa infections.


CHARACTERIZATION OF THE FORGOTTEN SLYA IN SHIGELLA FLEXNERI

Natasha Weatherspoon-Griffin1 and Helen J. Wing1

1University of Nevada Las Vegas School of Life Sciences Las Vegas, NV 89154-4004 The transcriptional regulator SlyA has been extensively characterized in enteric bacteria such as Escherichia coli and Salmonella species. Originally identified for its ability to induce hemolytic, cytotoxic and apoptotic phenotypes (Libby et al, 1994; Ludwig et al, 1995), SlyA has also been shown to be a major contributor to virulence in these organisms (Libby et al, 1994). While the slyA gene was identified in Shigella flexneri nearly two decades ago (Ludwig et al, 1995), no further analysis regarding the role of SlyA in Shigella has been reported. Thus, we hypothesize that slyA is expressed in S. flexneri and contributes to virulence similar to its close relatives. Here, we begin the initial characterization of SlyA in S. flexneri. We demonstrate that in S. flexneri (1) slyA is expressed, (2) exogenous expression of SlyA can activate a virulence gene encoded on its large virulence plasmid, and (3) exogenous expression of SlyA can induce a cytotoxic phenotype despite previous reports demonstrating that the cytotoxin homologous to the E. coli and Salmonella cytotoxins is non-functional (del Castillo et al, 2000; von Rhein et al, 2008). This study presents the first line of evidence to demonstrate that SlyA may have a functional role in Shigella. References: del Castillo FJ, Moreno F, del Castillo I.(2000) Characterization of the genes encoding the SheA haemolysin in Escherichia coli O157:H7 and Shigella flexneri 2a. Res Microbiol,151:229-30. Libby SJ, Goebel W, Ludwig A, Buchmeier N, Bowei F, Fang FC, Guiney DG, Songer JG, and Heffron F. (1994) A cytolysin encoded by Salmonella is required for survival within macrophages. Proc Natl Acad Sci USA, 91:489-493. Ludwig A, Tengel C, Bauer S, Bubert A, Benz R, Mollenkopf HJ, Goebel W. (1995) SlyA, a regulatory protein from Salmonella typhimurium, induces a haemolytic and pore-forming protein in Escherichia coli. Mol Gen Genet, 249:474 486. von Rhein, Bauer S, Simon V, and Ludwig A. (2008) Occurrence and characteristics of the cytolysin A gene in Shigella strains and other members of the family Enterobacteriaceae. FEMS Microbiol Lett.287:143-8.


Thiol Biochemistry in Methane-Producing Archaea

Jennie L. Catlett1, and Nicole R. Buan1 1University of Nebraska-Lincoln Department of Biochemistry Lincoln, NE, 68588-0664 Two Gigatons of methane is produced annually by anaerobic archaea called methanogens (Thauer, R.K. et al. 2008). Harvesting biologically produced methane from methanogens growing in anaerobic digesters is an increasingly valuable method for obtaining renewable methane for heat, electricity, and transportation fuel uses. Methanogens conserve energy and produce methane via the metabolic pathway known as methanogenesis. Essential steps in methanogenesis require two low-molecular-weight thiol cofactors, 2-mercaptoethane sulfonate, Coenzyme M (CoM-SH), and 7-mercaptoheptanoyl threonine phosphate, Coenzyme B (CoB-SH). CoM is a methyl carrier, and CoB is the terminal electron donor in methanogenesis. In the last step of the pathway, CoM-SH and CoB-SH form a heterodisulfide, CoM-S-S-CoB, which is the terminal electron acceptor. CoM-SH and CoB-SH thiols are regenerated by the terminal oxidase enzyme, heterodisulfide reductase (Hdr). We observed that methanogens usually have multiple Hdr genes in their genomes, suggesting there is an evolutionary selection for modulating Hdr enzyme activity to respond to changes in substrate availability (Buan, N.R. and W. W. Metcalf. 2010). To test this idea, we overexpressed a cytoplasmic Hdr from a strong constitutive promoter in Methanosarcina acetivorans and measured Hdr activity in cell extracts, growth rate, and the rate of methane production. We discovered that overexpressing Hdr does not affect growth compared to the parent strain, but has the unexpected effect of increasing the rate of methane production by 30% (US Provisional Patent application 61/980,656) . Our data suggests that Hdr overexpression partially uncouples methane production from generation of a transmembrane ion gradient, but that the loss in ATP is offset by an increased rate of substrate uptake and catabolism. This interpretation is consistent with the generalist growth strategy employed by M. acetivorans. Our results are expected to lead to tactics for increasing the rate of methane produced from renewable biomass in anaerobic digesters.


USE OF BIOINSPIRED TOPOGRAPHIES TO REDUCE BACTERIAL SURFACE CONTAMINATION AND

MEDICAL DEVICE RELATED INFECTIONS Ethan E. Mann1, Rhea M. May1, Anthony B. Brennan2 and Shravanthi T. Reddy1 1Sharklet Technologies, Inc, Aurora, CO 80045 2University of Florida, Departments of Materials Science & Engineering and Bioengineering, Gainesville, FL, 32611 Surface roughness affects wettability, which has motivated the study of surface topography to control bio-adhesion. The structure of shark skin matches roughness estimates for an effective anti-fouling surface, and as a result, the Sharklet micro-topography was developed as a non-toxic, non-biocidal surface designed to inhibit surface contamination of undesirable microorganisms. The initial goal of this biomimetic strategy was to reduce the occurrence of algae and barnacle growth on underwater surfaces to potentially replace existing toxic solutions. As predicted, the Sharklet micropattern was demonstrated to be effective against marine algal zoospores, barnacle cyprids and marine bacteria. Since then, the Sharklet micropattern has been evaluated for use in consumer products, such as chair levels and iPhone cases, and a variety of medical devices, including urinary catheter, central venous catheters, and endotracheal tubes, to reduce medical device related infections without the use of anti-microbial agents. Our studies show that the Sharklet micropattern performs against numerous clinically relevant pathogens on several materials and in a range of conditions. The pattern has been shown to inhibit microbial attachment of eight common nosocomial pathogens by up to 99% (p<0.05) when compared to unpatterned controls. It reduces the migration of several species, including Escherichia coli by up to 92% (p<0.05) and decreases the area coverage of Pseudomonas aeruginosa biofilms by as high as 96% (p<0.05). The reduction in microbial attachment, migration, and biofilm formation may have a major impact on decreasing rates of nosocomial and medical device related infections if the Sharklet micropattern is incorporated on to commonly-contaminated products.


INTRICATE REGULATION PROVIDED BY SMALL RNAS AND SMALL PROTEINS

Gisela Storz1 1Eunice Kennedy Shriver National Institute of Child Health and Human Development Cell Biology and Metabolism Program Bethesda, MD 20892-5430 I will discuss the regulatory roles of small RNAs and small proteins of less than 50 amino acids. These two types of molecules have generally been overlooked because they are not detected in biochemical assays and the corresponding genes are poorly annotated and missed in genetic screens. However, mounting evidence suggests that both types of small molecules play important regulatory roles. Bacterial small RNAs regulate gene expression through base pairing with mRNAs or bind to and modulate the activity of proteins. While the majority of the small RNA regulators characterized thus far are encoded as separate genes, it has recently been found that some base pairing small RNAs are derived from the 3’ ends of protein coding genes. For example, in Escherichia coli a third σE-dependent small RNA, MicL, is transcribed from a promoter located within the coding sequence of the cutC gene. MicL possesses features typical of base pairing small RNAs, but surprisingly only targets a single mRNA. The sole target however encodes the outer membrane lipoprotein Lpp, the most abundant protein of the cell. Interestingly, the copper sensitivity phenotype previously ascribed to inactivation of the cutC gene is actually derived from the loss of MicL and elevated Lpp levels. This observation raises the possibility that other phenotypes currently attributed to protein defects are due to deficiencies in unappreciated regulatory RNAs. Most bacterial small proteins contain a single transmembrane helix and co-fractionate with membranes. Increasing evidence suggests that these proteins serve as important regulators of larger membrane proteins. For example, the 49-amino acid AcrZ protein associates with the AcrAB-TolC multidrug efflux pump, which confers resistance to a wide variety of antibiotics and other compounds in E. coli. Co-purification of AcrZ with AcrB in the absence of AcrA and TolC, two-hybrid assays and suppressor mutations indicate that this interaction occurs through the inner membrane protein AcrB. Mutants lacking AcrZ are sensitive to many, but not all, of the antibiotics transported by AcrAB-TolC. This differential antibiotic sensitivity suggests that AcrZ enhances the ability of the AcrAB-TolC pump to export certain classes of substrates. Preliminary evidence suggests that other small proteins similarly modulate the activities of a range of transporters.


Oral Transmission of Mycobacteria: Investigations of M. marinum Association with and Breaching the Gut Epithelia of Fish

Martin Cheramie

1, Amrita Mallick

1, Lisa Shirreff

1, Meagan Bahlinger

1, Irene Salinas-Remiro

2

and Don G. Ennis1

1Department of Biology, University of Louisiana, Lafayette, LA 70504,

2Department of Biology,

University of New Mexico 87131.

Mycobacterium marinum (Mm) is an intracellular pathogen with greater than 85% sequence identity in more than 3,000 orthologous genes of Mycobacterium tuberculosis (Mtb). Like human Mtb infections, Mm survives and multiplies within host macrophages, produces granulomas in host tissues, the hallmark lesion of human Mtb and mounts life-long chronic infections in many fish. Because of the similar disease presentation in fish and the close genetic relatedness between this pathogen and Mtb complex, Mm has been used as a surrogate model to study aspects of human TB. This pathogen is a leading cause of fish mycobacteriosis (or “fish TB”), which burdens hundreds of fish species in both freshwater and saltwater systems. Mm infections can impact aquatic animals in the wild (bass and carp), in aquaculture (tilapia and striped bass) as well as research fish colonies (medaka, zebrafish and Xenopus). Mm occasionally causes human infections, most often by entry into cuts or wounds in the cooler extremities typically as a result of handling infected fish. We have shown that Japanese Medaka (Oryzias latipes) can be chronically infected by Mm and we have used this small laboratory fish as a host to model aspects of TB infections. Despite the substantial infectious burden wild fish stocks and frequent outbreaks in fish colonies that cost billions of dollars annually, the mode(s) of Mm transmission remained speculative. We have worked to elucidate transmission amongst fish and found that ingestion of infected food items is a major route Mm infection. We have shown that ingestion of small prey items carrying Mm, such as aquatic insect larvae or consumption of tissues from dead and infected fish are very efficient modes of infection. Mosquito larvae are constantly ingesting bacteria in the water and in biofilms, accumulating large amounts of Mm in the gastro-intestinal (GI) tracts; these larvae can in turn, be consumed by fish. We have shown that mosquito larvae not only act as efficient vessels for delivery of Mm to the GI tracts of fish. We discovered that the passage of through the larval gut increases virulence of Mm by approximately 100-100 fold compared to bacteria grown in laboratory media. Passage through the caustic digestive tracts of larvae induces a highly virulent metabolic state for Mm. We are employing transcriptomic analyses to identify the Mm virulence gene set that are induced with exposure to the larval gastric fluids. We also found that infected fish will carry Mm in their GI tracts months after ingesting infected larvae and these infected fish will continuously shed highly infectious mycobacteria into the surrounding water. We are currently examining the status of the GI tracts of fish following oral dosing by Mm-laced larvae. We found that these infected fish present similar histopathology and timing of colonization of target organs (e g., spleen, liver and kidney) as we observed by intraperitoneal injections (IP). A comparable disease progression by either IP or larval exposures suggested that crossing the gut mucosal membrane by Mm may be a rapid process. To gain insights into how this pathogen is able to cross the gut epithelia, orally-infected fish were dissected, and prepared for frozen sections and fluorescent microscopy. Our studies revealed that Mm expressing fluorescent reporters were attached to the tips of villi. We also determined that colonization and spread to the underlying tissues occurred within a week. These studies using the Mm-medaka model are expected to offer insights into how other mycobacteria like M. avium-M. paraturburculosis (MAP) complex which are also able to traverse mammalian gut mucosal membranes of humans and ruminents.


Fatty acid alterations protects Enterococcus faecalis from membrane damaging agents

including the antibiotic daptomycin Holly E. Saito1, John R. Harp1, Abigail Tester2, Shawn Campagna2, and Elizabeth M. Fozo1

1University of Tennessee Department of Microbiology Knoxville, TN 37996 2University of Tennessee Department of Chemistry Knoxville, TN 37996 Enterococcus faecalis, an intestinal commensal, is one of the leading causes of hospital acquired infections and many isolates are resistant to antibiotics. The bacterium is able to survive a wide variety of stressful environments and this ability contributes to its pathogenesis. Previous work by other groups have shown that some bacterial pathogens incorporate fatty acids from host fluids into their cellular membrane altering their physiology and ability to survive stressful conditions. Given that E. faecalis is stress-resistant and a human pathogen, we examined whether it is capable of incorporating exogenous fatty acids into its membrane and how this may impact its ability to survive membrane damage. When grown in media supplemented with bovine bile or human sera, the organism incorporated fatty acids from the exogenous sources into its membrane. Furthermore, supplementation with 0.2% bile protected E. faecalis when challenged with either 10% bile or 0.1% SDS; unsupplemented cultures were highly sensitive to this membrane challenge. We confirmed that these differences were not due to genetic changes, but rather physiological adaptation. To verify that the effects were due to fatty acid incorporation and not other components within bile or sera, we supplemented cultures with individual fatty acids found in bile or sera or those naturally produced by the bacterium. Of those examined, supplementation with oleic acid (C18:1 cis 9) significantly enhanced survival when challenged with 10% bile, despite that all fatty acids examined were incorporated readily in the membrane. Interestingly, supplementation with bile, sera, oleic acid or linoleic acid (C18:2

cis 9, 12; found in sera) protected cells from the membrane damaging antibiotic daptomycin. This is the first report that altered membrane fatty acid composition can protect cells from daptomycin. Preliminary studies indicate that supplementation with bile or oleic acid leads to increased levels of cardiolipin within the membrane, and cardiolipin synthase has been linked to daptomycin-resistant clinical isolates. Further work is aimed at examining how these alterations specifically lead to daptomycin resistance and potential alternative treatment options.


Deletion of an ABC Transporter Renders Enterococcus faecalis Susceptible to Lysozyme

Sriram Varahan, Kara Hinshaw and Lynn E. Hancock University of Kansas, Department of Molecular Biosciences, Lawrence, Kansas, 66045

Enterococcus faecalis is an opportunistic pathogen found in the gut of most mammals including human beings and is one of the leading causes of nosocomial infections. Previous studies have shown that the presence of lysozyme leads to the activation of SigV, an extracytoplasmic function (ECF) sigma factor in E. faecalis, and that the deletion of sigV increases the susceptibility of the bacterium toward lysozyme. It is known that the activation of ECF sigma factors involves a multi-step proteolytic degradation of the anti-sigma factor that sequesters the sigma factor and renders it inactive in the absence of a given stress. Our laboratory recently showed that a membrane embedded zinc-metalloprotease called Eep is one such protease that is essential for the activation of SigV under lysozyme stress. It is known that sigV autoregulates its own expression by recognizing and binding to its own promoter. We performed a transposon mutagenesis screen using a novel reporter strain in which lacZ was expressed under the sigV promoter to identify other candidate genes essential for the activation of SigV under lysozyme stress. Here we describe an ABC transporter that is essential for the activation of SigV under lysozyme stress. Deletion of this ABC transporter resulted in a 10-fold increased susceptibility to lysozyme compared to the wild-type. This phenotype could be complemented by expressing the transporter in the deletion mutant as a single copy in its native locus. Using a luciferase reporter system, we also demonstrate that the sigV promoter is inactive in the transporter deletion mutant in the presence of lysozyme. The exact mechanism by which this transporter contributes to the activation of SigV is currently being elucidated.


