Final Program and Abstracts

5th ASM Conference on Cell-Cell in

October 18 – 21, 2014 San Antonio, Texas © 2014 American Society for 1752 N Street, N.W. Washington, DC 20036-2904 Phone: 202-737-3600 World Wide Web: www.asm.org

All Rights Reserved Printed in the United States of America Table of Contents

ASM Conferences Information...... 2

Conference Organization...... 3

Acknowledgments...... 3

General Information...... 4

Travel Grants...... 5

Scientific Program...... 7

Speaker Abstracts...... 14

Poster Abstracts...... 35

Index...... 99

5th ASM Conference on Cell-Cell Communication in Bacteria 1 ASM Conferences Committee

Sean Whelan, Chair Petra Levin Harvard Medical School Washington University in Saint Louis

Victor DiRita Gary Procop University of Michigan Cleveland Clinic

Joanna Goldberg* Curtis Suttle Emory University University of British Columbia

Lora Hooper Theodore White University of Texas Southwestern University of Missouri – Kansas City Medical Center

*Indicates Committee Liaison for this Conference

ASM Conferences Mission

To identify emerging or underrepresented topics of broad scientific significance.

To facilitate interactive exchange in meetings of 100 to 500 people.

To encourage student and postdoctoral participation.

To recruit individuals in disciplines not already involved in ASM to ASM membership.

To foster interdisciplinary and international exchange and collaboration with other scientific organizations.

2 ASM Conferences Program Committee

Program Chairs Program Committee Marvin Whiteley; Co-chair, University Caroline Harwood; University of of Texas at Austin, Austin, TX Washington, Seattle, WA Eric Stabb; Co-chair, University of Steve Diggle; University of Nottingham, Georgia-Athens, Athens, GA Nottingham, United Kingdom Michael Federle; University of Illinois at Chicago, Chicago, IL George Salmond; University of Cambridge, Cambridge, England Karine Gibbs; Harvard University, Cambridge, MA Kendra Rumbaugh; Texas Tech Health Sciences Center, Odessa, TX

Acknowledgments

he Conference Program Committee and the American Society for Microbiology Tacknowledge the following for their support of the 5th ASM Conference on Cell- Cell Communication in Bacteria. On behalf of our leadership and members, we thank them for their financial support:

Platinum Patrons Gold Patrons Burroughs Wellcome Fund NIAID, National Institutes of Health* Gordon and Betty Moore Foundation National Science Foundation

Silver Patron Bronze Patron Society for General Microbiology Cystic Fibrosis Foundation

* Research reported in this publication was supported by the National Institute of Allergy And Infectious of the National Institutes of Health under Award Number R13AI114226. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

5th ASM Conference on Cell-Cell Communication in Bacteria 3 General Information

REGISTRATION AND NAME BADGES posters (2,4,6…) will be officially ASM Staff will be available at the presented in Session B on Monday, registration desk in the Grand Ballroom October 20. Foyer on the 3rd Floor at the San Antonio Marriott Rivercenter during Please check your assigned number in the posted registration hours. Participants abstract index. The same number is used may collect name badges and program for the presentation and board number. materials at the registration desk. A NETWORKING MEALS AND Social name badge is required for entry into all Events sessions and meals. Each participant may register one guest to attend the welcome Registration includes attendance at reception. Guest tickets are $50. Guests the Welcome Reception on Saturday, may not attend sessions, poster sessions, Networking Lunches on Sunday and lunches or coffee breaks. Monday, and Poster Session Mixers on Sunday and Monday. Ample time has GENERAL SESSIONS also been scheduled for participants to All general sessions will be held in the network during coffee breaks. Salon E of the Grand Ballroom at the San certificate of attendance Antonio Marriott Rivercenter. Certificates of Attendance can be found POSTER SESSIONS in the registration packet received at the Poster boards are located in the Grand registration desk. Ballroom at the San Antonio Marriott Note: Certificates of Attendance do not Rivercenter. Posters will be available list session information. for viewing informally throughout the CAMERAS AND RECORDINGS conference, with official poster sessions POLICY scheduled on Sunday and Monday. Audio/video recorders and cameras All posters should be mounted on Sunday are not allowed in session rooms or in morning, October 19, between 7:30 and the exhibit and poster areas. Taking photographs with any device is prohibited. 8:30 am, and should be removed by 1:00 pm on Tuesday, October 21. Posters CHILD POLICY which are not removed in time will be discarded. Children are not permitted in session rooms, poster sessions, conference meals Odd-numbered posters (1,3,5…) will or social events. Please contact the hotel be officially presented in Session A on concierge to arrange for babysitting Sunday, October 19, and even-numbered services in your hotel room.

4 ASM Conferences Travel Grants

ASM TRAVEL GRANTS ASM encourages the participation of graduate students and new postdocs at ASM Conferences. To support the cost of attending the conference, ASM has awarded travel grants of $500 to each of the following individuals:

Kyle Asfahl Bassam Elgamoudi Rikky Rai Heiko Babel Eran Even-Tov Rebecca Scholz Pengbo Cao Jake Everett Joe Sexton Brinta Chakraborty James Gurney Pratik Shah Angel Cueva Aiqun Jia Jiayue Yang Arup Dey Zohra Kajee Liqin Zhou Omar El-Halfawy Sabina Leanti La Rosa

NIH TRAVEL GRANTS The NIAID – NIH has provided a conference grant to ASM to support travel of junior investigators, postdoctoral fellows and students. The following individuals have received travel grants administered by ASM which are supported by NIH funds:

Gilles Brackman Beth Lazazzera Julie Valastyan Daniel Cornforth Charlotte Michelsen Karina Xavier Michael Federle Philip Rather Maria Hadjifrangiskou Lynne Turnbull

5th ASM Conference on Cell-Cell Communication in Bacteria 5 Travel Grants

NSF TRAVEL GRANTS The National Science Foundation has provided a conference grant to ASM to support travel of junior investigators, postdoctoral fellows and students. The following individuals have received travel grants administered by ASM which are supported by NSF funds:

Arpan Bandyopadhyay Jodi Connell Alice Min Ishay Ben-Zion Sylvie Estrela Matthew Powers Arunima Bhattacharjee Karine Gibbs Amy Schaefer Ilka Bischofs Lisa Hawver Beth Shank Sarah Jung Burkhard Hense Patricia Silva Allie Clinton Lucy McCully

SGM TRAVEL GRANTS The Society for General Microbiology (SGM) has sponsored travel grants for the following individuals to support their participation in the conference:

Luke McNally Roman Popat Aled Roberts Rita Monson

6 ASM Conferences Scientific Program

Saturday, October 18, 2014

6:00 – 7:30 pm Opening Session Grand Ballroom, Salon E

6:00 – 6:15 pm Welcome Remarks Marvin Whiteley, Eric Stabb, Joanna Goldberg

6:15 – 6:30 pm Tribute to Woody Hastings Pete Greenberg, University of Washington, Seattle, WA

6:30 – 7:30 pm Keynote Lecture Bonnie Bassler, Princeton University, Princeton, NJ

7:30 – 9:00 pm Session 1: Signal Generation and Perception Grand Ballroom, Salon E

7:30 – 8:00 pm Title to be announced Steve Winans, Cornell University, Ithaca, NY

8:00 – 8:20 pm Screening and Mutual Information Theory Reveal Single Nucleotides in Qrr4 Important for LuxR Repression Julie Valastyan, Princeton University, Princeton, NJ

8:20 – 8:40 pm Phase Variation Controls Quorum Sensing in baumanii Philip Rather, Emory University, Atlanta, GA

8:40 – 9:00 pm Quorum Sensing Control of a Lantibiotic Cluster in Streptococcus pneumonia Beth Lazazzera, University of California, Los Angeles, CA

9:00 – 10:00 pm Welcome Reception Sazo’s Latin Grill

5th ASM Conference on Cell-Cell Communication in Bacteria 7 Scientific Program

Sunday, October 19, 2014

8:30 – 11:40 am Session 2: Interference of Signaling Grand Ballroom, Salon E

8:30 – 9:00 am Title to be announced Michael Givskov, University of Copenhagen, Copenhagen, DENMARK

9:00 – 9:20 am How Does the Quorum Sensing Inhibitor Hamamelitannin Increase Staphylococcus aureus Susceptibility Towards Glycopeptides Gilles Brackman, Ghent University, Gent, BELGIUM

9:20 – 9:40 am Growth Substrate Topography Switches an Interspecies Signaling Pathway in Co-Culture Arunima Bhattacharjee, University of California, Irvine, Irvine, CA

9:40 – 10:00 am Combinatorial Quorum Sensing Can Allow Bacteria to Resolve Their Social and Physical Environment Daniel Cornforth, The University of Texas at Austin, Austin, TX

10:00 – 10:30 am Coffee break Grand Ballroom, Salons A & B

10:30 – 11:00 am Synthetic Ligands for the Interception of Bacterial Communication Helen Blackwell, University of Wisconsin, Madison, WI

11:00 – 11:20 am Cell-Cell Communication Among the Streptococci: Rgg , Inhibitors and Regulated Behaviors Michael Federle, University of Illinois at Chicago, Chicago, IL

11:20 – 11:40 am Investigating the Quorum Quenching Ability of Bacteria from Gastrointestinal Tracts of Cultured Ornamental Freshwater- Fish and Marine Farmed Fish Zohra Kajee, University of KwaZulu-Natal, Durban, SOUTH AFRICA

8 ASM Conferences Scientific Program

12:00 – 1:30 pm Lunch Grand Ballroom, Salon M

1:30 – 4:20 pm Session 3: Bacterial Development and Signaling Grand Ballroom, Salon E

1:30 – 2:00 pm Cell-cell Recognition by the Polymorphic Myxobacterial TraA Leads to Beneficial and Adversarial Interactions Dan Wall, University of Wyoming, Laramie, WY

2:00 – 2:20 pm Intercellular Signalling Using Extracellular DNA Facilitates Active Biofilm Expansion Lynne Turnbull, University of Technology Sydney, Sydney, AUSTRALIA

2:20 – 2:40 pm Characterization of Quorum Sensing-Regulated Gas Vesicle Morphogenesis in Enterobacteria Rita Monson, University of Cambridge, Cambridge, UNITED KINGDOM

2:40 – 3:10 pm Coffee break Grand Ballroom, Salons A & B

3:10 – 3:40 pm Jekyll and Hyde: Microbial Metabolites that Both Kill and Stimulate Biofilm Formation Beth Shank, University of North Carolina, Chapel Hill, NC

3:40 – 4:00 pm Kin-Specific Binding Between Two Proteins Correlates with in vivo Self Identity and Wild-Type Expansion in Proteus mirabilis Karine Gibbs, Harvard University, Cambridge, MA

4:00 – 4:20 pm Staphylococcus aureus Enhance Growth and Tolerance in a Human Host Adapted Lineage Charlotte Michelsen, Technical University of Denmark, Kgs. Lyngby, DENMARK

4:30 – 6:30 pm Poster Session A and Networking Mixer Grand Ballroom, Odd-numbered posters will be formally presented Salons A & B

5th ASM Conference on Cell-Cell Communication in Bacteria 9 Scientific Program

Monday, October 20, 2014

8:30 – 11:40 am Session 4: Host-Pathogen Interactions and Signaling Grand Ballroom, Salon E

8:30 – 9:00 am The Curious Case of Death by Seduction: How the Siren Song of Commensal E. faecalis Drove VRE to Suicide Michael Gilmore, Harvard Medical School, Boston, MA

9:00 – 9:20 am Based Biosensors for the Detection of Peptide Production and Cell-Cell Communication in Enterococcus faecalis Sabina Leanti La Rosa, Norwegian University of Life Sciences, Aas, NORWAY

9:20 – 9:40 am Understanding the Role of Diffusible Signalling Factor (DSF) in of Xanthomonas Plant Pathogens Rikky Rai, CDFD, Hyderabad, INDIA

9:40 – 10:00 am Integration of Multiple Sensory Inputs in the cholerae Quorum-Sensing Circuit and the Impact on Virulence and System Robustness Sarah Jung, Tufts University, Boston, MA

10:00 – 10:30 am Coffee break Grand Ballroom, Salons A & B

10:30 – 11:00 am Unravelling the Interplay Between Yersinia and Caenorhabditis elegans during Quorum Sensing-dependent in vivo Biofilm Formation Paul Williams, University of Nottingham, Nottingham, UNITED KINGDOM

11:00 – 11:20 am Resolving the Contribution of Quorum Sensing and Twitching in the Systemic Spread of Pseudomonas aeruginosa in Burn Wound Infections Jake Everett, Texas Tech University Health Sciences Center, Lubbock, TX

10 ASM Conferences Scientific Program

11:20 – 11:40 am The PmrB histidine Controls Expression of the qseBC Two-Component System via a Cognate and a Non-Cognate Response Regulator: Cross-Regulation or a Four-Component System? Maria Hadjifrangiskou, Vanderbilt University School of Medicine, Nashville, TN

12:00 – 1:30 pm Lunch Grand Ballroom, Salon M

1:30 – 4:20 pm Session 5: Systems and New Technologies Grand Ballroom, Salon E

1:30 – 2:00 pm Opening Lines of Communication: Quorum Sensing at the Intersection of Synthetic Biology and Microelectronics William Bentley, University of Maryland, College Park, MD

2:00 – 2:20 pm A Systems Analysis of Dual Signaling Control of Conjugative Drug Resistance Transfer in Enterococcus faecalis Arpan Bandyopadhyay, University of Minnesota, Minneapolis, MN

2:20 – 2:40 pm A Modular View on Cell Density Encoding Schemes in Bacterial Quorum Sensing Ilka Bischofs, University of Heidelberg, Heidelberg, GERMANY

2:40 – 3:10 pm Coffee break Grand Ballroom, Salons A & B

3:10 – 3:40 pm Title to be announced Peter Dorrestein, University of California, San Diego, CA

3:40 – 4:00 pm Electrochemical Quantification of Secondary Metabolites Produced by 3D-Printed Bacterial Aggregates Jodi Connell, The University of Texas at Austin, Austin, TX

5th ASM Conference on Cell-Cell Communication in Bacteria 11 Scientific Program

4:00 – 4:20 pm Quorum Sensing of Pseudomonas putida IsoF Burkhard Hense, Helmholtz Zentrum Muenchen, Neuherberg/ Munich, GERMANY

4:30 – 6:30 pm Poster Session B and Networking Mixer Grand Ballroom, Even-numbered posters will be formally presented Salons A & B

Tuesday, October 21, 2014

8:30 – 10:30 am Session 6: and Grand Ballroom, Salon E

8:30 – 9:00 am SypF, An Unusual Two-Component Regulator of Biofilm Formation and Colonization Karen Visick, Loyola University Chicago, Maywood, IL

9:00 – 9:20 am Interaction between Pseudomonas fluorescens Pf0-1 and Pedobacter sp. V48 Induces Social Motility Lucy McCully, University of Massachusetts Dartmouth, North Dartmouth, MA

9:20 – 9:40 am Manipulating the Interspecies Quorum Sensing Signal AI-2 in the Mouse Gut Karina Xavier, Instituto Gulbenkian de Ciencia, Oeiras, PORTUGAL

9:40 – 10:00 am A Plant-Responsive LuxR Homolog in a Cottonwood Tree Root Endophyte Amy Schaefer, University of Washington, Seattle, WA

10:00 – 10:30 am Coffee break Grand Ballroom, Salons A & B

12 ASM Conferences Scientific Program

10:30 am – 12:30 pm Session 7: Evolution and Signaling Grand Ballroom, Salon E

10:30 – 11:00 am Title to be announced Pete Greenberg, University of Washington, Seattle, WA

11:00 – 11:20 am The Evolutionary Origins of Quorum Sensing Luke McNally, University of Edinburgh, Edinburgh, UNITED KINGDOM

11:20 – 11:40 am The Evolution of Quorum Sensing in a Naturally Co-Evolved Host-Pathogen Model Liqin Zhou, Imperial College London - Silwood Park Campus, Ascot, UNITED KINGDOM

11:40 – 12:00 pm Exploiting Combinatorial Signalling - A New Type of Strategy in Pseudomonas aeruginosa James Gurney, University of Nottingham, Nottingham, UNITED KINGDOM

12:00 – 12:30 pm Facultative Cheating Drives the Maintenance of Quorum- Sensing Pherotype Diversity in Structured Populations Avigdor Eldar, Tel Aviv University, Tel Aviv, ISRAEL

12:30 – 1:00 pm Closing Remarks

5th ASM Conference on Cell-Cell Communication in Bacteria 13 Speaker Abstracts n OS:1 repression of LuxO. Comparison of these two data sets aided in defining sequences important B. Bassler; for target binding, but revealed significant Department of Molecular Biology, Howard functional throughout the rest of Hughes Medical Institute-Princeton Univer- the sequence. While our studies were able to sity, Princeton, NJ. ascribe function to most of the bases deter- mined to be important, some remain elusive. n S1:1 However, we were able to show that double mutant analysis may allow even deeper under- S. Winans; standing of the mechanism by which a single Cornell University, Ithaca, NY. mutation disrupts Qrr4 function. Additionally, expanding this analysis to other targets and n S1:2 the other Qrr sRNAs will elucidate more fully Screening and Mutual Information the individual roles of these highly conserved Theory Reveal Single Nucleotides in small RNAs, aiding in our understanding of Qrr4 Important for LuxR Repression their role in regulation of quorum sensing. J. S. Valastyan, S. T. Rutherford, T. O. Taille- fumier, C. G. Callan, N. S. Wingreen, B. L. n S1:3 Bassler; Phase variation controls quorum Princeton University, Princeton, NJ. sensing in Acinetobacter baumanii Five small regulatory RNAs, the Qrr sRNAs, K. M. Clemmer1, P. N. Rather2; are expressed at low cell density in Vibrio har- 1Atlanta VA Medical Center, Atlanta, GA, veyi and regulate multiple RNA targets whose 2Emory University, Atlanta, GA. encoded proteins function in the quorum- The Gram-negative bacterium Acinetobacter sensing pathway. While the Qrr sRNAs are baumannii has become a major cause of highly conserved and additively suppress nosocomial infections, which are exceedingly production at low cell density, their difficult to treat due to the extensive array expression levels and roles in the regulation of antibiotic resistance present in this of specific targets are divergent. We sought bacterium. In A. baumannii, a LuxI/R pair to more fully understand the contribution of of proteins designated AbaI/AbaR mediate individual nucleotides within one Qrr family a quorum sensing response via the signaling member - Qrr4 - to the repression of LuxR, the molecule 3-OH C -HSL. Our lab has found master regulator of the lux . To this end, 12 that the highly virulent A. baumannii strain we used saturating mutagenesis, fluorescence- AB5075 exhibits two colony opacity pheno- activating cell sorting, and mutual information types, opaque and translucent. Opaque and analysis to identify individual mutations that translucent variants interconvert at frequen- strongly inactivate Qrr4. Further analysis of cies ranging from 1/103-104 indicating a phase the mutants determined to have high mutual variable mechanism was responsible. Using information revealed differential roles for an Agrobacterium tumefaciens traG-lacZ various bases in Qrr4 function; we identified biosensor, the translucent colony variants bases important for base-pairing with the target were shown to produce greatly reduced levels RNA, forming stem-loops, and interacting of 3-OH C -HSL, which are restored in the with the chaperone Hfq. Complementary stud- 12 opaque form. However, the translucent colony ies elucidated nucleotides important for Qrr4’s

14 ASM Conferences Speaker Abstracts was not converted to opaque by biosynthesis operon. We further found that this the addition of exogenous 3-OH C12-HSL. tprA/phrA system is under strong catabolite Therefore, the translucent colony phenotype repression, such that PhrA peptide signaling does not result from decreased signal produc- only occurred in the presence of a poor carbon tion and it is hypothesized that 3-OH C12-HSL source, galactose. As galactose is primarily signal production is coordinately regulated encountered by S. pneumoniae in the naso- with the opaque/translucent switch by a global pharynx, and is a sugar encountered in regulatory mechanism. Consistent with this, the lungs and the bloodstream, our data sug- semi-quantative RT-PCR demonstrated trans- gest that this quorum-sensing cassette may be lucent colonies exhibited reduced expression important during colonization of the nasophar- of the abaI gene. An abaR mutant was capable ynx. Our currently model is that the metabolic of colony switching, although at significantly signals of the nasopharynx combined with a reduced frequencies indicating that quorum high cell density signal, mediated by phrA, in- sensing reciprocally regulates phase variation. duce production of a lantipeptide that allows S. Whole sequencing and RNA-Seq pneumoniae to compete with other analysis are currently being used to determine of the nasopharynx . the genetic and expression differences between each colony type. n S2:1 n S1:4 M. C. Givskov; Intl. Hlth. Immunol. and Microbiol., Univ. of Quorum Sensing Control of Copenhagen, Copenhagen, DENMARK. a Lantibiotic Gene Cluster in Streptococcus pneumoniae n S2:2 1 2 2 S. Hoover , T. Ho-Ching , M. Winkler , B. How does the quorum sensing 1 Lazazzera ; inhibitor hamamelitannin 1 2 Univ. of California, Los Angeles, CA, Indiana increase Staphylococcus aureus University, Bloomington, IN. biofilm susceptibility towards Quorum sensing in Gram-positive bacteria glycopeptides is mediated by small-secreted peptides that G. Brackman, F. Van den Driessche, diffuse through the environment. One such T. Coenye; group of quorum sensing peptides is the Phr Lab Pharm. Microbiology, Ghent University, family of peptides, originally characterized Gent, BELGIUM. in subtilis. We have identified in Streptococcus pneumoniae gene cassettes that Background: Biofilm-associated infections appear to encode Phr peptides. Here, we report caused by Staphylococcus aureus are often the characterization of one of these Phr peptide very difficult to treat and novel targets are cassettes, which like the others, encodes a needed to combat these infections. We have gene for a secreted Phr peptide (phrA) and a previously shown that the quorum sensing putative factor (tprA). The TprA (QS) modulator 2’,5-di-O-galloyl-D-hama- protein shares the greatest similarity with the melose (hamamelitannin, HAM) increases the PlcR of B. cereus, which susceptibility of S. aureus biofilms towards is activated by a Phr peptie. In contrast, TprA vancomycin (VAN) in vitro as well as in was found to acts as a transcriptional repressor vivo. However, the mechanism of action of and the PhrA peptide antagonized its activity. HAM at the molecular level has not yet been The tprA/phrA was found to include elucidated. Methods: Two parallel strategies the phrA gene itself and a putative lantibiotic were followed in order to gain insights in the

5th ASM Conference on Cell-Cell Communication in Bacteria 15 Speaker Abstracts way HAM affects QS. First, we evaluated Bacterial biofilms are sources of persistent the effect of HAM on the susceptibility of infection and resist antibiotic treatment in biofilms of S. aureus strains with mutations medical environments. P. aeruginosa is an in the QS systems (e.g. agrBCDA, trap, luxS) opportunistic pathogen associated with lung or in genes involved in biofilm formation and infections in cystic fibrosis and infections of virulence (e.g. icaA, sarA, codY). Secondly, indwelling device. Medical and environmental using illumina sequencing we identified genes biofilms are often multi- communi- that were differentially expressed in untreated ties of bacteria which are maintained through biofilms and biofilms treated with VAN alone chemical signaling and metabolite exchange. or in combination with HAM. Results obtained Most studies of multispecies interactions are with both strategies were further investigated in isotropic or homogeneous environments. using the appropriate tools. Results: No loss Here we demonstrate that growth substrate in HAM activity was observed for most of topography is capable of switching an interspe- the mutants. In contrast, HAM did not affect cies chemical signaling pathway in co-culture biofilm susceptibility of S. aureus strains with biofilms composed of and mutations in agrA or trap gene. This suggests Pseudomonas aeruginosa. Typically, an E. coli that these genes are involved in mediating receptor, SdiA, recognizes quorum sensing the activity of HAM. Using sequencing, we (QS) compounds from P. aeruginosa and trig- identified a large number of genes that were gers a biofilm dispersal response, resulting in differentially regulated after treatment. Treat- P. aeruginosa colonization of the growth sub- ment with HAM (alone or in combination with strate. Periodically structured substrates, on the VAN) resulted in a downregulation of genes other hand, are shown to modulate this path- involved in biosynthesis of lysine, diami- way by metabolite accumulation in engineered nopimelate and D-alanine and an upregulation microenvironments. Substrate structures of genes involved in glutamine consuming induce changes in E. coli biofilm morphology, pathways. In addition, the upregulation of which in turn increase the concentration of in- genes encoding virulence factors (e.g. tst, hla, dole, a constitutively produced metabolite and yent1, hlgB) during VAN treatment was not ob- active signaling molecule, within the biofilm. served when VAN was combined with HAM. The extent of indole accumulation is responsi- Conclusion: HAM reduces the upregulation of ble for modulating the dispersal response of E. peptidoglycan biosynthesis normally observed coli to P. aeruginosa QS signals. In contrast to after treatment with VAN. This possibly leads transient surface chemistry or antibiotic leach- to the increased susceptibility of S. aureus ing strategies, substrate topography generates biofilm cells towards VAN. Our results further persistent, physical cues that are promising indicate that combination therapy could posi- for long-term control of biofilm structure tively affect morbidity since the upregulation and properties. We show that these materials of virulence factors observed for VAN treat- are able to produce E. coli biofilms that are ment are not observed when VAN is combined resistant to P. aeruginosa colonization for at with HAM. least three weeks. Moreover, these surfaces render the resulting biofilm, almost exclusively n S2:3 composed of E. coli, more susceptible to anti- biotic treatment than co-cultures grown on flat Growth Substrate Topography surfaces. These results suggest the potential Switches an Interspecies Signaling for physical structures to modify signaling Pathway in Co-Culture Biofilms dynamics in multi-species communities and A. Bhattacharjee, A. Hochbaum; to engineer structure-property relationships in University of California, Irvine, Irvine, CA. biofilms to exhibit useful behavior.

16 ASM Conferences Speaker Abstracts n S2:4 n S2:5 Combinatorial quorum sensing can Synthetic Ligands for the allow bacteria to resolve their Interception of Bacterial social and physical environment Communication D. Cornforth1, R. Popat2, L. McNally2, J. H. E. Blackwell; Gurney3, T. C. Scott-Phillips4, A. Ivens2, S. P. University of Wisconsin, Madison, WI. Diggle3, S. P. Brown2; We are developing chemical tools that attenu- 1The University of Texas at Austin, Austin, ate cell-cell communication pathways in bac- TX, 2The University of Edinburgh, Edinburgh, teria. Many bacteria communicate using small UNITED KINGDOM, 3University of Not- organic molecules and peptides to monitor tingham, Nottingham, UNITED KINGDOM, their population densities in a process called 4Durham University, Durham, UNITED “quorum sensing.” At high cell densities, KINGDOM. bacteria use this signaling network to switch Many bacterial species engage in a form of from an isolated, nomadic existence to that of a cell-cell communication known as quorum multicellular community. This lifestyle switch sensing (QS). Despite great progress in unrav- is significant; only in groups will pathogenic eling the molecular mechanisms of QS, con- bacteria turn on virulence pathways and grow troversy remains over its functional role. There into drug-impervious communities called is disagreement over whether QS surveys biofilms that are the basis of myriad chronic bacterial cell density or rather environmental infections. In turn, certain symbiotic bacteria properties like diffusion or flow, and moreover will only colonize their hosts and initiate ben- there is no consensus on why many bacteria eficial behaviors at high population densities. use multiple signal molecules. We develop and Our research is broadly focused on the design, test a new conceptual framework for bacterial synthesis, and characterization of non-native cell-cell communication, demonstrating that ligands that can intercept quorum sensing bacteria can simultaneously infer both their and provide new insights into its role in host/ social (density) and physical (mass-transfer) microbe interactions. These molecules provide environment, given combinatorial (nonaddi- a novel approach to study quorum sensing tive) responses to multiple signals as well as with both spatial and temporal control in a distinct half-lives of the signals. We test these range of settings. We have developed a series predictions using the opportunistic pathogen of efficient synthetic methods that provide us Pseudomonas aeruginosa and demonstrate with straightforward access to these ligands. In significant differences in signal decay between addition, we have applied our quorum sensing its two primary signal molecules, as well as antagonists and agonists in vitro and in vivo to diverse combinatorial responses to dual-signal investigate quorum sensing as an anti-infective inputs. We also demonstrate that secretome target. This talk will introduce our research genes are preferentially controlled by synergis- approach and highlight recent results. tic “AND-gate” responses to multiple signal inputs which may help adaptively limit secre- tions to high cell density/ low mass-transfer environments.

5th ASM Conference on Cell-Cell Communication in Bacteria 17 Speaker Abstracts n S2:6 and long chain AHLs, respectively. Only 101 Investigating the quorum QQ isolates displaying no AHL-based QS were quenching ability of bacteria from further investigated for substrate specificity. gastrointestinal tracts of cultured Complete degradation of medium to long chain ornamental freshwater-fish and AHLs were most commonly observed with marine farmed fish majority of isolates degrading N-octanoyl- DL-homoserine lactone (96%) and N-(3- Z. Kajee, H. Y. Chenia; oxodecanoyl)-L-homserine lactone (68.31%). University of KwaZulu-Natal, Durban, The use of AHL-degrading enzymes to inhibit SOUTH AFRICA. QS displays great potential as these enzymes are not bactericidal and may thus reduce or While many bacterial pathogens in aquaculture inhibit the QS-regulated pathogenesis factors environments use quorum sensing (QS) for and need to be examined further. The QQ the regulation of processes including patho- enzymes from these isolates could replace tra- genesis, competitor bacteria can inhibit QS via ditional since the QQ strategy is not enzymatic quorum quenching (QQ). Gram- aimed at killing the pathogen or limiting cell negative bacteria generally communicate growth but rather is focused on shutting down using N-acyl homoserine lactones (AHLs) that the expression of pathogenicity genes. vary in structure but which are susceptible to degradation by enzymes resulting in QS inhibi- tion. QQ holds high value as an alternative n S3:1 mechanism in combatting bacterial infections Cell-cell recognition by the without selecting for drug-resistant cells and polymorphic myxobacterial TraA thus, the QQ ability of gastrointestinal tract receptor leads to beneficial and (GIT) bacteria from cultured fish environments adversarial interactions were investigated as potential anti-virulence D. Wall; resources. Bacteria were isolated, enriched and University of Wyoming, Laramie, WY. purified from GIT of Malawian cichlids, koi carp, tank biofilms and GIT of marine cultured Cooperative behavior among individuals dusky kob using standard microbiological as- often involves resource sharing which in turn says. Isolated bacteria were subjected to quali- provides fitness advantages to the community. tative T-streak assays to identify potential QS Myxobacteria are a microbial example where activity, using three AHL biosensor systems individuals share their resources and build to detect a wide spectrum of AHL molecules, cooperative multicellular communities. In the viz., Chromobacterium violaceum CV026, case of Myxococcus xanthus, cells will tran- VIR07, and Agrobacterium tumefaciens A136. siently fuse their outer membranes (OMs) and Biosensor sandwich assays were used as a exchange their OM proteins and lipids, a pro- preliminary, qualitative screen to investigate cess that is mediated by the TraA polymorphic the quenching activity. AHL degradation and the TraB cohort pro- assays were used to investigate QQ substrate tein. Resource sharing within diverse microbial specificity of selected isolates using six differ- communities found in native soil environ- ent synthetic AHLs (C6 - C12). The ability to ments raises interesting questions about how quorum sense, using AHL molecules of vary- partner cells are identified and whether such ing chain lengths, was observed for 51.72% interactions are regulated. Through a series of of Gram-negative isolates. The qualitative QQ chimeric and site directed mutagenesis studies sandwich assays identified 87.96%, 78.24%, we identify particular regions and amino acid and 19.91% of isolates which were capable of residues within TraA that govern the specificity quenching short chain, medium to long chain for cell-cell fusion. We also provide evidence

18 ASM Conferences Speaker Abstracts that among environmental populations there Introduction: -mediated are hundreds of different TraA recognition/ biofilm expansion is a complex, multicellular social groups. The ability of Tra-dependent behavior that enables the active colonisation interactions to lead to beneficial outcomes of surfaces by many species of bacteria. Com- was also tested. Here, a healthy population munication between individual bacteria must was tested for their ability to repair a damage occur for successful active biofilm develop- population. This was done with a conditional ment. Both active biofilms and hydrated sessile lpxC allele, an essential gene for lipopolysac- biofilms of many bacterial species have been charide biosynthesis. The cell viability and shown to contain complex polysaccharide ma- antibiotic sensitivity defects of lpxC mutants trix components including DNA. The role of were rescued when mixed with healthy tra+ extracellular DNA (eDNA) in early hydrated donor cells, but not when mixed with healthy biofilms and active biofilm expansion is cur- tra- donors. This result suggests OM exchange rently unclear. Aim: In this study we aim to can rejuvenate a sub-population that contains explore the role of eDNA in actively expand- a defective cell envelope by exchanging ing biofilms of Pseudomonas aeruginosa. corresponding wild-type components from Methods: We have used high resolution phase healthy cells. Although Tra-dependent interac- contrast time-lapse microscopy and developed tions can lead to beneficial outcomes, in other sophisticated computer vision algorithms to cases they led to antagonism. The antagonistic track and analyse individual cell movements interactions were triggered by disparate gliding during expansion of P. aeruginosa biofilms in motility among sibling strains. the presences and absence of eDNA. Results: Thus mixtures of nonmotile mutants with Our analyses reveal that at the leading edge of motile strains resulted in the latter strain being the actively expanding biofilm, highly coherent inhibited for motility and subsequently killed. groups of bacteria migrate across the surface In summary, OM exchange represents a new of the semi-solid media. This leads to the paradigm for how bacterial cells communicate emergence of a network of trails that guide the and interact. These interactions are complex; mass transit of cells toward the leading edges in some cases they are constructive and lead of the biofilm. We have determined that eDNA functional communities or tissues. In other facilitates efficient traffic flow throughout cases Tra-dependent interactions are antagonis- the trail network by maintaining coherent tic, suggesting populations regulate or ‘police’ cell alignments between neighbouring cells, their behaviors. thereby avoiding traffic jams and ensuring an efficient supply of cells to the migrating n S3:2 front. Our analyses reveal that eDNA also co- ordinates the movements of cells in the leading Intercellular signalling using edge vanguard rafts and is required for the as- extracellular DNA facilitates active sembly of cells into the “bulldozer” aggregates biofilm expansion that forge the interconnecting trails. Conclu- L. Turnbull1, E. S. Gloag1, A. Huang1, P. sions: Our observations have revealed that Vallotton2, R. Prabhakar3, M. P. Wand1, C. B. large-scale self- organization of multicellular Whitchurch1; communities occurs through construction of an 1University of Technology Sydney, Sydney, intricate network of trails that is facilitated by AUSTRALIA, 2CSIRO, Sydney, AUSTRALIA, eDNA. This reveals a new form of intercellular 3Monash University, Melbourne, AUSTRALIA. communication by bacteria using eDNA as a passive physical signal.

5th ASM Conference on Cell-Cell Communication in Bacteria 19 Speaker Abstracts n S3:3 to enhance structural rigidity. GVs produced Characterization of quorum heterologously in E. coli display the same sensing-regulated gas vesicle collapse pressure as those formed in S39006, morphogenesis in enterobacteria suggesting that they are physically similar. We have used pressure nephelometry to calculate R. E. Monson1, Y. Tashiro2, J. Ramsay3, G. P. the instantaneous turgor pressure within both Salmond1; S39006 and E. coli. Finally, we have demon- 1University of Cambridge, Cambridge, strated that GVs can make up ~20% of the cell UNITED KINGDOM, 2Graduate School of volume in S39006 and ~50% of the volume Engineering, Shizuoka University, Hama- of an E. coli cell expressing the S39006 GV matsu, JAPAN, 3Curtin University, Perth, cluster. This suggests that, while GVs within AUSTRALIA. S39006 and E. coli appear to be structurally similar, production of GVs may be “deregulat- Gas vesicles (GVs) are hollow, proteinaceous ed” in the synthetically engineered host where intracellular organelles produced by some organelle morphogenesis may be liberated bacteria and that facilitate flotation from the natural physiological control signals because GVs are permeable to gas but not to (such as quorum sensing or oxygen status sens- liquid. Strains capable of producing GVs can ing) operating in the cognate bacterium. use controlled production to manage verti- cal movement in an aquatic environment, presumably providing an adaptive competi- n S3:4 tive advantage for colonization of zones in a Jekyll and Hyde: Microbial water column. Recently, it was discovered that metabolites that both kill and GVs are produced in Serratia sp. ATCC39006 stimulate biofilm formation (S39006) - the first time such organelles were E. A. Shank; observed, naturally, in any enterobacterium. University of North Carolina, Chapel Hill, NC. The production of GVs in S39006 is positively regulated in a density-dependent manner by a In , bacteria are rarely found in isolation; LuxIR-type quorum sensing system: SmaIR. they are most often surrounded by other micro- SmaI produces N-butanoyl-L-homoserine organisms. We are interested in how microbes lactone (BHL) and, at high cell densities, in these complex communities alter their BHL derepresses the transcriptional regulator physiology and development in response to SmaR, leading to GV biogenesis. GV produc- metabolites secreted by their microbial neigh- tion in S39006 is also regulated by the small bors. To explore these interspecies interactions, RNA-binding protein RsmA, and production we focus on the ability of Bacillus subtilis to increases in oxygen-depleted environments, respond to bacteria from its natural environ- suggesting that GVs promote and maintain the ment, the soil. B. subtilis is ideal for these presence of S39006 at air-liquid surfaces. The studies because of its ability to differentiate 39006 GV cluster can be heterologously ex- into a variety of distinct cell types that can be pressed in Escherichia coli to engineer floating monitored using fluorescent reporters. Using recombinants. We have attempted to physically a co-culture screen, we previously identified characterize GVs in S39006 to understand numerous soil microbes secreting compounds more about their development and their role in that affect the differentiation of B. subtilis into the cell. GVs are sensitive to external pressure biofilm-forming cells. One of the microbes that and can have different structural robustness. activated B. subtilis biofilm Using pressure nephelometry, the collapse was Bacillus cereus. Using imaging mass spec- pressure of GVs within S39006 has been deter- trometry, we identified the thiocillins, a group mined and the protein GvpC has been shown of thiazolyl peptide antibiotics, as potential

20 ASM Conferences Speaker Abstracts biofilm-inducing compounds produced by B. ence motility, and unlike many T6S-associated cereus. Using bioassays and flow cytometry, proteins, do not impact cell viability. IdsD and we confirmed that purified thiocillin increased IdsE bind each other, and this interaction ap- the biofilm-expressing population of B. subtilis pears to be allele-specific, chiefly limited to the cells. We also showed that biofilm-induction variants originating from the same strain. The appears to be a general phenomenon common specificity for binding between these proteins to a variety of structurally diverse thiazolyl is primarily encoded in a stretch of distinctive peptides, and that biosynthesis genes encoding amino acids, i.e. a variable region, within each putative thiazolyl peptides were present in protein. Exchange of these sequences to those many bacterial species’ . Notably, al- of another strain changes the binding specific- though multiple alterations to the backbone of ity in vitro. Moreover, the in vitro strain- thiocillin abolished its ability to induce biofilm specific binding interactions between these gene expression in B. subtilis, a mutation that two proteins strongly correlate with in vivo self eliminated its antibiotic activity did not. The identity and wild-type swarm expansion. Lack antibiotic activity and biofilm-induction activi- of binding in vitro corresponded to atrophied ty of thiocillin are therefore independent of one swarm expansion. These two self-identity another. Our results thus indicate that thiocillin proteins, IdsD and IdsE, are examples of T6S- possesses dual bioactivities: acting not only as associated proteins that cause a behavioral a killing agent, but also as a specific modulator change (i.e., altered swarm expansion) within of microbial cellular phenotypes. Future work a clonal population that is not associated with is needed to determine whether other microbial cell death. Moreover, the communication of metabolites initially identified as antibiotics identity among cells likely via the Ids system might similarly possess dual bioactivities. indicates that self-recognition events between kin can influence not only boundary formation, n S3:5 but also group motility. Kin-specific binding between two proteins correlates with in vivo n S3:6 self identity and wild-type swarm Staphylococcus aureus enhance expansion in Proteus mirabilis growth and antibiotic tolerance in a human host adapted Pseudomonas L. Cardarelli, C. Saak, K. A. Gibbs; aeruginosa lineage Harvard University, Cambridge, MA. C. F. Michelsen1, M. S. Bojer2, H. Ingmer2, The ability to distinguish kin from others L. Jelsbak1; underlies many group behaviors, including 1Technical University of Denmark, Kgs. Lyn- territoriality. For the bacterium Proteus mirabi- gby, DENMARK, 2University of Copenhagen, lis, where self versus non-self recognition is Frederiksberg C, DENMARK. exhibited through the physical exclusion of one strain from surfaces occupied by another, Interactions among members of polymicro- the Ids system contributes to this behavior and bial infections can result in altered pathogen is associated with a type VI secretion (T6S) behaviours such as enhanced virulence, biofilm system. We have previously shown that of the formation or antibiotic tolerance, which may ids genes, idsD and idsE encode strain-specific influence the phenotype and clini- identifiers; however, it was unknown if and cal outcome of the infection. Pseudomonas how the encoded proteins interact to define aeruginosa and Staphylococcus aureus are strain identity. Here we present evidence that important opportunistic human pathogens and idsD and idsE encode a T6S-associated protein are both part of the polymicrobial infection pair that together determine identity and influ- communities in human hosts. Previous studies

5th ASM Conference on Cell-Cell Communication in Bacteria 21 Speaker Abstracts have revealed that cell-cell communication can Enterococci are highly adapted members of play a major role in the interaction behaviors the gastrointestinal tract consortia of many between these bacteria and subsequently can animals, from man to insects. It is very likely affect the behavior of the individual strains. that they were members of the commensal con- However, the extent to which evolutionary sortia of the last common ancestor of terrestrial processes may remodel interspecies interac- life, which existed about 450 MYA, and may tions and cell-cell communication during the go back further. However, in the antibiotic course of infection and therapy is currently not era, strains of enterococci have evolved into understood. Therefore, the aim of this study leading causes of multidrug resistant hospital was to analyze the in vitro interaction between acquired infection. One of our main research a community-associated methicillin-resistant goals is to understand how human use of an- S. aureus (CA-MRSA) and a collection of tibiotics has led to the changes in enterococci P. aeruginosa isolates representing different that have allowed them to become leading evolutionary steps of a dominant lineage, DK2, agents of hospital acquired infection. Compar- that have evolved through decades of growth ative genomics is showing us that enterococcal in chronically infected patients. Although, the adaptation to the hospital environment has early-adapted P. aeruginosa DK2 strains out- involved the loss of mechanisms for protecting competed S. aureus, we found that the majority the fidelity of its genome, such as the CRISPR- of the P. aeruginosa DK2 strain collection cas system, and the concomitant acquisition of showed a mutualistic interaction with S. aureus new traits, including antibiotic resistances and during co-culturing on agar plates, where the pathogenic traits, as well as phages, on mobile cell density of P. aeruginosa was increased genetic elements. This has occurred to the ex- in the presence of S. aureus. Furthermore, S. tent that the genomes of many multidrug resis- aureus derived extracellular compounds were tant hospital isolates are now more than 25% found to suppress P. aeruginosa autolysis, larger than commensal counterparts. Because and to protect P. aeruginosa from being killed of the large accumulation of mobile elements by clinically relevant antibiotics most likely in these strains, it was of interest to know to through a selection for small-colony variants what extent these hospital strains were capable (SCVs). The S. aureus derived extracellular of competing with commensal strains in the compounds were proteins controlled by the absence of antibiotic pressure. These studies virulence regulators, SarA and agr, as well showed that a prototype VRE E. faecalis strain as by the proteolytic subunit, Clp, of the Clp could no longer compete in a healthy GI tract protease. Our results suggest that P. aerugi- consortium, and in fact they were unexpectedly nosa host adaptation may be accompanied observed to be killed by it. The mechanism of with remodeling of interspecies interactions, killing was found to require the presence of which further can affect bacterial signaling and commensal E. faecalis; and commensal E. fae- development. calis were found to be capable of effecting this killing in pure culture. The mechanism of kill- n S4:1 ing was further found to depend on the produc- tion of a pheromone signal by the commensal The Curious Case of Death by E. faecalis, which induced the suicidal death Seduction: How the siren song of of the VRE. That suicide required the presence commensal E. faecalis drove VRE to in the VRE of an integrated genetic element suicide in its chromosome. This result shows that this M. S. Gilmore; VRE strain cannot co-exist with commensal Ophthalmology, Harvard Med. Sch., Boston, enterococci, meaning that in the patient, either MA. the VRE colonize a distinct microniche, or that they can only colonize patients in whom the

22 ASM Conferences Speaker Abstracts commensal enterococci have been eliminated expressed bioluminescence in the presence altogether. This work highlights the importance of GBAP and CylLS both in the supernatant of maintaining healthy commensal flora during of liquid cultures and in agar overlay assay in antibiotic therapy, as well as new strategies as little as three hours with high sensitivity. for the elimination of this multidrug resistant The biosensors were employed to investigate hospital pathogen. interaction between the fsr and cyl systems, Portions of this work were conducted by: Mar- revealing that fsr antagonizes CylLS activity cus Rauch, Lynn Hancock, Matthew Ramsey, by 75%. This effect towards cytolysin produc- Paul Himes, Sriram Varahan, and Francois tion was mediated by the metalloprotease Lebreton. GelE, presumably by degradation of CylLS. Furthermore, we show that the clinical isolate n S4:2 E. faecalis T2 acts as a biological cheater producing cytolysin only upon sensing CylLS Bioluminescence based biosensors producers in its environment. This isolate was for the detection of peptide thus able to enhance its virulence during poly- pheromone production and cell- microbial systemic infection in the Galleria cell communication in Enterococcus mellonella model. Conclusions: The reporter faecalis systems presented here are the first example of S. Leanti La Rosa, D. Diep, I. Nes, D. Brede; a bioluminescence-based screening method ap- Norwegian University of Life Sciences, Aas, plied to the detection of cell-cell communica- NORWAY. tion and virulence determinants in E. faecalis. These biosensors will be a very useful means Background: Once considered a harmless to study the complex mechanisms underly- commensal of the gastrointestinal tract, Entero- ing virulence in this prominent nosocomial coccus faecalis has become over the past three pathogen. decades a challenging nosocomial problem due to the emergence of multi-antibiotic resistant isolates refractory to therapies and equipped n S4:3 with a plethora of pathogenicity determinants. Understanding the role of The bacterium uses quorum-sensing systems to Diffusible Signalling Factor (DSF) regulate physiological processes among which in virulence of Xanthomonas plant the expression of virulence traits to adapt and pathogens proliferate in the host. Aim. This study was R. Rai, S. javvadi, S. Chatterjee; carried out to develop two bioluminescence CDFD, Hyderabad, INDIA. based reporter systems for the direct detection of the quorum-sensing regulated gelatinase Xanthomonas oryzae pv. oryzae (Xoo) and biosynthesis activating pheromone (GBAP) Xanthomonas oryzae pv. oyzicola (Xoc) are and cytolysin small subunit (CylLS) in natural important members of the genus Xanthomonas isolates. Methods: The two biosensors were which causes serious disease of rice. In constructed by cloning the cytolysin promoter Xanthomonas group of plant pathogens, a and the CylLS responsive regulatory genes secreted fatty acid signalling molecule known (Pcyl-cylR1R2) or the gelatinase promoter as Diffusible Signalling Factor (DSF) regulates (PgelE) into a luxABCDE-containing vector. diverse virulence associated functions. Our The resulting plasmids, pSL101cylR2R1Pcyl studies have shown that DSF regulates viru- and pSL101PgelE were introduced in the Cyl- lence associated traits in an atypical fashion strain E. faecalis JH2-2 and the Gel- strain in rice pathogen Xoo. In Xoo, DSF promotes V583fsrB*, respectively. Results: E. faecalis biofilm formation and regulates transition from cells containing the constructs conditionally planktonic to biofilm lifestyle. In Xoc, DSF

5th ASM Conference on Cell-Cell Communication in Bacteria 23 Speaker Abstracts deficient mutants exhibit reduced virulence LuxO. V. cholerae strains expressing only and in planta growth deficiency. To understand one of these four receptors are QS proficient, how DSF promotes in planta growth, we displaying LuxO-dependent gene regulation characterized the DSF deficient mutant of Xoc. with distinct temporal dynamics. Moreover, Evidence will be provided which indicates that these single-receptor-expressing mutants are DSF in Xoc promotes virulence by regulating capable of colonizing animal hosts as well as ferric iron uptake. We further show that ferric the wild type. In contrast, mutants lacking all uptake is critical for virulence in Xoc. This is four receptors are phenotypically identical in contrast to Xoo, which is a vascular patho- to LuxO-defective mutants both in vitro and gen wherein, ferrous uptake system contributes in vivo. We suggest that these four parallel to virulence. Our study revealed that closely signaling systems integrate into the V. cholerae related rice pathogens (Xoo and Xoc) fine tunes QS circuit to facilitate temporal coordination DSF mediated signalling to utilize alternate of multiple sensory inputs and modulate QS- source of iron to suite their different lifestyle. dependent gene expression. We hypothesize that the presence of multiple functionally n S4:4 redundant receptors controlling a single com- mon regulator increases the overall robustness Integration of multiple sensory of the system. To test this, we studied how inputs in the Vibrio cholerae LuxO-dependent gene expression is affected quorum-sensing circuit and the by exogenously adding a surplus amount of impact on virulence and system CAI-1, which inhibits LuxO activation by robustness CqsS. We found that the overall QS response S. A. Jung, W. Ng; is unaffected by excess CAI-1 in wild type V. Tufts University, Boston, MA. cholerae or in mutants missing single receptors LuxQ, VC1831, or VpsS. In contrast, the QS Bacteria use quorum sensing (QS) for response is significantly induced by CAI-1 in a cell-cell communication to carry out robust triple receptor mutant lacking LuxQ, VC1831, group behaviors. This inter-cellular signal- and VpsS. Therefore, by integrating multiple ing process relies on cell-density dependent sensory inputs, the V. cholerae QS circuit has production and detection of chemical signals, evolved to be refractory to sporadic fluctua- called (AIs). Vibrio cholerae, the tion in the external environment, thus allowing causative agent of cholera, controls biofilm robust and synchronous expression of QS- formation in the environment and virulence dependent genes. factor production in the host via QS. Two QS sensor histidine (HKs), CqsS and LuxQ, have been previously characterized and n S4:5 they detect two distinct AIs, CAI-1 and AI-2, Unravelling the interplay between respectively. These two receptors function in Yersinia and Caenorhabditis elegans parallel to activate the key regulator of the V. during quorum sensing-dependent in cholerae QS response, LuxO, which is essen- vivo biofilm formation tial for virulence. Surprisingly, mutants lacking P. Williams; CqsS and LuxQ display a normal cell-density Inst. of Infection, Immunity and Inflamma- dependent QS response and remain virulent tion, University of Nottingham, Nottingham, in vivo, suggesting that LuxO is activated by UNITED KINGDOM. additional unidentified signaling pathways. We show that similarly to CqsS and LuxQ, two The formation of an incapacitating biofilm on other HKs, VC1831 and VpsS, act upstream in Caenorhabditis elegans by Yersinia pseudotuber- the central QS circuit of V. cholerae to activate culosis that blocks nematode feeding, represents

24 ASM Conferences Speaker Abstracts a tractable model for investigating the genetic Thermal injuries represent a very unique type basis for host-pathogen interplay during the of physical trauma that are highly associated biofilm-mediated infection of a living surface. with the development of bacterial sepsis. Pseu- N-Acylhomoserine lactone-dependent quorum domonas aeruginosa is a gram-negative, op- sensing (QS) via two pairs of LuxRI orthologues portunistic pathogen that is exceptionally well (YpsR/I and YtbR/I) and multiple AHLs plays a adapted at establishing systemic infections in critical role in this process. Y. pseudotuberculo- thermally-injured patients. Previous studies sis strains expressing an AHL-degrading enzyme have described the importance of the bacterial or in which the AHL synthase (ypsI and ytbI) communication conduit, quorum sensing (QS), or response regulator (ypsR and ytbR) genes in P. aeruginosa burn wound infections. These have been mutated, are attenuated for biofilm studies largely evaluated the QS mutant JP2, formation. To gain more detailed information which is deficient in the LasI and RhlI QS sys- on the response of C.elegans to the presence of tems. This QS-deficient mutant was much less Yersinia biofilms and on the Yersinia QS regula- virulent in thermally-injured mice compared to tory hierarchy involved transcriptomic, reporter burn mice infected with the wild-type strain, gene fusion and promoter pull down assays PAO1. Additionally, numbers of bacteria were employed in conjunction with electron recovered at distal locations within the burn and fluorescence microscopy. For C. elegans, wound and from the spleen were significantly infection with Y.pseudotuberculosis resulted in lower in JP2-infected burn mice compared to the differential regulation of numerous genes, those infected with PAO1. However, later char- including a distinct subset of nematode C-lectin acterization of JP2 revealed that, in addition and fatty acid desaturase genes, the mutation to defective QS, JP2 also exhibits defective of which resulted in the abrogation of biofilm twitching motility, which is unrelated to the formation. For Yersinia, we have discovered that lasI and rhlI mutations. Given the importance the QS-regulatory hierarchy is itself controlled of twitching motility in bacterial transloca- via the sensing of N-acetyl glucosamine while in tion, biofilm formation, and chemotaxis, it turn regulating the flagellar regulatory cascade is necessary to reevaluate the independent in a pathway involving the histidine utilization contributions of QS and twitching motility to (hut) genes such that QS, flagellar pathway, the virulence of P. aeruginosa in burn wound hut and nag mutants all fail to form biofilms on infections. Here, using a QS mutant with fully C.elegans. Collectively this work indicates that intact twitching motility, JM2, we report and biofilm formation by Y. pseudotuberculosis on compare the importance of QS and twitching C. elegans is a sophisticated interactive process motility in the local and systemic spread of P. during which the initial attachment/recogni- aeruginosa in burn wound infections. In vivo tion of Yersinia to/by C. elegans is followed by examination revealed that thermally-injured biofilm development during which QS plays a mice infected with either JM2 or JP2 exhibited central role. delayed mortality compared to burn mice infected with PAO1, supporting the previous n S4:6 findings stressing the importance of QS in thermal infections and that twitching motil- Resolving the Contribution of ity is not necessary for this to cause Quorum Sensing and Twitching systemic infections. These results, in combina- Motility in the Systemic Spread of tion with the ongoing evaluation of bacterial Pseudomonas aeruginosa in Burn chemotaxis and dissemination of JP2 and Wound Infections JM2 in thermally-injured mice, should help us J. A. Everett, K. P. Rumbaugh; understand the individual and combined effects Texas Tech University Health Sciences Center, of QS and twitching motility on the systemic Lubbock, TX. spread of P. aeruginosa in burn wounds.

5th ASM Conference on Cell-Cell Communication in Bacteria 25 Speaker Abstracts n S4:7 transcription depending on its concentra- The PmrB histidine kinase controls tion relative to QseB. When PmrA and QseB expression of the qseBC two- are in a 1:1 or 2:1 ratio, PmrA assists QseB component system via a cognate and binding to the qseBC promoter and serves as a non-cognate response regulator: a transcription enhancer. When PmrA is found cross-regulation or a four- in higher concentrations in the cell it occludes component system? QseB binding and inhibits qseBC expression. Parallel mutagenesis studies revealed that M. Hadjifrangiskou; abrogating the kinase activity of the QseC sen- Vanderbilt University School of Medicine, sor did not alter its ability to rescue the qseC Nashville, TN. deletion phenotype. Thus, while the QseC phosphatase function is crucial for regulating Although bacterial two-component systems are QseB activity, the kinase function of QseC largely isolated from each other to avoid signal is dispensable, possibly due to the overlap- confusion, some flexibility must exist to allow ping kinase activity of PmrB. Taken together, bacteria to respond to thousands of signals, our findings show that in UPEC QseBC and using only the finite number of two-component PmrAB are physiologically connected via systems harbored in their genomes. PmrAB non-partner interactions that impact target and QseBC are two signaling modules that are gene expression. We are now investigating the conserved in many Gram-negative bacteria. global effects of QseBC-PmrAB interactions in PmrAB responds to ferric iron and other response to signals. signals and directs the expression of genes that are important for LPS modification. QseBC has been reported to respond to epinephrine n S5:1 and norepinephrine and deletion studies have Opening Lines of Communication: implicated QseBC in the virulence of several Quorum Sensing at the Intersection pathogens. Uropathogenic E. coli (UPEC) of Synthetic Biology and mutants deleted for the QseC sensor are drasti- Microelectronics cally attenuated in murine models of urinary W. E. Bentley; University of Maryland, Col- tract infection and this attenuation stems from lege Park, MD. the constitutive of the QseB response regulator by the PmrB histidine Synthetic biology provides a means for kinase. PmrB phosphorylates the non-cognate articulating concepts into new products and QseB at rates that are comparable to QseC- products. Its toolbox is extensive, including the mediated phosphotransfer, but unlike QseC the ability to create synthetic genomes and tailor phosphatase kinetics of PmrB towards QseB their regulation. Early successes augmented are slow. Loss of QseC also leads to the de- the cell’s biosynthetic capacity and rewired its regulation of known PmrA-regulated targets. regulation, transforming our ability to produce We thus hypothesized that PmrAB and QseBC products ranging from small molecules to fully are physiologically connected. Stimulation functional therapeutic proteins at high yield. of wild-type UPEC with ferric iron, but not Also, the theoretical formalisms of metabolic epinephrine, induced the expression of qseBC engineering provided a basis for optimally and this signal-dependent activation required routing its biochemical flux. These activi- both QseB and PmrA. Biochemical and in vivo ties focused largely on the cell’s intracellular analyses revealed that QseB and PmrA directly biochemical network and relied less on mo- control transcription of the qseBC operon. lecular cues from its immediate surroundings. While QseB activates qseBC expression, PmrA The emergence of quorum sensing (QS) as a exerts a positive or negative effect on qseBC model for signal transduction has enabled a

26 ASM Conferences Speaker Abstracts reexamination of metabolic flux and regula- transfer in plasmid-containing donors; and an tion by hardwiring population-scale biological inhibitor peptide encoded in the plasmid and function to extracellular cues. Regulatory and produced by donor cells serves to modulate fabrication modules are feasible, owing to a donor response in accordance with the relative few relatively simple QS signal transduction abundance of donors and recipients. The dual cascades. QS provides a context for entirely signaling system allows the donor to calibrate new products and processes that consider the its response to the probability of successful individual or small populations of cells. E. transfer and economize its resources. A multi- coli were engineered to swim towards and scale mathematical model was developed to interrogate receptor density on nearby cancer include intracellular molecular mechanisms as cells, and based on threshold levels, induce well as population level interactions between heterologous protein synthesis. Indeed, new donors and recipients. Kinetic parameters opportunities are emerging by understanding for the model were estimated from literature and manipulating such communication net- values and augmented by RNA-Seq data and works that exist between cells, however there binding constant measurements using surface are limited means for actuating and control. By plasmon resonance and isothermal titration connecting biological systems and their molec- calorimetry. The model was further verified ular based communication networks to those by comparing the model-simulated dynamic of microfabricated devices, new synergies are response to experimentally measured transcript envisioned. We are developing methods to profiles. While a calibrated response by donor interrogate QS and other biological signaling cells can avoid wasteful induction of conjuga- phenomena by connecting to microfabricated tive transfer system, a delay of transfer may devices enabling bi-directional communication imperil cell survival under some conditions. and control. Some examples will be discussed, We hypothesize that a mechanism that allows including a new redox capacitor that enables for heterogeneous response by donor cells will storage and readout of bioelectric functions. safeguard against such peril. Simulation of the induced expression profiles using the model n S5:2 suggested plasmid copy number distribution in donor cells and stochastic behavior as possible A systems analysis of dual signaling sources of the heterogeneity in donor response control of conjugative drug to the dual signals. We are currently using resistance transfer in Enterococcus newly developed fluorescent reporter fusion faecalis constructs to further examine and refine the A. A. Bandyopadhyay, C. Shu, Y. Chen, A. model. Barnes, G. M. Dunny, W. Hu; University of Minnesota, Minneapolis, MN. n S5:3 Enterococcus faecalis, a major causative agent A modular view on cell density of hospital-acquired infections, is resistant to encoding schemes in bacterial many known antibiotics. Its ability to laterally quorum sensing transfer conjugative plasmids encoding anti- B. Drees1, M. Reiger2, K. Jung2, I. Bischofs1; biotic resistance and virulence determinants 1University of Heidelberg, Heidelberg, to other pathogens in the environment poses GERMANY, 2Ludwig-Maximilians-Universität a serious therapeutic concern. Two antago- München, Munich, GERMANY. nistic signaling peptides controls the transfer of tetracycline-resistance plasmid pCF10: Nature has evolved and exploits a rich spec- a peptide pheromone produced by plasmid- trum of architecturally diverse quorum sensing free recipient cells triggers the conjugative systems across and even within species. This

5th ASM Conference on Cell-Cell Communication in Bacteria 27 Speaker Abstracts diversity comprises both the way how cell den- cells sense small molecules to interact with sity information is encoded into a concentra- the environment and each other. Although it tion of signaling molecules as well as how this is clear that spatial relationships may impact information is decoded into a cellular response. how microbes perceive their surroundings, Here we focus on the encoding process and studying the molecular processes that govern investigate theoretically how differences in these interactions in a relevant context to signal transport, signal modification and site of nature remains challenging due to the techni- signal detection shape the encoding function cal difficulties associated with manipulating and affect the sensitivity and the noise charac- small populations. Micro-3D printing is a teristics of the cell-density-encoding process. lithographic technique capable of arranging We find that different network modules are bacteria in situ by printing protein-based walls capable of implementing both fairly basic around individual cells or small populations. as well as more complex encoding schemes, This 3D-printing strategy can organize bacteria whose qualitative characteristics vary with cell in complex arrangements to investigate how density and are linked to network architecture, spatial and environmental parameters influence providing the basis for a hierarchical classifi- social behaviors within small populations. cation scheme. We exploit the tight relation- Here, we introduce a combined approach, ship between encoding behavior and network where micro-3D printing and scanning electro- architecture to constrain the network topology chemical microscopy (SECM) are coupled to of partially characterized natural systems, create a quantitative spatial map of , and verify one such prediction by showing a quorum sensing (QS)-regulated redox-active experimentally that is capable metabolite produced by Pseudomonas aerugi- of importing 2. The framework nosa, in real time within groups of <105 cells. developed here may serve to guide the reverse We describe the process of merging these tech- engineering of natural systems and generally nologies for the specific application of measur- facilitates a better understanding of the com- ing pyocyanin production as a proxy for QS plexities arising in the quorum-sensing process activity within P. aeruginosa aggregates. We due to variations in the physical organization show that QS occurs within clusters as small as of the encoder network module. 500 cells, and characterize the requirements for neighboring communities to sense and respond n S5:4 to one another. These studies establish the value of this novel approach for determining P. C. Dorrestein; how spatial parameters influence bacteria. Univ. of Calif. San Diego, La Jolla, CA. n S5:6 n S5:5 Quorum sensing of Pseudomonas Electrochemical quantification of putida IsoF secondary metabolites produced by 3D-printed bacterial aggregates B. A. Hense; Institute for Computational Biology; Helm- J. L. Connell, J. Kim, A. J. Bard, M. Whiteley; holtz Zentrum Muenchen, Neuherberg/Munich, The University of Texas at Austin, Austin, TX. GERMANY. Bacteria are regularly found in nature as dense Quorum sensing (QS) regulates cooperative aggregates of ~101-105 cells. These commu- behaviour in a number of bacterial species. It nities are highly organized, and the spatial is increasingly recognized that environmental configuration within a cluster and between conditions influence quorum sensing systems neighboring microcolonies can influence how in many species. Some experimental setups,