Regulation of a Dual Diguanylate Cyclase-Phosphodiesterase (GGDEF/EAL) protein in A.

tumefaciens Nathan Feirer1, Jing Xu1, Benjamin J. Koestler2, Christopher M. Waters2, and Clay Fuqua.1 1Department of Biology, Indiana University, Bloomington, IN 47405 2Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824 One of the primary factors mediating surface attachment in A. tumefaciens is an adhesin called the unipolar polysaccharide (UPP), which localizes to a single pole of the cell that contacts surfaces. UPP production is observed to be coincident with surface contact. A transposon mutant library was screened to isolate mutants with aberrant UPP production. Mutants with transposon insertions or in-frame deletions in Atu3495 exhibited dramatically elevated UPP production and uncoupling of UPP synthesis from surface contact. DcpA (diguanylate cyclase phosphodiesterase A, Atu3495) encodes a predicted membrane-anchored protein that contains both diguanylate cyclase (DGC) and phosphodiesterase (PDE) domains. DGC and PDE domains synthesize and degrade, respectively, the second messenger cyclic-dimeric guanosine-monophosphate (c-di-GMP), which plays a prominent role in regulating the motile-to-sessile transition in a wide diversity of bacteria. A dcpA mutant exhibits increased biofilm formation and UPP levels, phenotypes that are indicative of elevated c-di-GMP concentrations. This implicates that DcpA negatively regulates attachment and has a dominant PDE activity in A. tumefaciens. Ectopic expression of dcpA was also able to decrease biofilm levels in an unrelated elevated biofilm mutant, further demonstrating the PDE activity. In contrast, when DcpA was expressed in E. coli intracellular concentrations of c-di-GMP increased dramatically and this was entirely dependent on a functional GGDEF catalytic motif. DcpA DGC activity can be observed in A. tumefaciens when the EAL catalytic motif of the PDE domain is mutated, indicating a possible tight regulation of the ratio of PDE and DGC activities. Interestingly, DcpA DGC activity is also observed in a strain deleted for qene designated pruA (pteridine reductase regulator of UPP A, Atu1130) that was found in the original transposon screen. Pteridine reductases are cytoplasmically localized enzymes that catalyze oxidation-reduction reactions, often with small metabolites known as pterins as substrates. Site-specific mutagenesis of predicted catalytic site residues for PruA suggested that its enzymatic activity is essential for the regulation of both UPP production and DcpA activity. Studies are ongoing to determine if PruA affects pterin pools in A. tumefaciens. A candidate protein for linking PruA-modified pterin levels to DcpA enzymatic activity is pruR (pteridine reductase regulator of UPP Receptor, Atu3496). PruR is directly upstream of dcpA and has homology to pterin binding proteins. It is hypothesized to be an intermediate in the PruA-DcpA regulatory pathway since DcpA exhibits DGC activity in a ∆pruR, background, replicating the response seen in the ∆pruA strain. Future work is focusing on how this novel regulatory cascade controls the dual enzymatic activity of DcpA, and thereby the surface interactions of A. tumefaciens.


Sick and Twisted: A Structure-Function Analysis of the Helicobacter pylori Stomach

Colonization Factor Csd4 Kris M. Blair1, Anson Chan2, Michael Murphy2, and Nina R. Salama1 1University of Washington Molecular and Cellular Biology Program Fred Hutchinson Cancer Research Center Seattle, WA 98100 2University of British Columbia, Vancouver Life Sciences Centre Vancouver, BC Canada V6T 1Z3 Helicobacter pylori is a carcinogenic bacterial pathogen that chronically infects the human stomach of more than half the global population and is a major risk factor for the development of stomach cancer in a subset of those infected. Infection is acquired primarily during childhood and can persist for decades. H. pylori has a helical cell morphology and colonization is facilitated by a postulated corkscrew mechanism that enables the bacteria to escape the inhospitable acidic lumen, traverse through the viscous mucus lining of the stomach and intimately associate with the epithelial surface of the stomach. Non-helical H. pylori mutants are defective for efficient colonization and our lab has identified many proteins that contribute to normal helical cell shape. These cell-shape-determinant (Csd) proteins modify the bacterial cell wall through direct and indirect actions on peptidoglycan (PG); PG is a singular molecule comprised of a repeating disaccharides that encase the cell due to peptide crosslinks that connect individual strands and which determines the shape of nearly all bacteria. Null mutations of one of these proteins, Csd4 (and others also), give rise to non-helical, straight-rod shaped bacteria. Csd4 is a metal- dependent carboxypeptidase that cleaves uncrosslinked PG tripeptides into dipeptides within the PG sacculus that can no longer participate in crosslinking. Interestingly, the Csd4 active site contains a catalytic glutamine in place of a highly conserved histidine in related carboxypeptidase M family proteins. In vitro activity assays using purified enzyme variants have demonstrated that the Csd4-H46 variant has reduced activity relative to the Csd4-Q46 enzyme. We also found that the activity of the Csd4-Q46 protein is pH responsive with peak activity at pH6.0, whereas the activity of the H46 variant does not change. H. pylori bacteria expressing this Csd4-Q46H show perturbed shape distinct from the null mutant. We hypothesize that the catalytic glutamine in Csd4 arose to confer maximum activity of the enzyme in the slightly acidic local environment of the bacterial periplasm. We are currently exploring how variation in activity and localization of activity drive morphology changes in the cell wall and thus the cell body.


Biogenesis of BamA (Omp85), a core protein of the β-barrel assembly machine of Gram

negative bacteria, mitochondria and chloroplasts Rajeev Misra, Ryan Stikeleather and Rebecca Gabriele Arizona State University School of Life Sciences Tempe, AZ 85287 Assembly of the β-barrel outer membrane proteins (OMPs) is an essential cellular process in Gram negative bacteria and in the mitochondria and chloroplasts of eukaryotes—two organelles of bacterial origin. Central to this process is the conserved β-barrel OMP that belongs to the Omp85 superfamily. In Escherichia coli, BamA is the core β-barrel OMP, and together with four outer membrane lipoproteins, BamBCDE, constitute the β-barrel assembly machine (BAM). In this paper, we investigated the roles of BamD, an essential lipoprotein, and BamB in BamA biogenesis. Depletion of BamD caused impairment in BamA biogenesis and cessation of cell growth. These defects of BamD depletion were partly reversed by single amino acid substitutions mapping within the β-barrel domain of BamA. However, in the absence of BamB, the positive effects of the β-barrel substitutions on BamA biogenesis under BamD depletion conditions were nullified. By employing a BamA protein bearing one such substitution, F494L, it was demonstrated that the mutant BamA protein could not only assemble without BamD, but it could also facilitate the assembly of wild-type BamA expressed in trans. Based on these data, we propose a model in which the Bam lipoproteins, which are localized to the outer membrane by the BAM-independent Lol pathway, aid in the creation of new BAM complexes by serving as outer membrane receptors and folding factors for nascent BamA molecules. The newly assembled BAM holocomplex then catalyzes the assembly of substrate OMPs and BamA. These in vivo findings are corroborated by recently published in vitro data.


Regulation of agglutination in Staphylococcus aureus

Heidi A. Crosby1 and Alexander R. Horswill1 1University of Iowa Department of Microbiology Iowa City, IA, 52242 Staphylococcus aureus causes a wide range of diseases, ranging from minor skin infections to life threatening conditions such as septicemia and endocarditis. One of the hallmarks of S. aureus is its ability to coagulate blood and agglutinate (form clumps) by adhering to fibrinogen, a component of human plasma. This clumping allows S. aureus to form vegetations on heart valves, leading to endocarditis. Binding to fibrinogen is mediated by the cell surface proteins clumping factor A (ClfA) and clumping factor B (ClfB), which are covalently attached to the S. aureus cell wall. Our laboratory recently demonstrated that clumping of S. aureus is regulated by the two-component system ArlRS. An arlRS mutant fails to form clumps in the presence of human plasma or fibrinogen, and the mutant is attenuated in a rabbit model of infective endocarditis. Surprisingly, ArlRS does not appear to regulate the expression of clfA or clfB. Instead, ArlRS represses the expression of ebh, which encodes a giant ~1.1 MDa membrane-anchored surface protein. The function of Ebh is unknown, but it appears to block clumping, as deleting ebh in an arlRS mutant partially restores its ability to clump. Not all strains of S. aureus encode intact versions of ebh, but the presence of a complete ebh gene correlates with the clumping phenotype of an arlRS mutation. In this regard, several clinical isolates with disrupted ebh genes show no clumping defect when the arlRS locus is deleted. Preliminary evidence suggests that ebh is regulated by several other factors in addition to ArlRS, including the global regulator MgrA. Ebh expression is increased in an mgrA mutant, and, similar to an arlRS deletion strain, an mgrA mutant fails to clump in the presence of human plasma. Taken together, these results suggest that ebh is expressed at lower levels, but when full-length Ebh is produced it can impact disease progression by blocking agglutination.


Triclosan Induces Staphylococcus aureus Nasal Colonization

Adnan K. Syed1, Sudeshna Ghosh2, Nancy G. Love2, Blaise R. Boles1

1University of Michigan Department of Molecular Cellular and Developmental Biology Ann Arbor, Michigan, 48109 2University of Michigan Department of Civil and Environmental Engineering Ann Arbor, Michigan 48109

The biocide triclosan is used in many personal care products including toothpastes, soaps, clothing, and medical equipment. Consequently it is present as a contaminant in the environment and has been detected in some human fluids including serum, urine, and milk. Staphylococcus aureus is an opportunistic pathogen that colonizes the nose and throat of approximately 30% of the population. Colonization with S. aureus is known to be a risk factor for several types of infection. Here we demonstrate that triclosan is commonly found in the nasal secretions of healthy adults and the presence of triclosan trends positively with nasal colonization by S. aureus. We demonstrate that triclosan can promote the binding of S. aureus to host proteins such as collagen, fibronectin, and keratin as well as inanimate surfaces such as plastic and glass. Lastly, triclosan exposed rats are more susceptible to nasal colonization with S. aureus. These data reveal a novel factor that influences the ability of S. aureus to bind surfaces and alters S. aureus nasal colonization.


Peptidoglycan fragments as a potential signal for biofilm dispersal

David E. Payne1, Blaise R. Boles1 1University of Michigan Molecular, Cellular, and Developmental Biology Ann Arbor, MI, 48109 Abstract: Staphylococcus aureus is a commensal and pathogen capable of forming biofilms on host tissues and implanted medical devices. S. aureus biofilms exhibit extraordinary resistance to antimicrobial killing, limiting the efficacy of antibiotic therapy and often leaving surgical removal of infected tissues or implanted devices as the only treatment option. An improved knowledge of environmental conditions and bacterial molecular mechanisms controlling the process of biofilm formation and disassembly is needed to improve treatment strategies. In previous work, we demonstrated that tannic acid is a potent biofilm inhibitor, effective at preventing biofilm formation at nanomolar concentrations. We further showed that commonly ingested plant products that contain tannic acid, such as black tea, inhibit S. aureus biofilm formation in vitro. We developed a throat colonization model using cotton rats and showed that rats treated with tea are rapidly decolonized. Both in vitro and in vivo inhibition by tannic acid depended on IsaA, a lytic transglycosylase that cleaves peptidoglycan. This work aims to further elucidate the mechanism by which tannic acid acts together with IsaA to inhibit biofilm formation. To this end, we sequenced the genomes of 6 isolates from an in vivo biofilm that resisted tannic acid treatment. One of these isolates contained a SNP in pknB, a sensor kinase that modulates gene expression in response to peptidoglycan fragments. Mutating pknB in a clean background confirmed that this mutation is sufficient to prevent inhibition by tannic acid and IsaA. Taken together, these data begin to demonstrate a novel mechanism for signaling through small fragments of peptidoglycan within the S. aureus biofilm.


Role and Regulation of Extracellular Proteases in Staphylococcus epidermidis Biofilm Formation

Alexandra E. Paharik, Megan R. Kiedrowski, Michael E. Olson, and Alexander R. Horswill University of Iowa Department of Microbiology Iowa City, IA 52242 Staphylococcus epidermidis is a commensal organism that colonizes the skin and mucous membranes of humans. Although it is part of the normal flora of healthy individuals, S. epidermidis is responsible for a significant portion of healthcare-associated infections (HAI). It is the most frequent cause of central line-associated bloodstream infections and the second most frequent cause of surgical site infections. A major S. epidermidis virulence determinant is its ability to form a biofilm. Although the polysaccharide intercellular adhesin (PIA) has been thoroughly investigated as a critical mediator of S. epidermidis biofilm development, a number of studies show that 30-50% of S. epidermidis clinical isolates do not produce PIA. In PIA-negative strains, a cell surface-bound adhesin called Aap (accumulation-associated protein) is required for biofilm formation. To promote biofilm development, Aap must be proteolytically processed, but the mechanism employed by S. epidermidis to process Aap and facilitate PIA-independent biofilm formation is not clear. Using a microtiter plate assay to quantify PIA-negative biofilm formation, we found that deletion mutants of the secreted proteases Ecp and SepA exhibit diminished biofilm formation (p < 0.05). We have also shown by Western blot that ecp and sepA mutants have decreased processing of Aap. Further, the addition of the S. aureus protease homologs of Ecp and SepA to S. epidermidis cultures results in dramatically increased biofilm formation in an Aap-dependent manner. In studies of Ecp regulation, we have demonstrated by microarray analysis and activity assays that Ecp is positively regulated by the staphylococcal quorum-sensing system Agr. These findings indicate that secreted proteases are critical for S. epidermidis PIA-independent biofilm formation and that, unlike in S. aureus, the addition of proteases enhances biofilm formation. Further, the results suggest that the Agr system is required for S. epidermidis biofilm maturation, a phenomenon that has not been observed in S. aureus. , On the whole, this study identifies a novel role for the Agr system and secreted proteases in promoting staphylococcal biofilm development.


The Disulfide Bonding System Suppresses an Alternate Cellulose Activation Pathway.

David A. Hufnagel1 and Matt R. Chapman1

1University of Michigan Department of Molecular, Cellular, and Developmental Biology Ann Arbor, MI, 48109 Escherichia coli reside in the lower intestinal tract of many animals. Upon passage from the host, E. coli encounters new environmental conditions including: aerobiosis, varying temperatures, and nutrient limitation [1]. E. coli uses various strategies to combat these new conditions, including forming a biofilm. Biofilms are a ubiquitous slow-growing lifestyle of microbes that provides resistance to various environmental insults [2]. E. coli’s biofilm formation process is dependent on production of an extracellular matrix, predominantly composed of the amyloid protein, curli, and the polysaccharide, cellulose [3,4]. The biofilm master regulator, CsgD, positively regulates csgBA, which encodes curli fibers, and adrA, which encodes a diguanylate cyclase that produces the secondary messenger, c-di-GMP, which in turn activates the cellulose synthase [5,6]. Expectedly, CsgD is very tightly regulated and only produces curli and cellulose in low salt, low sugar, low nutrient, and low temperature conditions. We discovered that mutations to the disulfide bonding (DSB) system cause an alternate cellulose production (ACP) pathway that is independent of CsgD and AdrA. The ACP pathway causes cellulose promotion in many conditions where cellulose is normally inhibited, including high salt conditions, high sugar conditions, high nutrient conditions, and at higher temperatures. We discovered that the ACP pathway is dependent on the diguanylate cyclase, YfiN. YfiN is negatively regulated by the periplasmic protein, YfiR. We found that YfiR has conserved cysteine residues and contains a disulfide bond that is necessary for its stability. In DSB mutants, YfiR is unstable, causing activation of YfiN and promotion of cellulose production. Our work also unveiled that YfiN and YfiR regulate normal CsgD protein levels, and thus also control cellulose production in WT cells. 1. Winfield MD, Groisman EA (2003) Role of nonhost environments in the lifestyles of Salmonella and Escherichia

coli. Appl Environ Microbiol 69: 3687-3694. 2. Costerton JW (1995) Overview of Microbial Biofilms. Journal of Industrial Microbiology 15: 137-140. 3. Chapman MR, Robinson LS, Pinkner JS, Roth R, Heuser J, et al. (2002) Role of Escherichia coli curli operons in

directing amyloid fiber formation. Science 295: 851-855. 4. McCrate OA, Zhou XX, Reichhardt C, Cegelski L (2013) Sum of the Parts: Composition and Architecture of the

Bacterial Extracellular Matrix. J Mol Biol 425: 4286-4294. 5. Hammar M, Arnqvist A, Bian Z, Olsen A, Normark S (1995) Expression of two csg operons is required for

production of fibronectin- and Congo red-binding curli polymers in Escherichia coli K-12. Mol Microbiol 18: 661-670.