28 ASM Conferences Speaker Abstracts e.g. batch cultures, inherently cause changes of n S6:1 the environmental conditions as depletion of SypF, an unusual two-component nutrients and accumulation of waste products. regulator of biofilm formation and Alternatively, approaches based on chemostats colonization or microfluidic devices have been used, the lat- ter focussing on attached, growth of attached K. L. Visick1, A. Norsworthy2; colonies instead of . A comparison of 1Microbiology and Immunology, Loyola the outcome of such different approaches will University Chicago, Maywood, IL, 2Loyola support the understanding of purpose and func- University Chicago, Maywood, IL. tionality of the signalling. We thus conducted Biofilm formation and colonization by Vibrio a series of studies investigating the dynamics fischeri depends on the expression of the syp of acyl homoserine lactone signal production (symbiosis polysaccharide) locus, a set of 18 and degradation in Pseudomonas putida IsoF. genes that encode proteins involved in synthe- The main hypothesis was that the QS system sizing, modifying, and exporting the polysac- works differently under changing environ- charide component of the matrix. A complex mental conditions resp. life styles (plankton phosphorelay comprised of multiple two- versus biofilm). a) The bacteria were grown component regulators dictates whether biofilm in batch cultures until their entry into the formation occurs. In culture, biofilm formation stationary phase. b) The behaviour under more depends on overexpression of one or another continuous conditions was investigated under of these regulators. Specifically, the unlinked chemostat conditions using different dilution sensor kinase RscS induces syp transcription rates. Time courses of cell density and con- in a mechanism that depends on the presence centrations of acyl homoserine lactones resp. of SypG, the response regulator that serves as their degradation products are analysed both the direct transcriptional activator. Data to date in chemostat and batch culture experiments. c) suggest that RscS activates SypG and inacti- Induction behaviour of the QS system in cells vates SypE, a second response regulator whose growing in surface-attached microcolonies was default activity is to inhibit biofilm formation analysed under flow chamber conditions. The at a level below syp transcription. Alterna- dynamics of QS controlled GFP fluorescence tively, biofilm formation can be induced by in the cells resp colonies was measured. The overexpression of SypF*, a mutant version of values of quorum sensing parameters were the hybrid sensor kinase SypF with increased estimated by fitting the data to mathemati- activity. Here, we probed the specific role of cal models. The planktonic studies a) and b) SypF in biofilm formation. We found that the revealed a surprising constancy with respect to ability of RscS to induce biofilm formation quorum sensing parameters, i.e. non-induced depends upon the presence of SypF, suggest- and induced signal production rates per cell, ing that SypF functions at or below RscS. and signal threshold. Modelling indicated that Indeed, our data indicate that SypF functions the determined parameters values also fit to between RscS and the two response regulators, the induction dynamics in microcolonies in SypG and SypE, to promote biofilm formation. study c). Thus, the QS system is rather robust Although wild-type SypF can function as a to changing environments, at least in the inves- canonical hybrid sensor kinase in vitro, the first tigated range. Interestingly, the flow chamber two of its three conserved sites of phosphory- experiments indicated that communication is lation are not required for the ability of SypF focussed rather within colonies than between to promote RscS-induced biofilm formation. colonies, raising the question to which degree Instead, only the C-terminal HPt domain of all cells in planktonic populations communi- SypF is crucial for this phenotype, a result that cate with each other. suggests that the two sensor kinase proteins

5th ASM Conference on Cell-Cell Communication in Bacteria 29 Speaker Abstracts participate in an atypical phosphotranfer reac- which drives it. Transposon mutagenesis has tion. To determine the physiological relevance yielded three classes of mutants: non-motile, of these findings, we assessed the role of SypF slow moving, and fast moving when interacting in host colonization, which does not require with the wild-type partner. In P. fluorescens, overexpression of regulators. Deletion of the we isolated five mutants which cause reduced sypF gene severely impaired colonization, social motility, two of which have insertions but this defect could be complemented by a in genes associated with Type 6 Secretion. wild-type copy of the sypF gene present in Among 10 mutants leading to increased social single copy in the chromosome. The coloniza- motility, seven are required for flagellum or tion defect could also be complemented by a pilus assembly. Of note in Pedobacter, two null sypF derivative that expressed the HPt domain mutants have insertions in predicted polyketide alone. Together, these findings indicate that synthase genes, while six fast mutants have RscS and SypF interact in an atypical way to insertions in genes for polysaccharide or LPS control biofilm formation and host coloniza- production. Experimental evolution has yielded tion by V. fischeri. spontaneous mutants in sectors migrating rapidly from the main mixed colony. Evolved n S6:2 Pedobacter and P. fluorescens strains conferring increased social motility have growth defects in Interaction between Pseudomonas monoculture, suggesting a fitness tradeoff. Phe- fluorescens Pf0-1 and Pedobacter notypes of evolved Pedobacter strains include sp. V48 Induces Social Motility increased mucoidy and production of a putative L. M. McCully1, S. C. Seaton2, A. S. Bitzer1, S. surfactant. Mucoid strains have point mutations B. Levy3, M. W. Silby1; in the same polysaccharide locus identified 1University of Massachusetts Dartmouth, by transposon mutagenesis. The isolation of North Dartmouth, MA, 2University of North transposon and spontaneous mutants of both Carolina Asheville, Asheville, NC, 3Tufts Uni- species which impact social motility indicates versity School of Medicine, Boston, MA. both species contribute to the trait, and coupled with transcriptome data, suggests a two-way Soil bacteria live in multispecies communities communication which is partially dependent on in which interactions influence physiology and a P. fluorescens T6SS. Our data suggest that a behavior. Here we describe “social motility” signal from P. fluorescens triggers production across a hard surface, a phenotype which is of a polysaccharide and a surfactant which both apparent during interaction between Pseudomo- contribute to social motility, but the mechanism nas fluorescens Pf0-1 and Pedobacter sp. V48, by which Pedobacter influences P. fluorescens but not displayed by either bacterium alone. remains unknown. Social motility is observable only when the bacteria are grown close together, suggesting a contact-dependent cell-cell interaction is neces- n S6:3 sary. Transcriptome analysis reveals numerous Manipulating the interspecies gene expression changes in both species during quorum sensing signal AI-2 in the interaction. The social motility represents a mouse gut convenient phenotype with which to dissect the J. Thompson, A. Oliveira, K. B. Xavier; basis of interaction between P. fluorescens and Instituto Gulbenkian de Ciencia, Oeiras, Pedobacter. We have used reciprocal transpo- PORTUGAL. son mutagenesis screens and experimental evo- lution to explore the mechanistic basis of social The mammalian gastrointestinal tract harbours motility and the contact-dependent interaction a diverse and complex resident bacterial

30 ASM Conferences Speaker Abstracts community, called the commensal microbiota, deltoides. We have shown that quorum sensing which interacts with the host and have been signal synthase (luxI homologs) and recep- implicated in many physiological processes of tor (luxR homologs) genes are prevalent in great importance to host health. The ability of isolated from Populus roots. bacteria to communicate within and between Interestingly, many of these isolates encode species through small signalling molecules to an orphan or solo LuxR homolog, which is regulate behaviours at the community level closely related to OryR from the rice pathogen is well-established. We hypothesise that such Xanthomonas oryzae. OryR does not respond communication, known as quorum sensing, to acyl-homoserine lactone quorum sensing plays a role in the inter-species interactions signals, instead it detects an unknown plant occurring in the establishment and mainte- compound. We discovered an OryR homolog nance of the gut microbiota. The quorum in an endophyte isolated from Populus roots, sensing signal autoinducer-2 (AI-2) is a strong Pseudomonas sp. GM79. We created a reporter candidate for this communication because it that was responsive to the GM79 OryR homo- can mediate inter-species quorum sensing and log and used it to show that, like the X. oryzae is produced by many members of the com- OryR, its activity increased in the presence of mensal microbiota. We have previously shown plant leaf macerates, but it was not influenced that the enteric bacterium Escherichia coli has by acyl-homoserine lactones. We examined the a mechanism to sequester and process AI-2 genomic region surrounding the GM79 plant- produced by a variety of species present in the responsive oryR homolog and found genes environment and thus can influence AI-2-de- annotated as peptidases and genes coding for a pendent bacterial behaviours in these neigh- putative ABC-type peptide transporter. The X. bouring bacteria. We are using engineered E. oryzae oryR is also flanked by a peptidase gene coli mutants to manipulate AI-2 levels in the and genes annotated as amino acid transport- mouse gut and determine the effect of this ers. Strains with mutations in the coding for manipulation within the antibiotic-treated the putative peptidases showed increased microbiota. Our results showed that increas- responses to plant macerates. A strain with ing AI-2 following antibiotic-induced gut a mutation in a gene coding for the putative dysbiosis had a measurable effect in the most ABC-type peptide transporter did not respond abundant bacterial Phyla present in the mouse to plant leaf macerates. We hypothesize that gut. These Phyla are known to be important for the plant signal(s) enters the bacterial cells by protection against pathogens, chemical agents, activate transport and that the peptidases affect and inflammatory bowel diseases. the activity of the signal. We purified the two peptidases and demonstrated they are most n S6:4 active against N-terminal proline and alanine dipeptides, suggesting that the signal may be a A Plant-Responsive LuxR Homolog in proline and/or alanine-containing peptide. We a Cottonwood Tree Root Endophyte have partially purified active signal(s) and the A. L. Schaefer1, Y. Oda1, J. Weiburg1, G.B. purified material can be partially inactivated by Hurst2, K. G. Asano2, V. Venturi3, C. S. Har- one of the peptidases. We believe that a better wood1, and E. P.Greenberg1; understanding of these OryR-type LuxR homo- 1University of Washington, Seattle WA, 2Oak logs is of general importance as they occur in Ridge National Laboratory, Knoxville TN, dozens of bacterial species that are associated 3ICGEB, Trieste, Italy. with economically important plants. We are interested in the root of the fast-growing Eastern cottonwood tree, Populus

5th ASM Conference on Cell-Cell Communication in Bacteria 31 Speaker Abstracts n S7:1 n S7:3 E. P. Greenberg; The evolution of quorum sensing in a Dept of Microbiology, University of Washing- naturally co-evovled host-pathogen ton, Seattle, WA. model L. Zhou1, L. Slamti2, C. Nielsen-LeRoux2, D. n S7:2 Lereclus2, B. Raymond1; 1 The Evolutionary Origins of Quorum Imperial College London - Silwood Park Sensing Campus, Ascot, SL5 7PY, UNITED KING- DOM, 2INRA, UMR1319 Micalis Génétique L. McNally, L. Chandler, S. P. Brown; Microbienne et Environnement, La Minière University of Edinburgh, Edinburgh, UNITED 78285 Guyancourt cedex, FRANCE. KINGDOM. Bacteria can engage in multicellular behaviour Quorum sensing (QS) communication in bac- via quorum sensing (QS), whereby bacteria teria has been heavily researched owing to its monitor population density through the secre- important role in regulating virulence in many tion of small, diffusible signal molecules. species. However, we know little regarding However, the evolutionary forces that maintain the evolutionary origins of QS systems: how QS have rarely been investigated in natu- does QS initially evolve and what did it evolve rally co-evolved host-pathogen systems. We to regulate? The evolutionary emergence of investigated the evolutionary ecology of the signalling systems presents a chicken and egg PlcR-PapR QS system in Bacillus thuringi- problem as a functioning signalling system ensis, in which the PlcR regulon controls the requires both the production of signals and production of various extracellular proteins, response to the signals of others: why signal if often involved in virulence, in response to an no one can respond, and why respond if no one autoinduced heptapeptide signal PapR. We is signalling? One solution to this paradox is tested the hypothesis that both signal produc- if secreted functional molecules first serve as tion and PlcR regulated gene expression are cues for behavioural responses in others, and social traits, and measured the invasion of are then subsequently co-opted (‘ritualised’) isogenic mutants at varying pathogen doses as true signal molecules. We use a phyloge- and mutant frequencies. Productivity in host netic comparative analysis across the diversity and infection success was positively correlated of QS systems in bacteria to show that most with the abundance of wild type in inocula. quorum signals appear to have initially been However, mutants could not outcompete extracellular public goods, suggesting that QS wild type bacteria in vivo. Experiments with initially evolves by co-opting public goods to homogenized insects indicate that mutants can regulate the expression of other traits. Further- outcompete wild type bacteria in homoge- more, using a combination of a phylogenetic neous environment. Microscopic observation comparative analysis and a meta-analysis of of insect sections with fluorescent QS cells transcriptomic studies of diverse QS systems showed that, in the midgut, bacteria population we show that the primary evolutionary func- was founded on isolated patches of 1 to 3 indi- tion of QS is to regulate the production of vidual cells 24 hours post ingestion. However, secreted public goods. Overall, our analyses a mixed population consisted of wild type and suggest that QS evolved via the co-opting of mutants was evident 48 hours post ingestion. secreted public goods to regulate other secre- The results suggested that spatial structure and tions coded in the genome. population bottleneck imposed by the midgut barriers limited invasion of QS mutants. The results confirm that clinical interventions to

32 ASM Conferences Speaker Abstracts prevent QS might be beneficial, as diverse arise during a long term selection experiment virulence factors are often regulated by QS. If in which P. aeruginosa PAO1 was cycled QS regulated virulence factors are required for between a private and public goods media. We an essential part of infection, inhibiting these found that individuals arose that lost the ability behaviours can be helpful. to respond synergistically to signal molecules, and we examined the relative fitness of these n S7:4 in competition experiments with the ancestor strain that maintains combinatorial signalling. Exploiting combinatorial signalling Our work therefore shows that by exploiting - a new type of cheating strategy in combinatorial signalling, P. aeruginosa can Pseudomonas aeruginosa overcome metabolic incentives to cooperate. J. Gurney, S. P. Diggle; More generally, we show that there are differ- University of Nottingham, Nottingham, ent types of cheating strategy, and we add to UNITED KINGDOM. the growing body of evidence that a number of factors are required to work together to control The notion of exploiting sociality and social social cheating in natural environments. traits in bacteria has received wide attention in recent years in the context of evolutionary dynamics and understanding the evolution of n S7:5 virulence, and ultimately infection. A recurring Facultative cheating drives the question in the literature focuses around how maintenance of Quorum-Sensing do bacteria in natural environments maintain pherotype diversity in structured social traits when cheats have a higher relative populations fitness? One way to achieve this is to link the A. Eldar; acquisition of private goods to the same sys- Tel Aviv University, Tel Aviv, ISRAEL. tem that produces public goods. We recently showed that the opportunistic pathogen Pseu- Bacteria utilize quorum-sensing (QS) to regu- domonas aeruginosa can sense and respond late the secretion of other molecules, which combinatorially and in some cases synergisti- serve as public goods, benefiting all members cally to two signal molecules. By producing of the community. In sufficiently well mixed and responding to multiple signals, bacteria conditions, such behavior is susceptible to can use quorum sensing (QS) to correctly invasion by QS reception “cheater” mutants determine a combination of population density that avoid the costs of quorum-response but and other environmental factors. Correctly benefit from the response of others. In wild responding to a particular environment allows isolates, QS reception mutants are rare, sug- bacteria to maximise their fitness whilst main- gesting that either QS drives the production of taing social traits. However this introduces an private goods or that cooperation is maintained interesting conundrum that potentially allows by the high relatedness of cooperating cells. bacteria to cheat while still producing a certain In contrast, many bacterial species show high level of public goods. By losing the synergistic intra-specific allelic divergence of their QS response to a combination of molecules, an locus, forming multiple pherotypes where a individual would still be able to survive during signal from one pherotype specifically acti- the restrictions levied through private goods vates its cognate receptor but not the receptor metabolism and would have a higher relative of another pherotype. Often, these pherotypes fitness during public goods production as they show a phylogenetic distribution indicating benefit from those in the population producing their horizontal transfer (HGT) within the public goods at the higher synergistic level. species. It remains unclear what evolutionary We tested whether such signal synergy mutants forces select for pherotypes divergence, its

5th ASM Conference on Cell-Cell Communication in Bacteria 33 Speaker Abstracts maintenance and its horizontal spread. Here we in and that under these conditions, combine bioinformatics, mathematical model- a minority pherotype invades into the majority ing and experimentation to demonstrate that pherotype. Importantly, we show that selec- facultative cheating between pherotypes can tion for the minority pherotype occurs also in select for rapid even spatially structured populations that prevent under conditions where relatedness between the invasion of receptor mutants. Our results cells is high enough to prevent the spread therefore support a social role for QS and illus- of cheaters. We show that the comQXP QS trate the complexity of evolutionary dynamics system of B. subtilis has a cooperative function of bacterial social system.

34 ASM Conferences Poster Abstracts n 1 tions with the methylation susbrate SAM and Mechanism for the specific the methylation product SAH (Garcia-Fontana, targeting of methyltransferases Corral Lugo, & Krell, 2014). The titration of to chemoreceptors in Pseudmonas the four CheR paralogues with any of the three aeruginosa PAO1 above mentioned pentapeptides revealed a single interaction, namely that between CheR2 A. Corral Lugo, C. García Fontana, M. Espi- and the GWEEF pentapeptide, which was nosa Urgel, T. Krell; derived from the McpB chemoreceptor. Inter- Consejo Superior de Investigaciones Científi- estingly, the genes encoding CheR2 and McpB cas, Granada, SPAIN. are vicinal on the genome and form both part of the che2 chemosensory pathway. This Bacteria constantly sense and adapt to interaction was also detected for the titration of changing environmental conditions to assure full-length McpB with CheR2. In vitro assay survival. This important function is primar- shows that McpB is exclusively methylated by ily mediated by one-component systems, CheR2. Deletion of the terminal pentapeptide two-component systems, and chemosensory from McpB abolished both, the interaction pathways. Core proteins of chemosensory and methylation of McpB. When clustered pathways are the CheA sensor kinase, CheW according to protein sequence, bacterial CheR coupling protein, CheY response regulator, proteins form two distinct families-those that CheR methyltransferase, CheB methylesterase, bind pentapeptide: containing chemoreceptors and chemoreceptors. Methyltransferases of the and those that do not. These two families are CheR family and methylesterases of the CheB distinguished by an insertion of three amino family control chemoreceptor methylation, and acids in the β-subdomain of CheR. Deletion of this dynamic posttranslational modification is this insertion in CheR2 prevented its interac- necessary for proper chemotaxis of bacteria. tion with and methylation of McpB. Data Studies with enterobacteria that contain a sin- suggest that the CheR2-McpB interaction is a gle CheR and CheB show that, in addition to strict requirement for any methylation activity. binding at the methylation site, some chemore- Pentapeptide-containing chemoreceptors are ceptors bind CheR or CheB through additional common to many bacteria species; thus, these high-affinity sites at distinct pentapeptide short, distinct motifs may enable the specific sequences in the chemoreceptors. Pseudomo- assembly of signaling complexes that mediate nas aeruginosa PAO1 has five gene clusters different responses. encoding chemosensory signaling proteins that assemble into four chemosensory pathways, termed Che, Che2, Wsp, and Chp. Apart from n 2 the 26 chemoreceptor genes of P. aerugi- Evolution of resistance to a last- nosa this strain has four CheR paralogous resort antibiotic in Staphyloccocus methyltransferases (CheR1, CheR2, CheR3, aureus via bacterial competition and WspC). Three of these chemoreceptors G. Koch, D. Lopez; have C-terminal extensions with terminaal University of Wuerzburg, Wuerzburg, pentapeptides of sequence GWEEF, EVELF GERMANY. or GVEQA. We produced the four CheR methyltransferases of P. aeruginosa as purified Antibiotic resistance is a key medical concern, recombinant proteins, and their functionality with antibiotic use likely being an important was validated using microcalorimetric titra- cause. However, here we describe an alterna-

5th ASM Conference on Cell-Cell Communication in Bacteria 35 Poster Abstracts tive route to clinically-relevant antibiotic pathogen Staphylococcus aureus. We have resistance that occurs solely due to competitive performed a number of biochemistry and cell interactions between bacterial cells. We consis- biology approaches to ultimately understand tently observe that isolates of Methicillin-resis- the influence of FloA in the activation of the tant Staphylococcus aureus diversify spontane- diverse signal transduction pathways that are ously into two distinct, sequentially arising related to the development of staphylococcal strains. The first evolved strain outgrows the infections. Further characterization of these parent strain via secretion of surfactants and interactions led us to find sophisticated regula- a toxic bacteriocin. The second is resistant tory mechanisms in signaling transduction, to the bacteriocin. Importantly, this second which should directly influence an infection strain is also resistant to intermediate levels of process. Overall, the discovery of lipid rafts in vancomycin. This so-called VISA (vancomy- bacteria reveals an unexpected level of sophis- cin-intermediate S. aureus) phenotype is seen tication in signal transduction and membrane in many hard-to-treat clinical isolates. This organization that is unprecedented in bacteria strain diversification also occurs during in vivo and shows that bacteria as more complex infection in a mouse model, consistent with the organisms than previously appreciated. fact that both coevolved phenotypes resemble strains commonly found in clinic. Our study n 4 shows how competition between coevolving A novel function to a known bacterial strains can generate antibiotic resis- compound: How Pseudomonas tance and recapitulate key clinical phenotypes. protegens inhibits biofilm formation in Bacillus subtilis n 3 M. J. Powers1, E. Sanabria-Valentín2, A. Bow- Structural and functional ers3, E. A. Shank1; characterization of the raft- 1Department of Biology, University of North associated protein Flotillin from Carolina at Chapel Hill, Chapel Hill, NC, 2De- the bacterium Staphylococcus partment of Microbiology and Molecular Ge- aureus netics, Harvard Medical School, Boston, MA, S. T. Stengel, G. Koch, D. Lopez; 3Division of Chemical Biology and Medicinal University of Würzburg, Würzburg, GER- Chemistry, University of North Carolina at MANY. Chapel Hill, Chapel Hill, NC. Bacterial membranes contain discrete micro- Interspecies signaling has redefined our domains that are structurally and functionally understanding of how bacteria live and survive similar to the lipid rafts of eukaryotes. An im- in the world. Here, we focus on an interaction portant structural component of the lipid rafts between Bacillus subtilis and Pseudomonas is the membrane-bound protein Fotillin, which protegens, two common soil bacteria known to is a chaperone responsible for the recruit- occupy the same plant-root niche in the natural ment of other proteins to lipid rafts, thereby environment. P. protegens was identified in facilitating raft-harbored protein interactions. a co-culture screen designed to discover soil The ease with which bacteria can be geneti- bacteria that inhibit biofilm formation in B. cally modified offers a tractable model to study subtilis. This screen used a B. subtilis reporter the physiological role of Flotilllin within the strain in which a biofilm-specific promoter lipid rafts, which has complicated studies in drives the transcription of proteins that inhibit their eukaryotic counterparts. Here we present the transcription of the yellow fluorescent a structural and functional characterization protein. Thus, B. subtilis cells that are not of Flotillin protein FloA from the human producing biofilm can be easily identified by

36 ASM Conferences Poster Abstracts their fluorescence. In order to explore how P. 6h of starvation. The C-signal is a 17 kDa cell- protegens inhibited B. subtilis biofilm gene surface associated protein (p17) generated by expression, we used its conditioned medium N-terminal proteolytic processing of a 25kDa to perform a series of biological assays and precursor protein (p25), encoded by csgA chemical fractionations. In combination with gene. p25 is present in both vegetative and analytical LC-MS and NMR, we identified starving cells, however it is only cleaved to the compound responsible for the observed generate p17 during starvation. The molecular biological activity as 2,4-diacetylphloroglu- mechanism underlying the regulated proteoly- cinol (DAPG). DAPG is a known compound sis of p25 is based on regulated secretion of secreted by P. protegens; however, it has not the subtilisin-like serine protease PopC. PopC previously been shown to inhibit biofilm is retained within the cytoplasm of vegetative formation in any bacterium. To determine how cells and is slowly secreted only during starva- DAPG affects biofilm formation in B. subtilis, tion thus restricting p25 cleavage to starving we characterized its effect on pellicle forma- cells. Recent work has defined the regulatory tion both phenotypically and with flow cytom- mechanism by which PopC secretion is acti- etry using transcriptional reporters for various vated. PopC secretion is controlled post-trans- developmental processes in B. subtilis. This lationally by a cascade involving PopD and approach allowed us to quantify B. subtilis cel- (p)ppGpp synthase RelA. During vegetative lular development at the single-cell level, and growth, PopC and PopD form a cytoplasmic examine the heterogeneity of the cells present complex, which blocks PopC secretion. In in pellicles and biofilms in response to DAPG. response to starvation, RelA is activated and We are currently exploring the genetic pathway induces the proteolytic degradation of PopD through which DAPG acts in B. subtilis, as leading to release of PopC for secretion. Regu- well implementing an in vivo model to observe lated proteolysis of PopD appeared to require these bacterial interactions on plant roots. By ATP-dependent metalloprotease activity with understanding how DAPG inhibits biofilm some evidence indicating integral membrane formation in B. subtilis, we will gain insights AAA+ protease FtsHD. Recently, we have iso- into how bacterial interspecies signaling can lated a collection of non-developing mutants impact processes important to both plant and affected in PopC secretion and thus in genera- human health. tion of active C-signal. Complete genome sequencing revealed point mutations in LonD, n 5 another ATP dependent metalloprotease. In our current studies, we are investigating the role of LonD is required for regulated LonD in PopC secretion and in development of secretion of the signalling protease Myxococcus xanthus in general. PopC in Myxococcus xanthus M. A. Polatynska, A. Konovalova, L. Søgaard- n 6 Andersen; Molecular Basis of TraA Recognition Max Planck Institute Marburg, Marburg, that Governs Outer Membrane GERMANY. Exchange and Social Behaviors in The intercellular C-signal plays a fundamen- Myxobacteria tal role in fruiting body morphogenesis in P. Cao, D. Wall; M. xanthus. It induces and coordinates three University of Wyoming, Laramie, WY. elementary processes involved in fruiting body formation: rippling, aggregation and sporula- Kin recognition plays a key role in how tion. It is also required for the expression of free-living microbes identify sibling cells to developmental genes that are turned on after mediate beneficial social behaviors. Myxococ-

5th ASM Conference on Cell-Cell Communication in Bacteria 37 Poster Abstracts cus xanthus, a soil-dwelling bacterium, serves n 7 as an important model system for studying RNA-seq-based transcriptome complex multicellular interactions in bacteria. analysis of quorum sensing Previously we described a process whereby effecting on Staphylococcus aureus myxobacteria transiently fuse and exchange biofilms their outer membrane (OM) proteins and lip- ids. Two genetic determinants required for OM X. Tan1, N. Qin2, X. Peng1, A. Jia1; exchange are called TraA and TraB. Studies of 1Nanjing University of Science & Technol- OM exchange among M. xanthus environmen- ogy, Nanjing, CHINA, 2Zhejiang University, tal isolates suggested that TraA functions as Hangzhou, CHINA. a homophilic cell surface receptor to identify Background: Expression of most virulence sibling cells to engage in OM exchange. Here factors in Staphylococcus aureus is controlled cellular “goods” are shared among individuals by the accessory gene regulator (agr) locus, that carry the same or very similar traA alleles. which encodes a two-component signaling To elucidate the molecular mechanism of TraA pathway whose active is a bacterial recognition, we sought to identify key sub- quorum sensing peptide (autoinducing peptide regions and amino acids within TraA which [AIP]) encoded by agr. The agr locus consists govern specificity. To do this we created a of four genes agrBDCA. A polymorphism in series of chimeric traA alleles within the poly- the amino acid sequence of AIP and of its cor- morphic region (PA14-like domain) that de- responding receptor (AgrC) divides S. aureus termines specificity. Recognition between the strains into four major groups, agr-(I-IV). All chimeric alleles and parental alleles were then agr-(II-III) isolates were defective in agrDCA tested by an extracellular complementation and consequently they do not have a functional assay whereby motility mutants can be rescued AIP. In addition, agr-III strains are able to by OM transfer of deficient motility proteins. transcribe icaR, sarA, and rsbU at the late- and Strikingly, from these chimeric and subsequent post-exponential phases. agr-(I-IV) variants site directed mutagenesis studies, one particu- have functional agr-locus. Recent reports lar amino acid position was shown to govern indicated that all glycopeptides intermediate cell-cell specificity among a sub-group of traA S. aureus strains (GISA) examined belong- alleles. In these studies, recognition could be ing to agr-II were defective for agr function. switched by simply changing a single amino In contrast, these strains were strong biofilm acid. These findings support our model that kin producers. In this work, we confirm that S. recognition in OM exchange is governed by aureus ATCC25923, a quality control strain TraA homotypic interactions between adjacent in antimicrobial susceptibility test, is capable cells. Additionally, these findings provide a of forming biofilm in vitro, which belongs to foundation for predicting uncharacterized TraA agr-III strain. Therefore, these findings lead recognition groups and for engineering new to a hypothesis that S. aureus may have an recognition groups de novo. enhanced ability of forming a thick biofilm due to agr-locus inactivation, which is unrelated to resistance to antibiotics. Methods: To inves- tigate the possible mechanisms of methicillin- susceptible S. aureus biofilm formation, firstly, we used crystal violet staining assays and SEM images to measure the impacts of two compounds (ursolic acid and resveratrol, which could inhibit MRSA biofilm) on S. aureus ATCC25923 biofilm. Next, we used

38 ASM Conferences Poster Abstracts high-throughout Illumina sequencing of cDNA tunistic pathogen, Pseudomonas aeruginosa (Illumina RNA-seq) to study the differentially is one of the well-known bacteria in the study expressed genes of S. aureus ATCC25923 of MVs. In this bacterium, the Pseudomonas biofilm by addition of two compounds and quinolone signal (PQS), quorum-sensing compared with differentially expressed genes signal of P. aeruginosa, is reported to be one of MRSA biofilm supplemented with the of the major factors that induce MV forma- same compounds as previously described by tion in previous studies (Whitely et al. 2005, our group. Results and Discussion: Crystal Tashiro et al. 2010). In addition, we found that violet staining assays and SEM images showed P. aeruginosa produces MVs under anaero- that S. aureus ATCC25923 biofilm is thicker bic condition and MVs formation is induced than MRSA’s and its biofilm growth is faster. through SOS response, which is relative to However, only ursolic acid could inhibit its repairing DNA damage (Toyofuku et al. 2013). biofilm formation. RNA-seq transcriptome Further studies revealed that the production of analysis indicated that the MSSA may have an pyocin, which is induced by SOS response, is enhanced ability of forming a thick biofilm due involved in MV formation. In this study, we to agr-locus inactivation, which is unrelated to examined how pyocin production induces MV resistance to antibiotics. In addition, the data formation. As a result, the mutant of PA0614 showed that once sensitive strain formed bio- and PA0629, which encode putative holin and film, such sensitive strain would become drug- endolysin, respectively, formed less MVs than resistant strain. Meanwhile, sensitive strains WT. Furthermore, MV production increased biofilms resistance to antibiotics may be higher by the expression of PA0614 and PA0629 in than some drug-resistant strains biofilms due to Escherichia coli, however, no increase of agr-locus inactivation. Therefore, the infection MV production was observed by PA0629 from antimicrobial sensitive clinical S. aureus that carried a point mutation in the catalytic can not be ignored. Further studies are neces- site. Hence, holin-lysin system, the system sary to identify the correlation mechanism of considered to be involved in bacteriophage or other clinical S. aureus biofilm formation and bacteriocin release by the degradation of the agr-regulated quorum sensing system. peptidoglycan, is considered to be involved in MV formation. Generally, Holin-lysin system n 8 is known to be widely conserved in bacteria. Therefore, we suggest that this mechanism is Membrane vesicle formation possibly a common MV formation mechanism via “Holin-Endolysin” system in in bacteria. Pseudomonas aeruginosa PAO1 M. Kurosawa, M. Toyofuku, N. Nomura; n 9 University of Tsukuba, Tsukuba, JAPAN. Toxin YafQ and Phosphodiesterase Bacteria can adapt to surrounding environ- DosP Increase Persister Cell ment and communicate with other cells using Formation by Reducing Indole various cellular materials. Membrane vesicles Signaling (MVs) are naturally produced in Gram- T. K. Wood; negative bacteria, and considered to deliver Pennsylvania State University, University cellular contents including proteins, DNA and Park, PA. signaling molecules to prokaryotic and eukary- otic cells. Although their biological functions Persister cells survive stresses by slowing me- of MVs in the bacterial communities have tabolism. Since toxins of toxin/antitoxin (TA) become recognized to date, how MV formation systems increase persister cell formation, we is regulated is poorly understood. The oppor- investigated the influence of toxin YafQ of the

5th ASM Conference on Cell-Cell Communication in Bacteria 39 Poster Abstracts

YafQ/DinJ E. coli TA system on persister cell increased persistence and reduced indole levels formation. Under stress, toxin YafQ alters me- similarly to DosP. Therefore, phosphodiester- tabolism by cleaving transcripts with in-frame ase DosP increases persistence by reducing the 5’-AAA-G/A-3’ sites. We found that produc- interkingdom signal indole via reduction of the tion of YafQ increased persister cell formation global regulator cAMP. Hence, two distinct dramatically with multiple antibiotics, and methods were used to show that the inter- through proteomics, found that YafQ reduced kingdom signal indole is inversely related to tryptophanase levels (TnaA mRNA has 16 pu- persistence. We previously demonstrated that tative YafQ cleavage sites). Consistently, TnaA indole (i) is primarily active at low tempera- mRNA levels were also reduced by YafQ. tures whereas AI-2 is the primary signal in the Tryptophanase is activated in the stationary GI tract, (ii) reduces the pathogenicity of cells phase by the stationary-phase sigma factor that do not synthesize it, (iii) influences biofilm RpoS, which was also reduced dramatically formation, (iv) and tightens epithelial cell upon production of YafQ. Tryptophanase con- junctions in the GI tract. verts tryptophan into indole, and as expected, indole levels were reduced by the production n 10 of YafQ. Corroborating the effect of YafQ Effect of c-di-GMP on biofilm on persistence, addition of indole reduced formation and motility of persistence. Furthermore, persistence increased Campylobacter jejuni upon deleting tnaA, and persistence decreased upon adding tryptophan to the medium to B. A. Elgamoudi, J. Ketley; increase indole levels. Also, YafQ production University of Leicester, Leicester, UNITED had a much smaller effect on persistence in KINGDOM. a strain unable to produce indole. Therefore, Cyclic Diguanosine Monophosphate (c-di- YafQ increases persistence by reducing indole, GMP) is a naturally occurring small molecule and TA systems are related to . that acts as an intracellular second messenger Furthermore, given that the population of per- regulating important signalling systems in bac- sisters increases in biofilms and that c-di-GMP teria, including motility and biofilm formation. is an intracellular signal that increases biofilm The role of c-di-GMP in Campylobacter jejuni formation, we sought to determine whether is unknown. In this study, the biofilm inhibi- c-di-GMP has a role in bacterial persistence. tion assay was used along with Confocal Laser By examining the effect of 30 genes from Scanning Microscopy (CLSM) to investigate E. coli, including diguanylate cyclases that the effect of different concentrations of extra- synthesize c-di-GMP and phosphodiesterases cellular c-di-GMP on C. jejuni. We found that that breakdown c-di-GMP, we determined that extracellular c-di-GMP significantly reduced DosP (direct oxygen sensing phosphodiester- (>50%) biofilm formation compared to the ase) increases persistence by over a thousand untreated control with no effect on growth. We fold. Using both transcriptomic and proteomic also found that c-di-GMP promotes chemotac- approaches, we determined that DosP increases tic motility as judged by a plate-based swarm persistence by decreasing tryptophanase activ- assay. This observation may indicate a role in ity and thus indole. Despite the role of DosP the transition between the sessile and motile as a c-di-GMP phosphodiesterase, the decrease lifestyle of C. jejuni. These results suggest in tryptophanase activity was found to be a that c-di-GMP inhibits biofilm formation by result of cyclic adenosine monophosphate modulating the transition between biofilm to (cAMP) phosphodiesterase activity. Corrobo- planktonic forms. rating this result, the reduction of cAMP via CpdA, a cAMP-specific phosphodiesterase,

40 ASM Conferences Poster Abstracts n 11 sium leakage, which was previously suggested Quantitative in vivo functional to be induced by surfactin, a cyclic lipopeptide assays suggest how a antibiotic. transmembrane histidine kinase KinC becomes active to induce n 12 cannibalism and biofilm formation in Evidence that autophosphorylation Bacillus subtilis of the major sporulation kinase in S. N. Devi, M. Vishnoi, B. Kiehler, S. Thri- Bacillus subtilis occurs in trans kawala, L. Haggett, M. Fujita; B. Kiehler, S. N. Devi, S. Thrikawala, L. University of Houston, Houston, TX. Haggett, M. Fujita; University of Houston, Houston, TX. Upon starvation, Bacillus subtilis cells dif- ferentiate into different subsets, undergoing Entry into sporulation in Bacillus subtilis is cannibalism, biofilm formation, or sporulation. governed by a multi-component phosphorelay, These processes require a multiple component a complex version of two-component system phosphorelay, wherein the master regulator which includes five histidine kinases (KinA- Spo0A is activated upon phosphorylation by KinE), two phosphotransferases (Spo0F and one or a combination of five histidine kinases Spo0B), and a response regulator (Spo0A). (KinA-KinE) via two intermediate phos- Among five histidine kinases, KinA is known photransferases, Spo0F and Spo0B. While as the major sporulation kinase, where it is KinA and KinB are known to be primarily autophosphorylated with ATP upon starva- responsible for sporulation, KinC has been tion and then transfers phosphate group to the demonstrated to contribute for triggering bio- downstream components (Spo0F, Spo0B and film formation and cannibalism. At present, it Spo0A) in a His-Asp-His-Asp signaling path- remains unknown how KinC becomes active. way. The recent results of the computational In this study, we established an in vivo quan- approach suggest that the autophosphoryla- titative assay system for KinC activity using tion reaction of KinA occurs within the same an IPTG-inducible expression system. Using protomer in the homodimer in a cis fashion. this system, we found that the N-terminal However, no direct experimental data is avail- transmembrane domain is dispensable but the able to support this notion. Our recent study PAS domain is needed for the kinase activ- demonstrated that KinA forms a homotetramer, ity. Second, an in vivo chemical cross-linking not dimer, mediated by the N-terminal domain, experiment demonstrated that the soluble and as a functional unit. Furthermore, when the functional KinC (KinC∆TM1+2) forms a N-terminal domain was overexpressed in the tetramer. Third, genetic experiments reveal that wild-type strain cultured under starvation con- KinC activity and the membrane localization ditions, sporulation was impaired. This impair- are independent of both the lipid raft marker ment of sporulation can be explained by form- proteins FloTA and cytoplasmic potassium ing non-functional heterotetramer of KinA, concentration, which were previously shown to resulting in the reduced level of Spo0A~P. be required for the kinase activity. Finally, we These results suggest that autophosphoryla- demonstrated that KinC controls cannibalism tion of KinA occurs in a transphosphorylation and biofilm formation in a manner dependent manner. To test this hypothesis and provide on phosphorelay. Based on these results, direct evidence whether the autophosphoryla- we propose a revised model in which KinC tion of KinA occurs in a cis- or a trans-fashion, becomes active by forming a homotetramer via we generated a series of strains expressing the N-terminal PAS domain, but its activity is homogeneous or heterogeneous protein com- independent of both lipid raft and the potas- plexes consisting of various combinations of

5th ASM Conference on Cell-Cell Communication in Bacteria 41 Poster Abstracts the phospho-accepting histidine point mutant the biofilm development in the long run, but protein and the catalytic ATP-binding domain may not of benefit in a short time window. In point mutant protein. Each of the purified addition, our model suggests that the induction homo- and heterotetramers was incubated with of quorum sensing in biofilm is sensitive to [γ-32P]ATP in vitro and the reaction mixtures hydrodynamic stress. were analyzed on SDS-PAGE followed by au- toradiography. The results suggested that ATP n 14 initially binds to one or more protomers within The role of Vibrio cholerae Qrr1- the tetramer complex and then the γ-phosphate 4 sRNAs in maintaining community is transmitted to another in a trans fashion. stability through quorum sensing We further found the ATP-binding deficient point mutant is complemented in vivo by the L. A. Hawver, W. Ng; phosphorylation deficient point mutant. Taken Tufts University, Boston, MA. together, these in vivo and in vitro results rein- Vibrio cholerae (Vc), the causative agent of force evidence that KinA autophosphorylation the acute diarrheal disease cholera, utilizes occurs in a trans fashion. a highly regulated quorum sensing (QS) circuit to control the expression of many n 13 genes including those involved in virulence A Three-dimensional Hydrodynamic and biofilm formation. The switch between Model for biofilms coupled with low cell density (LCD) and high cell density Quorum Sensing (HCD) gene expression in Vc is assumed to center on the reciprocal production of two J. Zhao, Q. Wang; transcriptional regulators AphA and HapR. University of South Carolina, Columbia, SC. At LCD, four small RNAs (sRNAs) Qrr1-4 Biofilms are , where bacteria are made and they activate and repress the stay together, embedded in glue-like extra- expression of AphA and HapR, respectively. At cellular polymeric substances (EPS). It is HCD, Qrr1-4 are not made, thus, AphA expres- observed that bacteria in biofilms communicate sion is repressed and production of HapR is and cooperate by sensing the density of signal- activated. Aside from virulence and biofilm ing molecules, which phenomenon is common formation, QS is believed to be important for known as quorum sensing. Recently, we have maintaining the stability of a bacterial com- developed a 3d hydrodynamic model to ac- munity through synchronizing gene expression count for the bacterial cell’s quorum sensing according to cell density. However, the genes ability by classifying the bacterial cells into that are important for maintaining community downgraded and upgraded quorum sensing stability and the mechanism of QS control are cells, as well as non quorum sensing cells, in not well defined. We hypothesize that, because which quorum sensing molecule is introduced QS is employed to monitor cell density and to regulate the quorum sensing activity. We species complexity, QS is likely involved in implement the model in full three dimension in coping with stress caused by overpopulation space to arrive at a numerical solver studying and resource deprivation. Therefore, there biofilm development under quorum sensing are additional roles for QS in adapting to the regulation, as well as hydrodynamic effects on fluctuations in the environment that occur quorum sensing induction. Our model predicts when exogenous nutrients are consumed and that quorum sensing regulation contributes to metabolized. To test this idea, we studied heterogeneous structure development during how different Vc QS mutants behave in the biofilm formation. Besides, our model have presence of metabolic stresses. We show that a shown that QS regulation is beneficial for genetically LCD-locked Vc mutant constantly

42 ASM Conferences Poster Abstracts producing Qrr1-4 is sensitive to accumulation signal factors GABA and succinic semial- of by-products of carbohydrate metabolism, dehyde (SSA) activated gabT expression. while a genetically HCD-locked Vc mutant GABA- and SSA-inducible gabT transcription producing no Qrr1-4 is not. By testing mutants was controlled by Sigma 54 and activated by defective in different downstream QS pathway GabR, but the induction of gabT transcription components, we show that Qrr1-4 sRNAs, by GABA and SSA is not the result of Sigma but not AphA nor HapR, contribute to the 54 or GabR induced expression. Deletion of hypersensitivity to carbohydrate metabolism. the PAS domain of GabR resulted in increased Our findings suggest Vc enlists an sRNA- gabT transcription activity, both in the pres- dependent, HCD-specific gene expression pro- ence or absence of GABA. It suggests that the gram to resolve metabolic stress. We further PAS domain of GabR acts as a signal sensor tested the idea that the ability to withstand the domain that in the presence of GABA and accumulation of toxic by-products is essential SSA relieves GabR enhancer transcriptional for maintaining a stable community of Vc, activity. and observed that the inability to withstand such toxicity can cause an entire population to n 16 collapse over time. Uncovering the mechanism Membrane vesicle trafficking of of QS control of population stability during a long-chain acyl-homoserine resource deprivation will help us to better lactone, C16-HSL understand the role that cell signaling plays in cell survival when faced with other potential M. Toyofuku1, Y. Hashimoto2, H. Inaba2, N. stress conditions. Nomura1; 1University of Tsukuba, Tsukuba, JAPAN, n 15 2Sumitomo Heavy Industries, Yokosuka, JAPAN. Activation of gab cluster transcription in Bacillus N-Acyl-homoserine lactone (AHL) is one of thuringiensis by γ-aminobutyric acid the most common classes of signals produced or succinic semialdehyde is mediated in Gram-negative bacteria. The acyl side chain by the Sigma 54-dependent activator of the AHLs varies in length that confers the GabR specificity to the signal. Paracoccus denitri- ficans is a Gram-negative bacterium capable Q. Peng, M. Yang, J. Zhang, F. Song; of thriving under aerobic and anoxic condi- Institute of Plant Protection,Chinese Academy tions. This bacterium has been reported to of Agricultural Sciences, Beijing, CHINA. produce a long chain acyl-homoserine lactone GabR is a Sigma 54 (C16-HSL) that is predicted to be highly (encoded by sigL)-dependent activators that hydrophobic. A previous report has suggested has three typical domains, an N-terminal that the majority of the C16-HSL produced regulatory domain Per-ARNT-Sim (PAS), a in P. denitrificans is associated with the cell central AAA+ (ATPases associated with differ- membrane (Schaefer et al., J. bac. (2002)), ent cellular activities) domain and a C-terminal questioning how the signal is excreted to the helix-turn-helix (HTH) DNA binding domain. extracellular environment. Here we show that GabR positively regulates the expression of in a Paracoccus denitrificans closely related the gab gene cluster, which is responsible for strain, Paracoccus sp. N11 that we isolated the γ-aminobutyric acid (GABA) shunt. Puri- from an activated sludge, C16-HSL is packed fied GabR was found to specifically bind to a in membrane vesicles (MV). Paracoccus sp. repeat region that mapped 58 bp upstream of N11 produced MVs under normal culture the start codon of the gabT gene. The specific conditions that were examined by transmission

5th ASM Conference on Cell-Cell Communication in Bacteria 43 Poster Abstracts electron microscopy. C16-HSL production it dampened the maturation of mushroom in N11 strain and its presence in MVs were structure and crumbled biofilm at late stage, confirmed by MS analysis. Our results dem- making flat biofilm. Anthranilate was able to onstrate that once packed in MVs, C16-HSL crumble the pre-formed biofilm and extracel- is able to freely diffuse in aqueous environ- lular polymeric substance (EPS) staining also ments while C16-HSL alone is absorbed to showed the biofilm-crumbling effect of anthra- hydrophobic surfaces. Interestingly, MVs were nilate. To investigate the interplay of anthra- also isolated from activated sludge, suggesting nilate with indole in the biofilm formation, we their active roles in the natural environment. co-treated anthranilate with indole and found MVs have been shown to carry Pseudomo- that the addition of anthranilate abolished the nas quinolone signal (PQS) in Pseudomonas biofilm-enhancing effect of indole. Interest- aeruginosa (Mashburn et al., Nature (2005)), ingly, the anthranilate degradation pathway and our results strengthen the role of MVs in was synergistically activated by co-treatment cell-cell communication, and further imply that of anthranilate and indole. HPLC analysis other hydrophobic long-chain AHLs may be shows that the anthranilate accumulation in P. carried by MVs. aeruginosa decreased by the indole-treatment, demonstrating that the indole-activation of n 17 the anthranilate degradation pathway reduced the anthranilate level. Based on these results, Anthranilate, an intermediate we suggest two points: 1) anthranilate has a of tryptophan metabolism crumbling effect on biofilm formation of P. antagonizes biofilm formation and aeruginosa and can be a promising anti-biofilm indole signaling in Pseudomonas agent. 2) the biofilm-enhancing effect of indole aeruginosa may come from the reduction of anthranilate L. Xi-Hui, K. Soo-Kyoung, L. Joon-Hee; level. Pusan National University, Busan, KOREA, REPUBLIC OF. n 18 Anthranilate and indole are aromatic com- The ornithine lipid metabolism pounds that are alternatively produced from influences the biofilm formation tryptophan degradation according to bacterial and virulence of Pseudomonas species. P. aeruginosa produces anthranilate aeruginosa by modulating quorum from tryptophan degradation. While the effects sensing of tryptophan and indole on the biofilm forma- K. Soo-Kyoung, L. Xi-Hui, L. Joon-Hee; tion of bacteria have been reported, the influ- Pusan National University, Busan, KOREA, ence of anthranilate on biofilm formation was REPUBLIC OF. not addressed. In this study, we investigated the anthranilate effects on the P. aeruginosa Ornithine lipids (OLs) are a component of biofilm formation. While indole enhances and bacterial membrane. OLs are widely found in accelerates the biofilm formation of P. aerugi- outer membrane of many gram-negative bac- nosa throughout development, the anthranilate teria, but not detected in Eukarya and Archaea. effect on biofilm formation was differentially Pseudomonas aeruginosa, an opportunistic exerted depending on the developmental stage pathogen has olsBA genes that constitute an and the presence of shear force. Anthranilate operon to function the ornithine lipid biosyn- a bit accelerated the initial attachment of P. thesis. olsBA encodes acyltransferases and aeruginosa at the early stage of biofilm devel- works in two steps for the OL biosynthesis, opment and appeared to build more biofilm in which OlsB transfers an acyl group to or- without shear force, but with shear force, nithine to make lyso-ornithine lipid and OlsA

44 ASM Conferences Poster Abstracts converts the lyso-ornithine lipid into ornithine regulator QsmR functions to downregulate lipid by another acyl-group transfer. OLs are glucose uptake, substrate level and oxida- reported to increase in phosphorus-free culture tive phosphorylation, and de novo nucleotide condition and olsBA operon of P. aeruginosa biosynthesis in the rice pathogen Burkholderia is induced in phosphate-limiting condition. glumae. Systematic analysis of core primary While OLs were suggested to reduce the toxic metabolite levels showed that quorum sensing effect of endotoxin probably functioning as an deficiency caused perturbation of nutrient antagonist, a recently study showed that the acquisition and energy and nucleotide metabo- mutation of this operon had no effect on the lism of individuals within the group. The quo- virulence of P. aeruginosa. In this study we rum sensing mutants grew faster than the wild found that the overexpression of olsBA operon type at early exponential stage in Luria-Bertani modulated some virulence related-phenotypes medium and outcompeted the wild type in co- of P. aeruginosa, including quorum sensing culture. Quorum sensing-mediated metabolic regulation, biofilm formation, and motility. We slowing of individuals indicates that quorum found that the overexpression of this operon sensing functions as a metabolic switch to modulates quorum sensing by reducing the ensure efficient energy and resource utilization quorum sensing signal production. Interest- of individuals in crowded environments. ingly, the olsBA-overexpressing P. aeruginosa cells induced calcium release of animal cells, n 20 implying that it may modulate the physiology Stigmergic behaviour leads to the of host cells. self-organisation of bacterial biofilms n 19 E. S. Gloag1, A. Javed2, H. Wang3, M. L. Gee3, Bacterial Quorum Sensing and S. A. Wade2, L. Turnbull1, C. B. Whitchurch1; Metabolic Slowing in a Cooperative 1University of Technology Sydney, Sydney, Population AUSTRALIA, 2Swinburne University of Tech- J. An, E. Goo, I. Hwang; nology, Hawthorn, AUSTRALIA, 3University of Seoul National University, Seoul, KOREA, Melbourne, Parkville, AUSTRALIA. REPUBLIC OF. Introduction: Many bacterial pathogens have Quorum sensing regulates bacterial social the capacity to actively expand their biofilm behaviors, such as virulence, motility, biofilm communities via complex multi-cellular be- formation, and toxin production, in response haviours. We have observed that when the bio- to cell density. Acyl-homoserine lactone-medi- films of Pseudomonas aeruginosa are cultured ated quorum sensing controls cooperativity of at the interstitial surface between a coverslip individual cells in many species of Proteobac- and solidified nutrient media, the resulting teria. These cooperative activities are costly at biofilms are characterised by an extensive an individual level but provide benefits to the pattern of interconnected trails that emerges as group. One long-standing question is whether a consequence of the active expansion of these quorum sensing controls nutrient acquisition communities. and contributes to maintain homeostatic pri- Aim: To identify the factors governing mary metabolism of individuals in a coopera- emergent pattern formation during P. aeru- tive population. In crowded but cooperative ginosa biofilm expansion. Experimental populations, quorum sensing might coordinate methods: Bacterial biofilms were cultured at nutrient utilization and homeostatic primary the interstitial space between solidified growth metabolism of individual cells. We show that media and a glass coverslip. Biofilm expansion the QS-dependent LysR-type transcriptional was observed using phase contrast time-lapse

5th ASM Conference on Cell-Cell Communication in Bacteria 45 Poster Abstracts microscopy and the topography of the underly- (QS) controls exopolysaccharide production, ing media was imaged using atomic force mi- the secretion of which during the late stages of croscopy (AFM) and 3D optical profilometry infection blocks water transport in the xylem. after the cells where removed by washing the The key master QS regulator in P. stewartii samples with water. Results: Our observations is EsaR. At low cell densities EsaR represses have revealed that during the migration of P. or activates expression of a number of genes aeruginosa biofilms, aggregates of cells at the in the absence of its acyl homoserine lactone advancing edge forge furrows as they migrate (AHL) ligand. At high cell densities binding of across the semi-solid media. The formation AHL inactivates EsaR leading to deactivation of a series of interconnecting furrows and the or derepression of its direct targets. Prior work reinforcing effect of cells traversing these fur- has demonstrated that the QS response orches- rows leads to extensive remodelling of the sub- trates three major physiological responses in P. stratum. Our analyses indicate that whilst the stewartii: capsule and cell envelope biosyn- furrows are shallow relative to the height of thesis, surface motility and adhesion, and the bacterial cells, this appears to be sufficient stress response. It is hypothesized that there is to confine cells within the furrows. The gen- coordinate expression of these outputs through eration and maintenance of the interconnected feed-forward and feed-back regulation modu- furrow network therefore accounts for the lated by the regulators RcsA, LrhA and UspA extensive large-scale patterning that is charac- present downstream in the QS regulon. RNA- teristic of these bacterial biofilms. Conclusion: Seq is being used to examine the physiological Our observations indicate that self-organised impact of deleting the genes encoding these pattern formation during biofilm expansion three regulators. In addition, the role of these across semi-solid media is a stigmergic phe- and other QS-controlled genes is being tested nomenon. The concept of describes in plant virulence assays to establish a better processes of self-organised group behaviours understanding of the key factors necessary for that occur through indirect communication the pathogenesis of this model xylem-dwelling via persistent cues in the environment left by bacterium. Preliminary findings support the individuals that influence the behavior of other existence of multi-layered regulatory control of individuals of the group at a later point in time. virulence during QS in P. stewartii. Stigmergy has been extensively examined in higher organisms and non-living systems. We n 22 propose that stigmergy can be included in the Differential RNA-seq analysis of repertoire of systems used by bacteria to coor- Vibrio cholerae identifies the VqmR dinate complex multicellular behaviours. sRNA as a regulator of collective behaviors n 21 K. Papenfort1, K. U. Förstner2, J. Cong1, C. M. Coordination of Key Physiological Sharma2, B. L. Bassler1; Outputs During Quorum Sensing 1Department of Molecular Biology, Princeton in the Phytopathogen Pantoea University, Princeton, NJ, 2University of Würz- stewartii burg, Centre for Infection Diseases (ZINF), A. Kernell Burke, A. Duy Duong, R. V. Jensen, Würzburg, GERMANY. R. Ramachandran, A. M. Stevens; The causative agent of cholera is Vibrio chol- Virginia Tech, Blacksburg, VA. erae which colonizes the small intestine and Pantoea stewartii subsp. stewartii is a Gram- causes profuse diarrheal symptoms. Regulation negative proteobacterium that infects corn of V. cholerae virulence mainly occurs through plants causing wilt disease. Quorum sensing cell-to-cell signaling, i.e. Quorum Sensing

46 ASM Conferences Poster Abstracts

(QS). In this study we performed differen- expression of QstR in response to autoinduc- tial RNA-sequencing (dRNA-seq) analyses ers. Transcription factor TfoX is expressed in of wild-type and low-cell-density locked V. the presence of environmental chitin. CytR cholerae. Our analyses identified a total of (cytidine repressor) and CRP (cAMP receptor 7641 transcriptional start sites (TSS) at all protein) are active during nucleoside and car- conditions and ~40% of these TSSs initiated bon starvation, respectively. Although each of antisense transcripts. We further identified 107 these regulators is required for natural compe- non-coding transcripts and genome-wide TSS tence, the mechanism by which each promotes mapping in combination with phylogenetic transcription of competence apparatus genes comparisons allowed the re-annotation of ~130 is currently unknown. Through a combina- genes on both chromosomes of V. cholerae. tion of genomic analyses, genetic screens, and Our analyses also revealed expression of the gene reporter assays we show that CytR and previously unidentified VqmR sRNA upstream CRP positively regulate expression of multiple of the vqmA gene. Transposon mutagenesis competence apparatus genes, such as comEA, identified VqmA as the transcriptional activa- pilA, vc0857, as well as the competence tor of VqmR and microarray-based target regulator qstR. Our expanded analysis revealed gene searches revealed eight mRNA targets distinct classes of competence gene promoters; of VqmR. We confirmed direct regulation one class that requires only TfoX, another that by VqmR for all target genes including the needs CytR as well, and finally a third class mRNAs of the rtxBA toxin genes and the vpsT that also requires QstR (and therefore HapR). transcriptional regulator as well as additional Screening of > 150,000 transposon mutants mRNAs relevant for the chemotaxis, sidero- identified no negative regulators of compe- phore uptake and metabolism. In summary, our tence, but an on-going complementary screen data provide a high-density map of the V. chol- for positive regulators has revealed several erae transcriptome and highlight the impor- putative targets that might serve as activators tance post-transcriptional control for collective of the competence pathway. It has been hy- behavior of this major human pathogen. pothesized that the coupling of quorum sensing with genetic control of natural competence n 23 promotes transfer of beneficial mutations, by coordinating the timing of gene expression Defining the regulatory mechanisms to favor acquisition of extracellular DNA of natural competence in Vibrio provided by ‘kin’. A deeper insight into the cholerae regulatory mechanisms of natural competence S. S. Watve, J. Thomas, B. K. Hammer; in V. cholerae will enable a better understand- Georgia Tech, Atlanta, GA. ing of the way in which cell to cell communi- cation in many bacteria enables rapid evolution In the waterborne human pathogen Vibrio by promoting horizontal gene transfer. cholerae, quorum sensing mediates several critical behaviors such as virulence, biofilm formation, and natural competence. Natural competence enables V. cholerae to horizon- tally acquire genetic material through DNA uptake and recombination. Synthesis of the components of the DNA uptake apparatus is controlled by several transcriptional regulators that respond to specific environmental cues. HapR, the master regulator of quorum sens- ing, is active at high cell density and induces

5th ASM Conference on Cell-Cell Communication in Bacteria 47 Poster Abstracts n 24 n 25 PhrA controls population The TonB-dependent receptor Oar heterogeneity in B. subtilis by and the associated lipoprotein MlpA intercellular signaling are important for secretion of a signaling protease in Myxococcus S. Trauth1, M. Ziesack1, A. Mutlu1, J. Ber- xanthus geest2, N. Harder2, K. Rohr2, I. B. Bischofs1; 1University of Heidelberg (ZMBH) / BioQuant, N. Gomez-Santos, L. Søgaard-Andersen; Heidelberg, GERMANY, 2University of Heidel- Max Planck Institute for Terrestrial Microbiol- berg (IPMB) and German Cancer Research ogy, Marburg, GERMANY. Center (DKFZ) / BioQuant, Heidelberg, In response to starvation cells of Myxococcus GERMANY. xanthus initiates a developmental program that Quorum sensing is commonly associated with culminates in the formation of -filled fruit- the coordination of a synchronized homoge- ing bodies. The intercellular C-signal is essential neous population-wide response. In contrast, for fruiting body formation and sporulation and still relatively little is known about whether acts as a morphogen to induce distinct responses and how intercellular signaling contributes to at discrete thresholds during fruiting body for- the development of phenotypic diversity in mation. The C-signal is a cell surface-exposed the population. Sporulation in Bacillus subtilis protein. Consistently, C-signal transmission in- has emerged as a model system for studying volves a contact-dependent mechanism in which heterogeneous population development. Sporu- C-signal on one cell interacts with a receptor on lation is controlled by a complex circuitry a neighboring cell. The C-signal is generated that involves extracellular signaling peptides by proteolytic cleavage of a 25 kDa precursor from the Phr-family. Phrs are produced by an protein (p25) by the secreted protease PopC. export-import circuit and have been suggested Cleavage of p25 by PopC generates a 17 kDa to serve in quorum sensing or alternatively, to protein (p17), which is the bona fide C-signal. implement an autocrine-like time-delay circuit p25 as well as p17 are anchored in the outer on the level of the individual cell. Here, we an- membrane. PopC as well as p25 accumulate in alyzed the effects of perturbed PhrA-signaling vegetative cells; however, PopC only cleaves on population development by using quantita- p25 in starving cells. We have previously shown tive fluorescence timelapse microscopy assays. the restriction of p25 cleavage by PopC in starv- Our data demonstrates that PhrA can serve in ing cells relies on regulated secretion of PopC: global cellular communication: it functions In vegetative cells, PopC is complexed by the as a diffusive signaling molecule that causes PopD protein and PopC secretion is blocked; in a concentration dependent population-level response to starvation, and in a RelA-dependent response by adjusting the ratio of sporulat- manner, PopD is degraded and PopC secretion is ing and non-sporulating cells. One can mimic induced. Once secreted, PopC is able to cleave the effects of external PhrA stimulations by p25 at the cell surface giving rise to the forma- varying the size of the sending and receiving tion of p17. The mechanism underlying PopC populations -or their ratio- during development secretion remains an open question. Here we re- respectively. Thus, PhrA signaling exhibits port the identification of two proteins important some traits that are characteristic for quorum for PopC secretion, the TonB-dependent recep- sensing. However, rather than coordinating tor Oar and the lipoprotein MlpA. Oar and MlpA a homogeneous population response, PhrA mutually stabilize each other, and secretion of seems to act as a potent regulator of population PopC during development is strongly impaired diversity. in an oar mutant. Further detailed results will be presented.