6. Zogaj X, Nimtz M, Rohde M, Bokranz W, Romling U (2001) The multicellular morphotypes of Salmonella typhimurium and Escherichia coli produce cellulose as the second component of the extracellular matrix. Mol Microbiol 39: 1452-1463.


MICROBE VS. MICROBE AT THE OK CORRAL; GLOBAL CHANGE AND THE

BIOGEOGRAPHY OF SOIL MICROBES Ferran Garcia-Pichel 1Arizona State University School of Life Sciences Tempe, AZ 85226

Shifts in plant and animal geographical ranges are one of the most commonly predicted outcomes of global climate change. On warming, species are faced with either migrating towards cooler areas or sustaining range losses. Such predictions have so far excluded microorganisms, not only because their geographic distributions remain largely undefined, but also because it is unknown if environmental parameters sensitive to global change, such as temperature or water availability, help at all in shaping their large scale distributions. We analyzed DNA-based, continental-scale compositional surveys of North American biocrusts and found that a clear replacement in dominance exists between the cyanobacteria Microcoleus vaginatus and M. steenstrupii, key pioneers to biocrust formation. Statistical analyses of climatic, geographic, edaphic and microbial compositional data, indeed point to temperature as a major driver of the segregation of M. vaginatus dominance to colder climates. Moreover, the responses to temperature of enrichment cultures and of a collection of cultivated strain representative of these two taxa clearly support this contention, with M. vaginatus strains being more psychrotolerant and less thermotolerant than those of M. steenstruppi, regardless of their geographic origin. In view of predicted climate drift in the US Southwest, M. steentrupii dominance is likely to replace that of M. vaginatus in much of the studied area within the next few decades, with yet to be determined ecological consequences for soil fertility and erodibility. M. steenstripii now becomes a priority target for ecophysiological and genetic research. An interesting and hardly predictable outcome results from adding a third species, however… (to be continued)


GALLERIA MELLONELLA AS AN INFECTION MODEL FOR GROUP A STREPTOCOCCUS

Scott V Nguyen1, Kimberly McCullor1, Cathy King1, Maliha Rahman1, and W. Michael McShan1

1University of Oklahoma Health Sciences Center Department of Pharmaceutical Sciences Oklahoma City, OK, 73162 Our laboratory has characterized and identified several Streptococcus phage-like chromosomal islands that confer a complex mutator phenotype onto its host through dynamic excision and reintegration. The S. pyogenes chromosomal island M1 (SpyCIM1) is the best characterized chromosomal island (CI) of the group and controls the expression of DNA mismatch repair (mutL), a multidrug efflux pump (lmrP), UV repair (ruvA), and base excision repair (tag). As a phage-like CI, SpyCIM1 utilizes helper bacteriophages to package itself into smaller bacteriophage particles for dissemination. We have evidence that not only do SpyCI resides in its host symbiotically through variable control of its mutator phenotype but may also provide additional benefits by enhancing S. pyogenes virulence. Using the caterpillar larvae of the greater wax moth, Galleria mellonella, as an in vivo infection model, we can determine SpyCIs’ impact on its host virulence. Overall, it is critical to understand the impact that these CI have on host mutator phenotype and virulence. The balance between integration and excision is tightly controlled, as interfering with the SpyCI’s ability to excise results in sickly cells. Ultimately, the control of the DNA repair system by SpyCI is ideal for achieving a mutator phenotype while minimizing the risks of long-term hypermutability. S. pyogenes is still a major human pathogen despite decades of antibiotic therapy. The ability of the organism to colonize multiple niches within the human host and its ability to rapidly adapt have been key to its success. MMR chromosomal islands have been successful in colonizing pathogenic members of Streptococcus and understanding their impact on the fitness and adaptability they confer on their hosts will be key to combating this important human pathogen. This work was made possible by a Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR11-133 and by NIH Grant Number R15A1072718 to WMM.


A PROPOSED MODEL FOR THE REGULATION OF INCA/C PLASMID CONJUGATIVE

TRANSFER Kevin S. Lang1and Timothy J. Johnson1 1University of Minnesota Department of Veterinary and Biomedical Sciences Saint Paul, MN 55108 Multidrug resistant bacterial infections are a growing threat to public health. The alarming rate at which resistant organisms emerge and spread is, in part, due to horizontal gene transfer (HGT). Often times, HGT is mediated by conjugative plasmids. IncA/C plasmids are a group of plasmids able to be transferred and maintained in a broad range of Gram-negative bacteria. IncA/C plasmids have been significant contributors to the spread of clinically relevant resistance genes, including genes that confer resistance to third-generation cephalosporins and carbapenems. Despite the threat they impose, little is known about the basic biology of IncA/C plasmids. We set out to identify and characterize genes involved with the regulation of plasmid transfer. Using prototypical IncA/C plasmid pAR060302, we screened mutants using a conjugation assay that measures the frequency of transfer events. We identified ORFs 183-188 as being critical for transfer. Using a luxCDABE reporter plasmid, we assessed the ability of genes in this region to modulate transcription of promoters upstream of transfer associated genes. Our analyses suggest that ORF183 and ORF184 function in a dual repressor molecular switch. Furthermore, our data show ORF186 and ORF187 are the positive regulators of the genes encoding the conjugative machinery. Taken together, these data indicate that IncA/C plasmids encode a complex regulatory network that governs conjugative transfer. Further work is needed to elucidate the cues given by either the host microbe or environmental conditions where plasmid transfer is de-repressed. Understanding these biological processes and the mechanisms that drive them will aid efforts to curtail the spread of multidrug resistance.


Regulation of the alternative sigma factor, σX, during transformation in Streptococcus pneumoniae.

Yanina Tovpeko1, and Donald Morrison1. 1University of Illinois – Chicago Department of Biological Sciences – Lab for Molecular Biology. Chicago, IL 60607 Streptococcus pneumoniae is naturally competent but the mechanisms controlling rapid development and loss of competence are still imprecisely understood. This transient physiological state depends on an auto-inducing peptide (CSP), which activates a feedback loop and two “early” competence genes, comX and comW. Both are necessary for transcription of “late” genes. ComX is the alternative σ factor, σX, while ComW is a small protein whose function is unknown. ComW may act as an anti-anti-sigma or a pro-sigma. ΔcomW mutants are severely deficient (~1000-fold) in transformation. This severe defect in transformation allows a strong, but not absolute, enrichment for suppressor mutations restoring transformation in the absence of ComW. To seek possible ComW interaction partners in a ΔcomW background, we enriched for spontaneous mutants with increased transformation efficiency by three successive rounds of transformation. To avoid recovery of mutant “siblings,” the three compound enrichments were applied in parallel to 15 subclones of the parent ΔcomW strain. The transformation efficiency of recovered putative suppressor progeny was evaluated relative to ComW+ and ComW- strains. 60% of the recovered mutants transformed at elevated rates, from 1% to 10% when compared to a ComW+ strain. This indicates the presence of at least one gene involved in this competence induction pathway which, when mutated, relieves the defect caused by the absence of comW. To identify the unmarked suppressor mutations, we performed whole genome sequencing. All sequenced strains contained one of four mutations in a single gene. After back-cross to a clean WT background, each of the four mutations was independently sufficient to elevate transformation in a ΔcomW mutant to the level observed in the potential suppressor strains recovered after enrichments. Furthermore, the suppression was lost when the mutation was replaced with the wild type sequence within the recovered suppressor strains. The identity of the suppressor gene should provide a clue to the interactions of ComX and ComW with other factors during competence induction in S. pneumoniae. This material is based upon work supported in part by the National Science Foundation under Grant No. MCB-1020863.


Development of competence leads to mutagenesis in stressed Bacillus subtilis cells

Amanda A. Prisbrey1, Carmen Vallin1, John Creech2, Holly A. Martin3, and Eduardo A. Robleto1

1University of Nevada, Las Vegas School of Life Sciences Las Vegas, NV 89154 2Alamance Community College North Carolina 3Gillette College Gillette, WY 82718 Mutagenesis is central to the evolutionary process. We currently view evolution as a gradual process affecting all cells within a population. However, I aim to study an underappreciated part of the evolutionary process, mutations generated during stationary phase within a subpopulation. Stationary phase cultures of Bacillus subtilis develop subpopulations that exhibit different survival strategies. One of these subpopulations, known as competence, develops the ability to uptake exogenous DNA. During competence, new alleles can be acquired and recombine into the hosts genome leading to genetic diversity. Published results from my research group have shown that i) defects in genetic factors that control competence (ComK and ComA) result in decreases in mutagenesis in non-growing cells; and ii) the observed decrease is independent of recombination. We speculate that some other mechanism, activated during the K-state, regulated by the transcriptional activator ComK, in which more than just competence genes are activated, is responsible for most of the mutations seen during stationary phase. My project seeks to bring together these separate observations into a coherent understanding of how competence or the K-state leads to increases in mutagenesis. Here we test the hypothesis that the population of cells that develops competence experiences increased levels of mutagenesis during stationary phase.


Non-B DNA promotes genetic diversity in B. subtilis stationary phase cells Carmen Vallin, Amanda A. Prisbrey, Eduardo A. Robleto

University of Nevada, Las Vegas School of Life Sciences Las Vegas, NV 89154 It is widely accepted that mutations are generated during the process of DNA replication in actively dividing cells, however research dating as far back as 1955 has continued to build evidence for mutations arising in non-growing conditions, a phenomenon known as stationary-phase mutagenesis (SPM). In the Gram positive bacterium Bacillus subtilis, it has been proposed that the process of transcription influences stationary-phase mutagenesis. The specific mechanisms of how transcription mediates mutagenic events during stationary phase are currently under investigation. One interesting possibility is that the act of transcription promotes the formation of non-B DNA structures that prone DNA to damage and, subsequently through low-fidelity repair, to accumulate mutations (See Figure below). These mechanisms are novel and improve our understanding of evolution. Further, given that all organisms have sequences with potential to form non-B DNA these mutagenic mechanisms have been implicated in increasing genetic diversity in all domains of life.


An Iron Transporter Limits Salmonella Replication In Single-Infection But Is Required For

Mixed-Infection Of Mice And Macrophages Toni A. Nagy1, Sarah M. Moreland2 and Corrella S. Detweiler1 1University of Colorado- Boulder Department of Molecular Cellular and Developmental Biology Boulder, CO, 80309 2Temple University School of Medicine Philadelphia, PA, 19140 Bacteria harbor both ferrous and ferric iron transporters. Our current data indicates that infection of macrophages and mice with a Salmonella strain containing an inactivated feoB-encoded ferrous iron transporter results in increased bacterial replication, compared to infection with wild-type. Strains lacking other cation transporters, either SitABCD or MntH, have no apparent phenotype in macrophages or mice. Increased replication of the feoB mutant strain is not due to an intrinsically faster growth rate and instead correlates with increased expression of the fepB-encoded bacterial ferric iron transporter, and requires siderophores, which capture ferric iron, for the hyper-replication phenotype. Co-infection of mice with wild-type and a feoB mutant strain, however, yields a different outcome: FeoB is clearly required for tissue colonization. In co-infected classically activated primary mouse macrophages, wild-type out-competes the feoB mutant strain only in macrophages that have engulfed erythrocytes, a model of hemophagocytosis. These observations suggest that spontaneous inactivation of feoB during infection would be maladaptive despite increased replication upon single infection, and that ferrous iron uptake is important specifically in hemophagocytes.


Ironing it Out: the Role of the Yersinia Type III Secretion System in the Gut

Leah Schwiesow1, Halie Miller

1, Walter Adams

1, Winnie Au-Yueng

1, and Victoria Auerbuch

1

1University of California, Santa Cruz Department of Microbiology and Environmental

Toxicology Santa Cruz, CA, 95064

Inflammatory bowel disease (IBD) is thought to arise from the imbalance of the intestinal immune response to the gut microbiota. The enteropathogenic Yersinia species and other type III secretion system (T3SS)-expressing pathogens are transient members of the gut microbiota, a few of which have been associated with IBD. The T3SS is a needle like structure that is used to inject bacterial effector proteins into eukaryotic cells. It is known that the mammalian innate immune system can recognize the T3SS, leading to secretion of inflammatory cytokines and other immune mediators. Given that the enteropathogenic Yersinia T3SS induces several inflammatory mediators involved in IBD, we investigated the innate immune response of intestinal epithelial cells to the Yersinia T3SS. Expression of both the chemokine IL-8 and the prostaglandin synthase Cox2 was increased in HT29 and Caco-2 human intestinal epithelial cells (IEC) infected with a Y. pseudotubercolosis strain expressing a functional T3SS, but lacking the T3SS effector proteins YopHEMOJT. The T3SS effector protein YopJ partially inhibited this MAP kinase-dependent expression of IL-8 and Cox2. Importantly, a Y. pseudotuberculosis mutant expressing a non-functional T3SS did not induce IL-8 or Cox2 expression. These data suggest that IECs can respond to a functional T3SS and that Yersinia can manipulate this response through its T3SS effector proteins. However, it is not known whether IECs can be targeted by the Yersinia T3SS in vivo. Our group recently identified a novel component of the Y. pseudotuberculosis T3SS regulatory cascade, IscR. IscR in E. coli is an iron-sulfur [2Fe-2S] cluster coordinating transcriptional regulator that has been shown to bind different DNA target sequences dependent upon [2Fe-2S] coordination. We found that IscR likely controls expression of the Yersinia T3SS through direct regulation of LcrF, the T3SS master regulator. Furthermore, Y. pseudotuberculosis displayed decreased expression of iscR, lcrF, and T3SS genes in iron overloaded mouse tissues compared to normal tissues, suggesting that increasing iron bioavailability dampens T3SS gene expression through IscR and LcrF. Interestingly, enteropathogenic Yersinia experience large changes in iron bioavailability upon transit from the gut to deeper tissues and once disseminated, Yersinia can traffic back to the gut. We hypothesize that enteropathogenic Yersinia use IscR to dampen T3SS gene expression in iron-rich intestinal tissue in order to prevent unnecessary T3SS deployment that may lead to expression of inflammatory mediators. To test this hypothesis, we are currently developing Yersinia reporter strains to monitor expression of iscR, lcrF, and the Yersinia T3SS throughout infection. Through this analysis, we aim to better understand the relationship between the Yersinia T3SS, iron, and gut inflammation.


Nutritional stress activates the Salmonella pathogenicity island 2 type III secretion

system intracellular virulence program. Timothy Tapscott1, Joseph Westrich2, Matthew Crawford2, Lin Liu2, Jessica Jones-Carson3, Andres Vázquez-Torres1,2,4 University of Colorado School of Medicine 1Molecular Biology Program, 2Department of Microbiology, and 3Division of Infectious Diseases, Aurora, CO 4Veterans Affairs Eastern Colorado Health Care System, Denver, CO Salmonella quickly responds to a myriad of environmental conditions within epithelial cells and

macrophages. In particular, nutritional starvation elicits a spectrum of changes in gene

expression collectively known as the stringent response, which is mediated by the RNA

polymerase-binding protein DksA and the alarmone guanosine tetraphosphate (ppGpp). We

show herein that DksA and ppGpp play an essential role in Salmonella pathogenesis by

transcriptionally activating the intracellular expression of the type III secretion system encoded

within the Salmonella pathogenicity island 2 (SPI2). We discovered dksA and ppGpp-null

Salmonella exhibited a functional loss of the SPI2 type-III secretion system in macrophages.

Moreover, dksA and ppGpp-null mutants lost the SPI2-dependent ability to divert away host

antimicrobial defenses from mannose-6P+ endolysosomal vesicles. DksA and ppGpp directly

activated SPI2 in vitro gene transcription. A murine competitive index assay showed that a

Salmonella strain deficient in the stringent response DksA regulatory protein was equally

attenuated than an isogenic SPI2 mutant, supporting the model that most contributions of DksA

to Salmonella pathogenesis are mediated through the activation of SPI2. Our results

demonstrate a direct regulation of Salmonella pathogenesis by an effector of the classical

stringent response to nutritional starvation.


Multiple flagellins in Agrobacterium tumefaciens: function and coordination

Bitan Mohari and Clay Fuqua

Indiana University Bloomington, Department of Biology, Bloomington, IN-47405, USA.

Agrobacterium tumefaciens, the causative agent of crown gall disease, swims via

clockwise rotation and bundling of a tuft of lophotrichous flagella. Flagella are helical filaments composed of the bent flexible hook that forms a universal joint between the basal body complex and the more rigid filament. In A. tumefaciens flagella are thought to be comprised of four different flagellins - FlaA, FlaB, FlaC and FlaD, the first three of which are quite similar in length (306-320 aa) and sequence, and are encoded together in a single gene cluster. In contrast FlaD is divergent in sequence, significantly larger (430 aa), and not genetically linked to the other three flagellins. Previous studies suggest that all the flagellin genes appear to have individual promoters and are independently transcribed, although this does not rule out the possibility that flaA, flaB and flaC might also be co-expressed in a single transcript. In order to explore the role and contribution of multiple flagellins, single and multiple in-frame deletion mutants in the corresponding genes were created and evaluated using swim agar assays and electron microscopy. Mutants for FlaA form non-helical flagella, and are severely compromised in motility. Other single flagellin mutants are unaffected for motility. However FlaA alone is not sufficient for motility as ∆flaBCD mutants are non-motile and make very few normal flagella. Cells with FlaA and one other secondary flagellin appear to be completely proficient for synthesis of flagella and motility. Mutants that lack all but either of the secondary flagellins FlaB or FlaC individually make straight, truncated and non-functional filaments, and those with FlaD alone exhibit much smaller, minute rods attached to curved hooks. Mass spectrometric analysis of wild type filament preparations indicates that FlaA is the most abundant flagellin followed by FlaB, FlaC and finally FlaD which appears to be in much lower proportion to the other flagellins. Incorporation of a cysteine residue into the FlaA flagellin allows fluorescent labeling of flagellar filaments, and real time detection of the flagellar bundle. Within the flagellin gene cluster there is an annotated hypothetical gene, Atu0544 in line with and between flaA and flaB. Atu0544 encodes an 85 aa acid predicted protein with no known function, but may be a novel contributor to flagellin function. Regulation of the flagellin gene cluster (flaA-Atu0544-flaBC) and flaD is not well understood. The regulators FlbT and FlaF have been reported to function as checkpoints of flagellin regulation, and A.

tumefaciens ΔflaF and ΔflbT mutants are non–motile. FlbT appears to positively

regulate expression of flaA, flaB and flaC and FlaF appears to regulate only flaA expression. The role of these regulators in orchestrating expression and assembly of the multiple flagellins is currently being investigated.


FlgM is secreted in Bacillus subtilis

Rebecca A. Calvo1 and Daniel B. Kearns1 1Indiana University Department of Biology Bloomington, IN, 47403 Spontaneous assembly of a proteinaceous, trans-envelope machine, such as the flagellum, requires bacteria to coordinate gene expression with assembly. The flagellum rotates to allow motility of diverse bacterial species and is composed of three major structural elements: a basal body, a hook, and a filament. A dedicated flagellar export apparatus, housed within the basal body, secretes hook and filament protein subunits through the basal body where they are assembled on the outside of the cell. To coordinate assembly of the flagellum, genes encoding components of the hook, basal body, and export apparatus are transcribed by the housekeeping sigma factor σA, and the gene encoding the flagellar filament (flagellin) is transcribed by the alternative sigma factor σD. B. subtilis is unique in that the motility sigma factor σD is required not only for flagellar assembly but also for separation of daughter cells following cell division. The activity of σD is inhibited through direct protein-protein interaction by the anti-sigma factor FlgM prior to HBB assembly. We show that FlgM is secreted outside the cell through the fully assembled HBB, and infer that the resulting drop in intracellular FlgM relieves inhibition of σD to permit transcription of flagellin. In addition, we find that FlgM is specifically degraded extracellularly by the secreted proteases WprA and Epr. Secretion, however, may not be sufficient to regulate FlgM activity in B. subtilis. All mutants defective in HBB assembly abolish

D. Using a forward genetic approach, we identified six alleles of FlgM that constitutively inhibit σD (flgM*). Two flgM* alleles were not secreted, which supports the hypothesis that secretion regulates FlgM activity. Four flgM* alleles, however, were secreted as well as wild-type FlgM. We hypothesize that FlgM may require activation by a HBB component to function as an anti-sigma factor. In S. enterica, FlgM is thought to play a critical role during flagellar morphogenesis in that it couples flagellin gene expression to the assembly state of the flagellum. In B. subtilis, this

D prior to HBB completion to prevent the transcription of D regulon. What is the function of FlgM in B. subtilis D also regulates cell wall remodeling enzymes, perhaps FlgM is necessary to coordinate flagellar assembly with cell division.


Characterization of swrD function in Bacillus subtilis swarming motility

Ashley N. Roarty1, Rebecca Calvo1 and Daniel Kearns1

1Indiana University Department of Biology Bloomington, IN, 47405 The flagellum is a molecular machine composed of three main parts: the basal body, hook, and filament. In Bacillus subtilis, genes required to build the hook-basal body are encoded in the

fla/che operon. The fla/che operon also encodes the alternative sigma factor, D, which is required for transcription of the flagellar filament gene and the flgM gene. FlgM is an anti-sigma

factor that inhibits D prior to hook-basal body completion. B. subtilis produces multiple flagella to mediate both swarming motility, which occurs on a surface, and swimming motility, which occurs in liquid. Swarming motility, unlike swimming, requires a higher number of flagella, a surfactant, and the ability to sense a surface. The fla/che operon also encodes a small 213bp gene, swrD, that is required for swarming motility. We show that a mutant containing an in-frame deletion of the swrD coding region

(swrD) can produce approximately wild-type numbers of basal bodies, hooks, and flagellar

filaments. We isolated swarming-proficient suppressors of swrD and show that many swrD suppressors have mutations in flgM. We show that mutation of flgM can partially suppress the

swarming defect of the swrD mutant, but that increasing the activity of D is not sufficient to

fully overcome the swarming defect of the swrD mutant. Lastly, we show evidence that SwrD probably does not inhibit FlgM directly. It is possible that SwrD plays a role in the regulation of FlgM, in surface sensing, or in a yet unidentified requirement for swarming motility.


Surfing on the bacterial chromosome: the dynamics of plasmid partition promoted by

ParA ATPases Barbara E. Funnell

University of Toronto Department of Molecular Genetics Toronto, Ontario M5S 1A8 Canada In bacteria, low-copy-number plasmids as well as many chromosomes rely on active partition systems for faithful segregation to daughter cells. The partition cassette on the P1 plasmid in Escherichia coli consists of a centromere-like partition site (parS), a site-specific DNA binding protein called ParB that recognizes parS, and an ATPase called ParA. P1 ParA is a member of a larger class of ATPases, defined by a specific variant of the Walker ATP binding motif, that are responsible for localization of DNAs and large protein machineries in bacteria. These ATPases form patterns in vivo on a surface (the nucleoid for ParA; the membrane for some members) in conjunction with a partner protein that stimulates their ATPase activities (ParB for ParA). We have focused on reconstitution of steps in the partition pathway in vitro, using several biochemical approaches as well as direct visualization in a cell-free TIRF (total internal reflection fluorescence) microscopy system. ATP binding and hydrolysis control the interconversion of different forms of ParA during the pathway, and the kinetics of conversion are critical to mechanism. We have also exploited interesting phenotypes of parA and parB mutants to define these steps. For example, using synthetic peptides as well as ParBs with mutations in the N-terminus, we show that this region of ParB is responsible for stimulation of ParA ATPase as well as interconversion of active to inactive ParA in the absence of hydrolysis. Our results support the idea that plasmids, via bound ParB, essentially surf over the surface of the nucleoid by both promoting and following a dynamic wave of ParA protein. Further, we suggest that this molecular mechanism is shared by other Walker ParA-like proteins that promote localization of plasmid or protein cargo in bacterial cells.


POSTERS

PHYSIOLOGICAL AND BIOCHEMICAL CHARACTERIZATION OF DEAMINATED- DNA REPAIR PATHWAYS IN Bacillus subtilis.

Víctor M. Ayala-García1 and Mario Pedraza-Reyes1 1Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, Guanajuato 36050, Mexico. DNA contains amino groups in three of the four bases (cytosine, adenine and guanine). Deamination of these DNA bases can occur in a spontaneous manner and at a physiologically significant rate via a hydrolytic reaction generating the base analogues uracil, hypoxanthine and xanthine, respectively1. As deaminated bases affect the pairing properties in DNA, if left unrepaired, these lesions are not only mutagenic but also potentially lethal. To avoid the noxious effects of amino group’s loss in DNA, cells possess excision repair pathways to prevent mutagenesis and proteins belonging to these pathways are highly conserved in all three domains of life2. Recently, in our laboratory, it was reported that in the spore-forming bacterium Bacillus subtilis, uracil in DNA can be processed by: i) Base excision repair system (BER) by the action of uracil-DNA glycosylase; ii) Mismatch repair pathway (MMR) through MutSL complex, and, iii) Alternative excision repair (AER) whose pathway apparently is initiated by the enzyme Endonuclease V (YwqL)3. In Escherichia coli, Endonuclease V (Nfi) is able to process, uracil and other deaminated bases including hypoxanthine and xanthine as well as other detrimental DNA lesions4, presumably through an alternative repair pathway that requires the 3’-5’ exonuclease activity of DNA polymerase I followed by gap-DNA synthesis5. Interestingly DNA polymerase I of B. subtilis lacks a proofreading 3’-5’ exonuclease activity; therefore, in addition to analyzing the spectrum of lesions that are recognized and processed by YwqL, we are investigating the mechanism by which the AER pathway commenced by this enzyme proceeds in this Gram-positive bacterium. Since our results have shown that ywqL as well as yxlJ (the latter encoding a protein homologous to mammalian 3-methyladenine DNA glycosylase involved in processing hypoxanthine6) are expressed during sporulation and that deletion of ywqL decreased the survival of spores, we are also analyzing the role played by these proteins in protecting these differentiated cells from the genotoxic effects of the deaminating agents nitrous acid and sodium bisulfite.

58th Annual Wind River Conference, June 4-8, 2014

1. Lindahl, T. Instability and decay of the primary structure of DNA. Nature 362, 709–715 (1993).

2. Lindahl, T. & Wood, R. D. Quality control by DNA repair. Science 286, 1897–1905 (1999).

3. López-Olmos, K. et al. Roles of Endonuclease V, Uracil-DNA Glycosylase, and Mismatch Repair in Bacillus subtilis DNA Base-Deamination-Induced

Mutagenesis. Journal of Bacteriology 94(2), 243-252 (2012).

4. Yao, M. et al. Purification and characterization of a novel deoxyinosine-specific enzyme, deoxyinosine 3’ endonuclease from Escherichia coli. J. Biol. Chem

269, 16260-16268.

5. Lee, C. et al. The excision of 3’ penultimate errors by DNA polymerase I and its role in endonuclease V-mediated DNA repair. DNA Repair 12, 899– 911

(2013)

6. Aamodt, R.M. et al. The Bacillus subtilis counterpart of the mammalian 3-methyladenine DNA glycosylase has hypoxanthine and 1,N6-ethenoadenine as

preferred substrates. J. Biol. Chem 279, 13601-13606 (2004).

Work supported by CONACYT (grant 205744) and University of Guanajuato (grant DAIP-324-2013). V. M. A-G was supported by a scholarship from CONACyT.


INCREASED EXPRESSION OF RIBONUCLEOTIDE REDUCTASE NrdEF PROMOTES SPONTANEOUS AND STATIONARY PHASE ASOCIATED MUTAGENESIS IN Bacillus

subtilis. Karla Castro-Cerritos

1, Ronald E. Yasbin

2, Eduardo Robleto

3 and Mario Pedraza-Reyes

1

1Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, Guanajuato 36050,

Mexico; 2College of Arts and Sciences, University of Missouri –St.Louis, MO 303, USA;

3School of Life Sciences,

University of Nevada—Las Vegas, Las Vegas, Nevada 454004, USA.

Ribonucleotide reductases (RNRs) are a family of enzymes that play an essential role catalyzing the synthesis of deoxyribonucleotides (dNTPs) required for DNA replication and repair. In bacteria, expression of RNR-encoding genes is regulated by the transcriptional repressor NrdR that senses dATP/ATP pools and binds to tandem repeat sequences, called NrdR boxes, located in or near to the promoter regions(1). Transcriptional regulation of RNRs is essential to a faithful DNA synthesis; thus, elevated dNTPs levels has been associated with a mutator state in E. coli increasing spontaneous and induced mutagenesis in this bacteria(2). Moreover, in yeast, a high activity of RNR has been linked to survival after DNA damage with a subsequent increase in mutation rates (3). In B. subtilis the RNR is coded by the nrdEF genes(4), and the candidate NrdR ortholog is encoded by ytcG (5). YtcG amino acid sequence is 49% similar to E. coli NrdR; it is predicted that YtcG keeps a dATP/ATP cone and a zinc finger characteristic of this type of repressors. In addition, tandem NrdR boxes have been predicted upstream of the B. subtilis nrdEF operon(5).

In this work, using a B. subtilis strain harboring a transcriptional nrdE-lacZ fusion, we

demonstrated that deletion of ytcG increased ~6-7 times the β-galactosidase levels in this

strain. Furthermore, during growth, ytcG deletion promoted spontaneous mutagenesis; whereas, the genetic inactivation of nrdEF significantly decreased this process. In experiments of stationary-phase-associated mutagenesis, performed with strain B. subtilis YB955 (his-, met-, leu-) (6), a similar behavior was observed; namely, the reversion rates of the his, met and leu alleles was proportional to the expression level of the nrdEF genes. Interestingly, 24% of the Leu+ revertants in ytcG strain also showed prototrophy for his and met compared with only 0.2% of these phenotypes observed in the parental strainYB955.

In conclusion, we propose that high expression levels of the nrdEF genes, and therefore elevated dNTPs levels, promote stationary phase-associated mutagenesis probably through the generation of a hypermutable state.

1. I. Grinberg et al., J Bacteriol 188, 7635 (Nov, 2006).

2. S. Gon, R. Napolitano, W. Rocha, S. Coulon, R. P. Fuchs, Proc Natl Acad Sci U S A 108, 19311 (Nov 29, 2011).

3. A. Chabes et al., Cell 112, 391 (Feb 7, 2003). 4. C. Scotti, A. Valbuzzi, M. Perego, A. Galizzi, A. M. Albertini, Microbiology 142 ( Pt 11), 2995 (Nov, 1996). 5. D. A. Rodionov, M. S. Gelfand, Trends Genet 21, 385 (Jul, 2005). 6. H. M. Sung, R. E. Yasbin, J Bacteriol 184, 5641 (Oct, 2002).

Work supported by CONACYT (grant 205744) and University of Guanajuato (grant DAIP-324-2013). C. C-C. was supported by a scholarship from CONACyT.