48 ASM Conferences Poster Abstracts n 26 ing of links between relatedness, ecology and Kin discrimination in Bacillus intraspecific social interactions. subtilis n 27 P. Stefanic1, B. Kraigher1, J. Planinc1, N. Lyons2, R. Kolter2, I. Mandic Mulec1; The production of outer membrane 1University of Ljubljana, Ljubljana, SLOVE- vesicles in quorum sensing mutants NIA, 2Harvard University, Boston, MA. of Burkholderia glumae Y. Kang, E. Goo, J. An, I. Hwang; Individuals of many animal species have re- Seoul National University, Seoul, KOREA, markable abilities to behave differently at first REPUBLIC OF. encounter towards non related groups. Bacillus subtilis, a soil dwelling bacterium, which The rice pathogen, Burkholderia glumae shows diverse social behaviors including quo- possesses one LuxI-R type quorum sensing rum sensing, biofilm formation and swarming, system, TofI-R. The complex of C8-HSL and also responds differently to nonrelated groups. its cognate receptor, TofR, activates expres- For example, genetic difference at the comQX- sion of toxin, flagella genes and an IclR type PA quorum sensing locus results in selective transcriptional regulator gene, qsmR. During induction of QS response only in isolates of growth in Luria-Bertani medium, B. glumae the same pherotype (communication specific- faces ammonia-mediated alkaline toxicity ity group) that carry genetically similar QS as a result of amino acid catabolism. In the loci. Here we address another, possibly more wild-type, quorum sensing activates oxalate general kin discrimination mechanism among biosynthesis genes to avoid alkaline toxic- swarms of highly related isolates of B. subtilis ity. However, quorum sensing mutants do not inhabiting soil at micrometer distances. In produce oxalate, which causes cell death at addition we explore interdependences between stationary phase. Quorum sensing mutants their genetic / ecological distance, pherotype secreted abundant outer membrane vesicles association and ability to discriminate between (OMVs) into culture supernatant whereas kin and non- kin. We show for the first time the wild-type did not produce OMVs during that B. subtilis indeed possess a remarkable kin growth. We purified OMVs from quorum discrimination mechanism, which manifests sensing mutants using density-gradient sedi- in the formation of a clearly visible boundary mentation technique. Four membrane proteins line between swarms growing on a semi-solid (type VI secretion system component protein, agar media. Frequency of kin discrimina- ABC transporter protein, outer membrane tion increased with genetic distance among protein, phage integrase family protein) and strains and was always detected when strains three periplasmic proteins (protease Do, were ecologically distinct. The boundary line, lipoprotein, transglutaminase) were identi- however, was never visible between two clonal fied from the OMVs with high confidence by swarms and was significantly less frequent LC-MS/MS analyses. To answer the question among relatives that shared the ecotype and/or why quorum sensing mutants produce OMVs pherotype. Using MALDI imaging we investi- into environment, we hypothesized that the gated ions found in the boundary line and with vesiculation in quorum sensing mutants might transposon mutagenesis we identified potential give a short-term protection against envelope genetic determinants that may contribute to stress. According to metabolome analyses in B. this kin discrimination phenomenon during glumae, certain metabolites such as glutamate swarming or surface growth. Our results dem- were accumulated higher in quorum sensing onstrate a novel social behavior of B. subtilis mutants than in the wild-type in exponential that contributes significantly to the understand- phase. This indicated that abnormal concen-

5th ASM Conference on Cell-Cell Communication in Bacteria 49 Poster Abstracts tration of specific metabolites might cause a a minimal number of mutant nonself cells are turgor pressure problem to bacterial envelope. also needed: high self-to-nonself ratios result We propose that OMVs of B. glumae might in inhibition of neither strain. Second, using be induced to relieve turgor stress caused by single-cell analysis of ids promoter activity imbalanced metabolism. Proteome profiles and using a plasmid-based fluorescence reporter, physiological nature of B. glumae OMVs pro- we confirmed that expression of the ids system vide a basis on further study to determine how is affected by the social environment: the quorum sensing is related to producing OMVs ids promoter is upregulated by the presence and what biological functions and properties of of nonself. Finally, using in situ single-cell OMVs are. fluorescence microscopy of active swarms, we have begun to analyze cell fates throughout n 28 a complete swarm cycle, chronicling the role of Ids-mediated self recognition in popula- A new role for self recognition in tion growth and migration. Here, we present bacterial populations: assessing evidence that BB2000 uses the Ids system to local environments assess and potentially communicate about its M. J. Tipping, K. Little, K. A. Gibbs; local social environment, raising the tantaliz- Harvard University, Cambridge, MA. ing possibility of similar signaling events in other social bacteria with non-lethal T6S- Swarming populations of the bacterium associated factors. Proteus mirabilis can recognize foreign isolates of the same species, often resulting in a macroscopic boundary. In mixed swarms, n 29 self-recognition behavior can also be observed Overexpression of a small gene by the dominance of one strain at the expand- in the dev-operon can inhibit ing edges, while the other is inhibited to the Myxococcus xanthus Development swarm interior. In the model P. mirabilis strain R. Rajagopalan, L. Kroos; BB2000, self recognition is partially mediated Michigan State University, East Lansing, MI. by an Ids system that requires an active type VI secretion (T6S) system. Intriguingly, not When cells of Myxococcus xanthus are starved all cells in a clonal swarm appear to express of nutrients they aggregate and from fruit- Ids proteins at any given time. Moreover, ids ing bodies, in which rod-shaped cells morph deletion mutants are temporally inhibited in into rounded cells, eventually forming . coswarms and form boundaries against the Positional information is transmitted by C- wild-type parent, despite the Ids proteins signaling via transcription factor FruA, while having no measured lethal activity. Given the starvation information may be transmitted via complexity of the Ids system, we have queried MrpC. MrpC and FruA bind cooperatively to whether ids expression may govern individual the promoter regions of gene loci such as the cell fates within clonal and mixed swarms. dev operon, and regulate transcription. The Using quantitative fluorescence microscopy dev operon regulates expression of fruA, and and physiological analysis, we have begun to of other downstream loci such as exo and nfs, dissect the role of Ids-mediated self recogni- which are required for spore morphogenesis and tion by surveying both an entire swarm and cell shape maintenance. Mutants of dev operon the life-cycles of individual cells. Our primary genes such as devS, devR and devT are develop- input is to alter the initial ratios of BB2000 to mentally impaired, and qPCR analysis showed an Ids-deficient mutant strain. First, we found that devS and devR negatively autoregulate Pdev that initial thresholds of ids expressing self expression 10-fold. The first gene of the dev cells are required for inhibition. Furthermore, operon, MXAN7266, is predicted to code for a

50 ASM Conferences Poster Abstracts

40-residue peptide, and it was shown to be ex- n 30 pressed in a lacZ-translational fusion. Deletion Association of Antibiotic of DNA spanning -38 to +19 of the dev promot- Resistance and Capsule switching er, or a clean deletion of MXAN7266, resulted in the Emergence of Vaccine Escape in no change in the developmental phenotype Recombinant Streptococcus of wild type DK1622 but restored normal pneumoniae 7B Clone in absence of development in a devS, devR or devT mutant. Pneumococcal Vaccination The devS MXAN7266 double mutant (∆devS ∆MXAN7266) was successfully complemented M. Rahman; International Centre for Diarrhoeal Disease with a fragment of Pdev containing MXAN7266. We propose that MXAN7266 negatively Research,Bangladesh, Dhaka, BANGLADESH. regulates expression of genes important for Background: Antibiotics act as cell to cell sporulation, and is countered by DevS, DevR, communication molecules that induce compe- and DevT. Co-development of ∆devS (which tence for genetic transformation and fratricide overexpresses MXAN7266), with the wild type, in Streptococcus pneumoniae to acquire exog- did not affect development of either strain. This enous DNA resulting genomic and phenotypic indicates that MXAN7266 is likely not secreted diversity. Aims & Methods: Studied capsule extracellularly, but acts in a cell-autonomous switching, seroconversion and genomic manner. To test whether MXAN7266 codes for a homology among antibiotic-resistant pneu- protein or an RNA product, a single thymidine mococci from children with invasive diseases was added between the original +54 and +55 by antibiotic resistance, serotyping, MLST DNA bases in the reading frame of MXAN7266 and eBURST analysis in Bangladesh where in a ∆devS background. If MXAN7266 codes pneumococcal vaccines are not used routinely. for a protein product, this would result in the Results: Among 136 pneumococci, 11 com- creation of a stop codon within its reading mon serogroups (77% of invasive-isolates) frame, whereas the structure of an RNA mol- were 6, 14, 19, 5, 12, 1, 7, 45, 2, 9 and 23 (in ecule might not be significantly altered by an descending order), where as 11 most common additional nucleic acid base. The developmental serogroups of colonized-isolates comprising phenotype of the resultant MXAN7266-stop 78% were 6, 19, 14, 23, 9, 7, 13, 15, 21, 22 codon mutant in ∆devS background function- and 37. 71% were antibiotic resistant, MDR ally resembled that of ∆devS ∆MXAN7266. This in 12%. Macrolide resistance was detected in indicates that MXAN7266 likely codes for a pro- six isolates (four 7B, one 9V and one 18C). tein, rather than for a small RNA product. Rela- Four 7B isolates were MDR; three had ST tive P transcript level in ∆devS ∆MXAN7266 dev 1553 and one ST 1586, a single locus variant was about 5-fold higher than wild type, but low- of ST 1553. 9V isolate were MDR and ST er than the 10-fold increase observed in ∆devS. 1553 indicating genomic homology with 7B This suggests that MXAN7266 may have a pos- and capsule switching where prevalent MDR itive regulatory effect on the dev-promoter, and 9V acquired 7B capsule resulting in serocon- DevS may act in part by inhibiting MXAN7266 version (9V 7B). eBURST analysis of MLST expression. Expression from P and P were nfs exo 1586 and 1553, and available pneumococcal respectively three to fifteen fold lower in ∆devS MLST database (www.mlst.net) indicated that and ∆devS ∆MXAN7266, suggesting that DevS ST 1553 and ST 1586 were not closely associ- and MXAN7266 positively regulate expression ated with any other clone. Conclusion: Thus, of exo and nfs. Taken together, our results sug- newly emerged vaccine escape recombinant gest that further investigation of MXAN7266 MDR pneumococcus 7B originated by 7B is key to understanding regulation of the dev caps locus acquisition by 9V preferably by operon, and its role in sporulation. antibiotic-induced cell-cell signaling and

5th ASM Conference on Cell-Cell Communication in Bacteria 51 Poster Abstracts caused invasive diseases. To our knowledge, selective pressure acts to reinstate motility in this unique pneumococcal clonal complex was the biofilm. detected for the first time. n 32 n 31 Quorum sensing regulation Conflicting messages: Signals from of antimicrobials protects the extracellular matrix maintain public-goods cooperation from motility within the biofilm exploitation by other bacterial species N. Steinberg, Z. Porat, I. Kolodkin-Gal; Weizmann Institute of Science, Rehovot, N. E. Smalley1, J. R. Chandler2, A. A. Dan- ISRAEL. dekar1; 1University of Washington, Seattle, WA, 2Uni- Bacteria in nature are usually found in com- versity of Kansas, Lawrence, KS. plex multicellular structures, termed biofilms, which protect them from numerous environ- Quorum sensing (QS) is a cell-cell commu- mental threats. The biofilm structure is the end nication system that can regulate cooperative result of of different cell behaviors in bacterial populations. Pseudomo- types in a coordinated fashion, achieved by nas aeruginosa has a complex QS system that the accurate regulation of dedicated genetic regulates the production of several secreted programs. So far, it was well established that in products, including proteases, which are shared the single-cell level once a bacterial cell com- among the whole population. Such products mits to the biofilm state it represses motility are termed “public goods.” We, and others, both transcriptionally and post-translationally. have previously shown that QS in P. aeru- However, at the population level, we found ginosa can serve as a model to study public a motile cell sub-population that is tightly goods cooperation. P. aeruginosa requires retained inside the biofilm of Bacillus subtilis, the production of a QS-regulated secreted enabling fast occupation of new niches, as protease, elastase, to grow when casein is the well as invasion of neighboring colonies of sole carbon and energy source. In this setting, foreign bacterial species. This dogma-breaking QS mutant “social cheaters” arise that avail phenomenon depends on a unique signal origi- themselves of the carbon liberated by the nating in the extracellular matrix (ECM) that elastase produced by the QS-intact coopera- surrounds the cells in the biofilm, maintaining tors. Other bacterial species may also reap the a motile cell sub-population. This ECM-based benefits of the nutrients liberated by elastase, signal acts to induce motility, as mutants defi- and these may be viewed as another type of cient in ECM production show lower numbers cheater. P. aeruginosa QS also controls antimi- of motile cells. When the ECM extracted from crobials -- such as , phenazines, a wild type biofilm was externally added to the hydrogen cyanide, and quinolones -- that can ECM mutants, motility level was restored. In inhibit growth of other bacteria. We asked if particular, TasA, the proteinaceous component QS-produced antimicrobials prevent inva- of B. subtilis biofilm, appears to be a specific sion or “cheating” by other species against P. chemical signal that strongly induces bacterial aeruginosa. As a competitor strain, we used motility. Another line of evidence for the im- Burkholderia multivorans strain AMT0468-1, portance of maintaining motility in the biofilm which exhibits similar growth kinetics as P. ae- emerges from the highly frequent evolution ruginosa PAO1 in nutrient-rich conditions. We of hypermotile suppressors in ΔtasA mutant observed that P. aeruginosa inhibits growth colonies. We suggest that in the absence of of B. multivorans in nutrient-rich conditions, ECM-based induction of motility, a strong and that this is due to the combined effects of

52 ASM Conferences Poster Abstracts three QS-produced antimicrobials; phenazine, chronically colonized with P. aeruginosa and hydrogen cyanide and rhamnolipids. In casein LasR mutants accumulate over time. We have media, where growth requires QS-controlled also observed CF isolates with intact quorum elastase, wild-type P. aeruginosa similarly systems have small quorum-sensing out-competed B. multivorans. Our strain of B. as compared to environmental isolates. We multivorans did not grow to high density alone tested the hypothesis that, when quorum-sens- in casein media, likely because it does not pro- ing-regulated factors are required for growth duce the necessary protease required to liberate of P. aeruginosa, the quorum regulon will be nutrients from casein. Strikingly, however, B. adaptively reduced. We grew P. aeruginosa multivorans grew well in co-culture with an on casein and adenosine, a condition in which antimicrobial-deficient P. aeruginosa mutant quorum sensing is required for growth and and could invade the P. aeruginosa population emergence of LasR mutants is constrained. even from a very low starting density. This After over 1000 generations of growth, the result shows that QS-controlled antimicrobi- frequency of LasR mutants remained below als protect the P. aeruginosa population from 1%. We developed a method to assess the cheating by Burkholderia. These findings add quorum sensing regulon on a population level to the increasing body of work supporting the by growing evolved populations in the pres- view that QS is important for competition in ence of absence of an AHL-degrading enzyme mixed-species environments. Furthermore, the and using RNA-seq to identify genes that were results show that by co-regulating proteases affected by signal. After 160 days (roughly 900 and antimicrobials, QS may protect and stabi- generations), the number of quorum-activated lize the cooperative production of proteases. genes was reduced by 40% as compared to the progenitor. Our data suggest that when bacteria n 33 grow under conditions that require quorum sensing, there is a relatively rapid reduction Evolutionary pressure on the in the number of genes directly or indirectly Pseudomonas aeruginosa quorum regulated by quorum-sensing transcription sensing regulon factors. These data also imply that the large A. A. Dandekar1, D. Müller2, A. L. Schaefer1, quorum regulon in environmental isolates of E. P. Greenberg1; P. aeruginosa reflects varied and numerous 1University of Washington, Seattle, WA, 2ETH, selective factors. Finally, our results suggest Zurich, SWITZERLAND. LasR mutants and isolates with relatively small quorum sensing regulons may reflect The opportunistic pathogen Pseudomonas environmental conditions where this bacterium aeruginosa regulates the expression of dozens depends on quorum-regulated factors for either of genes via two acyl-homoserine lactone growth or survival. (AHL)-responsive quorum-sensing transcrip- tion factors, LasR and RhlR. Many quorum- controlled genes code for “public goods,” n 34 which are shared among the population. Thus QS systems can accumulate as quorum sensing can be a cooperative behavior. selfish genetic elements Experiments show this leaves P. aeruginosa E. Even-Tov, S. Omer, A. Eldar; quorum sensing systems susceptible to “social Tel-Aviv University, Tel-Aviv, ISRAEL. cheating” by LasR mutants. Such cheaters avail themselves of public goods, but do not The Rap-Phr family of quorum sensing sys- incur the metabolic cost of quorum sensing tems is found in the Bacillus subtilis and ce- and therefore can outcompete cooperators. In reus groups. The peptide signal of this system, one human disease, cystic fibrosis, patients are which is encoded by the phr gene, de-represses

5th ASM Conference on Cell-Cell Communication in Bacteria 53 Poster Abstracts the activity of the Rap receptor. In the absence sole carbon and energy source, and over time of signal, most studied Rap receptors repress LasR mutants emerge and invade populations the activity of either the Spo0A or ComA of LasR-intact cooperators. These mutants response regulators. A typical B. subtilis strain are “social cheaters” that reap the benefit of contains 8 paralogs of this system, where the cooperator-produced proteases without each paralog encodes for a different signaling being burdened by the metabolic costs of peptide which specifically interact with its public goods production. We and others have cognate receptor but no other (non-orphan) observed that these social cheaters come to receptors. In addition, all rap-phr systems are an equilibrium with cooperators and do not also similarly regulated transcriptionally. It is cause the population to collapse, as would therefore unclear what drives the evolution- occur if the number of cooperators fell below ary accumulation of these redundant systems. the quorum threshold. Here we probe the Here we demonstrate both experimentally and molecular basis of the equilibrium. We hy- theoretically that the accumulation of rap-phr pothesized that cooperators can police cheaters systems might be the result of a cheating be- by intoxicating them. RhlR activates genes havior. In a co-culture where only part but not coding for production of several reactive toxic all of the population had acquired an additional compounds, including peroxides and hydro- rap-phr system, there will be less of the novel gen cyanide. We believe RhlR also induces phr signal than the one which is produced by immunity to these factors. We reasoned that the entire population. Thus, leading to less de- RhlR-activated genes allow P. aeruginosa repression of the cognate novel rap gene which cooperators to police cheaters, and that LasR+, results in decreased cooperative behavior (i.e. RhlR- cooperators are “policing mutants.” cheating). Therefore we suggest that the ac- Policing mutants will not have a capacity to cumulation of rap-phr systems is not the result limit invasion by LasR-, RhlR- cheaters and we of added group benefit but rather due to their expect population crashes for failure to achieve selfish element like behavior. a quorum. This prediction was borne out by experiments where LasR-, RhlR- social cheaters n 35 rapidly arose to high frequencies and caused a population crash. We then showed that polic- Pseudomonas aeruginosa quorum ing involves cyanide production by wildtype sensing: cooperator policing of P. aeruginosa by following social evolution in social cheaters an HcnC- cooperator, which cannot synthesize M. Wang1, A. L. Schaefer2, A. A. Dandekar2, E. hydrogen cyanide. Like the RhlR- cooperators, P. Greenberg2; populations of HcnC- cooperators were over- 1Zhejiang Gongshang University, Hangzhou, run by LasR mutants. To test the hypothesis CHINA, 2University of Washington, Seattle, that wildtype P. aeruginosa and LasR- cheat- WA. ers coexist by virtue of RhlR-activated toxin secretion, and not a direct cell-cell interaction, There are two acyl-homoserine lactone quorum we used dialysis membranes to separate LasR+, sensing (QS) circuits in P. aeruginosa, LasR-I RhlR- cooperator populations from either and RhlR-I. LasR-I activates RhlR-I and thus LasR+, RhlR+ or HcnC- cooperators. Over 48 serves as a master QS regulator. Together, hours, the cell yields of LasR+, RhlR- coopera- these circuits activate hundreds of genes, many tors with the wildtype were two logs lower of which code for production of secreted or ex- than when they were when grown with the creted factors. These factors are called “public HcnC- cooperators. Our studies support the no- goods” and can be shared among individuals in tion that groups of cooperating P. aeruginosa the group. P. aeruginosa requires QS-activated can police social cheaters and we provide a secreted proteases for growth on casein as the plausible mechanism for this phenomenon.

54 ASM Conferences Poster Abstracts n 36 they protect only the producing cells. During Quorum sensing promotes defense growth in mixed microbial communities, quo- and self-preservation during rum sensing-controlled defense strategies may interspecies competition serve to restrain the emergence of quorum- defective social cheaters. K. Hinshaw, J. R. Chandler; University of Kansas, Lawrence, KS. n 37 Many Proteobacteria use acyl-homoserine lac- Acyl-HSL quorum sensing control tone mediated quorum sensing to control the of an extracellular product in a production of public goods such as antibiotics synthetic system or proteases whose benefits are shared among R. L. Scholz, E. P. Greenberg; all of the cooperating members of the popula- University of Washington, Seattle, WA. tion. Because quorum sensing systems regulate public goods, they may be susceptible to In Pseudomonas aeruginosa, quorum sensing invasion by social cheaters, such as a quorum- (QS) controls and coordinates the production defective mutant, which can benefit from the of many secreted and excreted products, for public goods but do not incur the cost of pro- example proteases, which can be shared by ducing them. In some species quorum sensing all members of a community. QS allows a also controls production of efflux pumps and production delay of these products until cells other factors that may protect the cells from are at a sufficient density to benefit from them. antimicrobials produced by other species; these The P. aeruginosa LasR-LasI QS system is at are not believed to be shared among all of the the top of a QS-signaling cascade. LasI is a members of the population making them pri- 3-oxo-C12-HSL synthase and LasR is a 3-oxo- vate goods. Quorum regulation of such defen- C12-HSL responsive transcriptional activa- sive strategies may be important for protecting tor. We aim to study the costs and benefits a niche or competing for limited resources of QS control of extracellular products in a in multispecies bacterial communities. To simplified synthetic recombinant Escherichia investigate the importance of quorum sensing coli system. To do this we have developed a in defense during inter-species competition, we recombinant E. coli enterochelin mutant (entF) used a model of competition between two soil with chromosomal copies of lasR, lasI, and saprophytes, Chromobacterium violaceum and a lasI-promoter-controlled entF. The E. coli Burkholderia thailandensis, that we previously entF mutant does not grow in iron-restricted described. We demonstrate that C. violaceum medium, whereas the genetically engineered quorum sensing mutants are more sensitive QS E. coli and the wildtype grow to similar than wild type to a potent bactobolin antibiotic densities. The entF mutant can obtain iron and produced by B. thailandensis. C. violaceum grow together with the wildtype or the QS con- quorum sensing mutants have a growth advan- trolled entF strain on iron-restricted medium. tage over their quorum-intact parent in labora- However, the Ent- freeloaders do not increase tory growth media. However in co-culture with in frequency when grown together with B. thailandensis, quorum-intact C. violaceum enterochelin-producing strains. We are now outcompetes the quorum-defective cells. This in position to compare the costs and benefits result was dependent on the production of bac- of QS vs constitutive control of an extracel- tobolin by B. thailandensis. Our results suggest lular product during growth of E. coli growing that quorum sensing is important for defense under a variety of conditions. during interspecies competition and that the defensive strategies are likely private because

5th ASM Conference on Cell-Cell Communication in Bacteria 55 Poster Abstracts n 38 n 39 A design principle of quorum A quorum sensing molecule PQS, sesning regulation privatises iron for Pseudomonas aeruginosa I. Ben-Zion, A. Eldar; Tel Aviv University, Tel Aviv, ISRAEL. R. Popat1, L. McNally1, F. Harrison2, P. Wil- liams2, S. Brown1, S. Diggle2; Quorum sensing (QS) signaling systems 1University of Edinburgh, Edinburgh, UNITED typically regulate cooperative traits that are KINGDOM, 2University of Nottingham, Not- costly to the individual cell but beneficial to tingham, UNITED KINGDOM. the population. A common network structure of QS systems is a positive feedback loop In addition to signalling, quorum sensing that is manifested by QS dependent activation molecules often have secondary properties. In of the QS signal. The prevalent view is that Pseudomonas aeruginosa, the Pseudomonas regulation by QS is advantageous because QS quinolone signal (PQS) binds iron with a high activates cooperative behaviors only in dense affinity causing an iron starvation response. populations, in which they are beneficial. This PQS:iron complex can then be used as an In addition, the positive feedback structure iron source to support the growth of P. aeru- is typically regarded as a mechanism that ginosa via the production of iron scavenging sharpens the threshold-like behavior of QS . Here we investigate the possibil- response. Other aspects, relating to the social ity that the production of PQS enhances the fit- nature of cooperative traits and the possibility ness of P. aeruginosa when in competition for of exploitation by cheaters, are often over- iron with other species. We present a simple looked. In our work, we ask how the regula- evolutionary model for PQS production which tory structure of cooperative traits affects the predicts that (a) PQS increases competitive evolutionary stability of cooperation. In this ability in an iron-dependent manner and (b) framework, we use mathematical modeling to that high PQS production necessitates corre- show that QS regulation is beneficial since it spondingly high investment in iron scavenging grants a better resistance against cheaters in a siderophores. We present experimental support structured population. It does so by allowing for both predictions. Firstly, a meta-analysis cooperators to sense nearby signaling mutants, of previous work as well as new experiments, and to reduce cooperative effort accordingly. indicate that the iron binding property of PQS Moreover, we demonstrate that unlike regular reduces competitor growth, favouring P. aeru- QS regulated cooperators that are susceptible ginosa in competition. Secondly we show that to cheating by receptor mutants, a positive PQS production and production feedback on the QS signal provides resistance are positively correlated in natural isolates of against these mutants. More generally, our P. aeruginosa indicating that PQS induced iron results provide a condition for the evolution- sequestration must be accompanied by invest- ary stability of cooperation, which general- ment in iron acquisition. Finally we show that izes Hamilton’s rule for cases of regulated PQS, whilst beneficial for competition with cooperation. Furthermore, we are testing our other species, can result in intensified competi- predictions in a synthetic system of feedback tion with con-specific cheats that do not invest regulated cooperative behavior in Bacillus in iron acquisition. Our results are suggestive subtilis. Preliminary experimental results will of the possibility that PQS acts to privatise be presented. iron, reducing its availability to other species. Such investment in the future availability of nutrients presents novel behavioural strategy to overcome heterospecific competition.

56 ASM Conferences Poster Abstracts n 40 invade but they have a window of increased Evolutionary tricks to constrain QS opportunity to revert into WT. Our results signalling in Bacillus subtilis indicate that the ComQ dependent intracel- lular link couples prudent QS response with A. Oslizlo, I. Dogsa, P. Stefanic, I. Mandic- QS signalling, providing constrains on signal Mulec; production. Evolutionary stability of signal- University of Ljubljana, Biotechnical Facoulty, ling in B. subtilis could be then explained by Ljubljana, SLOVENIA. direct benefits, because the signal serves as a coercion to non-senders while the sender is Quorum sensing (QS) signals serve as spa- equipped with the negative feedback regula- tiotemporal triggers of cooperation among tion of the QS response. In addition, signal microbes, however evolutionary stability of deficiency of B. subtilis in the presence of WT signalling has not received much experimental simultaneously translates into decreased fitness insight. Bacillus subtilis uses evolutionary and increased recombination rate. This sup- ancient ComQXPA QS system where the pep- ports long-standing hypothesis on fitness as- tide signal ComX undergoes posttranslational sociated recombination as a way to maximize isoprenylation by ComQ. At critical concen- the adaptive potential of genetic competence tration ComX activates its cognate receptor within species. ComP which thorough transcriptional activator ComA triggers expression of lipopeptide antibiotic surfactin (srfABCD) and genetic n 41 competence for transformation. Multiple Non-social adaptation defers a sequence alignment of comQXPA loci from tragedy of the commons in quorum- soil isolates confirmed that signal linked- sensing bacterial populations (comQ and comX) and receptor-encoding K. L. Asfahl1, J. Walsh1, K. Gilbert2, M. genes (comP) coevolved. We asked to which Schuster1; extent signalling and response to ComQXPA 1Oregon State University, Corvallis, OR, QS are co-dependent and how signal defi- 2Donald Danforth Plant Science Center, St. cient cells with functional ComP respond to Louis, MO. ComX produced by the wild type (WT) in homogeneous environment. We monitored In a process termed quorum sensing (QS), the QS response of the WT (srfA-cfp) and the opportunistic pathogen Pseudomonas aeru- signal-deficient (∆comQ; srfA-yfp) strains in ginosa uses diffusible signaling molecules to 1:1 co-culture by fluorescence microscopy and regulate the expression of numerous secreted the response of these strains in monocultures factors or public goods that are shared within supplemented with ComX. We discovered that the population. But not all cells respond to QS the mutant overly responds to ComX, which signals. These social cheaters typically harbor leads to increased production of surfactin a mutation in the QS receptor gene lasR and and increased genetic competence. We find exploit the public goods produced by coopera- also that surfactin promotes the DNA release tors. Here we show that non-social adaptation for intraspecific genetic exchange. Paradoxi- under growth conditions that require QS- cally therefore, non-signalling mutants act as dependent public goods increases tolerance to both hypercooperators - because they share cheating and defers a tragedy of the commons. surfactin more generously than the wild type The underlying mutation is in the transcrip- despite the high fitness costs of its production; tional repressor gene psdR. This mutation has and as hypercheats - because they take up no effect on public goods expression but in- more exogenous DNA than WT under selective stead increases individual fitness by derepress- conditions. Consequently, the mutants cannot ing growth-limiting intracellular metabolism.