Identification of Proteins that Interact with The Pseudomonas aeruginosa PrrF and PrrH Small RNAs

Jonathan Osborne1, Louise Djapgne1, Bao Tran1, Young Ah Goo1, and Amanda Oglesby-Sherrouse1,2 1University of Maryland School of Pharmacy, Department of Pharmaceutical Sciences, 2University of Maryland School of Medicine, Department of Microbiology and Immunology, Baltimore MD 21201 Pseudomonas aeruginosa is a gram-negative bacterium and opportunistic pathogen that infects people with compromised immune systems. P. aeruginosa is highly resistant to multiple antibiotics, in part due to its ability to form biofilms. Iron is required for P. aeruginosa virulence and biofilm formation, but can also be toxic to the bacteria. Therefore, iron acquisition and utilization are tightly regulated in response to iron availability. Iron homeostasis is maintained in part by the production of two small RNAs (sRNAs), PrrF1 and PrrF2. Under iron-depleted conditions, the prrF genes are expressed, leading to the repressed expression of iron containing proteins. This mechanism is used by P. aeruginosa to “spare” iron when this nutrient becomes scarce. PrrF1 and PrrF2 are encoded in tandem on the P. aeruginosa genome, allowing for the expression of a third sRNA named PrrH. Expression of PrrH is regulated by heme (Oglesby-Sherrouse and Vasil, 2010), an abundant source of iron during infection. However, it is unknown how heme regulation of PrrH occurs. To identify factors required for heme-regulated PrrH expression, we designed a method for identifying proteins that interact with the PrrF and PrrH sRNAs in vivo. Bacteria were grown in low iron conditions and irradiated to irreversibly crosslink RNA-protein complexes. RNA was isolated from the cells, and PrrF- and PrrH-protein complexes were enriched using cDNA “bait”. Enriched RNA was analyzed by LC-MS/MS mass spectrometry (MS) to identify associated protein binding partners. Using this method, we identified Hfq, an RNA-binding protein that stabilizes many bacterial sRNAs, as a potential PrrF- and PrrH-binding protein. Hfq was identified in 75% of the MS analysis with the PrrF cDNA “bait”, but only in 37% of the MS analysis using PrrH cDNA “bait”. These data suggest Hfq has a stronger binding affinity for the PrrF sRNAs as compared to the PrrH sRNA. We hypothesize the weak binding of Hfq to PrrH is indicative of the role of this protein in stabilizing PrrH in the absence of heme. We are currently using gel-shift assays to determine the binding affinity of Hfq for the PrrF and PrrH sRNAs under different in vitro conditions. Identifying factors required for the synthesis and regulation of these unique sRNAs may provide new targets for therapeutics.


Temperature-dependent Post-transcription Regulation of Shigella ompA Expression by an RNA Thermometer

Amanda E. Dunson1, Andrew B. Kouse1, Kevin F. Gross1, Erin R. Murphy Ph.D.2 1Ohio University Biological Sciences Department Athens, OH, 45701 2Ohio University Heritage College of Osteopathic Medicine Athens, OH, 45701 Bacteria of the genus Shigella cause shigellosis, a severe diarrheal disease that sickens 90 million people each year and claims nearly a million lives annually. Following ingestion and transit through the human gastrointestinal tract Shigella initiate an infection by invading, replicating within and spreading from one cell to the next within the colonic epithelium. The processes of eukaryotic cell invasion, intracellular replication and intercellular spread are all essential for the establishment and progression of a productive infection. As such, any factor that facilitates one or more of these processes is considered to be a virulence determinant. It has recently been demonstrated that the outer-membrane protein OmpA is required for cell-to-cell spread of Shigella, marking this protein as an important virulence factor in these pathogens. Given the newly identified role of OmpA in Shigella virulence we began an investigation into the regulatory mechanisms controlling its production. Our initial studies demonstrated that, as in Escherichia coli, expression of ompA in S. dysenteriae is thermo-regulated and that this regulation is mediated by a post-transcriptional mechanism. Further investigation revealed the presence of a putative 4U RNA thermometer within the 5’ untranslated region of ompA; a regulatory element that if present could mediate the observed temperature-dependent post-transcriptional regulation of ompA expression. Cloning of the putative ompA RNA thermometer between a constitutive plasmid promoter and a gfp reporter gene demonstrated that the regulatory element is sufficient to confer temperature-dependent post-transcriptional regulation. Finally, site-specific mutagenesis of the ompA RNA thermometer confirmed that it is a function ribo-regulator. Experiments are ongoing to determine the precise role of the ompA RNA thermometer is controlling Shigella virulence. Given the conservation among enteropathogens, the development of a drug to stabilize the inhibitory structure within the ompA RNA thermometer might ultimately function to treat infection with Shigella and related pathogens.


Characterizing the sRNA RyfA from Shigella flexneri and Escherichia coli Megan E. Fris1, William H. Broach2, Sarah E. Klim1, and Erin R. Murphy2

1Ohio University Biological Sciences Athens, OH 45701 2Ohio University Heritage College of Osteopathic Medicine Department of Biomedical Sciences Athens, OH 45701 Bacteria of the genus Shigella (S. dysenteriae, S. flexneri, S. sonnei and S. boydii) all cause shigellosis, a severe bloody diarrheal disease in humans. Shigella primarily affects children of developing countries; specifically those that lack access to proper sanitation and clean water sources. Deaths caused from shigellosis are results of severe dehydration. Currently, there is no vaccine for Shigella, and many species are rapidly evolving antibiotic drug resistance. Researching virulent mechanisms is key to identifying new targets for vaccines or drugs to treat infection with Shigella and related pathogens. Recently, it has been demonstrated that S. dysenteriae produces two nearly identical twin regulatory small RNA molecules, RyfA1 and RyfA2. RyfA1 and RyfA control S. dysenteriae virulence in vastly different and complex ways, despite the fact that they are 95% identical to each other. Interestingly, the other Shigella species, as well as Escherichia coli each possess a singlet copy of ryfA, which vary slightly in their sequences from each other and from those encoded by S. dysenteriae. Here, we begin the characterization of the differences between the singlet RyfA molecules found in E. coli and S. flexneri, and the twin RyfA molecules found in S. dysenteriae. Eventually, these studies will lead to an understanding to how each RyfA molecule impacts the physiology and virulence of the bacterium in which it is produced.


Functional Characterization of Genes Required for Lignocellulose Degradation in Cellvibrio

japonicas Jeffrey G. Gardner1 1University of Maryland - Baltimore County Department of Biological Sciences Baltimore, Maryland, 21250 The complex assortment of polysaccharides that comprise the plant cell wall, collectively known as lignocellulose, is potentially a large reservoir of carbon and energy for soil microbial communities. However, these polysaccharides are exceedingly difficult to degrade, and therefore few bacteria are able to use this prospective energy source. The genomes of lignocellulose-degrading bacteria often encode for hundreds of enzymes that can modify and degrade plant polysaccharides. Regulation of this many genes requires sophisticated management, however the regulatory networks controlling expression of lignocellulose degrading enzymes are poorly defined, with little data on the physiological (in vivo) functions of the encoded proteins. Recently, lignocellulose-degrading microbes are of great interest to further understand the global carbon cycle, human microbiome nutritional contributions, and sustainable energy development. Our laboratory uses systems biology approaches to assign physiological function to predicted carbohydrate modifying enzymes in the lignocellulose degrading bacterium Cellvibrio japonicus. Gene expression profiling of C. japonicus during growth in lignocellulose revealed that despite having over two hundred genes annotated as contributing to plant cell wall deconstruction, only a subset are highly expressed using these substrates. Gene deletion and growth experiments indicated that only a few genes are essential for plant polysaccharide depolymerization. Heterologous expression of the critical genes in a non-cellulolytic bacterium showed that they were sufficient for the degradation of plant polysaccharides. Collectively, this work has begun to unravel the complex network of lignocellulose degrading enzymes that C. japonicus uses for the consumption of plant polysaccharides.


MOLECULAR ROADBLOCK APPROACH TO FURTHER UNDERSTAND VIRB ANTAGONISM OF

H-NS-MEDIATED REPRESSION OF ICSP IN SHIGELLA FLEXNERI Daren R. Ginete1, Hiromichi S. Park2 and Helen J. Wing1 1University of Nevada, Las Vegas School of Life Sciences Las Vegas, NV, 89154 2Touro University Nevada College of Osteopathic Medicine Henderson, NV, 89014 Shigella flexneri is an intracellular pathogen that causes bacillary dysentery in humans. Many of the genes important for virulence are encoded by its large virulence plasmid. These genes are repressed by the histone-like nucleoid structuring protein (H-NS) and derepressed by the virulence plasmid-encoded regulator VirB. The mechanism by which VirB alleviates H-NS-mediated repression remains elusive. Using the VirB-dependent PicsP as a model, we previously identified regions specific for its regulation. An inverted repeat located over 1 kb upstream and a region located between -838 and -436 relative to the transcription start site, are essential for VirB-dependent regulation and H-NS-dependent repression of the PicsP, respectively. In this study, we tested the hypothesis that a molecular roadblock between the regions required for regulation by VirB and H-NS will interfere VirB-dependent regulation of the PicsP. Our data show that when the molecular roadblock is introduced in this region, VirB-dependent regulation of the PicsP is lost. This is consistent with our overall hypothesis that VirB oligomerizes along the DNA, like its closest homolog ParB, and that this alleviates H-NS-mediated repression. Future work aims to test whether VirB oligomerization occurs which will aid in understanding the mechanism of VirB alleviation of H-NS-mediated repression, ultimately gaining more insight into virulence gene regulation in S. flexneri.


Identification of a Metabolic Repair Pathway in Salmonella enterica Jake J. Flood, Laurence S. Prunetti, Valerie De-Crecy Legard, Shelley D. Copley University of Colorado Boulder Department of Molecular, Cellular and Developmental Biology Boulder, CO 80309 Metabolites are subject to damage that can lead to the accumulation of useless or toxic products. Metabolite damage has been shown to result from promiscuous enzyme activities and non-enzymatic chemical reactions. Analogous to DNA or protein repair, cells have metabolic repair pathways to convert these detrimental metabolites back into useful ones. Glycolaldehyde (GAD) is a toxic aldehyde shown to result from lipid peroxidation. In Escherichia coli, an enzyme annotated as low-specificity threonine aldolase (LtaE) catalyzes an aldol condensation between GAD and glycine to form L-4-hydroxythreonine (4HT), another toxic metabolite. A similar enzymatic activity is present in Salmonella enterica. Here, we describe a metabolite repair pathway present in S. enterica that converts 4HT into a precursor of pyridoxal-5’-phosphate (PLP). We found that an enzyme of unknown function, DUF1537, is capable of phosphorylating 4HT with a kcat/Km of 5.4 x 105 M-1s-1. A second enzyme encoded within the same operon oxidizes 4-phosphohydroxythreonine with a kcat/Km of 8.2 x 103 M-1s-1. The high activities of these enzymes suggest that they act as dedicated metabolic repair enzymes that recycle 4HT back into mainstream metabolism.


Heme-binding NirD is a transcriptional regulator of acetoclastic methanogenesis in

Methanosarcina acetivorans Aileen R. Lee1, Mitchell T. Shea1, Nathan Sindt1 and Nicole R. Buan1 1University of Nebraska-Lincoln Department of Biochemistry Lincoln, NE, 68588-0664 Abstract 1.2 Gigatons of methane is produced globally from acetate by methane-producing archaea (methanogens). The eight-subunit membrane-bound protein complex homologous to the Rhodobacter nitrogen fixation (Rnf) complex found in Bacteria is essential for growth of the methanogen, Methanosarcina acetivorans, when acetate is the sole carbon source. Rnf is thought to be involved in the transport of electrons from ferredoxin to methanophenazine and the generation of a sodium ion-gradient which contributes to ATP-synthesis. Rnf expression is controlled by availability of carbon source, acetate, by an unknown mechanism. Rnf is expressed 10-fold greater when M. acetivorans is grown with acetate relative to cells are grown on methanol. We have identified a transcriptional repressor of the rnf locus from methanol-grown cell extract using high performance liquid chromatography (HPLC), electromobility shift assays (EMSA), and peptide mass spectrometry. We identified a heme-binding NirD homolog, MA0574, as binding to the rnf promoter region. Stronger EMSA shifts occurred when the protein was treated with dithiothreitol (DTT), however, when hematin was added to the DTT-treated protein, DNA-binding decreased. Binding of the DNA is more favorable under reduced conditions while the presence of heme blocks the protein’s ability to bind to DNA. This suggests that during times of oxidative stress or high levels of free heme, NirD protein is unable to bind to the DNA, resulting in the expression of rnf. Based on our work and recent reports, we propose a mechanistic model whereby metabolic flux through the alternative heme biosynthetic pathway in methanogens could coordinate redox sensing and the switch to acetate catabolism.


SHARKLET MICROPATTERNED SURFACE LIMITS BACTERIAL CONTAMINATION Ethan E. Mann1, Ryan M. Mettetal1, Rhea M. May1, Anthony B. Brennan2 and Shravanthi T. Redddy1 1Sharklet Technologies, Inc, Aurora, CO 80045 2University of Florida, Departments of Materials Science & Engineering and Bioengineering, Gainesville, FL, 32611 Microbial contamination of surfaces is a significant problem in healthcare and public spaces. Existing technologies to reduce surface contamination are limited to the use of chemicals, toxic metals, or antibiotics. Novel and innovative technologies for self-cleaning surfaces are needed to limit contamination by diverse pathogens. Therefore, several HAI-causing organisms were included in testing: Staphylococcus aureus, methicillin-resistant S. aureus, Staphylococcus epidermidis, Enterococcus faecium, Escherichia coli, and Acinetobacter baumannii. Surfaces were challenged with inoculum via spray or touch to mimic real-world scenarios of surface contamination. In all tests, the Sharklet MP reduced bacterial contamination by as much as 98% (p < 0.05) compared to smooth control surfaces. Importantly, the Sharklet MP was more effective than a 99.9% pure copper alloy C110 at reducing surface contamination of S. aureus (MSSA and MRSA). MSSA was reduced by the Sharklet MP as much as 98% (p < 0.005) and MRSA was reduced as much as 94% (p < 0.005) compared to smooth controls. In the same conditions, antimicrobial copper had no significant effect on MSSA contamination but reduced MRSA contamination by 80% (p < 0.005). The data presented here support the use of the Sharklet MP for controlling surface contamination. Evidence of antimicrobial copper successfully being implemented to decrease the HAI rates combined with head-to-head comparison of the Sharklet MP and copper using in vitro tests suggests that similar clinical implementation of the Sharklet MP has potential to reduce the incidence of HAIs as well.


Exploring the mechanistic contribution of the leucine aminopeptidase (LAP) to Staphylococcus aureus virulence

Devon N. Marking, Ronan K. Carroll, Frances E. Rivera, Stanley M. Stevens, and Lindsey N. Shaw University of South Florida Department of Cell Biology, Microbiology, and Molecular Biology Tampa, FL, 33620 Staphylococcus aureus is a highly pathogenic bacterium capable of causing a wide range of diseases in humans. It is arguably one of the most dangerous pathogens, responsible for the greatest number of hospital and community-acquired infections worldwide by a single bacterium. The main factors attributing to the pathogenesis of S. aureus are hemolysins, nucleases, and proteases. Previously, we identified an intracellular leucine aminopeptidase (LAP, pepZ) which is necessary for virulence in S. aureus. Intracellular aminopeptidases are responsible for catalyzing the cleavage of N-terminal amino acids from peptides and have rarely been shown to be involved in virulence in Gram-positive bacteria; which makes LAP a very distinctive protein. An intracellular and extracellular proteome analysis previously performed using a LAP mutant revealed large alterations in protein abundance and secretion of virulence factors. Interestingly, a foldase, PrsA, known to facilitate secretion of virulence factors in various Gram-positive bacteria, was significantly decreased in abundance in the mutant strain, leading us to hypothesize that the avirulent phenotype observed in the pepZ mutant could be mediated, in part, by PrsA; as a substrate of LAP. As such, to explore further the avirulent phenotype of the LAP mutant, we used the cutting edge proteomic tool, N-terminomics, to identify protease substrates and their cleavage sites in the presence and absence of LAP. Through this approach, we have identified a number of key LAP targets that appear to explain the role of this enzyme in virulence. In addition, we have also investigated the role of PrsA trafficking and function using western blot analysis and comparative proteomic techniques. Collectively, these results further our understanding of the contribution of this unique intercellular aminopeptidase to S. aureus virulence.


NOVEL MICRO-PATTERNED SURFACES REDUCE BIOFILM FORMATION OF Staphylococcus aureus and Pseudomonas aeruginosa

Rhea M. May1; Matthew G. Hoffman1; Melinda J. Sogo1; and Shravanthi T. Reddy1 1Sharklet Technologies Inc., Aurora, Colorado, USA

Introduction: The Sharklet micro-pattern is a specific ordered micro-topography that mimics shark skin and has been shown to alter adhesion of prokaryotic and eukaryotic cells. This pattern may provide an alternative strategy for reducing medical device-related infections as it has been shown to reduce microbial attachment and migration of several pathogens without the use of antimicrobial agents. The goal of this study was to evaluate this novel surface technology for biofilm reduction against two common pathogens: Methicillin-Resistant Staphylococcus aureus (MRSA) and Pseudomonas aeruginosa. Methods: P. aeruginosa: Patterned and un-patterned silicone samples (n=3) were immersed with 10^6 CFU/ml hyper-biofilm forming variant (PA14 ∆bifA) in arginine minimal medium for 24hrs at 37°C. MRSA (ATCC 700698): Patterned and un-patterned silicone samples (n=3)

were immersed with 10^6 CFU/mL in TSB over four days at 37⁰C with daily media replenishment. All samples were rinsed, fixed, dehydrated, and stained with propidium iodide for imaging via confocal laser scanning microscopy. Image stacks were obtained in three pre-selected sites per sample. Semi-volumetric analysis was achieved by totaling biofilm area coverage in each z-stack. Each experiment was replicated three times and significance was assessed by student t-test or ANOVA. Results: The micro-pattern reduced P. aeruginosa biofilm formation by an average of 52% (p=0.05) when compared to control surfaces. MRSA biofilms were reduced by an average of 67% (p=0.12). Conclusions: The micro-pattern surface modification inhibits the formation of MRSA and P. aeruginosa biofilms, as demonstrated via semi-volumetric quantification. These data suggest that the micro-pattern may reduce infection rates without the use of antimicrobial agents if applied to medical device surfaces.


THE EFFECT ON HELPER PHAGE REPLICATION BY STREPTOCOCCUS PYOGENES

CHROMOSOMAL ISLAND SPYCIM1 Kimberly A. McCullor1, Scott V. Nguyen1, Catherine J. King1, Chad Euler2, and William M. McShan1 1University of Oklahoma Health Sciences Center Department of Pharmaceutical Sciences Oklahoma City, OK. 73104 2Laboratory of Bacterial Pathogenesis and Immunology, The Rockefeller University, New York, NY 10065 The World Health Organization’s estimates from 2005 place the burden of Group A streptococcus (GAS; Streptococcus pyogenes) infections worldwide at approximately 18.1 million, with an estimated 1.78 million of new cases expected annually. GAS infections can cause a range of symptoms from sore throat to life threatening necrotizing fasciitis. Most strains of GAS contain lysogenic bacteriophages from the family Siphoviridae, which encode exotoxins such as the scarlet fever or Scarlatina toxin. We study Streptococcus Pyogenes Chromosomal Island M1 (SpyCIM1), which confers a mutator phenotype upon its host cell, and evidence suggests that a helper prophage (SF370.1) is used for packaging and dissemination of this chromosomal island. The specific aim of this study was to quantify the ability of S. pyogenes phage SF370.1 to infect a susceptible host strain at various stages of bacterial growth and to determine whether the presence or absence of SpyCIM1 interfered with the replicative cycle of this phage. Unlike many other bacteria, S. pyogenes fails to create a uniform lawn of growth on agarose plates, making it difficult to perform plaque assays. To overcome this limitation, our approach was to tag the helper phage SF370.1 with erythromycin resistance gene (ermB) through homologous recombination with the phage encoded pyrogenic exotoxin C gene (speC). This selectable marker enabled us to quantify the helper phage population by counting erythromycin resistant CFU following infection. Utilizing this marker, we were able to demonstrate that helper phage infections occurred predominantly during early log phase with significantly less infections occurring during the late log to stationary phase of bacterial growth. There was a decrease in helper phage population size when the phage lysate was harvested from wild type SF370 compared to lysate harvested from a SpyCI M1 knockout SF370 derived strain (CEM1∆4). The noticeable decrease in helper phage population size when SpyCI M1 was present suggests that SpyCI M1 suppresses the helper phage’s ability to replicate within the bacterial host during the lytic cycle. This work was made possible by a Oklahoma Center for the Advancement of Science and Technology (OCAST) grant HR11-133 and by NIH Grant Number R15A1072718 to WMM.


Identifying Genes Expressed by Salmonella Persister Populations with In-Vitro and In-Vivo

Platforms Erin M. McDonald1, Eugenia Silva-Herzog1, Zachary W. Bent2, and Corrella Detweiler1 1University of Colorado Boulder Department of Molecular, Cellular & Developmental Biology Boulder, CO, 80503 2Sandia National Laboratory, Livermore, CA Chronic infections are a significant threat to worldwide health, yet the mechanisms by which microbes escape immune detection and establish persistent infections are not well understood. Salmonella enterica subspecies Typhi causes typhoid fever in humans. Up to five percent of infected people become carriers, i.e., Salmonella are able to infect the mesenteric lymph nodes, bone marrow and gall bladder for the rest of the carrier’s life. A key question is how pathogens are able to maintain a chronic infection state. "Persisters" are phenotypic variants that have low metabolic activity and are believed to contribute to chronic infection because they survive under conditions that kill the vast majority of bacteria. Persisters are revealed in response to antibiotic or physiologic stress. We confirm the presence of Salmonella enterica serovar Typhimurium (STm) persisters after treatment with antibiotics or under stress conditions that mimic the host microenvironment. We are taking two approaches to identify genes important for the persister state. First, we have performed RNA-sequence analyses on persisters that are tolerant to ampicillin and have identified genes expressed at higher levels in response to this particular stress. Second, we are collaborating with Sandia National Laboratories to use a novel capture-based technique to enrich for pathogen RNA sequences in our murine chronic infection model. The capture-based enrichment strategy has identified STm genes that were also expressed at higher levels in persisters harvested after ampicillin treatment. We are at present determining which of these genes are expressed by the bacteria during infection of macrophages, a cell type in which STm resides in mice. Our long-term goal is to identify bacterial pathways that establish and maintain the persister state and thereby allow for chronic infection.


Targeted Gene Replacement in Streptococcus pneumonaie: Transformation Efficiency Analysis Samim Taraji and Donald A. Morrison University of Illinois at Chicago Department of Biological Sciences Chicago, IL 60607 Streptococcus pneumoniae (pneumococcus) is known for its competence for natural genetic transformation—the ability to uptake foreign DNA from the environment. Competence development is transient but can affect all cells throughout a culture under appropriate conditions. The high rate of pneumococcal transformation reported in literature prompted us to explore the upper limits of this mechanism, as a very high efficiency would provide a powerful tool for marker-less gene modification or other genetic manipulation in S. pneumoniae. To explore the efficiency of gene replacement under optimal conditions, we used synthetic pheromone peptide and a pheromone-deficient comA strain as recipient. Efficiency was estimated by observing the proportion of cells within a competent population that took up a gyrase gene marked by a novobiocin-resistance single-base mutation centered within an 8-kb donor PCR product. The fraction of cells with at least one gene copy replaced varied between 80 and 99%, confirming pervasive competence development. To determine how many copies of the recipient gene were replaced in these cells, we allowed the culture to grow and segregate for three or more generations after exposure to donor DNA. At low donor concentrations, an average of 1 out of every 5 genes was replaced in a transformed CFU. At saturating donor DNA levels (100-500 ng/ml DNA), 80-90% of gene copies were replaced in each transformed CFU, indicating an average transformation event could replace at least four ss-DNA gene copies in a single CFU. We conclude that under optimal conditions marker-less gene modification can be rapid and efficient when using large pure donor DNA gene donors.


Competitive adaptation of iron regulatory pathways during chronic Pseudomonas aeruginosa

infection of the cystic fibrosis lung Angela T. Nguyen1, Jace W. Jones1, Maureen A. Kane1, and Amanda G. Oglesby-Sherrouse1,2 1University of Maryland School of Pharmacy, Department of Pharmaceutical Sciences, Baltimore, MD 21201 2University of Maryland School of Medicine, Department of Microbiology and Immunology, Baltimore, MD 21201

Cystic fibrosis (CF) is a hereditary disease that affects more than 27,000 people in the United States and predisposes these individuals to chronic pulmonary infections. Early infection of the CF lung is polymicrobial and includes many bacterial species, amongst them Staphylococcus aureus, Burkholderia cepacia, Haemophilus influenzae, and Pseudomonas aeruginosa. As lung function declines, P. aeruginosa eventually becomes the predominant resident in the CF lung. Many factors likely contribute to P. aeruginosa’s ability to compete in this distinct niche. One such factor is the Pseudomonas Quinolone Signal (PQS), which is involved in several processes, including quorum sensing, lysis of other bacteria, and transport or capture of iron. In particular, PQS contributes to S. aureus lysis, potentially providing an iron source for P. aeruginosa in the CF lung. Previously, Oglesby et al. (2008), showed that the iron-regulated PrrF small RNAs are required for PQS production in low iron conditions. This occurs by repression of antA, which encodes an enzyme that degrades anthranilate for use as an energy source. Alternatively, anthranilate can serve as the precursor for PQS. Thus, PrrF repression of antA spares anthranilate for PQS production. PQS is one of many 4-hydroxy-alkylquinolines (HAQs) produced from anthranilate and secreted by P. aeruginosa. Here we show that iron mediates the production of several of these HAQs in laboratory strains and CF isolates, potentially via the PrrF sRNAs. Additionally, we show that PrrF regulation is altered over the course of infection in at least one longitudinally isolated CF strain. We hypothesize that changes in iron-regulated production of HAQs allows P. aeruginosa to maintain iron homeostasis throughout CF lung infection. Currently, we are testing this hypothesis by identifying iron-regulated HAQs using LC-MS/MS and analyzing PrrF-regulated gene expression in longitudinal CF isolates. Combined, these approaches will determine how iron-regulated production of HAQs adapts during CF lung infection.


The Effects of Increased Thiol levels on Methane-producing Archaeon Methanosarcina

acetivorans Alicia M. Ortiz1, Jennifer Catlett1 and Dr. Nicole R. Buan1 1University of Nebraska-Lincoln Department of Biochemistry Lincoln, NE, 68588-0664 Prokaryotes represent half of all biomass on Earth, and the majority of these organisms are strict anaerobes with poorly characterized physiology and metabolism. The methane-producing archaea, methanogens, are strictly anaerobic microorganisms that produce methane as a metabolic byproduct. Methane is a high-energy combustible fuel that comprises up to 90% of natural gas. Half of all methane on earth is produced by methanogens, while the other half is geologically produced. A clear understanding of methanogen gene expression is essential to engineer optimal strains for methane production in industrial settings. The genes MA3298 (comDE, sulfopyruvate decarboxylase) and MA3297 (thrC, threonine synthase) are involved in the biosynthesis of coenzyme M (CoM) in the methanogen, Methanosarcina acetivorans. These genes are essential because CoM is an obligate C1 methyl carrier in the methanogenesis pathway, and the availability of CoM determines the rate at which methane is produced. Our hypothesis is that if CoM biosynthesis is increased in the cell, the rate of substrate catabolism would also increase. Our preliminary data suggests overexpression of MA3297 and MA3298 does not affect the growth of wild-type cells, but surprisingly, rescues the growth

phenotype of a coenzyme M-coenzyme B heterodisulfide reductase deletion mutant (hdrABC), and may have other unexpected effects on methanogen physiology.


The role of ATP in regulated intramembrane proteolysis of Pro-σK by SpoIVFB during Bacillus

subtilis sporulation Daniel D. Parrell1 and Lee Kroos1,2 1Michigan State University Department Microbiology and Molecular Genetics East Lansing, MI 48824 2Michigan State University Department of Biochemistry and Molecular Biology East Lansing, MI 48824 An important event during Bacillus subtilis sporulation is the activation of the mother cell-specific sigma factor σK, through regulated intramembrane proteolysis by SpoIVFB. During sporulation SpoIVFB is held inactive in the membrane until a series of proteolytic signals relieve inhibition by the proteins SpoIVFA and BofA. Once active, SpoIVFB is able to cleave membrane-tethered Pro-σK and release active σK into the mother cell, promoting the final stages of gene expression that lead to a mature spore. Another potential regulatory feature of SpoIVFB is an ATP-binding cystathione-β-synthase (CBS) domain. To investigate the role of ATP binding to the CBS domain and to address the effects of ATP levels on SpoIVFB activity during sporulation, two paths of research are being pursued. First, mutational and biochemical analysis of ATP binding to the CBS domain is underway. Predicted ATP-binding residues are being substituted with alanine and the ability to cleave Pro-σK in vivo and bind ATP in vitro will be determined. Second, a method to measure the ATP concentration in the mother cell and in the forespore during sporulation is being developed. Relative ATP concentrations will be measured using a FRET-based sensor. Progress on these two aims will be reported. We hope to gain an understanding of how CBS domains can contribute to the regulation of intramembrane proteolysis, possibly linking cellular energy status to progression of sporulation, and we hope to develop the use of FRET-based ATP sensors in bacteria.


ROLE OF Mfd AND BASE EXCISION REPAIR IN PROCESSING OXIDATIVE-INDUCED DNA DAMAGE DURING Bacillus subtilis SPORULATION

Adriana G. Patlán-Vázquez1, Fernando H. Ramírez-Guadiana1, Ronald E. Yasbin2, Eduardo Robleto3 and Mario Pedraza-Reyes1 1Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, Guanajuato 36050,

Mexico; 2College of Arts and Sciences, University of Missouri –St.Louis, MO 303, USA;

3School of Life Sciences,

University of Nevada—Las Vegas, Las Vegas, Nevada 454004, USA.

The successful accomplishment of the sporulation program in Bacillus subtilis may potentially be endangered by spontaneous or induced chromosomal damage occurring in either sporangia compartment. In line with this concept, we recently showed that the nucleotide excision repair (NER) and transcription coupled repair (Mfd) pathways play fundamental roles in eliminating distorting DNA lesions induced by ultraviolet light and mitomycin-C that compromise B. subtilis sporulation1.

Interestingly, our studies have also revealed that the sole absence of Mfd affected sporulation suggesting that spontaneous DNA lesions occurring in sporulating cells could affect the expression of genes that are specifically required to successfully completing spore morphogenesis. We speculate that oxidative-induced DNA damage could be involved in this phenotype.

Non-distorting DNA lesions including 8-oxo-guanine (8-oxo-G) and uracil are processed by the base excision repair pathway (BER), which requires the strict participation of apurinic/apyrimidinic (AP) endonucleases and B. subtilis counts with different proteins with AP-endonuclease activity, including Nfo, ExoA and Nth to eliminate these genotoxic lesions2, 3.

To investigate whether repair of spontaneous DNA lesions by the BER pathway is performed with participation of Mfd a collection of B. subtilis strains deficient for nfo, exoA, nth and/or mfd was constructed. Our studies with these strains revealed that although the absence of Mfd made the sporulating cells of B. subtilis more sensitive to hydrogen peroxide, the deficiency of both, the BER system and Mfd increased much more this effect. Notably, sporangia of the nfo exoA nth mfd strain showed deficiencies in sporulation that were more severe than those presented by the single mfd mutant.