5th ASM Conference on Cell-Cell Communication in Bacteria 57 Poster Abstracts

Even though psdR mutant populations remain producers were assessed at each successive susceptible to invasion by isogenic psdR lasR selection round. A planktonic control popula- cheaters, they are able to bear a higher cheater- tion (absence of plastic beads) was also tested. load, and they are completely resistant to inva- Our results show that PEL and PSL provide sion by lasR cheaters with functional psdR. a direct fitness benefit in biofilm populations Mutations in psdR also sustain cooperation whilst having zero benefit (or cost) in plank- at wild-type levels when paired with certain tonic populations. Furthermore, it was evident partial loss-of-function lasR mutations. Tar- that the production of both PEL and PSL was geted sequencing of multiple evolved isolates not exploitable by non-producing “cheaters” revealed that mutations in psdR arise before due to the loss of non-producers in low-related mutations in lasR, and rapidly sweep through populations after four and six rounds of selec- the population. Our results indicate that strong tion, respectively. Additionally, the loss of selection for QS can lead to adaptations in PEL non-producers two selection rounds prior non-social, intracellular traits that increase the to PSL non-producers suggests this particular fitness of cooperating individuals and thereby extracellular polysaccharide is less socially ex- contribute to population-wide maintenance of ploitable by cheaters than the latter. Our results QS and associated cooperative behaviors. suggest that non-producing mutants would not survive in the environment (i.e. during chronic n 42 infection), or that their frequency would be extremely low. Furthermore, it is possible that Self-produced PEL and PSL are only PEL and PSL production are non-social due to of direct benefit in Pseudomonas the positive feedback regulatory circuit which aeruginosa biofilms would directly benefit producing cells more. A. E. Roberts1, Y. Irie2, S. West3, S. Diggle1; 1University of Nottingham, Nottingham, n 43 UNITED KINGDOM, 2University of Bath, Thrombocytopenia and Prolonged Bath, UNITED KINGDOM, 3Oxford Univer- Prothrombin Time in Neonatal sity, Oxford, UNITED KINGDOM. Septicemia Pseudomonas aeruginosa is known to as- A. S. Oluremi; sume a surface attached lifestyle and grow as Lautech, Ogbomoso, NIGERIA. multicellular communities known as bio- films during chronic infection. These sessile Septicemia in neonates refers to generalized cells are surrounded by a hydrated matrix of bacterial infection documented by positive polysaccharides, of which PEL and PSL are blood culture in the first 28days of life and is the main structural constituents of non-mucoid one of the leading causes of neonatal mortal- strains. These self produced extracellular ity in sub-Sahara Africa. Thrombocytopenia polysaccharides act as signals during biofilm in newborns is a result of increased platelet formation (indirectly increasing c-di-GMP consumption; sepsis was found to be the most levels), however it is unclear as to wether the common risk factor. The objective of the study effects of PEL and PSL are exploitable by was to determine if there are organism-specific social cheating. We carried out in vitro selec- platelet responses among the 2 groups of tion experiments using plastic beads to allow bacterial agents: Gram-positive and Gram- biofilm formation of PEL/PSL producers and negative bacteria, and also to examine the non-producers in both high- and low-related association of platelet count and prothrombin environments (high and low strain diversity, time with neonatal septicemia. 232 blood respectively). The ratio of producers and non- samples were collected for this study. The blood culture was performed using Bactec

58 ASM Conferences Poster Abstracts

9050, an instrumented blood culture system. two-component signaling pathways (TCS) and The platelet count and prothrombin time were their cognate signals (host metabolites), which performed using Abacus Junior5 hematology regulate toxin production in Vibrio cholerae analyzer and i-STAT 1 analyzer respectively. and other enteric bacteria by mutually exclu- Of the 231 neonates hospitalised with clinical sive and novel non-canonical mechanisms. sepsis, blood culture reports were positive in We also report two novel high-throughput and 51 cases (21.4%). Klebsiella spp. (35.3%) and systems-biology portable assays to measure Staphylococcus aureus (27.5%) were the most host and bacterial RNA expression profiles and common Gram-negative and Gram-positive metabolites of infected mice, and generate a isolates respectively. Thrombocytopenia was “molecular signature” of diarrheal diseases. observed in 30(58.8%) of the neonates with We first utilized high-throughput screening to septicemia. Of the 9(17.6%) patients with identify bacterial TCS pathways regulating severe thrombocytopenia, seven (77.8%) cholera toxin production, followed by genom- had Klebsiella spp. septicemia. Out of the ics, digital gene-expression technology, RNA- 21(63.6%) of thrombocytopenia produced by Seq, metabolomics, proteomics and animal Gram-negative isolate, 17(80.9) had increased models to decipher their detailed mechanism. prothrombin time. In conclusion, Gram- One TCS pathway acts a phosphorylation-me- negative organisms showed the highest cases diated switch between bacterial virulence gene of severe thrombocytopenia and prolonged PT. expression and host metabolism. It activates This study has helped to establish a distur- bacterial toxin production during hypoxia via bance in hemostatic systems in neonates with a non-phosphorylated response regulator, and septicemia. Further studies, however, may be represses host metabolic process detrimental required to assess other hemostasis parameters to pathogenesis in it’s phosphorylated form. in order to understand their interaction with the A second TCS pathway senses host potassium infectious organisms in neonates. levels, and activates toxin production via tyro- sine phosphorylation of its response regulator. n 44 Thus, this pathway can switch between aspar- tate and tyrosine phosphorylation of its cog- New Signaling Pathways and nate response regulator to modulate bacterial Metabolites for Host:Pathogen virulence and pathogenesis. Using sequencing Communication During and 2D LC/ESI/MS/MS proteomics, I identify Gastrointestinal Infections protein-interacting partners and DNA binding P. Shah, D. Hung; sites of these TCS. By characterizing a battery Broad Institute/Harvard University, Boston, of locked, null, constitutively actively genetic MA. point mutants under standard laboratory condi- tions, followed by profiling spatiotemporal Background: Precise and regulated activation kinetics of host immunity and metabolism and production of bacterial virulence factors regulated by these pathway using digital gene- during gastrointestinal infection is a critical expression technology and high-throughput determinant for diarrhea. Signaling molecules, metabolomics in a mouse model, I confirm receptors, downstream regulators and in vivo phenotypes and mechanisms of these signaling spatiotemporal expression kinetics for several modalities. Significance: My data integrates virulence factors, and subsequent host respons- bacterial signaling and host responses in vivo es to infections are also not properly under- to identify a new landscape of host-pathogen stood and defined. These processes can serve interactions, metabolites and mechanisms for as targets for prophylactic and therapeutic infectious diseases. I also report two novel interventions against infectious diseases. Find- RNA and metabolic profiling approaches that ings: We report discovery of several bacteria

5th ASM Conference on Cell-Cell Communication in Bacteria 59 Poster Abstracts can be ported into systems-biology platforms, other P. aeruginosa lineages. We show that the which will have wide utility in microbiology transferred regions provide enhanced R5 pyo- research. cin resistance to DK1/2. Our data suggest that the within-host genetic interactions between n 45 co-infecting DK1 and DK2 strains could be driven by super-infections with R5 producing Microbial interactions and genotypes. To more systematically explore evolution in chronic cystic fibrosis interactions between DK1 and DK2, we are infections mapping phenotypic interactions between 100 C. I. Amador, T. Markussen, L. Jelsbak; DK1 and DK2 isolates sampled from multiple DTU Systems Biology, Lyngby, DENMARK. patients. For that purpose we are currently per- forming a pairwise screening on agar surfaces Chronic cystic fibrosis (CF) airway infec- using differentially fluorescent-tagged strains tions provide opportunities for fundamental to assess neutral, negative or positive effect. investigations related to microbial evolutionary Our results point towards an unexplored area dynamics, diversity, and interactions within a for novel interference treatment strategies in natural polymicrobial ecosystem. Patients with relation to microbe-microbe interactions. CF are predisposed to airway infections from a wide range of microbial species of which Pseu- domonas aeruginosa is the major contributor to n 46 patient morbidity and mortality. It is well- Within-host evolution of established that diverse factors such as the Pseudomonas aeruginosa toward host defense, antibiotic treatment in the clinic, iron acquisition from hemoglobin in and a heterogeneous distribution of nutrients polymicrobial CF infections drive P. aeruginosa evolutionary adaptation S. Khademi, R. Marvig, S. Damkiær, L. to the lung environment. However, whether Jelsbak; interactions with other infecting microbes is Technical University of Denmark, Lyngby, also an evolutionary driver is not well under- DENMARK. stood. To begin to explore the relationship between evolution and microbial interactions, Bacterial pathogens require iron to survive we have focused on two distinct P. aeruginosa and colonize a human host but their access to lineages (called “DK1” and “DK2”) that have free iron is often limited by iron-withholding transmitted among and evolved in CF patients process where free iron is bound by proteins in Denmark during the last 40 years. Although such as hemoglobin. Although most patho- most patients are infected with either clone- gens have developed tactics to acquire iron type, DK1 and DK2 have also been co-existing from host proteins, little is known about how in many patients for extended periods. Here, evolutionary processes modulate bacterial iron we focused on a single patient with a mixed acquisition systems in chronic, polymicrobial infection containing both clone types. To infections where interspecies competition for investigate interactions between DK1 and DK2 limited iron could be an evolutionary driver. we sequenced the genomes of isolates sampled To begin to address this issue, we use chronic from the patient over 15 years. Surprisingly, airway infections in patients with cystic fibro- we identified isolates with mosaic genomes: sis (CF) as a model to investigate evolutionary These isolates (which we call “DK1/2”) had adaptation to an iron-limited environment in DK2-based genomes but containing regions a polymicrobial context. Here, we investigate of DK1 DNA acquired by horizontal gene the within-host evolution of the transmis- transfer and recombination. DK2 isolates are sible P. aeruginosa DK2 lineage sampled sensitive towards R5 pyocins produced by from (CF) airway infections over a period of

60 ASM Conferences Poster Abstracts several decades. We find a positive selec- by influenza infections affecting the innate tion for promoter mutations in P. aeruginosa immune response and cellular clearance DK2 leading to increased expression of the mechanisms in the lung. Using an in vivo phu (Pseudomonas heme utilization) system. model of subsequent infections with influenza By mimicking conditions of the CF airways A virus and S. pneumoniae via the intra- in vitro, we experimentally demonstrate that tracheal infection route we show that sublethal increased expression of phuR confers a growth influenza infections clearly predispose for advantage in the presence of hemoglobin, thus severe pneumococcal infections even at low suggesting that P. aeruginosa evolves towards bacterial doses. Coinfected C57BL/6 mice are iron acquisition from hemoglobin. We also find more susceptible to pneumococcal infection similar adaptive mutations in the genomes of than single-infected mice, resulting in drasti- two additional P. aeruginosa lineages ruling cally less survival and earlier development of out the specificity of these mutations to this pneumonia and bacteremia. Despite an upregu- particular lineage. Furthermore, in all three lin- lation of the endogenous TLR inhibitor A20 at eages phuR promoter mutations coincide with 24 hours post secondary infection in lungs of the loss of pyoverdine production, suggest- coinfected mice, analyses show a sig- ing that within-host adaptation towards heme nificant increase of proinflammatory utilization is coupled to the loss of pyoverdine and increased numbers of neutrophils in the production. We hypothesize that this particular airways of co-infected compared to single- adaptation in P. aeruginosa DK2 has an impact infected mice. This enhanced inflammation on interspecies interaction with other members associated with tissue damage contributes to of the CF polymicrobial community capable of the severity of secondary bacterial pneumonia heme utilization. We are currently testing this suggesting that influenza-induced A20 is either hypothesis by exploring competition for iron not sufficient to suppress the inflammation or from hemoglobin between P. aeruginosa DK2 is dysfunctional. and Staphylococcus aureus that are frequently co-isolated from CF infections. n 48 Regulatory Characteristics of n 47 Vibrio vulnificus gbpA, Encoding Innate immune dysfunction is N-acetyl Glucosamine Binding associated with elevated A20 Protein and Essential for expression during Influenza A Pathogenesis virus/S. pneumoniae coinfection in K. Jang1, S. Gil2, J. Lim1, Y. Bang1, S. Choi1; mice 1National Research Laboratory of Molecular V. Sender, A. Pathak, B. Henriques-Normark; Microbiology and Toxicology, Department of Karolinska Institutet, Stockholm, SWEDEN. Agricultural Biotechnology, Center for Food Safety and Toxicology, and Research insti- Both, influenza A virus and S. pneumoniae tute for Agriculture and Life Sciences, Seoul are a leading cause of morbidity and mortality National University, Seoul 151-921, KOREA, worldwide. However, most influenza-related REPUBLIC OF, 2LG Health Care, Daejeon, mortality is not due to the viral infection KOREA, REPUBLIC OF. alone but rather secondary bacterial infec- tions, mainly caused by S. pneumoniae. The Mucin glycoprotein is a major component of mechanisms driving virulent influenza coinfec- mucus layer that is the major site of entry for tion are poorly defined, making it difficult to most pathogens and serves as the initiation develop effective therapeutic strategies. This surfaces for host-microbe interactions. Vibrio study investigates signaling events evoked vulnificus, a model human enteric pathogen, is

5th ASM Conference on Cell-Cell Communication in Bacteria 61 Poster Abstracts a causative agent of fatal septicemia and uti- of diverse that can be readily lizes the mucin to survive and cause disease in isolated from most environments allowing for host. From RNA-seq analysis of V. vulnificus numerous interactions with hosts and other grown with mucin-containing media, numer- bacteria. pv. lachry- ous genes which were induced by mucin were mans 107 contains a 1Mb cryptic megaplasmid identified. Among them, a gbpA gene encoding that can be easily transferred across Pseudo- an N-acetyl glucosamine binding protein monas species. We have yet to discover a clear GbpA, a homologue of V. choelrae GbpA, was benefit to acquiring the pMP, but strains that selected and further studied. The influence of have recently acquired the megaplasmid dem- global regulatory proteins on the expression onstrate lowered growth rates, reduced biofilm of gbpA was examined, and quorum sensing production, decreased antibiotic resistance, regulator SmcR and Fe-S cluster regulator and decreased thermotolerance. Despite these IscR were found to downregulate and upregu- costs the pMP is stably maintained within the late the gbpA expression at the transcriptional recipient strain, potentially due to high rates of level, respectively. Primer extension analysis transfer. One intriguing and, to our knowledge, revealed that the transcription of gbpA begins unprecedented cost of megaplasmid acquisi- at a single site. Direct bindings of SmcR and tion is the sensitivity to an unknown agent IscR to PgbpA were demonstrated by EMSA. produced by various Pseudomonas species at The binding sites for SmcR and IscR were stationary phase that inhibits growth specifi- mapped based on a deletion analysis of the Pg- cally of pMP strains. Our goal is to better un- bpA and confirmed by DNase I protection as- derstand how acquisition of a large, self-trans- says. A mutational analysis demonstrated that missible plasmid with such a cost could shift GbpA contributes to the ability of adherence to interactions within bacterial communities and mucin-secreting HT-29 MTX cells as well as further alter evolutionary dynamics in natural mucin. In addition, the gbpA mutant exhibited environments. With increased knowledge of reduced intestinal colonization and virulence in this unknown inhibitory agent, it is possible mice. The combined results proposed a model that an interesting microbial community in which SmcR and IscR cooperate for precise dynamic where pathogens forced to uptake control of the expression level of GbpA during the pMP could be targeted and treated with infection. extracted supernatant leading to novel forms of agricultural and medical treatment exists. n 49 A Moving Target: Acquisition of a n 50 Cryptic Megaplasmid Results in Pseudomonas aeruginosa quorum Sensitivity to Unknown Inhibitory sensing in chronic wound infections Agent A. R. Cueva1, A. Armstrong1, J. Everett1, D. B. A. Smith, D. A. Baltrus; Fleming1, K. Turner2, M. Whiteley2, K. P. University of Arizona, Tucson, AZ. Rumbaugh1; 1TTUHSC, Lubbock, TX, 2UT Austin, Austin, It is well known that microbes can alter sur- TX. rounding environments as well as interact with their host and fellow microorganisms. Pseudomonas aeruginosa is one of the most Therefore, to better understand complexes frequent causes of chronic wound infections. such as the microbiome, it is necessary to The production of most virulence factors that understand the dynamics of microbe-microbe contribute to the pathogenesis of P. aeruginosa interactions and potential drivers of microbial is regulated by cell to cell signaling, or quorum communities. The Pseudomonas genus consists sensing (QS). We have previously demon-

62 ASM Conferences Poster Abstracts strated that QS is essential to the pathogenesis Recently, the whole-genome sequencing of B. of P. aeruginosa in burn wound infections cepacia strain GG4, isolated from and quorum signals have also been detected of ginger, was performed. Preliminary studies in ischemic wounds; however, little has been showed that the wild type strain was capable elucidated about the role of QS in the chronic of AHL synthesis. We aimed to characterize wound environment. To determine the extent the putative luxI homologue and postulated of QS activity in P. aeruginosa-infected chronic similar AHL profile by the recombinant AHL wounds we: used LCMS to detect QS signals synthase. Methods: The genome of strain in murine chronic wound tissue; used fluores- GG4 was analyzed with RAST server and cent QS ‘reporter’ strains and confocal micros- BLAST2GO for gene predictions and an- copy to indirectly visualize the expression of notations. From the annotated genome, the P. aeruginosa QS genes in situ; analyzed the putative luxI homologue, burI, was identified global expression of P. aeruginosa in vivo with and cloned into pET28a for overexpression in RNAseq technology; and compared the infec- E. coli BL21. The recombinant BurI protein tion sequela of wild-type P. aeruginosa versus was purified and the production of AHL was a QS mutant in mouse chronic wounds. Our characterized using liquid data indicate that QS is active early in murine mass spectrometry (LC-MS) Results: In silico chronic wound infections, but quickly tapers analysis of the 6.6 Mb genome of strain GG4 off. We also observed that a QS mutant strain revealed the abundance of genes coding for of P. aeruginosa was significantly less tolerant carbohydrate, fats and protein metabolism, to gentamicin treatment in situ compared to a which suggests that the bacterium is well wild-type strain, and that the tolerance of the adapted to nutrient-rich rhizosphere environ- wild-type was reduced after early treatment ment. The burI gene which encodes the 25 with a QS inhibitor. Taken together, our data kDa putative AHL synthase was overexpressed indicate that QS may be important in the in E. coli. The protein sequence has a high formation of biofilms early in chronic wound degree of homology with AHL synthases from infections, which results in increased antibiotic other Burkholderia strains. LC-MS analysis tolerance. of the culture supernatant showed the pres- ence of 3-oxo-hexanoylhomoserine lactone, n 51 N-octanoylhomoserine lactone, 3-hydroxy- octanoylhomoserine lactone and N-nonanoyl- Whole Genome Sequencing Enables homoserine lactone. To our knowledge, strain Characterization of LuxI homologue GG4 is the first soil isolate among Burkhold- of Burkholderia cepacia GG4 eria strains to synthesize N-nonanoylhomoser- K. How, K. Hong, K. Chan; ine lactone. The results also indicates that BurI University of Malaya, Kuala Lumpur, MALAY- is indeed the AHL synthase as the AHL profile SIA. was found to be similar to its wild type strain. Conclusion: In this present study, the genome Background: Quorum sensing is a mechanism of strain GG2 is a significant addition of the for regulating proteobacterial gene expression genomic data from the Burkholderia genus and in response to changes in cell density. Such will therefore be a valuable resource for future cell-to-cell communication regulates a diverse investigation. We also reported the production array of physiological activities, ranging from of AHL by E. coli harboring the recombinant biofilm formation to . By far, BurI was indeed similar to the wild type. This the most extensively studied QS molecules is finding represents the initial step in elucidating N-acyl homoserine lactone (AHL). These sig- the role and the mechanism of the autoinducer nalling molecules are produced by LuxI homo- system possessed by strain GG4. logue or commonly known as AHL synthase.

5th ASM Conference on Cell-Cell Communication in Bacteria 63 Poster Abstracts n 52 and the production of virulence factors and Recombination drives Pseudomonas quorum sensing (QS) signals. Whole genome aeruginosa diversity during cystic analysis indicates high levels of intra-variant fibrosis infection diversity and that recombination and not spontaneous mutation is the dominant driver S. E. Darch1, A. McNally2, K. H. Turner1, F. of diversity in this population. Furthermore, Harrison3, J. Corander4, K. Paszkiewicz5, M. phenotypic differences between variants are Whiteley1, S. P. Diggle3; statistically associated with distinct recombina- 1University of Texas at Austin, Austin, TX, tion events rather than linked to mutations in 2Nottingham Trent University, Nottingham, known genes. Our results support a number of UNITED KINGDOM, 3University of Not- important and previously unobserved findings: tingham, Nottingham, UNITED KINGDOM, (i) the significant role of recombination in 4University of Helsinki, Helsinki, FINLAND, driving phenotypic and genetic diversification 5University of Exeter, Exeter, UNITED KING- during in vivo chronic respiratory infection; DOM. (ii) the potential impact of in vivo diversity on patient-to-patient transmission studies; (iii) the During chronic bacterial infection, evolu- complexity of in vivo genetic mutations and tion and diversification of founding clones their resulting effects on phenotype. facilitates adaptation to in vivo growth and the appearance of antibiotic resistance. The Cystic Fibrosis (CF) lung harbors a complex, n 53 polymicrobial ecosystem, in which Pseudomo- Dose-dependent effects of Bacillus nas aeruginosa is capable of sustaining chronic on colonic bacterial community in infections, which are highly resistant to mul- piglets challenged with Escherichia tiple antibiotics. It is now well established that coli, as revealed by deep 16S rRNA founder P. aeruginosa populations evolve over sequencing many years of chronic CF infection, leading to Q. Wu, J. Wang, Y. Zhu; high levels of temporal phenotypic and genetic China Agricultural University, Beijing, diversity within a single patient. Recent stud- CHINA. ies examining diversity within P. aeruginosa populations isolated from the CF lung have Probiotic could be a promising alternative described significant variation in antibiotic to antibiotics for the prevention of enteric susceptibility profiles in isolates, which vary in infections; however, further information on morphological appearance. However, no study the underlying mechanisms is required. Stable has conducted a detailed examination of a sin- commensal gut microbial community contrib- gle, morphologically homogeneous population utes to block pathogenic action in infection. of P. aeruginosa, nor has any study provided Understanding probiotic effects on bacterial a comprehensive map at the genome level of community in gut is essential to realize the full phenotypic variation within a CF lung popula- benefits and consequences of in-feed probi- tion. We address this gap in understanding of otics. In this study, we defined the lumenal how diversity evolves in the CF lung by inves- bacterial community from the colon, and char- tigating the phenotypic and genotypic diversity acterized the dose effects (3.9 × 109 CFU/d or of 44 morphologically identical Liverpool 7.8 × 109 CFU/d) of in-feed Bacillus mixture epidemic strain B58 P. aeruginosa ‘variants’ (Bacillus subtilis and Bacillus licheniformis) taken from a single CF patient sputum sample on the community in an F4 (K88)-positive at a single snapshot in time. Comprehensive Enterotoxigenic Escherichia coli infection phenotypic analysis of these variants reveals model of F4 receptor-negative piglets enteritis large variances and trade-offs in both growth (n = 6) using culture-independent Illumina

64 ASM Conferences Poster Abstracts high-through 16S rRNA V4 gene sequencing. ing by few isolates compared to other strains, After quality filter, 376,600 - 1,230,800 reads including TIGR4 and D39. Subsequent whole were used for further analysis. 347 - 516 op- genome sequence analyses revealed the erational taxonomic unit (OTU) were obtained. presence of two different pspC genes in the We found that the colonic bacterial commu- clinical isolates, pspC6.9 encoding a choline nity, regardless of the different treatment, was binding version and pspC9.4 encoding a cell mainly composed of members of the phyla wall anchored LPXTG protein. Interestingly, Bacteroidetes, Spirochetes, , Teneri- PspC9.4 displayed an overall low sequence cutes and Proteobacteria, presenting on > 90% homology to various PspC proteins which are of bacteria species. The collateral effects on known to interact with FH. Further investiga- the microbiota of Bacillus-fed animals caused tions showed that PspC9.4 binds human FH divergence from that of control animals, with through its amino terminal domain and a 9 notable changes being increases in the relative amino acid motif present in the N terminal abundance of lactobacillus and Bifidobacteria domain is directly involved in this interaction. populations and decreases in Escherichia coli Interaction kinetics between recombinant His- populations. Bacillus mixture increased the PspC9.438-434 and FH using surface plasmon community diversity as determined by shannon resonance displayed a significantly higher index. High dose of Bacillus mixture increased binding affinity in comparison to other PspC the number of class and decreased the family members. We here show that high FH number of class Mollicutes and Thermoplas- binding capability of clinical isolates of sero- mata. We suggested that up- and down- regu- type 6B strains is associated with the presence lation of specific bacterial species may be of an additional copy of PspC, pspC9.4. involved in colonization against Escherichia coli providing a potential therapeutic approach n 55 through specific manipulation of the intestinal The sialic acid N-acetyl neuraminic microbiome. Also, it adds further suggest a acid acts as a human-specific threshold reference for the use of probiotics in signal to enhance virulence in clinical practice. Key words: Bacillus, Micro- Streptococcus pneumoniae TIGR4 bial community, DNA sequencing, piglets K. Hentrich1, J. Löfling2, S. Normark1, B. n 54 Henriques Normark1; 1Department of Laboratory Medicine, Division High FH binding capability of of Clinical Microbiology, Karolinska Univer- clinical isolates of Streptococcus sity Hospital, Stockholm, SWEDEN, 2Depart- pneumoniae serotype 6B is ment of Microbiology, Tumor and Cell Biology, associated with the presence of an Karolinska Institutet, Stockholm, SWEDEN. additional copy of PspC Streptococcus pneumoniae is a pathogen A. Pathak, V. Sender, S. Normark, B. Hen- known to cause severe diseases like pneu- riques Normark; monia or septicemia. When entering the host Karolinska Institutet, Stockholm, SWEDEN. S. pneumoniae encounters a mucin-layer Pneumococcal surface protein C (PspC), a decorated with sialic acids. In most animals N- major virulence factor of Streptococcus pneu- acetylneuraminic acid (Neu5Ac) is converted moniae mediates evasion of the host immune to N-glycolylneuraminic acid (Neu5Gc) by the system by binding to human complement cytidine monophosphate-N-acetylneuraminic regulatory protein factor H (FH). Binding acid hydroxylase (CMAH). A mutation in the studies using different clinical isolates of CMAH gene, exclusively found in humans, serotype 6B strains showed high FH bind- leads to a loss of synthesis of Neu5Gc and to

5th ASM Conference on Cell-Cell Communication in Bacteria 65 Poster Abstracts an overproduction of Neu5Ac. In this study we ning electron microscopy of septal epithelia are investigating the role of sialic acids as sig- isolated from colonized mice. Production of naling molecules in host specificity and patho- neuraminidase (responsible for cleavage of SA genesis. We observed an increase in transcrip- from host cells) was assayed for a number of tion and enzyme activity of the pneumococcal clinical isolate strains using a standard fluoro- neuraminidase A (NanA) in response to the metric assay. Streptococcus pneumoniae bio- sialic acid Neu5Ac as compared to Neu5Gc. films were grown in the presence and absence After intranasal challenge, NanA mediates an of various carbohydrate sources on polystyrene earlier development of pneumonia, septicemia plates or glass slides and stained to determine and lower survival rates in CMAH-/- as com- levels of biofilm formation and overall biofilm pared to C57BL/6 wild-type mice. Here, we architecture. A mutant deficient in NanA was show that S. pneumoniae is able to decrease not attenuated in an in vitro biofilm model, but chemokine levels and neutrophil recruitment displayed a complete inability to form biofilms in the lungs of CMAH-/- mice. In conclusion, within the nasopharynx. Clinical isolate strains S. pneumoniae leads to increased virulence exhibited a wide range of neuraminidase enzy- in response to Neu5Ac, mainly abundant in matic activity, potentially contributing to the humans, and is able to alter the innate immune varying abilities of these strains to form bio- response. films in vivo. Both free SA and mucin-bound SA were able to increase biofilm formation n 56 in vitro in a modest fashion at low concentra- tions. The increase in biofilm formation in the Contribution of host presence of mucin-bound SA was abrogated in monosaccharides to biofilm a neuraminidase deficient mutant. Conversely, formation during Streptococcus addition of glucose and other preferred sugars pneumoniae nasopharyngeal was markedly inhibitory to biofilm formation. colonization This data suggests that site-specific nutrient K. Blanchette, R. Gilley, C. J. Orihuela; availability may have a profound affect on University of Texas Health Science Center at colonization by influencing the bacterial mode San Antonio, San Antonio, TX. of growth. Therefore, carbohydrate availability may act as an important signal during pneumo- Streptococcus pneumoniae is an important coccal biofilm formation and dispersal. etiologic agent causing otitis media, pneumo- nia, meningitis, and bacteremia. Asymptomatic nasopharyngeal colonization is a requisite step n 57 in the development of invasive disease, and is Bacterial fight-and-flight mediated by formation of immunoquiescent responses enhance virulence in a pneumococcal biofilms. Multiple investiga- polymicrobial infection tors have now suggested that pneumococcal A. Stacy1, J. Everett2, P. Jorth1, U. Trivedi2, K. neuraminidase (NanA) plays an important role P. Rumbaugh2, M. Whiteley1; during colonization. Within the nutrient sparse 1The University of Texas at Austin, Austin, TX, nasopharynx, neuraminidase may act to cleave 2Texas Tech University Health Sciences Center, sialic acid (SA) from host epithelial cells. The Lubbock, TX. utilization of SA may act as an important nu- tritional signal for colonizing bacteria. Herein, The oral pathogen Aggregatibacter actinomy- we examine the contribution of neuraminidase cetemcomitans (Aa) resides in infection sites and SA, as well as other carbohydrate sources, with many microbes including commensal to biofilm formation by S. pneumoniae. In vivo streptococci such as Streptococcus gordonii biofilm formation was examined using scan- (Sg). During infection, Sg promotes the viru-

66 ASM Conferences Poster Abstracts lence of Aa by producing its preferred carbon by increasing their tolerance to the oxidative source, L-lactate, a phenomenon referred stress generated by immune cells. Despite the to as cross-feeding. However, as with many recognized role of QS in enhancing the oxida- streptococci, Sg also produces high levels of tive stress response, the consequences of this the antimicrobial hydrogen peroxide (H2O2), relationship for the bacterial ecology remain leading to the question of how Aa deals with unexplored. In this work we demonstrate this potent antimicrobial during co-infection. that QS increases resistance also to osmotic, Here we demonstrate that Aa possesses thermal and heavy metal stress.Furthermore two complementary responses to H2O2: a a QS-deficient lasR rhlR mutant is unable to detoxification or “fight” response mediated by exert a robust response against H2O2 as it catalase (KatA) and a dispersion or “flight” has less induction of catalase and NADPH- response mediated by Dispersin B (DspB), an producing dehydrogenases. Phenotypic enzyme that dissolves Aa biofilms. Using a microarrays revealed that the mutant is very murine abscess infection model, we show that sensitive to several toxic compounds. As the both of these responses are required for Sg to anti-oxidative enzymes are private goods not promote Aa virulence. While the role of KatA shared by the population, only the individuals is to detoxify H2O2 during co-infection, three- that produce them benefit from their action. dimensional (3D) spatial analysis of mixed Based on this premise, we show that in mixed infections revealed that DspB is required for populations of wild-type and the mexR mutant Aa to spatially organize itself at an optimal (resistant to the QS inhibitor furanone C-30), distance (>4 µm) from Sg, which we propose treatment with C-30 and H2O2 increases the allows cross-feeding yet reduces exposure to proportion of mexR mutants; hence, oxidative inhibitory levels of H2O2. In addition, these stress selects resistance to QS compounds. In behaviors benefit not only Aa but also Sg, sug- addition, oxidative stress alone strongly selects gesting that “fight” and “flight” stimulate the for strains with active QS systems that are fitness of the community. These results reveal able to exert a robust anti oxidative response that an antimicrobial produced by a human and thereby decreases the proportion of QS commensal bacterium enhances the virulence cheaters in cultures that are otherwise prone to of a pathogenic bacterium by modulating its invasion by cheats. As in natural environments spatial location in the infection site. stress is omnipresent, it is likely that this QS enhancement of stress tolerance allows cells to n 58 counteract QS inhibition and invasions by so- cial cheaters, therefore having a broad impact Quorum sensing enhancement of in bacterial ecology. the stress response promotes resistance to quorum quenching and prevents social cheating n 59 Resistance against Quorum R. Garcia-Contreras1, L. Nuñez-López1, R. quenchers in Pseudomonas Jasso-Chávez1, B. W Kwan2, J. A Belmont1, T. aeruginosa clinical isolates Maeda3, T. K Wood2; 1National Institute of Cardiology, Mexico City, R. Garcia-Contreras1, B. Peréz-Eretza1, R. MEXICO, 2Pennsylvania State University, Coria-Jiménez2, M. Martínez-Vázquez3, G. Pennsylvania, PA, 3Kyushu Institute of Tech- Soberón-Chávez4, T. Maeda5, T. K. Wood6; nology, Kytakyushu, JAPAN. 1National Institute of Cardiology, Mexico City, MEXICO, 2National Institute of Pediatry, Quorum sensing (QS) coordinates the expres- Mexico City, MEXICO, 3Institute of Chemistry, sion of virulence factors and allows bacteria UNAM, Mexico City, MEXICO, 4Institute of to counteract the immune response, partly

5th ASM Conference on Cell-Cell Communication in Bacteria 67 Poster Abstracts

Biomedical Research, UNAM, Mexico City, mechanisms are also involved, since C-30 MEXICO, 5Kyushu Institute of Technology, resistant strains that are sensitive to antibiotics Kytakyushu, JAPAN, 6Pennsylvania State (hence the over-expression of efflux pumps is University, Pennsylvania, PA. not expected) were also identified. References 1. García-Contreras, R., T. Maeda, and T. K. Resistance against Quorum quenchers in Pseu- Wood. 2013. Resistance to quorum-quenching domonas aeruginosa clinical isolates. Quorum compounds. Appl Environ Microbiol 79:6840- quenching is a promising alternative to combat 6. 2. García-Contreras, R., M. Martinez- infections of several bacterial pathogens Vazquez, N. Velazquez Guadarrama, A. G. including Pseudomonas aeruginosa, one of the Villegas Paneda, T. Hashimoto, T. Maeda, H. advantages of these approach in comparison Quezada, and T. K. Wood. 2013. Resistance to with the utilization of antibiotics is that inter- the quorum-quenching compounds brominated fering with cell-cell communication attenuates furanone C-30 and 5-fluorouracil in Pseudo- virulence without affecting growth (at least in monas aeruginosa clinical isolates. Pathog Dis rich culture medium); hence it was postulated 68:8-11. 3. Maeda, T., R. García-Contreras, M. that the selection of clones resistant to quorum Pu, L. Sheng, L. R. Garcia, M. Tomas, and T. quenching will be minimum or even absent, K. Wood. 2012. Quorum quenching quandary: due the lack of a selective pressure that favor resistance to antivirulence compounds. ISME the spreading of the resistant clones; however, J 6:493-501. since in addition to control the expression of virulence traits quorum sensing also controls the expression of some private goods (such as n 60 the enzymes necessary to catabolize adenos- Interference of bacterial energy ine); conditions in which a strong selective generation and quorum-sensing pressure exists can be readily achieved. regulator folding by indole Recently, we demonstrated that when using J. Kim, W. Park; adenosine as sole carbon source the selection Korea University, Seoul, KOREA, REPUBLIC of clones resistant against the canonical quo- OF. rum quencher, the brominated C-30 furanone was possible, the selected clones were mutants Indole has received great attention because that over express the multidrug efflux pump of its extensive effects on various biological MexAB-OmpR, moreover we also discovered functions in the bacterial population, such that clinical isolates with similar mutations are as biofilm formation, antibiotic resistance also able to resist C-30 inhibition (of adenosine and virulence. Indole has been also reported based growth), even if those strains were not to function as an intercellular signal, like pre treated by the compound (1, 3). Our recent QS signals, in bacterial communities. We work was focused in the characterization of demonstrated that many genes involved in several P. aeruginosa clinical strains isolated energy generation and chaperones are highly from several sources, including blood, urine expressed under indole treatment in both Pseu- and catheter tips, and of strains isolated from domonas putida and Acinetobacter oleivorans. cystic fibrosis patients, among those strains P. putida hslU mutant study partially supported we found some isolates resistant against C-30 that chaperone is important for protecting and some of them also resistant against another cells under indole. Subsequent biochemical quorum quencher 5 fluorouracil (2) and we are analyses have shown that indole increases currently working in the characterization of the NADH/NAD(+) ratio and decreases the those strains in order to understand the subja- adenosine triphosphate (ATP) concentration cent resistant mechanisms, preliminary work inside cells, due to membrane perturbation and suggest that in addition to efflux other resistant higher expression of TCA cycle genes. This