Based on these results we postulate that repair of non-bulky lesions that compromise the temporal expression of genes required for an efficient sporulation is carried out by the BER pathway, an event that is possibly directed by the transcription-coupling factor Mfd. References 1 Ramírez-Guadiana, F.H., Barajas-Ornelas, R. C., Ayala-García, V. M., Yasbin, R. E., Robleto, E., and Pedraza-Reyes, M. (2013)

Transcriptional coupling of DNA repair in sporulating Bacillus subtilis. Mol Microbiol 90: 1088–1099. 2 Friedberg, E. C., Walker, G. C., Siede, W., Wood, R. D., Schultz, R. A., Ellenberger, T. (2006) DNA Repair and Mutagenesis. 2nd ed. ASM

Press, Washington, DC, USA. 3 Barajas-Ornelas, R. C., Ramírez-Guadiana, F. H., Juárez-Godínez, R., Ayala-García, V. M., Robleto, E., Yasbin

R. E. and Pedraza-Reyes,

M. (2014) Error-prone-precessing of apurinic/apyrimidinic (AP) sities by PolX underlies a novel mechanism that promotes adaptive in Bacillus subtilis (paper submitted).

Work supported by CONACYT (grant 205744) and University of Guanajuato (grant DAIP-324-2013). A. G. P-V. and F. H. R-G were supported by a scholarship from CONACyT.


PURSUIT OF THE TRANSRIPTIONAL CONTROL MECHANISM OF VIRULENCE BY VIRB IN SHIGELLA FLEXNERI

Michael A. Picker1, Dustin J. Harrison2 and Helen J. Wing1

1University of Nevada, Las Vegas School of Life Science Las Vegas, NV 89154-4004 2Research Department, Naval Medical Research Unit 2 Pacific, Pearl Harbor, HI, United States Shigella flexneri is an intracellular pathogen that causes bacillary dysentery in humans. It harbors a large virulence plasmid which is home to many virulence genes that are up-regulated by the essential transcriptional regulator VirB. Regulation of the icsP promoter (PicsP) by VirB requires an inverted repeat located an atypical distance upstream of the transcription start site (over 1 kb), yet mechanistic details of this regulation remain poorly understood. Hence, my overall goal is to elucidate the mechanism by which Shigella virulence is controlled by VirB. VirB is proposed to have similar DNA binding properties with its closest homolog, the plasmid partitioning protein ParB, despite differing cellular functions. ParB has been shown to oligomerize along DNA and affect transcription, as well as cause changes in DNA supercoiling. The work presented focuses on the hypothesis that VirB can cause changes in DNA supercoiling. Preliminary observations of our VirB-dependent icsP promoter lacZ reporter plasmid display a VirB-dependent change in its supercoiled state, which depends on an inverted repeat essential for VirB-dependent regulation. Currently, the reason for these changes are not fully understood, but future experiments will dissect the importance of supercoiling in the mechanism of VirB-dependent regulation.


Infection with Salmonella Stimulates Macrophages to Engulf Erythrocytes M. Carolina Pilonieta1, Christopher English2 and Corrella S. Detweiler1 1University of Colorado at Boulder Molecular, Cellular, Developmental Biology Boulder, CO, 80309 2Intelligent Imaging Innovations, Inc. Denver, CO, 80216 ABSTRACT Humans and mice infected with the bacteria Salmonella enterica accumulate hemophagocytes, macrophages that are "blood eating" and have engulfed erythrocytes and leukocytes. In mice, S. Typhimurium (Salmonella) can reside within hemophagocytes containing leukocytes. Here we show that Salmonella also resides within hemophagocytes containing erythrocytes. However, the relationship between hemophagocytosis and infection with Salmonella is not well understood. In a cell culture model of erythrocyte hemophagocytosis using bone marrow derived macrophages (BMMs), relatively few BMMs appear to have the capacity to engulf erythrocytes. Examination of BMM-derived hemophagocytes on a single-cell basis using flow cytometry demonstrates that BMM exposure to live Salmonella stimulates erythrophagocytosis but not engulfment of inert beads. Neither heat-killed Salmonella nor LPS stimulate erythrophagocytosis. Within a sample, the infected, but not the uninfected, BMMs have increased erythrophagocytosis. Finally, cultured hemophagocytes contain more Salmonella than BMMs in the same sample that did not engulf erythrocytes. These observations indicate that Salmonella has the capacity to stimulate hemophagocytosis.


The Role of Mfd in Oxidative Damage Repair Kate Porter, Amanda Prisbrey, Carmen Vallin, and Eduardo A. Robleto University of Nevada Las Vegas School of Life Sciences Las Vegas, Nevada, 89119 Since the 1950’s it has been shown that bacterial cells can accumulate mutations even in non-dividing conditions. However, how this type of mutation occurs is still highly debated. This is an underestimated area of evolution because although cells spend most of their time in non-replicating conditions, mutagenesis is studied primarily during exponential phase. Recent evidence in B. subtilis suggests that transcription factor Mfd mediates the formation of mutations in stationary-phase or non-replicating cells by interacting with different repair systems. Mfd is a part of transcription coupled repair, a pathway that preferentially targets transcribed genes. Here we examine the hypothesis that Mfd mediates the formation of mutations by interacting with cellular components that repair oxidative damage. We test this hypothesis by determining whether Mfd affects cell viability after exposure to hydrogen peroxide in stationary phase. Our experiments showed the following: 1) Deficiencies in Mfd result in significant loss of cell viability after exposure to hydrogen peroxide and 2) the level of transcription in the cell modulates the effect on viability. These results are significant because they suggest that: i) oxidative damage is an intermediate in the formation of stationary-phase mutations and ii) Mfd has different roles in DNA repair and mutagenesis.


TRANSCRIPTIONAL COUPLING OF DNA REPAIR IN

SPORULATING Bacillus subtilis CELLS

Fernando H. Ramírez-Guadiana1, Eduardo Robleto2, Ronald Yasbin3 and Mario Pedraza-Reyes1

1Department of Biology, Division of Natural and Exact Sciences, University of Guanajuato, Guanajuato 36050, Mexico; 2College of Arts and Sciences, University of Missouri –St.Louis, MO 303, USA; 3School of Life Sciences, University of Nevada—Las Vegas, Las Vegas, Nevada 454004, USA.

During sporulation, Bacillus subtilis replication becomes limited; consequently, this bacterium

must face non-appropriate conditions to process DNA damage that may compromise the transcriptional program that drives this developmental process. We have investigated the role of Mfd during B. subtilis sporulation; Mfd mediates transcription-coupled repair (TCR) by displacing a stalled transcription elongation complex at bulky or non-coding lesion and recruits the nucleotide excision repair machinery (NER), via its interaction with the UvrA subunit to the lesion site.

We found that mfd and uvrA are expressed in both compartments of the sporangia during B. subtilis sporulation. Importantly, Mfd and UvrA conferred protection to sporangia from the genotoxic effects of ultraviolet C (UV-C) irradiation and mitomycin-C (M-C). In fact, the absence of both Mfd and UvrA made B. subtilis sporangia extremely sensitive to both agents compared to the single mutants lacking mfd or uvrA. In addition of this protective role, we also found that Mfd is required for an efficient sporulation.

In sporulating cells, the absence of either Mfd or UvrA promoted mutagenesis following UV-C exposure and such effect was enhanced in the double mfd uvrA mutant strain. These findings suggest that in the absence of TCR and NER, there is a pathway that processes in an error-prone manner the genetic damage generated by UV-C irradiation in B. subtilis sporulating cells. Interestingly, gene inactivation in both yqjH and yqjW dramatically suppressed the UV-C-induced mutation frequency in sporangia of the mfd knock-out strain. These observations support the notion that in conditions of high levels of UV-C damage, translesion synthesis (TLS) promotes mutagenesis because the NER and TCR pathways become insufficient in processing pyrimidine dimers (PDs) in B. subtilis sporangia. Under this situation, it is possible that YwjD, an UV-damage-endonuclease present in sporulating cells, by incising DNA immediately 5’ to PDs, activates an alternative repair mechanism that is completed by the error-prone polymerases YqjH and/or YqjW to promote mutagenesis, a possibility that is currently being investigated in our laboratory.

In conclusion, in sporulating cells whose metabolic needs are highly dependent on a timely program of gene expression, Mfd not only prevents mutagenesis in transcribed genes involved in this differentiation program but also contributes to sporulation efficiency and spore survival.

Work supported by CONACYT (grant 205744) and University of Guanajuato (grant DAIP-324-2013). F. H. R-G was supported by a scholarship from CONACyT.


THE SOS RESPONSE IS REQUIRED FOR PROCESSING OF DNA DAMAGE DURING Bacillus subtilis SPORULATION

Fernando H. Ramírez-Guadiana1 and Mario Pedraza-Reyes1

1 University of Guanajuato, Department of Biology. Guanajuato, Gto., México. CP. 36050. Spontaneous or induced chromosomal damage occurring in either compartment of the sporangium may potentially endanger the successful accomplishment of Bacillus subtilis sporulation. Thus, it can be expected that sporulating cells may have developed mechanisms to detect, repair and even tolerate DNA damage in order to accurately complete their sporulation program. In line with these concepts, we have demonstrated that sporulating cells deploy distinct routes of DNA repair to process lesions that may potentially affect sporulation. These mechanisms include the nucleotide excision repair (NER) pathway that processes DNA-distorting or –crosslinking lesions produced by ultraviolet irradiation (UV-C) and mitomycin-C (M-C), respectively. Importantly, we have shown that the efficiency of the NER system is enhanced by the transcription-coupling repair (TCR) factor, Mfd, during B. subtilis sporulation. However, when high levels of genetic damage overwhelm the capacity of the general NER and TCR-dependent pathways, it is possible that processing of DNA lesions can be carried out through the UV-endonuclease, YwjD. This alternative excision repair pathway could be completed by the error-prone polymerases YqjH and/or YqjW, which also operate during sporulation.

Since some of these repair proteins belong to the SOS regulon and RecA is the master transcriptional activator of this response, in this work, we investigated if the SOS response is active during sporulation and the role played by RecA in providing resistance to sporangia against different DNA damaging factors.

Using a reporter strain containing a translational PrecA-gfpmut3a fusion to monitor the SOS response, we found that expression of the recA-gfp construct was elicited by DNA damage in sporulating cells of B. subtilis, as evidenced by the synthesis of the GFPmut3a protein in both compartments of sporangia after UV-C exposure.

Interestingly, the genetic disruption of recA delayed sporulation for ~2h, as shown by comparing the expression pattern of spoVFA (a gene which is exclusively expressed during B. subtilis sporulation) during this developmental process in the wild-type (WT) and ΔrecA genetic contexts. Finally, we found that sporangia deficient for RecA were more susceptible to UV-C irradiation and M-C treatments, as compared to the WT strain.

In summary, our results support the notion that RecA plays an important role during sporulation activating the SOS response to provide B. subtilis sporangia with the DNA repair machinery required to eliminate DNA lesions that compromise completion of this developmental process.

Work supported by CONACYT (grant 205744) and University of Guanajuato (grant DAIP-324-2013). F. H. R-G was supported by a scholarship from CONACyT.


A Microscopy-Based Screen to Identify Compounds That Disrupt Salmonella Host-Pathogen

Interactions Abigail Reens1, Raphael Urbani2, Dirk Bumann2, Corrella S. Detweiler1

1University of Colorado – Boulder Department of Molecular, Cellular & Developmental Biology Boulder, Colorado, 80309 2Biozentrum University of Basel Basel, Switzerland Antibiotics must satisfy a range of conditions in order to be effective in the clinic, including high potency, low toxicity, and correct localization. Many traditional screening strategies identify “hit” compounds based on a limited subset of characteristics, such as potency against the molecular target. As a result, the vast majority of compounds identified as hits through standard screens lack important characteristics and will not succeed in the clinic, despite extensive optimization. For example, a compound may be highly effective against bacteria grown in broth, but cannot gain access to intracellular bacteria due to integral chemical properties. We are attempting to identify new antibiotics by screening compounds in a system that incorporates the key hurdles an antibiotic must overcome. We have developed a medium-throughput assay to identify compounds with activity against a model Gram-negative bacteria, Salmonella enterica serovar Typhimurium, which causes a typhoid fever-like disease in mice and survives inside host macrophages. Following the work of Lieberman & Higgins (2009), we are using an automated microscopy approach to quantify bacterial load inside cultured macrophages after compound treatment, enabling identification of compounds with activity against intracellular bacteria. In addition, the method enables analysis of host cell viability after compound treatment, allowing identification and rejection of host-toxic compounds. We have validated this approach using a variety of antibiotics and toxic chemicals, and we are currently screening a library of drug-like compounds. We expect to identify a small number of hits with large potential to be developed into antibiotics that kill or inhibit intracellular bacteria with minimal host cell toxicity. We also anticipate identifying compounds that target the host macrophage to disrupt host-pathogen interactions. Reference: Lieberman LA, Higgins DA (2009) A small-molecule screen identifies the antipsychotic drug Pimozide as an inhibitor of Listeria monocytogenes infection. Antimicrob Ag Chemother 53:756-64.


FeuPQ is a potential regulator of the expression of ftrABCD, the genes encoding a ferrous iron specific transporter in Brucella abortus 2308, in response to acidic

pH Ahmed E. Elhassanny1 and R. Martin Roop II1

1Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, North Carolina 27834

Recently, we identified a Fe2+ transporter, FtrABCD, which is essential for the virulence

of Brucella abortus 2308 in mice. Expression of the ftrABCD operon is induced in response to low-iron conditions, and this response is mediated by Irr, the predominant iron-responsive

regulator in Brucella and the other -proteobacteria. The ftr locus is also induced by exposure to acidic pH in both the parental 2308 strain and an isogenic irr mutant, indicating that the iron- and pH-specific responses of these genes are independently regulated. We believe that the acid-responsive expression of ftrABCD is important because it potentially allows the brucellae to regulate the expression of their iron acquisition genes to adapt to the acidic environment they encounter in the endolysosomal Brucella-containing vesicles (eBCVs), where Fe2+ is thought to be a biologically relevant iron source. The Brucella two-component regulator FeuPQ shares significant amino acid homology with the recently-described Pseudomonas BqsRS, which senses extracellular Fe2+ and regulates the expression of a number of genes including itself. Since acidic pH favors the stability and solubility of ferrous iron, it is possible that the expression of the Brucella ftrABCD operon is induced in response to extracellular Fe2+ instead of acidic pH per se. An isogenic feuPQ mutant derived from B. abortus 2308 displays a prominent growth defect under iron-deprived conditions at acidic pH, and this mutant displays significantly reduced ftr expression in response to acidic pH under iron-replete growth conditions. Studies are underway to define the specific role that FeuPQ plays in the acid-responsive expression of the ftrABCD operon in B. abortus 2308, and more importantly, what role that this mode of regulation plays in the virulence of this strain.