68 ASM Conferences Poster Abstracts energy reduction leads to a reduction in cell n 61 size and an enhancement of biofilm formation Identification and characterization in P. putida. Interestingly, our in vitro protein- of the N-acylhomoserine lactone- refolding assay using malate dehydrogenase degrading gene from the coagulase- with purified GroEL/GroES demonstrated negative staphylococci (CNS) that indole interferes with protein folding. Indole enhanced P. putida biofilm formation T. Morohoshi1, R. Sato1, T. Yamaguchi1, N. and inhibited swimming motility, which were Someya2, T. Ikeda1; not observed when AHL was already bound to 1Utsunomiya University, Utsunomiya, Tochigi, the QS regulator, thereby suggesting that the JAPAN, 2National Agriculture and Food quorum sensing regulator PpoR-AHL complex Research Organization (NARO), Tsukuba, masks the effects of indole. Quorum sensing Ibaraki, JAPAN. (QS)-dependent biofilm formation and motility Many gram-negative bacteria have a quorum- were controlled by AqsR in A. oleivorans. sensing system and produce N-acylhomoserine QS-controlled phenotypes appeared to be lactones (AHLs) that they use them as a inhibited by indole and the aqsR mutant had quorum-sensing signal molecule. AHL-degrad- the same phenotypes. We demonstrated that ing genes have been cloned and characterized the turnover rate of AqsR became more rapid from various bacteria. Coagulase-negative without AHL signal. The addition of exog- staphylococci (CNS) are present on the skin of enous indole decreased the expression of two animals and considered to be less virulent. In AqsR-targeted genes. The overexpression of this study, the novel AHL-degrading gene was AqsR in Escherichia coli was impossible with screened from the genome sequences of CNS the indole treatment. Surprisingly, our [(35)S] species. In previous study, we have identified a methionine pulse-labelling data demonstrated novel AHL- gene (ahlS) from potato that the stability and folding of AqsR protein leaf-associated Solibacillus silvestris. The ahlS decreased under indole without changing homologues were present in the genome of aqsR mRNA expression in E. coli. Indole CNS strains, which were Staphylococcus car- also resulted in a loss of TraR-dependent traG nosus, S. haemolyticus, and S. saprophyticus. expression in an Agrobacterium tumefaciens To check the AHL-degrading activity, these indicator strain. However, when indole was three CNS strains were cultivated in LB me- added after incubation with exogenous AHL, dium containing 10 µM C6-HSL or C10-HSL. indole could not inhibit the TraR-dependent After incubation for 9 h, AHLs were complete- expression of the traG promoter. When P. aeru- ly degraded by these strains. The ahlS genes ginosa and Chromobacterium violaceum were on the genome of CNS strains were amplified tested to extend this conclusion, QS-dependent by PCR and cloned into the pHSG398 vector. production of pyocyanin and violacein was Escherichia coli harboring the ahlS-expressing inhibited by indole without reducing the QS plasmids showed AHL-degrading activity. signal production under indole treatment. Here, Therefore, it was revealed the ahlS homolo- we provided evidence showing that the indole hues on the genome of CNS strains function as effect on QS-controlled bacterial phenotypes AHL-degrading gene. We also isolated another is due to reduction of energy generation and AHL-degrading CNS strain, Staphylococcus inhibited QS regulator folding. sciuri StLB252, from the plant surface. To amplify the internal region of the ahlS homo- log from StLB252, degenerate PCR primers were designed based on the ahlS gene from S. haemolyticus. The ahlS gene on the genome of StLB252 was successfully amplified by

5th ASM Conference on Cell-Cell Communication in Bacteria 69 Poster Abstracts

PCR and showed AHL-degrading activity as is not able to response to PQS. Our research well as the ahlS genes from other CNS strains. imply that mucoid mutant may act as an unco- These results demonstrated that the ahlS genes operative strain to group behavior in bacterial are widely conserved in AHL-degrading CNS community because of its poor response ability strains. to PQS. n 62 n 63 Extracellular polysaccharide The interference of the alginate interfere with cell-cell intracellular signaling signaling molecule c-di-GMP on cell-to-cell communication J. Yang1, M. Toyofuku1, R. Sakai1, K. Tateda2, N. Nomura1; S. Ito, K. Kakihara, M. Toyofuku, N. Nomura; 1University of Tsukuba, Tsukuba, JAPAN, 2Toho Graduate School of Life and Environmental University School of Medicine, Tokyo, JAPAN. Sciences, University of Tsukuba, Tsukuba, JAPAN. Most bacteria exist as communities such as biofilms in the environment, and bacteria use Biofilm dispersion and cell-to-cell communica- signaling molecule to communicate with each tion are highly related. Nitric oxide (NO) is other in such kind of bacterial communities. one of the cues that has been shown to induce It has been reported that bacteria detect their biofilm dispersion in many bacteria including population by sensing the concentration of Pseudomonas aeruginosa. Therefore the accu- the signaling molecules and regulate the gene mulation of NO inside biofilms is important in expression of the community, which is called determining the timing of dispersion. Still how quorum sensing. Bacteria use quorum sensing the accumulation of NO is controlled is unde- to control the group behavior. Interestingly, the termined. We demonstrated one of the commu- frequency of spontaneous mutation in biofilms nication signal Pseudomonas Quinolone signal is very high, and various spontaneous mutants (PQS) in P. aeruginosa induces NO-mediated emerge in biofilms. However, the interaction biofilm dispersion. A delay in dispersion of between the mutants and the wild-type strain a ΔpqsA mutant of P. aeuginosa PAO1, that are still not fully understood. Therefore, the could not produce PQS was observed com- aim of this study is to investigate the cell-cell pared to the parent strain. In this process, PQS communication of spontaneous mutants. We promotes nirS gene expression during denitrifi- chose the mucoid mutant of Pseudomonas cation and accumulate Nitric Oxide. However, aeruginosa as our research object, which is no significant difference of nirS expression is well known to dominate in P. aeruginosa bio- detected in presence or absence of PQS when films in the lung of cystic fibrosis patients and cultured planktonically. Due to the difference overproduce extracellular polysaccharide algi- of nirS gene expression between biofilm and nate. In this study, we focused on the alginate planktonic cells, we considered that intracel- overproduction phenotype of mucoid mutant, lular second messenger, c-di-GMP is involved. and examined if alginate interfere the signaling Here we analyzed the effect of c-di-GMP on response of mucoid mutants. We compared cell-to-cell communication and demonstrate how the mucoid mutant and nonmucoid strain that c-di-GMP interferes PQS quorum sensing response to PQS. Also, the PQS response in a mediated regulation of denitrification gene.In nonmucoid strain was tested in the presence this study, we construct wspF deletion mutant and absence of exogenous alginate. We found which can overproduce c-di-GMP and thus that alginate may interfere the response to could mimic the biofilm state of cells. Besides, PQS. The results suggested that mucoid mutant we deleted the pel and psl genes to prevent

70 ASM Conferences Poster Abstracts aggregating of cells in liquid shaking culture AHL acylase, which hydrolyzes the amide as is used in other previous study. Hence, we bond of AHL. AmiE showed homology with a used ΔpelΔpsl and ΔpelΔpslΔwspF strains to member of amidases (EC 3.5.1.4) but not with compare denitrification gene expression with any known AHL acylase enzymes. To analyze or without PQS. Expression of nirS gene in- the substrate specificity of AmiE, we mixed a creased when PQS was added in the c-di-GMP cell suspension of Escherichia coli harboring overproducing strain ΔpelΔpslΔwspF, but not amiE gene with a wide range of AHLs. AmiE in the ΔpelΔpsl strain. No significant differ- degraded C8-HSL, C10-HSL, and C12-HSL ence in the expression of norB gene, which and showed the highest degrading activ- encodes NO reductase was observed among ity against C10-HSL, but it did not degrade the strains tested. Our data suggest that PQS C6-HSL. These results suggested that AmiE promotes accumulation of NO by upregulating has activity similar to those of known AHL ac- nirS gene expression in the presence of c-di- ylases. An amino acid sequence of AmiE from GMP. Our data further imply that c-di-GMP Ooi24 showed greater than 99% identities with modulates PQS regulon. uncharacterized proteins from Acinetobacter ursingii CIP 107286 and Acinetobacter sp. CIP n 64 102129 but it was not found in the genome sequences of other Acinetobacter strains. The AmiE, a novel N-acylhomoserine presence of transposase-like genes around lactone belonging to the amidase the amiE genes of these three Acinetobacter family, from the activated sludge strains suggests that amiE was transferred by a isolate Acinetobacter sp. Ooi24 putative transposon. Furthermore, the expres- S. Ochiai; sion of AmiE in Pseudomonas aeruginosa Utsunomiya University, Utsunomiya, JAPAN. PAO1 reduced AHL production and elastase activity, which was regulated by AHL-mediat- A number of gram-negative bacteria use N- ed quorum sensing. acylhomoserine lactones (AHLs) as quorum- sensing signal molecules. AHL-degrading genes have been cloned from various microor- n 65 ganisms and used to repress gene expression A small-molecule inhibitor of SmcR, via AHL-mediated quorum sensing. In previ- a qourum sensing master regulator, ous study, we have reported that Acinetobacter in Vibrio vulnificus strains, which isolated from activated sludge, Y. Bang, B. Kim, B. Kim, S. Choi; have AHL-degrading activity. For cloning of Seoul National University, Seoul, KOREA, novel AHL-degrading gene, pUC118-based REPUBLIC OF. genomic library of Acinetobacter sp. strain Ooi24 was prepared. When approximately Bacterial cell-to-cell communication known 10,000 clones were screened, one positive as quorum sensing (QS) is involved in diverse clone showed AHL-degrading activity and physiological processes including biofilm contained seven ORFs. Because sequencing development and pathogenesis. An opportu- analysis revealed that the fourth ORF, desig- nistic human pathogen Vibrio vulnificus also nated amiE, encoded the hydrolase family, we has a well conserved QS system consisting of next determined whether amiE works as an the homologues of V. harveyi Autoinducer-2 AHL-degrading gene. To determine whether (AI-2) signaling components. Among them, AmiE functions as an AHL-degrading enzyme, SmcR, a homologue of LuxR, plays a critical we used HPLC to analyze the structure role in regulation of various QS regulon as a of 3OC10-HSL digested by AmiE. HPLC master transcriptional regulator. Previously, we analysis revealed that AmiE functions as an determined the crystal structure of SmcR and

5th ASM Conference on Cell-Cell Communication in Bacteria 71 Poster Abstracts revealed that SmcR belongs to the TetR family Lysinibacillus sp.Gs50 treated C6-AHL could protein possessing putative ligand-binding be restored under acidic conditions indicated pockets. To identify the small molecules which the presence of putative AHL lactonase in inhibit the function of SmcR, we performed it. The isolate was able to degrade AHL of high-throughput screening of 8844 molecules various acyl chain lengths. Furthermore, the at 20 μM concentration using Escherichia enzyme was found to be localised into the coli dual plasmid system containing SmcR- membrane of the bacteria. To identify the gene expressing and SmcR-dependent reporter responsible for AHL degradation, primers plasmids. As a result, eight molecules were were designed against hypothetical protein screened out and verified. Among them, a Bsph_3377 [Lysinibacillus sphaericus C3-41] chemical 377B6 exhibiting the most effective (NCBI Reference Sequence: YP_001698999.1) SmcR-inhibition activity was selected as a for the putative AHL lactonase .Using these SmcR inhibitor. Effect of 377B6 on biofilm primers gene was amplified and cloned into development of V. vulnificus was assessed expression vector pET22b(+) which resulted in using an assay based on crystal violet staining the expression of 35kDa size protein. The soft at different time points. 377B6 significantly rot attenuation studies on potato by recombi- inhibited biofilm detachment of wild type V. nant E.coli BL21 against the plant pathogen vulnificus to the smcR mutant level. Further- Pectobacterium carotovorum carotovorum more, to determine the influence of 377B6 BR1 (PccBR1, laboratory isolate) and Mass on the expression of SmcR-dependent and spectrum analysis to ascertain the mechanism -independent genes, the transcriptomic changes of the enzyme are being conducted. Thus, this induced by 377B6 in V. vulnificus biofilm cells is the first report of the quorum quenching N- were analyzed by RNA-sequencing. In conclu- acyl homoserine lactonase from Lysinibacillus sion, the combined results suggested that this sp. and its application to quorum quenching as chemical could be a novel lead compound to biocontrol mechanism against plant pathogen interfere the QS of V. vulnificus. Pectobacterium carotovorum carotovorum BR1, the causal agent of soft rot. n 66 Quorum quenching N- Acyl n 67 Homoserine Lactonase from soil Degradation of N-Acylhomoserine isolate Lysinibacillus sp. Gs50 Lactone Quorum Sensing Signaling Molecules by Fish-Associated S. Garge, S. Ghosh, I. Ranadive, A. Nerurkar; Flavobacterium isolates M.S.University of Baroda, Vadodara, INDIA. H. Y. Chenia, N. Du Plooy; Quorum sensing is an example of cell-to-cell University of KwaZulu-Natal, Durban, communication in bacteria and depends on the SOUTH AFRICA. production, secretion and response to small, diffusible signal molecule called autoinducer Members of the genus Flavobacterium are which is Acyl Homoserine Lactone (AHL) often implicated in aquaculture disease in Gram negative bacteria. Lysinibacillus sp. outbreaks worldwide. Their biofilm-forming Gs50 isolated form soil was found to be the ability, virulence, recalcitrance to antimicrobial best among 97 isolates screened for their AHL therapy often leads to recurring outbreaks in degradation ability but not able to utilise it as fish farms and economic losses. Their ability to sole carbon source for growth. AHL-degrading communicate via quorum sensing (QS) and/or enzymes such as lactonase has been widely to quench QS was investigated in order to un- characterized in Bacillus species but has not derstand the role of cell-to-cell communication been yet reported in Lysinibacillus sp. Isolate in Flavobacterium pathogenicity and survival.

72 ASM Conferences Poster Abstracts

Twenty-eight Flavobacterium johnsoniae-like n 68 isolates from South African trout, salmon, eel Deciphering the molecular and fish tank biofilm and six Flavobacterium structure and function of VirB4-like spp. type strains were assessed for acyl-homo- ATPases in nucleoprotein import and serine lactone production (AHL) using Chro- export mobacterium violaceum CV026 and VIR07 and Agrobacterium A136 biosensor T-streak V. Hunsur Rajendra1, C. Gruber2, E. M. assays. Quorum quenching (QQ) was assessed Schrank3, S. Raffl1, K. Zangger3, E. L. Zech- using C. violaceum CV026 (short-chain AHL) ner1; and VIR07 (long-chain AHL) biosensor sand- 1Institute of Molecular Biosciences, University wich assays as well as AHL-degrading assays of graz, Graz, AUSTRIA, 2Institute of Molecu- (C4 – C12 AHLs). QQ was correlated with lar Biosciences, University of Graz, Graz, presence of lactonase gene, aidC, using primer AUSTRIA, 3Institute of Chemistry, University sets 1 (PS1) and 2 (PS2) and PCR amplifica- of graz, Graz, AUSTRIA. tion. No AHL production was observed with Bacterial type IV secretion systems (T4SS) all biosensor T-streak assays. QQ was only transfer macromolecules like nucleic acids observed for 17.9% (5/28) of F. johnsoniae- and proteins from bacteria to eukaryotes or like isolates using the short-chain C. violaceum between bacterial cells generally by a direct CV026 biosensor sandwich assay, while no contact dependent communication mecha- QQ was observed with the long chain VIR07 nism. VirB4-like proteins are associated with system. Both short- and long-chain AHL deg- all T4SSs described to date. These signature radation was observed for all F. johnsoniae- ATPases function in assembly of the T4SS like isolates using the AHL degradation assay, channel and biogenesis of extracellular pili in however, type strains did not display QQ Gram-negative systems. They are also required abilities. The AidC lactonase gene was ampli- for translocation of secretion substrates and fied from 35.7% (10/28) and 10.7% (3/28) of nucleoprotein uptake during pilus-mediated F. johnsoniae-like isolates using PS1 and PS2, phage infection. Very little is known about the respectively. While QS has not been observed regulation of VirB4-like ATPases in protein for Flavobacterium species, F. johnsoniae-like trafficking. It is also not known whether possess the ability to enzymatically degrade these proteins are involved in recognition of and prevent cell-to-cell signalling of other or- substrates destined for secretion. Our paradigm ganisms within their immediate environment. to study control of VirB4-like ATPases is TraC This would allow them to exclude competitors, of the F-like conjugative plasmid RI. TraC thus facilitating their survival. The lack of QS was modified with a strep-tag and its function might be attributed to limitations of biosensors verified by complementation of conjugative used and/or use of signalling molecules other transfer of a αtraC derivative of R1. However, than AHLs. While the aidC lactonase has been purified strep-TraC did not exhibit ATPase identified in some of the isolates tested, other activity in vitro. One possible explanation may QQ enzymes ( and//or acylases) be that ATPase activity is controlled by oligo- originating from diverse sources may be meric state. Hexamers of VirB4-like protein responsible for the observed QQ. TrwK hydrolyze ATP in vitro and ATP binding forces the formation of hexamers. Strep-TraC was incubated with a 20-fold molar excess of non-hydrolysable ATP and analyzed by gel filtration. The chromatograms indicate that strep-TraC behaves as a monomer in solution with and without ATP. The ATPase activity

5th ASM Conference on Cell-Cell Communication in Bacteria 73 Poster Abstracts might be controlled by interaction(s) of TraC antagonize QS are desired as they hold the with other relaxosome proteins. Possible inter- potential to control bacterial infections while action partners are currently under investiga- reducing the selective pressure incurred by tion. Enzymatic activity of VirB4-like ATPases traditional antibiotics. Previously we identified can also be controlled intra-molecularly. The several cysteine-based derivatives inspired by C-terminus of the VirB4-like protein TrwK natural products from the plant Petiveria allia- possesses alpha helices, which were shown cea, which are capable of antagonizing multiple to have an auto-inhibitory activity. Secondary different quorum sensing circuits. One of these structure prediction of TraC shows four con- compounds, S-phenyl-L-cysteine sulfoxide served alpha helices. The significance of these (compound 7), was shown to inhibit las-based alpha helices for enzyme regulation is under QS in both E. coli and P. aeruginosa-based re- investigation. Interestingly, truncated versions porters as well as reduce biofilm formation and of TraC, lacking individual alpha helices are increase Drosophila survival in a P. aeruginosa unable to complement the function of wild infection model. In the current work we have type traC in conjugative transfer and phage performed a global transcriptomic analysis of P. infections. To test the hypotheses that TraC is aeruginosa PAO1 when exposed to compound subject to auto-inhibition and that the alpha 7 using Illumina-based RNA-sequencing. helices mediate protein-protein interactions, Interestingly, many genes that were observed to truncated versions of TraC will be purified. be differentially regulated pertain to second- The ability of truncated TraC to hydrolyze ATP ary metabolism (e.g. anthranilate biosynthesis/ will be tested with and without other potential degradation) and iron starvation response. binding partners. Also, the purified truncated Many of these genes have also been shown to versions of TraC will be used in protein-pro- be regulated by QS. For example, phnAB (an- tein interaction studies with other relaxosome thranilate biosynthesis) and phzA-G (phenazine proteins. Furthermore, these TraC truncated biosynthesis) which are positively expressed in variants are used in phage-infection assays to the presence of 3-oxo-C12-HSL (the autoin- study nucleoprotein uptake by T4SS. ducer for the las pathway) are down-regulated in our experiments. Additionally, we observed n 69 that antABC (anthranilate degradation), which is repressed in the presence of 3-oxo-C12-HSL, A natural product-based cysteine were amongst the most highly up-regulated derivative inhibits quorum sensing genes when exposed to compound 7. Because and induces iron starvation of the potential anthranilate depletion that may response in Pseudomonas be suggested from these results, there may be aeruginosa less Pseudomonas Quinolone Signal (PQS) S. H. Kasper1, R. P. Bonocora2, J. T. Wade2, R. produced, as anthranilate is the precursor for A. Musah3, N. C. Cady1; PQS biosynthesis. Other highly up-regulated 1SUNY College of Nanoscale Science and genes include those involved in pyochelin and Engineering, Albany, NY, 2Wadsworth Center, pyoverdine biosynthesis (two iron-binding New York State Department of Health, Albany, siderophores) which suggests an iron starvation NY, 3University at Albany, State University of response. Although it is well established that New York, Albany, NY. QS and iron regulation are intricately inter- twined in P. aeruginosa, it is unclear exactly Pseudomonas aeruginosa is a Gram negative how compound 7 contributes to the observed opportunistic pathogen that utilizes multiple response. Investigations are currently underway quorum sensing (QS) pathways to coordinate to determine the molecular target of compound group behaviors such as biofilm formation and 7. It is anticipated that this information will en- virulence. Small molecule inhibitors that can able the design of more potent QS inhibitors.

74 ASM Conferences Poster Abstracts n 70 while V. ambigua and V. blumeiodes terpenoids Terpenoid components of African inhibited signal synthesis (LuxI). Vernonia Vernonia species as quorum sensing terpenoid components have the potential to inhibitors be novel therapeutic agents, which might be important in reducing virulence and pathoge- A. B. Aliyu, N. A. Koorbanally, B. Moodley, H. nicity of drug-resistant bacteria in vivo. Y. Chenia; University of KwaZulu-Natal, Durban, n 71 SOUTH AFRICA. Chivalry in Rivalry: An Insight into Despite the widespread use of the genus Verno- Interspecific and Intraspecific nia in traditional medicine, there is insufficient interactions in soil dwelling information on the mechanism/s by which they Bacillus cereus MSM-S1 exert a medicinal effect. Given the increasing B. Chakraborty, A. Sumana, T. Sengupta; incidence of multidrug-resistant pathogens, Indian Institute of Science Education and the medicinal potential of terpenoids from Research Kolkata, Mohanpur, INDIA. four Vernonia species was assessed as natural, plant-based anti-virulence compounds, by Bacteria have come a long way from be- inhibition of microbial quorum sensing (QS). ing considered as solitary recluses to social The antimicrobial activity of terpenoid compo- organisms. In a natural environment like soil, nents from four Vernonia species was assessed bacteria communicate with each other using against both Gram-positive and Gram-negative specialized chemical signaling process called bacteria using disc diffusion assays. Inhibi- quorum sensing (QS). The nature of commu- tion of QS-controlled violacein production in nication through QS does not always involve Chromobacterium violaceum was quantified co-operative signals rather they often evoke using the violacein inhibition assay (extracts conflicts within and between the species. In ranging from 0-9.5 mg/ml). Qualitative modu- spite of extensive research on bacterial interac- lation of QS activity and signal synthesis was tions, the basic question remains to answer is investigated using the agar diffusion double the detailed molecular mechanisms of signal ring assay and C. violaceum and Agrobacte- mediated interference in bacterial interactions. rium tumefaciens biosensor systems. Varying To address the problem, we have investigated antimicrobial activity was observed with V. the molecular mechanisms involved in soil blumeiodes extract being the most effective dwelling Bacillus cereus MSM-S1 during in- and V. ambigua the least effective. Inhibition ter- and intraspecific competitive interactions. of QS-controlled violacein production in Chro- We show that a single soil bacterium Bacillus mobacterium violaceum was quantified using cereus MSM-S1 adapt multiple strategies dur- violacein inhibition assays and was significant ing interspecific and intraspecific interactions. in the following order: V. glaberrima > V. For interspecific interaction, Bacillus cereus ambigua > V. blumeiodes = V. perrotetii. Inhi- MSM-S1 loses the battle against Pseudomonas bition was concentration-dependent, with the ≥ sp. MSM-M1 by a secreted antibacterial com- 90% inhibition being obtained with ≥ 0.6 mg/ pound of the later in the media. In the case of ml. Qualitative modulation of quorum sensing sibling rivalry between the colonies of Bacillus activity and signal synthesis was investigated cereus MSM-S1, the cells stop moving towards using the agar diffusion double ring assay. each other by inhibition in QS. Our investiga- Both LuxI and LuxR activity were affected tion involving Real Time PCR also reveals by V. glaberrima and V. perrotetii terpenoids differential expression of numbers of genes suggesting that they target both quorum in competing cells of Bacillus cereus colonies sensing signal synthesis and receptor activity, and such differentially expressed genes are

5th ASM Conference on Cell-Cell Communication in Bacteria 75 Poster Abstracts involved in spore formation, chemotaxis, We also saw that growth in albumin inhibited flagellar motions, cell division, production of the production of the acylated homoserine virulence factors and quorum sensing. Interest- lactones (AHL) 3OC12-HSL and C4-HSL, but ingly, comparison of cellular protein profiles promoted the production of quinolone-related between interacting and non-interacting edges autoinducers. Preliminary binding assays of Bacillus cereus MSM-S1 colonies show suggest that albumin can bind and sequester over-expression of a number of proteins and AHL-based autoinducers, which represses QS. mass spectrometry analyses confirmed that We are now investigating the physiological these are associated with bacterial stress relevance of these observations in albumin-rich response. Therefore, the present study revolves and albumin-deplete in vivo environments. around the ecological and evolutionary signifi- cance of understanding the molecular dynam- n 73 ics of signaling network involved in interspe- Effect of Static Magnetic Field in cific and intraspecific interactions in bacterial the autoaggregation phenomenon of world as the dynamics direct the bacteria to Enteropathogenic Escherichia coli choose between co-operation, co-existence and (EPEC) competition. M. A. Quiñones-Peña, B. González-Pedrajo, n 72 G. Tavizon, C. A. Eslava; UNAM, Mexico city, MEXICO. Pseudomonas aeruginosa quorum sensing is altered by serum albumin Enteropathogenic Escherichia coli (EPEC) constitute an important cause of infant diar- A. Clinton, K. P. Rumbaugh; rhea in developing countries. A three-stage Texas Tech University Health Sciences Center, EPEC pathogenesis model was described, Lubbock, TX. however, properties as autoaggregation, also Chronic wound infections cause high rates of are involved in the bacterial pathogenesis. The morbidity and mortality in a large patient pop- static magnetic field (SMF), has been used to ulation and are responsible for a large medical evaluate its effect on bacteria growth, but little cost burden in the US annually. Pseudomonas on pathogenicity properties. In this work was aeruginosa (PA) is one of the most common analyzed the SMF effect on EPEC autoaggre- bacterial pathogens in chronic wounds, and gation, considering time and intensity of ferrite much of the virulence associated with PA is or neodymium magnets in a static model. controlled by a system of cell-cell communi- Strains E coli HB101 and EAEC (enteroag- cation termed quorum sensing, or QS, which gregative E coli) 49766 (O?:H10), were used involves three distinct regulatory systems (Las, as negative and positive controls respectively. Rhl and PQS). QS can alter the expression of Strain E2348/69 EPEC wild type and mutant over 5% of PA genes in response to the amount strains in type III secretion system (T3SS), of specific chemical signals, or autoinducers, bundle-forming pilus fimbriae, flagella and within an environment. Our lab has previously EspC (autosecreted protein C) were used to shown that the blood protein albumin inhibits evaluate the magnet effects. An autoaggrega- the ability of PA to lyse Staphylococcus aureus tion activity is considered positive when auto- and we hypothesize this is due to an inhibition aggregation index is over 2.5% and negative of QS by albumin. In this study, we sought if the result is less. For autoaggregation assay to identify how albumin is altering QS. We measure the O.D 600 nm each culture, after observed that QS-controlled exoproducts, such each measurement the aliquot was vortexed for as LasA and LasB, were downregulated when 30 s while still in the cuvette and the O.D. 600 PA was grown in the presence of albumin. nm was measured again. The percent increase

76 ASM Conferences Poster Abstracts in O.D. 600 nm after vortexing was recorded sized and in vitro analyzed for their potential as a quantitative aggregation index. The results to induce QS phenomenon in agr mutant showed that a treatment during 30 min expo- C. perfringens. The 5-residue oligopeptide sure or more with 100 mT reduce significantly showed significantly stronger activity to in- the autoaggregation in E2348/69 wild type duce the transcription of perfringolysin O gene strain, when it´s compared with untreated (pfoA). Furthermore, based on the determined strain. The EPEC mutant in T3SS shows structure of AIP, we designed analogue inhibi- similar effects to the observed in the wild tors of AIP targeting two component (VirR/ type EPEC strain. However, bundle-forming VirS) regulatory system of C. perfringens pilus fimbriae, flagella and EspC mutants, the 13. As a result, we obtained several thio- autoaggregation activity is negative and SMF lactone oligopeptides which are suppressed treatment do not induce changes. Although, the transcription of pfoA around micromolar the specific is not clear the SMF mechanism of concentration. By using those oligopeptides as action, is possible that it is inducing interfer- leading compounds, we will develop an effec- ence in cell to cell interactions, or regulating tive antipathogenic drug targeting the cyclic the gene expression. peptide-mediated cell-to-cell communication system of C. perfringens 13. n 74 Identification of Autoinducing n 75 Peptide of Clostridium Perfringens Defining Ligand Specificity for the and Development of its Inhibitors LuxN Quorum-Sensing Receptor R. P. Singh1, K. Ohtani2, R. Yokohata1, K. X. Ke, B. Bassler; Sonomoto1, T. Shimizu2, J. Nakayama1; Princeton University, Princeton, NJ. 1Kyushu University, Fukuoka City, JAPAN, Quorum sensing (QS) is a process of cell-cell 2University of Kanazawa, Kanazawa, JAPAN. communication that bacteria use to control col- Quorum sensing (QS) system of Gram-positive lective behaviors. QS relies on the production, bacteria uses lactone and thiolactone autoin- release, and receptor-driven detection of extra- ducer peptide (AIP) to modulate expression cellular signal molecules called autoinducers. of certain gene(s), particularly associated with Gram-negative bacteria commonly use acyl virulence in case of pathogenic bacteria. C. homoserine lactones (AHLs) as autoinducers. perfringens 13 is an anaerobic pathogen, caus- The bioluminescent marine bacterium Vibrio ative agent for gas gangrene (myonecrosis) harveyi relies on N-(3-hydroxybutanoyl)-L- and produces various toxins, such as α, θ and homoserine lactone (3OH-C4 HSL) for QS. κ encoded by plc, pfoA, and colA, respectively. The cognate transmembrane histidine kinase It has been found that activation of VirR/VirS receptor, LuxN, is exclusively agonized by system enhances transcription of these toxins 3OH-C4 HSL. AHLs with differing chemi- which have been implicated in disease process. cal modifications at the C3 position (3-O and To identify AIP of the VirR/VirS system, we 3-H) and those with longer carbon tail lengths analyzed culture filtrate of C. perfringens 13 (C6 - C12) competitively antagonize LuxN. by liquid chromatography-mass spectrometry To identify residues in LuxN governing ligand and detected 5- and 6-amino acid residue specificity, we modified AHL structures by dehydrated oligopeptides whose molecular chemical synthesis and altered the companion weight were correspond to those deduced from LuxN receptor binding pocket by mutagen- amino acid sequence of AgrDCp. The 5- and esis. We pinpointed His210 as responsible 6-residue oligopeptides (cyclo(CLWFT) and for recognition of the C3 modification, and A-cyclo(CLWFT)) were chemically synthe- identified Leu166 as the determinant for chain-

5th ASM Conference on Cell-Cell Communication in Bacteria 77 Poster Abstracts

length preference. We also isolated a series of lsrR-lacZ fusion was assayed at OD600 = 0.6 in LuxN mutants that are constitutively biased parent and mutant (sirA, barA, csrB, csrC, cs- to the agonized state. These mutations reside rBC, and csrA) strains. As reported previously, in the region of the transmembrane domain the lsrD mutant was impaired in internalizing immediately following the ligand-binding site. AI-2, while an lsrR mutant was more efficient We propose that this region acts as the switch in AI-2 internalization than the parent strain that triggers signal transduction. Together, our at two hours of growth. Despite lsrD deletion, analyses allowed us to dissect how a histidine AI-2 concentration in the extracellular media sensor kinase differentiates between ligands, oscillated over time indicating an alternative and uses that information to regulate kinase transport system. Due to de-repression of the activity. lsr operon AI-2 was removed from the extra- cellular medium faster by the sirA, barA and n 76 csrBC mutants, and slower by a csrA mutant compared to parent strain at 3h of growth. Integration of SirA/BarA and lsr Mutants in sirA, csrB, and csrBC exhibited Regulatory Systems in decreased lsrR-lacZ expression while a csrA Typhimurium mutant showed higher expression compared E. Gart, S. Myers, E. Lee, S. Lawhon; to parent strain. These results imply that LsrR Texas A&M, College Station, TX. is normally repressed by CsrA, and the SirA/ BarA two-component system is involved in Quorum sensing is a method of bacterial lsr regulation by controlling lsrR expression. communication developed to regulate gene Therefore, a SirA-LsrR regulatory cascade expression in response to population density. could be important in coordinating Salmonella Salmonella Typhimurium uses autounducer-2 AI-2 signaling and virulence. (AI-2) signaling molecule to regulate the Sal- monella pathogenicity island-1 (SPI-1) Type 3 Secretion System (T3SS), flagellar gene n 77 expression, and biofilm formation. The AI-2 Detection of Autoinducers from is sensed by the lsr-encoded transport cassette Veillonella tobetsuensis and Their and is actively transported into the bacterial Roles in Biofilm Formation cell. Following intracellular phosphorylation, I. MASHIMA, F. NAKAZAWA; AI-2 negatively regulates the transcriptional Health Sciences University of Hokkaido, Hok- repressor protein LsrR, allowing the transcrip- kaido, JAPAN. tion of lsr- and SPI-1 – encoded genes. The global regulatory RNA-binding protein CsrA Backgrounds: It is suggested that oral Veil- also regulates the SPI-1 T3SS and is, in turn, lonella has important roles in the biofilm regulated through the SirA/BarA-CsrB/C-CsrA formation at early stage. Our previous study regulatory cascade. The interaction between demonstrated that the supernatants from V. the SirA/BarA two-component system and the tobetusensis had inhibiting effects for the bio- lsr operon are unknown. The goal of this study film formed by Streptococcus gordonii, but in was to determine whether SirA/BarA regulates case of existing bacterial cells of V. tobetsuen- lsr expression and AI-2 transport in Salmonella sis, the biofilm formation was promoted. It was Typhimurium. AI-2 production was measured suggested that some factors in supernatants in mutant (luxS, lsrR, lsrD, sirA, barA, csrA, may affect the biofilm formed by S. gordonii. and csrBC) and parent (ATCC14028) strains of Purpose: In this study, autoinducer-1 and Salmonella Typhimurium using the lumines- autoinducer-2 (AI-1 and AI-2) were focused as cent response of reporter V. harveyi to Salmo- one of the factors in the supernatants and tried nella AI-2. The β-galactosidase activity of the to detect and purify from the supernatants.