In Brucella abortus, Irr is the main iron-responsive transcriptional regulator and its activity is controlled by cellular iron levels, the enzymatic activity of ferrochelatase and an internal heme

binding motif David A. Martinson1 & Marty Roop1 1Brody School of Medicine, East Carolina University Department of Microbiology and Immunology Greenville, NC 27834 As an intracellular pathogen, B. abortus must overcome iron sequestration in the host cell by utilizing highly efficient iron transport systems. These systems must be tightly regulated, however, as excess intracellular iron is toxic to the bacterial cells. Most of the alpha-proteobacteria rely on a transcriptional regulator known as the iron response regulator (Irr) to control the expression of their iron metabolism genes. In these bacteria, Irr serves as an activator of genes involved in iron acquisition and a repressor of genes encoding for products that require high levels of iron for their function or serve as iron storage proteins. An isogenic B. abortus irr mutant produces significantly less siderophore when grown under iron limiting conditions compared to the parent strain. The irr mutant is also significantly less sensitive to the iron requiring antibiotic streptonigrin and exhibits a slower rate of radioactive iron uptake than the parent strain, indicating that the irr mutant is unable to efficiently internalize iron. Microarray analysis and real-time RT PCR have been used to show that essentially all of the known genes encoding for iron uptake systems are mis-regulated in the irr mutant strain, along with the genes encoding for cytochrome biosynthesis proteins and iron storage proteins. We have also shown that Irr directly binds to the promoter regions of these genes. The iron responsive activity of the B. abortus Irr protein is unique, in that when intracellular iron levels are high, Irr is degraded and it can no longer function as a transcriptional regulator. The iron responsive degradation of Irr is dependent on the enzymatic activity of ferrochelatase, the enzyme that catalyzes the last step in the biosynthesis of heme by incorporating iron into a protoporphyrin IX skeleton. We have experimentally determined that an internal HXH heme binding motif that is highly conserved among the alpha-proteobacteria is required for iron dependent degradation of Irr in B. abortus. We are presently exploring the mechanism behind the HXH and ferrochelatase-dependent iron responsive degradation of Irr in B. abortus in an effort to better understand how Irr coordinates the expression of iron metabolism genes.


The Disruption of Prenylation in Staphylococcus aureus Leads to Pleiotropic Rearrangements in Cellular Behavior and Virulence

Christina N. Krute1, Ronan K. Carroll1, Frances E. Rivera1, Ryan M. Young2, Mohsen Botlani-Esfahani1, Sameer Varma1, Bill J. Baker2 and Lindsey N. Shaw1 1Department of Cell Biology, Microbiology & Molecular Biology; and 2Department of Chemistry, University of South Florida, Tampa, FL, 33620, USA. Molecular regulation is a complex and multi-tiered process that proceeds via the control of gene expression (transcription factors), translation (sRNAs), and the functionality of newly synthesized proteins (post-translation modifications, PTMs). In bacteria, PTMs such as proteolysis and phosphorylation, and to lesser extents acetylation, lipidation and glycosylation, have thus far been explored. Interestingly, our group has noted that the machinery for an additional PTM, prenylation, which is well studied in eukaryotes, is also highly conserved in bacteria. It is believed that a typical eukaryotic cell has hundreds of prenylated proteins, which have been shown to serve major roles in cell signaling and development, and the progression of diseases such as cancer and Alzheimer’s. To explore the impact of prenylation in bacteria, we inactivated the geranyltranstransferase (IspA) of S. aureus, which synthesizes farnesyl pyrophosphates: a central initiating step for prenylation within cells. Strikingly, the ispA mutant displayed a novel small colony variant (SCV)-like phenotype, with reduced cell size and a complete lack of characteristic golden pigment. Transcriptomic analysis via RNA-seq revealed a global alteration in expression of >700 genes, involved in a wealth of key cellular processes. Further exploration of this data lead to the observation that ispA mutants are highly sensitive to numerous oxidative stressors and a range of cell wall-targeting antibiotics, whilst at the same time display marked resistance to cationic aminoglycosides and antimicrobial peptides. These findings appear to be due, at least in part, to differences in envelope composition, as we observed increase membrane fluidity in the ispA mutants, alongside marked alterations in fatty acid composition. Each of these observations manifests in a highly irregular and diffuse cell envelope, as determined by electron microscopy. In the context of pathogenesis, unlike other SCVs, which develop a persister-like phenotype, leading to chronic colonization, the ispA mutant of S. aureus was highly attenuated in organ dissemination using a murine model of sepsis. A bioinformatic investigation of the C-terminal CAAX-motif targeted by eukaryotic prenylation machinery identified only 2 such proteins in the S. aureus genome, which clearly do not explain the extensive phenotypic alterations observed in our ispA mutant. Accordingly, protein prenylation in prokaryotic cells seemingly proceeds via a novel mechanism that is distinct from eukaryotes, which our preliminary data appears to supports. As such, we suggest that prenylation is a highly important and largely overlooked process in bacteria that influences a wealth of biological functions central to cellular physiology and homeostasis; and, in the context of pathogenic organisms, the ability to establish and maintain infection.


The RelBE Toxin–Antitoxin family and Salmonella enterica Persistence. Eugenia Silva-Herzog, Erin M. McDonald , Corella Detweiler University of Colorado Boulder Department of Molecular, Cellular & Developmental Biology Boulder, CO, 80309 Persister cells are considered the basis of bacterial chronic infections caused by many bacterial pathogens. Persisters are phenotypic variants characterized by low metabolic activity and slow or no-replication. A key characteristic of persister cells is tolerance to multiple antibiotics. Among the mechanisms that establish a persister state is the induction of Toxin-Antitoxin systems. Toxins target essential metabolic functions, such as translation, transcription, DNA replication, or ATP synthesis, thereby reducing the metabolic state of the cell and providing protection from antibiotics or the immune response. Using bioinformatics tools, we identified 25 Toxin and Antitoxin genes in Salmonella enterica Typhimurium. However, only six TA pairs are conserved in S. enterica serotypes that are able to cause systemic infection in mammals. Four are from the RelBE family, one is in the VapBC family and one is in the HigAB family. In this study we show that ectopic expression of the toxin RelE results in growth arrest that is relieved by co-expression of the RelB antitoxin, thus demonstrating that RelBE is a Toxin – Antitoxin pair. Furthermore, ectopic expression of RelE , but not RelB increases the number of persister cells in response to antibiotics as well as to physiological stresses. These data suggest a link between the Toxin RelE and the formation or maintenance of the persistent state. The effect of Toxin-Antitoxin system expression during infection of Macrophages is being analyzed. Modulation of growth and metabolic state by Toxin-Antitoxin systems may be a way of bacteria to survive under adverse environmental conditions.


Extremely radiation resistant spore producing Bacillus from spacecraft assembly facility harbor novel genes and gene arrangement.

Madhan R Tirumalai1, George E Fox1 1University of Houston Department of Biology and Biochemistry Houston, Texas, 77204-5001 Bacterial endospores displaying hyper-resistance to ionizing radiation and peroxide treatments have been repeatedly isolated from the oligotrophic environments of Spacecraft Assembly Facilities (SAF). Understanding the causes and mechanisms of the resistance(s) is critical to planetary protection concerns, as well as, our search for potential life elsewhere. The genomes of two such endospore forming SAF contaminants, namely Bacillus pumilus SAFR-032 and B. safensis FO-36b, were compared with the very closely related type strain B. pumilus ATCC7061T that does not produce resistant spores. Forty SAFR-032 characteristic genes are entirely unique open reading frames, while, four genes are unique to the genomes of the resistant SAFR-032 and FO-36b, which may be responsible for the elevated resistances. Fifty three genes involved in spore coat formation, regulation and germination, DNA repair, and peroxide resistance, are missing from all three genomes. Furthermore, the comparative analysis revealed an extended region of nonhomologous genes, comprising an ICEBs1-like integrative conjugative element in SAFR-032. As compared to the ICEBs1 of B. subtilis, the SAFR-032 element shows extremely rapid flux in gene content, genome organization and sequence similarity. Some of the SAFR-032 unique genes are part of this element and hence these genes may be responsible for the rapid evolution that led to the extreme radiation and desiccation resistance of this organism’s spores.


pNEB193-derived plasmids for gene deletion and protein expression in the methane-producing archaeum, Methanosarcina acetivorans

Mary E. Walter, Mitchell T. Shea, Nikolas Duszenko, Anne-Lise Ducluzeau, Jared Aldridge, Shannon K. King, and Nicole R. Buan University of Nebraska-Lincoln Department of Biochemistry, Redox Biology Center Lincoln, NE 68588 Abstract Understanding metabolism and gene regulation in methane-producing archaea (methanogens) requires improved genetic methods for protein expression and purification. Methanogens use unique coenzymes and cofactors such as: coenzyme M, coenzyme B, methanopterins, methanophenazine, dimethylbenzimidazolyl cobamide, and deazaflavin F420. This makes expressing some proteins difficult or impossible in heterologous hosts that do not produce these cofactors. It is also necessary to purify protein from the native host in order to study post-translational modifications and their effect on gene expression or enzyme activity. We have created several new plasmids to complement existing genetic tools for use in the methanogen, Methanosarcina acetivorans. The commercially available plasmid, pNEB193, provided the foundation for creating our new plasmids. The pNEB193-derived plasmids can be propagated as small, high-copy plasmids in E. coli, can be transformed into standard E. coli host strains, and have ampicillin resistance cassettes for selection. Markerless deletion of the prenyl reductase gene (MA4421) from M. acetivorans chromosome proved the functionality of

the pNB723 plasmid, similarly the expression of tagged and untagged -glucuronidase (uidA) gene confirmed the pNB730 plasmid. The pNEB193-derived plasmids we have developed facilitate markerless gene deletion, gene transcription, protein expression, and purification of proteins with cleavable affinity tags from M. acetivorans.


Dual-regulation of shuT, a gene within the Shigella dysenteriae heme uptake locus

Yahan Wei1, Andrew B. Kouse1, Erin R. Murphy2 1Department of Biological Sciences, Ohio University, Athens, OH, 45701 2Department of Biomedical Sciences, Ohio University, Athens, OH, 45701 Shigella is a genus of gram-negative bacteria whose infection causes shigellosis, a form of severe dysentery in humans. Each year, Shigella is estimated to cause approximately 90 million infections and 0.7 million deaths globally. Survival of Shigella in the host, and thus its ability to cause disease, is dependent on the availability of the pathogen to acquire essential nutrients, such as iron. As a non-specific host defense, the concentration of bioavailable iron within the human body is maintained at a concentration that is too low to support the growth of bacterial pathogens, including Shigella. The low concentration of available iron within the human body is achieved, in part, by sequestration of the metal within iron-binding and iron-storage proteins. Remarkably, nearly 95% of all iron present within the human body is sequestered within heme, making heme a potently rich source of nutrient iron for invading pathogens. S. dysenteriae is able to utilize heme as a source of nutrient iron and this process is dependent upon the Shigella heme uptake (Shu) system. The Shu system is encoded by eight highly regulated genes contained within a single genetic locus. Our current research is focused on understanding the complex regulation controlling the expression of shuT, a gene encoding the periplasmic heme binding protein of the Shu system. Real-time PCR and Western blot analysis were used to study shuT regulation, allowing the comparison of shuT mRNA and ShuT protein levels in relation to environmental changes. Results obtained indicated that shuT is transcription is regulated in response to iron availability by the transcriptional repressor Fur and that shuT translation is modulated in response to temperature. To determine the mechanism(s) of temperature-dependent post-transcriptional regulation of shuT expression, the promoter and 5’ untranslated region of the gene was investigated. Rapid Amplification of cDNA ends (RACE) and reverse-transcriptase PCR were used to locate the shuT promoter region. These studies suggested that there are two active shuT promoters, an unexpected result that was confirmed by beta-galactosidase assays using transcriptional reporter plasmids each carrying a putative shuT promoter region fused to a promoter-less lacZ reporter gene. Studies are ongoing to determine details of the molecular mechanisms underlying the iron- and temperature-dependent regulation of shuT. Given the significance of iron acquisition to the survival of Shigella and the abundance of iron-bound heme in the human host, knowledge of the regulation of shu genes may provide valuable groundwork for the development of therapeutic agents to disrupt heme iron acquisition and by doing so eliminate or limit the ability of Shigella to cause disease.


List of participants Victor Ayala-Garcia Guanajuato Kris Blair Fred Hutchinson Cancer Research Institute [email protected] William Broach Ohio University [email protected] Nicole Buan University of Nebraska [email protected] James Budnick Virginia Tech [email protected] Rebecca Calvo Indiana University [email protected] Ronan Carroll University of South Florida [email protected] Karla Castro-Cerritos Guanajuato Clayton Caswell Virginia Tech [email protected] Nadja Chech University of North Carolina [email protected] Heidi Crosby University of Iowa [email protected]

Corrie Detweiler University of Colorado Boulder [email protected] Leticia Djapgne Guanajuato Louise Djapgne University of Maryland [email protected] Anne Dunn University of Oklahoma [email protected] Amanda Dunson Ohio University [email protected] Donald Ennis University of Louisiana [email protected] Nathan Feirer Indiana University [email protected] Jake Flood University of Colorado Boulder [email protected] Elizabeth Fozo University of Tennessee [email protected] Megan Fris Ohio University [email protected] David Fujimoto Monserate Biotechnology Group [email protected] Barbara Funnel University of Toronto [email protected]

Ferran Garcia-Pichel Arizona State University [email protected] Jeffery Gardner University of Maryland [email protected] Michael Gilmore Harvard Medical School [email protected]

Daren Ginete University of Nevada Las Vegas [email protected] Eugenia Herzog University of Colorado Boulder [email protected]

Alexander Horswill University of Iowa [email protected] David Hufnagel University of Michigan [email protected] Andrew Kouse Ohio University [email protected] Junitar Kurasz University of Nevada Las Vegas [email protected] Jeremy LaBarge Dupont [email protected] Kevin Lang University of Minnesota [email protected] Aileen Lee University of Nebraska [email protected]


Ethan Mann Sharklet [email protected] Devon Marking University of South Florida [email protected] Rhea May Sharklet [email protected] Kimberly McCullor University of Oklahoma [email protected] Erin McDonald University of Colorado [email protected] Michael McShan University of Oklahoma Health Science [email protected] Rajeev Misra Arizona State University [email protected] Bitan Mohari Indiana University [email protected] Charles Moran Emory University [email protected] Donald Morrison University of Illinois [email protected] Erin Murphy Ohio University [email protected]

Toni Nagy University of Colorado [email protected] Angela Nguyen University of Maryland [email protected] Scott Nguyen University of Oklahoma [email protected] Amanda Oglesby-Sherrouse University of Maryland Baltimore aoglesbycrx.umaryland.edu Alicia Ortiz University of Nebraska [email protected] Alexandria Paharik University of Iowa [email protected] Daniel Parrell Michigan State University [email protected] Adriana Patlan-Vazquez Guanajuato David Payne University of Michigan [email protected] Mario Pedraza-Reyes Guanajuato Michael Picker University of Nevada Las Vegas [email protected] Carolina Pilonieta University of Colorado

Amanda Prisbrey University of Nevada Las Vegas [email protected] Benjamin Pursley Michigan State University [email protected] Fernando Ramirez-Guadiana Guanajuato Abigail Reens University of Colorado [email protected] Alexandria Reinhart University of Maryland [email protected] German Rivera University of Nevada Las Vegas [email protected] Ashley Roarty Indiana University [email protected] Eduardo Robleto Biology UNLV [email protected] Marty Roop East Carolina University [email protected] John Ruedas Monserate Biotechnology Group [email protected]

[email protected]


Leah Schwiesow University of California SC [email protected] Lindsey Shaw University of South Florida [email protected] Lauren Sheehan Virginia Tech [email protected] Gisela Storz National Institutes of Health [email protected] Uldis Streips University of Louisville unstre01@lousiville Adnam Syed University of Michigan [email protected] Timothy Tapscott University of Colorado [email protected] Madhan Tirumalai University of Houston [email protected] Yanina Tovpeko University of Illinois [email protected] Carmen Vallin University of Nevada Las Vegas [email protected] Sriram Varahan University of Kansas [email protected]

Mary Walter University of Nebraska [email protected] Christopher Waters Michigan State University [email protected] Natasha Weatherspoon-Griffin University of Nevada Las Vegas [email protected] Yahan Wei Ohio University [email protected] Andy Weiss University of South Florida [email protected] Daniel Wozniak The Ohio State University [email protected] Binjie Xu The Ohio State University [email protected] Naomi Ward University of Wyoming [email protected]

mailto:[email protected]


Notes


notes


Notes

th Annual Wind River Conference on Prokaryotic Biologywriver.sites.unlv.edu/58th_WR_Complete_Prog_5.pdfAmerican Society for Microbiology UNLV, School of Life Sciences College of Arts

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