78 ASM Conferences Poster Abstracts

After purification, the effects of autoinducers n 78 on biofilm formation were examined. Method: A bioinformatic survey of The crude AI-1 was extracted by ethyl acetate distribution, amino acid from the culture supernatants of V. tobetsuen- conservation and probable sis. Then these extracts were evaporated to functions of LuxR solo regulators dryness after filtration. The residues dissolved in bacteria in ethanol were used in bioassay and thin layer chromatography using C. violaceum CV026 S. Subramoni1, V. Florez2, Z. Suárez-Moreno1; and R. radiobacter NTL4 (pZLR4) to detect 1Universidad de La Sabana, Chia, COLOM- AI-1. AI-2 activities in the supernatants were BIA, 2Universidad Nacional de Colombia, detected by using Vibrio assay as reported Bogota, COLOMBIA. previously. The supernatants were filtered LuxR solo transcriptional regulators contain through a 0.2-µm-pore-size filter and subse- both an autoinducer binding domain (N- quently through an Amicon Ultra Centrifugal terminal end) and a DNA binding domain Filters Ultracel-3K (Millipore). The filtrate (C-terminal end), but are not associated with were lyophilized, suspended in cold sodium a N-acyl homoserine lactone (AHL) synthase phosphate buffer, and chromatographed on a coding gene in the same genome. Although a C18 Sep-Pak reverse-phase column (Waters few LuxR solos have been characterized, their Co.) according to the manufacturer’s instruc- distributions as well as their role in bacte- tions. AI-2 activity in the column fractions was rial signal perception and other processes are followed by monitoring the induction of biolu- poorly understood. In this study we have car- minescence of Vibrio harveyi BB170 (sensor ried out a systematic survey of distribution of 1- sensor 2+). After this partial purification, the all LuxR transcriptional regulators harboring effects of AI-2 like substance for biofilm for- both domains available in the InterPro data- mation were examined by using wire method. base (IPR005143), and identified those lacking Results: AI-1 was detected in the supernatants a cognate AHL synthase. These proteins were from V. tobetsuensis when only R. radiobacter analyzed regarding their distribution, predicted NTL4 (pZLR4) was used. Also, AI-2 like ac- functions for neighboring genes, the presence tivity was detected strongly in the supernatants of AI-2 signaling or endogenous AHL-QS from V. tobetsuensis. Partial purification of systems. Our preliminary analyses reveal the AI-2 was succeeded and the bioluminescence presence of multiple predicted LuxR solos in level was about 10 times compared with that many proteobacterial genomes, most of them of the supernatants. Furthermore, AI-2 like harboring genes for one or more AHL-QS substance from V. tobetsuensis inhibited the circuits. Surprisingly, putative LuxR solos biofilm formed by S. gordonii. Discussion & were also found in a few non-proteobacterial Conclusion: According to prosperity of R. ra- genomes, which are not reported to have AHL- diobacter NTL4 (pZLR4) and the result of thin QS systems. Multiple predicted LuxR solos in layer chromatography, V. tobetsuensis may the same genome appeared to have different produce long chain N-acyl homoserine lactone levels of conservation of invariant amino as AI-1. It is the first report that AI-1and AI-2 acid residues of autoinducer binding domain from V. tobetsuensis has affected the biofilm questioning their response to AHLs. Finally, formation. presence of LuxR solos in bacteria occupy- ing diverse environments suggests potential ecological functions for these proteins beyond AHL and interkingdom signaling.

5th ASM Conference on Cell-Cell Communication in Bacteria 79 Poster Abstracts n 79 Qrrs are not the only sRNAs that participate in Characterization of a Quorum QS. My characterization of QsrA will elucidate Sensing Regulated Small RNA in finer details of the V. harveyi QS circuit and Vibrio harveyi could lead to novel ways to manipulate QS- mediated behaviors, such as biofilm production A. Min, K. Papenfort, B. L. Bassler; and virulence, in V. harveyi and possibly other Princeton University, Princeton, NJ. Vibrio species. At the heart of the Vibrio harveyi quorum sensing (QS) circuit lie five homologous small n 80 RNAs (sRNAs), Qrr 1-5 (quorum regulatory Indole Signal Induces Multidrug RNAs). The Qrr sRNAs control the levels of Tolerance in Uropathogenic AphA and LuxR, the master low cell den- Escherichia coli (UPEC) sity (LCD) and high cell density (HCD) QS K. Kurabayashi, S. Nishioka, T. Matsuzawa, N. regulators, respectively. Hence, the Qrrs are Matsumoto, S. Fueki, H. Hirakawa; key dictators of whether V. harveyi will engage ASRLD Unit, Gunma University, Maebashi in individual or group behavior. Given the Gunma, JAPAN. critical role that the Qrr sRNAs play in QS, I am investigating whether there exist additional Background: Some species of enterobacteria sRNAs that influence QS. Using high-through- including Escherichia coli use indole for their put whole genome microarrays and RNA-se- social behaviors. TnaA is a synthetic enzyme quencing, the Bassler group recently discov- for indole production from tryptophan, and its ered dozens of intergenic sRNAs in V. harveyi, expression is activated at high cell density, and which could play roles in QS. I study one of also by indole. Therefore, indole production these sRNAs, QsrA (quorum sensing-regulated can be auto-regulated like acyl-homoserine sRNA A), which exhibits more than 30-fold lactone quorum sensing signals. We previously gene expression change between LCD-locked studied on indole-mediated social behavior in and HCD-locked V. harveyi strains. Northern non-pathogenic E. coli laboratory strain. Re- blot analysis confirmed that QsrA exhibits sult: Here, we show roles of indole signal in cell density-dependent production, where it is uropathogenic E. coli (UPEC). We found that more highly expressed at HCD than at LCD. indole induces tolerance to several antibiotics Promoter-reporter fusion assays also show that including beta-lactams, fluoroquinolones and QsrA is directly activated by LuxR and that it fosfomycin. The indole-induced multidrug is repressed by AphA. Here, I explore whether tolerance is attributed to up-regulation of drug LuxR and AphA have specific requirements to efflux genes because UPEC cells grown in the bind to the QsrA promoter and regulate qsrA presence of indole had higher expression of expression. Additionally, I investigate the bio- drug efflux genes, acrD, mdtEF, yceE and yceL logical role of QsrA in V. harveyi. Toward this than those in the absence of indole. Wild-type end, I have identified putative QsrA targets by UPEC is able to produce 2 mM of indole at the comparing the transcriptomes of wildtype and stationary phase when grown in tryptophan- a qsrA knockout V. harveyi strain. Preliminary supplemented medium. We observed expres- analysis of the most highly regulated targets sion of these drug efflux genes were activated suggests that QsrA could be involved in the in a conditioned medium from the tryptophan- transition from the motile planktonic state to supplemented wild-type stationary phase the sessile biofilm state. Using fluorescence culture but not that from tnaA mutant culture and confocal microscopy, I test the effect because the tnaA mutant did not produce of QsrA on motility and biofilm formation. indole. We also found that induction of acrD Discovery of QsrA supports my thesis that the but not mdtEF, yceE and yceL requires

80 ASM Conferences Poster Abstracts two-component signal transduction system crystallographic), biochemical, and genetic (TCS), CpxAR and BaeSR. Conclusion: approaches to determine at the atomic level These results suggest that indole acts as a quo- how Gram-positive quorum-sensing recep- rum sensing signal instead of acyl-homoserine tors function and how the receptor activities lactones for UPEC, then induces drug efflux are modulated by oligopeptide pheromones to genes through TCS-dependent and indepen- regulate cell development. dent-pathways, results in increased tolerance to multiple drugs. We presume that indole-type n 82 quorum sensing may contribute to an innate Selective interaction of membrane bio-defense for UPEC against antibiotic pro- vesicles with bacterial cells ducers in nature. Y. Hasegawa, M. Shintani, K. Kimbara, H. n 81 Futamata, Y. Tashiro; Shizuoka University, Hamamatsu, JAPAN. Structure-Function Studies of Gram-positive Cell-Cell Signaling Many bacteria secrete membrane vesicles and Development (MVs) ranging from 20 to 200 nm in diameter. MVs contain bacterial important substances M. B. Neiditch, V. K. Parashar; including DNA, proteins and low-molecular Rutgers University - New Jersey Medical compounds at high concentrations. Since School, Newark, NJ. MVs deliver their content to prokaryotic Bacterial quorum sensing is a cell-cell com- cells as well as eukaryotic cells, they play munication process driven by pheromones. At an important secretion system for predatory, low cell density, in the absence of appreciable horizontal DNA transfer and quorum sensing, amounts of pheromones, bacteria act as indi- in microbial communities. However, little viduals. At high cell density, bacteria respond is understood the details about the fusion of to the pheromones by synchronizing the gene MVs with bacterial cells. In this study, we expression of the community. Thus, quorum aimed to investigate whether MVs selectively sensing allows groups of bacteria to coordinate interact with specific bacterial cells and obtain social behaviors including virulence factor ex- new insights into MV-mediated communica- pression, motility, biofilm development, biolu- tion between MV-forming bacteria and other minescence, antibiotic production, and genetic microbes. MVs were extracted from each competence. Typically,Gram-positive bacteria bacterial culture, labeled with a fluorescent use oligopeptides as pheromones. Oligopeptide reagent FM4-64 and incubated with bacterial pheromones are synthesized as immature pro- cells. After removing freely suspended MVs, peptides. The immature pro-peptides are se- interaction of MVs with bacterial cells was creted from the cell and subsequently undergo quantified as fluorescent intensities associated proteolytic maturation. Outside of the cell, with bacterial cells using a microtitre plate the mature peptides can bind to and regulate reader. MVs secreted from Pseudomonas aeru- membrane receptors. Alternatively, the mature ginosa PAO1, which is an opportunistic Gram- peptides can bind to oligopeptide permeases negative pathogen, interacted with all bacte- that import the oligopeptides into the cell. Here rial cells tested in this study. The interaction the oligopeptides bind to and regulate cytosolic between P. aeruginosa MVs and other bacterial receptors. Despite their obvious importance, cells were also confirmed through detecting the mechanistic basis of oligopeptide recep- the fluorescence of MVs using flow cytometry tor function and regulation in Gram-positive analysis, suggesting that MVs derived from P. species is largely unknown. We have used a aeruginosa non-specifically attach or fuse with combination of biophysical (mainly X-ray many kinds of bacterial cells. On the other

5th ASM Conference on Cell-Cell Communication in Bacteria 81 Poster Abstracts hand, MVs secreted from an enterobacterium the mammalian cells to produce this activity. Buttiauxella agrestis JCM 1090T, which is the Ongoing studies will identify the AI-2 mimic most highly MV-forming bacterium tested in and provide evidence that the host can engage this study, interacted with own cells at a high in a bi-directional small-molecule conversation frequency, while did not with other bacterial with its commensal counterparts. cells. These results provide striking examples of specific and non-specific interactions of n 84 MVs with bacterial cells, and indicate selective A hierarchy quorum sensing properties in MV-mediated cell-cell interac- signaling network controls tion. Elucidation of mechanisms for interaction diverse biological functions in of MVs with specific cells would contribute Burkholderia cenocepacia to understanding of the intracellular substance transfer through MVs in heterogeneous micro- Y. Deng1, S. Chen1, L. Zhang2; bial communities. 1Institute of Molecular and Cell Biology, Sin- gapore, SINGAPORE, 2Guangdong Province n 83 Key Laboratory of Microbial Signals and Disease Control, South China Agricultural Host-microbe small molecule University, Guangzhou, CHINA. signaling Many bacterial pathogens produce quorum A. S. Ismail, L. C. Miller, J. S. Valastyan, M. F. sensing (QS) signals in regulation of various Semmelhack, B. L. Bassler; biological functions. Burkholderia cenocepa- Princeton University, Princeton, NJ. cia, which is an important opportunistic human Mammals have coevolved with commensal bacterial pathogen, employs N-acyl homoser- bacteria, the majority of which reside in the ine lactone (AHL) and cis-2-dodecenoic acid gut. In a process called quorum sensing (QS), (BDSF) QS systems in regulation of bacterial bacteria communicate using chemical signal virulence. It is intriguing how these two QS molecules called autoinducers to synchronize systems act together in regulation of similar bi- group behaviors. Given the size of the com- ological functions. Our recent studies showed mensal population and its intimate contact that the two QS systems constitute a hierarchy with intestinal surfaces, it is likely that these signaling network in regulation of swarming bacteria profoundly influence many aspects of motility, biofilm formation and virulence in intestinal physiology. However, it is not known B. cenocepacia. We found that BDSF signal how mammals perpetuate beneficial relation- negatively controls the intracellular cyclic ships with gut symbionts, and we have a dimeric guanosine monophosphate (c-di-GMP) limited understanding of the role that QS plays level through a receptor protein RpfR, which during bacterial gut symbiosis. The goal of this contains Per/Arnt/Sim (PAS)-GGDEF-EAL project is to assess the role of quorum sensing domains. RpfR regulates the same phenotypes (QS) in host-microbial interkingdom signal- as BDSF signal. In addition, the BDSF− mu- ing. Preliminary studies show that mammalian tant phenotypes could be rescued by in trans host tissues produce a mimic activity that acts expression of RpfR, or its EAL domain that analogously to AI-2, the universal bacterial functions as a c-di-GMP phosphodiesterase. QS autoinducer. This signal differs structurally BDSF signal is shown to bind to the PAS from bacterial AI-2, but can agonize AI-2 re- domain of RpfR with a high affinity and stimu- ceptors to activate QS responses. Mammalian lates its phosphodiesterase activity through cells produce mimic when co-cultured in the induction of allosteric conformational changes. presence of live but not dead bacteria, impli- Interestingly, BDSF system also controls AHL cating an active role for bacteria in stimulating signal production through regulation of the

82 ASM Conferences Poster Abstracts

AHL synthase CepI expression at transcrip- studies and in vivo studies of promoter activi- tional level by modulating the intracellular ties. We demonstrate that cooperative binding level of the second messenger c-di-GMP. of the transcription factor at the promoter is Moreover, we showed that the BDSF and AHL required for pherotype signaling. Our results systems have a cumulative role in regulation of indicate that full promoter activity of natural various biological functions, including swarm- target genes requires a strain-specific quorum, ing motility, biofilm formation and bacterial suggesting that maximum investment into the virulence, and exogenous addition of either production of common goods is favored in BDSF or AHL signal molecules could only homogenous social situations. partially rescue the changed phenotypes of the double deletion mutant defective in BDSF and n 86 AHL signal production. In combination, our Acyl-homoserine lactone- work presents a unique and widely conserved independent activation of an orphan cis-unsaturated fatty acid type QS signal recep- LuxR in B. thailandensis and B. tor that directly links the signal perception to pseudomallei c-di-GMP turnover, and establishes a hierarchy QS signaling network in regulation of bacterial P. M. Silva1, T. Truong2, P. Greenberg2, J. physiology in B. cenocepacia. Chandler1; 1University of Kansas, Lawrence, KS, 2Wash- n 85 ington University, Seattle, WA. Pherotype and species-specific LuxR family members with no genetically signal integration at quorum linked acyl-homoserine lactone (acyl-HSL) sensing promoters signal synthase are called solos or orphans. Orphans are found in the genomes of many V. Rippa1, D. Wolf1, P. Sauer1, J. Mobarec2, L. Proteobacteria. Of those that have been Adlung2, S. Trauth1, P. Kolb2, I. Bischofs1; studied, the majority are acyl-HSL-responsive 1Ruprecht-Karls-University of Heidelberg, and thought to detect signals produced by Bioquant, Heidelberg, GERMANY, 2Philipps- acyl-HSL synthases encoded elsewhere in University of Marburg, Department of Phar- the genome or produced by other bacteria. A maceutical Chemistry, Marburg, GERMANY. subset of orphans do not respond to acyl- Quorum sensing is a common regulatory HSLs, and instead respond to as-yet unknown strategy to control the production of common signals derived from plants. However all of the goods. The underlying molecular signaling plant signal-responsive orphan LuxR family networks used for quorum sensing show often members have changes in two of the nine key puzzling complexity. Frequently several sig- residues attributed to many of the LuxR family nals converge to regulate target gene expres- proteins, both occurring in the acyl-HSL bind- sion. For example, in Bacillus subtilis strain- ing domain. We have found an orphan LuxR, specific pherotype signals and species-specific BtaR4 in Burkholderia thailandensis, with all signals converge to regulate the activity of the nine of the conserved LuxR-family residues transcription factor ComA by modulating its that does not require acyl-HSLs to activate phosphorylation state and its ability to bind its target genes. Previous work demonstrated DNA, respectively. How the different signals BtaR4 is required for virulence in C. elegans are integrated at the level of target promoters is and we show that BtaR4 directly regulates not well understood. Here, we elucidate mech- gene for the virulence factor malleilactone. anistic determinants of promoter activation Although B. thailandensis encodes three by pherotype and strain-specific signals using acyl-HSL synthases, these are not required a comprehensive set of in vitro DNA binding for BtaR4 to activate transcription of the mal-

5th ASM Conference on Cell-Cell Communication in Bacteria 83 Poster Abstracts leilactone biosynthetic genes. Instead, BtaR4 regulate production of the toxins remained activity correlates with its cellular level, which largely unknown until now. Our data reveal is influenced by the addition of particular that a novel quorum signal accumulates extra- growth-inhibitive antibiotics. BtaR4 activation cellularly during growth and induces toxin pro- of target genes requires a lux-box motif in the duction at high cell density when a threshold promoter region of the malliolactone operon signal concentration is reached. Using a classic (mal) promoter, similar to other LuxR-family quorum signaling bioassay, we determined proteins. BtaR4 is unique to B. thailandensis that cell-free stationary-phase supernatants and two of its close relatives, Burkholderia collected after 8 h of growth induce premature pseudomallei and Burkholderia mallei. The toxin production in low-density tester cells. B. pseudomallei BtaR4 homolog BpsR4 is This toxin-inducing activity, termed the TI also important for virulence in C. elegans and signal, was purified by acetone precipitation, regulates a malleilactone-like gene cluster in anion exchange, and HPLC. The purified TI response to growth-inhibitive antibiotics, simi- signal stimulated elevated toxin production lar to BtaR4. It is unclear if BtaR4/BpsR4 ac- in both low-density hypervirulent and non- tivates its target genes in a ligand-independent hypervirulent clinical C. difficile strains and manner or if it is activated by a ligand that is induced early transcription of the C. difficile not an acyl-HSL. Our finding that a conserved toxin genes. The molecular weight of the TI orphan LuxR does not require acyl-HSLs to signal was determined by MALDI-TOF/TOF activate its target broadens our current view of and MS/MS to be 612.37 Da. This is consistent the LuxR family. with the structure of a novel 5-residue cyclic peptide composed of Cys, Pro, Trp, and 2 Ile n 87 with a thioester linkage between the carboxyl group of the C-terminal Ile and the N-terminal Toxin Synthesis by Clostridium Cys; these five residues are found within the difficile is Stringently Regulated C. difficile agrD1 gene. A synthetic version Through Quorum Signaling of the TI signal induced toxin levels similar C. Darkoh, H. L. DuPont, S. J. Norris, H. B. to that of the purified signal. We constructed Kaplan; a Himar-based transposon vector to generate University of Texas Health Science Center, isogenic mutants defective in toxin production Houston, TX. (Tox-). One Tox- mutant contains an insertion in the agrA2 gene that is able to generate the Clostridium difficile infection (CDI) is the TI signal, but does not respond to the TI signal, most common cause of hospital-acquired and and is deficient in toxin synthesis. Response to antibiotic-associated diarrhea in the United the TI signal and toxin synthesis were restored States, with a total treatment cost estimated be- by complementation with wild-type C. difficile tween 1 to 4.8 billion U.S. dollars annually. C. agrA2. Finally, while testing diarrheal stools difficile, a Gram-positive multidrug-resistant for TI signal, we detected the TI signal only in pathogen, overpopulates the colon after the na- CDI-positive stools but not in CDI-negative tive microbiota has been altered by antibiotic stools. This underscores the clinical relevance therapy. Treatment of CDI is hampered by of the TI signal in C. difficile pathogenesis increased virulence, sporulation, recurrence, during infection. These findings provide direct and indiscriminate antibiotic use that destroy evidence that C. difficile toxin synthesis is the colonic microbiota. Pathogenic C. difficile regulated by an Agr quorum signaling and of- strains produce toxins A and B, which are fers new avenues for both rapid CDI detection directly responsible for disease. Despite the and development of quorum signaling-based enormous public health problem posed by non-antibiotic therapies to combat this life- this pathogen, the molecular mechanisms that threatening emerging pathogen.

84 ASM Conferences Poster Abstracts n 88 cell surface proteins of C. aggregans by pro- Exogenous protease released signal teases from other bacteria. To our knowledge promoting cell aggregation of a this is the first report that exogenous protease thermophilic filamentous bacterium released signal causing phenotypical change of bacteria. Thermo-stability of peptides may be S. Morohoshi, K. Matsuura, S. Haruta; a character common to intercellular signals in Tokyo Metropolitan University, Tokyo, JAPAN. high temperature environments. Cell to cell communication in thermophiles has yet to be identified, while mesophiles have n 89 been well characterized. Interspecies com- Integration of environmental munication at high temperature possibly works signals and cell density for in hot spring microbial mats composed of a efficient cell-cell communication in variety of bacteria with high cell density. An the Bacillus cereus group anoxygenic photosynthetic bacterium Chlo- L. Slamti, C. Lemy, D. Lereclus; roflexus aggregans, widely distributed in hot INRA, Jouy-en-Josas, FRANCE. spring microbial mats at 48-65 °C, has a rapid gliding motility and their filamentous cells dis- In Gram-positive bacteria, cell-cell commu- persed in liquid media collect together to form nication relies in part on cytoplasmic sensors dense aggregates. We have obtained an isolate regulated by secreted and re-imported signal- closely related to Bacillus licheiformis from ing peptides. The Bacillus cytoplasmic quorum the microbial mats dominated by C. aggregans sensors Rap, NprR, and PlcR were previously and found that its culture supernatant promoted identified as the first members of a new protein the aggregation rate of C. aggregans. This pro- family called RNPP. Except for the Rap pro- moting effect was suppressed by the addition teins, these RNPP regulators are transcription of a protease inihibitor. The aim of this study factors that directly regulate gene expression. is to understand how exogenous proteases Quorum sensing (QS) controls important bio- lead C. aggregans to promote cell aggregation. logical functions in bacteria of the Bacillus ce- It was confirmed that purified protease of B. reus group. PlcR was first characterized as the licheniformis showed the promoting effect main regulator of virulence in B. cereus and of cell aggregation. C. aggregans cells were Bacillus thuringiensis (Bt). NprR controls the removed after the protease treatment to obtain necrotrophic properties allowing the bacteria the fraction containing degradation products. to survive in the infected host. A recent report This fraction had the promoting activity even has shown that the global stationary phase after its protease activity was suppressed by regulator CodY, involved in adaptation to a protease inhibitor. We hypothesized that poor growth conditions, represses expression the degradation products derived from cell of nprR by directly binding to its promoter surface of C. aggregans promoted cell ag- region (1). It has also been shown that CodY gregation. This promoting activity was heat is required for expression of virulence genes tolerant at 98°C and effectively suppressed belonging to the PlcR regulon (2, 3). However by a peptidase, Chymotrypsin. Gel filtration the mechanism underlying this regulation has was conducted to achieve size fractionation. not been reported. Our experiments indicate SDS-PAGE analysis showed that active that CodY does not directly control expres- fractions contained bands with around M.W. sion of plcR. We showed that CodY regulates 3,000. These results suggest that extracel- expression of the virulence genes via PapR, lular peptides (M.W. ~3,000) work as a signal PlcR’s cognate signaling peptide. Using affecting the motility of C. aggregans. These synthetic peptides we demonstrated that PapR peptides were released through degradation of is not efficiently re-imported in ∆codY cells.

5th ASM Conference on Cell-Cell Communication in Bacteria 85 Poster Abstracts

We also showed that CodY positively regulates rsmA is the only known target to be controlled expression of the gene encoding the oligopep- by this system. RsmA is a small RNA-binding tide-binding protein allowing entry of PapR protein that binds to the mRNAs of multiple in the cell. Contrary to what was expected, genes affecting translation and suppressing this gene did not belong to the opp operon virulence. The deleterious effects on virulence encoding the pore required for PapR import. production are counteracted by the ExpS/ExpA Altogether, these data complete our model system (homologous to the GacA-GacS in of quorum sensing during the lifecycle of Bt Pseudomonas spp. and the BarA-UvrY in E. and indicate that environmental conditions, as coli) that upon activation initiates the produc- well as cell density, are integrated by bacteria tion of the small RNA (sRNA), RsmB, which to coordinate their behavior throughout the sequesters RsmA. Thus, Pectobacterium spp., infectious cycle via cell-cell communication. have developed a distinct alternative to control References 1. Dubois T, et al. Activity of the virulence where the two cell-cell signaling Bacillus thuringiensis NprR-NprX cell-cell networks are intrinsically linked to each other communication system is co-ordinated to the via RsmA. In this study, we demonstrate that physiological stage through a complex tran- the gene coding for RsmB, is also controlled scriptional regulation. Mol Microbiol. 2013 by the AHL system. Using both, biochemical Apr;88(1):48-63. 2. Frenzel E, et al. CodY and genetic assays, we also demonstrate that orchestrates the expression of virulence deter- the amount of PCWDEs is dependent on the minants in emetic Bacillus cereus by impacting quantity of RsmB rather than the quantity of key regulatory circuits. Mol Microbiol. 2012 RsmA being produced in response to AHLs. Jul;85(1):67-88. 3. Lindbäck T, et al. CodY, a These results provide an explanation on how pleiotropic regulator, influences multicellular AHLs and the ExpS/ExpA system converge to behaviour and efficient production of virulence exquisitely control the activity of RsmA and factors in Bacillus cereus. Environ Microbiol. consequent all the virulence RsmA-targeted 2012 Aug;14(8):2233-46. genes. n 90 n 91 Small RNAs are central in An integrated quorum sensing integrating quorum-sensing system and its clinical implications signals in the plant pathogen J. Lee, L. Zhang; Pectobacterium wasabiae Institute of Molecular and Cell Biology, P. Nadal-Jimenez, R. Valente, K. B. Xavier; Singapore, SINGAPORE. Instituto Gulbenkian de Ciencia, Oeiras, Pseudomonas aeruginosa is an opportunistic PORTUGAL. human pathogen that may cause severe noso- Quorum sensing (QS) controls the production comial infections in hospital patients and cys- of plant cell wall degrading enzymes (PC- tic fibrosis sufferers. Pathogenesis of P. aerugi- WDEs), the major virulence factors in plant nosa is complex and involves diverse virulence pathogens from the genus Pectobacterium (for- processes and mechanisms. One of the primary merly Erwinia spp.). In Pectobacterium spp., virulence regulatory mechanisms is the cell- two cell-cell signaling circuits control the pro- cell communication system termed quorum duction of PCWDEs: the N-acyl homoserine sensing (QS). The QS system of P. aeruginosa lactone (AHL) network and the ExpS/ExpA/ was previously known to be organized in a las, RsmA network. Unlike in other bacteria where pqs, rhl hierarchy, with the central las system the AHL system directly controls the expres- governing the entire chain of QS pathways. It sion of multiple genes, in Pectobacterium spp., is therefore perplexing that P. aeruginosa clini-

86 ASM Conferences Poster Abstracts cal isolates frequently harbor loss-of-functions we uncovered was the ability of nonmotile mutations in las. Our recent discovery of IQS cells to inhibit the movement of motile cells provides useful clues in solving this puzzle (1). when mixed. Interestingly, we found that a few IQS is a cell-cell communication signal ca- nonmotile cells could control the gliding motil- pable of integrating phosphate-depletion stress ity behavior of many cells, for example at a cue with bacterial quorum information, even 1:50 ratio, by a Tra-dependent mechanism. To in the absence of the central las QS system. assess the behavior outcome of mixed strains Given that phosphate limitation is frequently under different conditions they were labeled encountered by P. aeruginosa during infec- with fluorescent proteins or antibiotic markers. tions of hosts, the biological significance of Surprisingly, our results found that strains IQS has been investigated by using over 20 with a functional Adventurous gliding motility clinical isolates collected from lung and skin system were killed by nonmotile sibling cells. wound infections. Consistent with previous Importantly, killing was Tra-dependent. From reports, our results show that more than half a panel of test strains a single nonmotile strain of the clinical isolates have various mutations was found that did not inhibit swarm expan- in the las system, which lead to decreased sion and did not kill. Intrigued by this rare virulence factor productions under phosphate- behavior, we sequenced the genome of this rich conditions. In contrast, most of the clinical strain to gain insight into the killing mecha- isolates are capable of producing IQS signal nism. From this analysis a number of SNPs under phosphate-depletion conditions and IQS and Indels were identified in type VI secre- deletion mutants are attenuated in virulence. tion system genes. Subsequently we disrupted These findings suggest that IQS could play an several type T6SS genes in a nonmotile strain important role in clinical infections and more and found killing was reduced. Interestingly, work is warranted to investigate this intriguing when a T6SS mutant was mixed with an A- QS system. motile strain that contained a traA mutation, the tables were turned and the nonmotile strain n 92 was killed. In summary, our results indicate that OME plays a key role in ‘policing’ social Sibling rivalry in myxobacteria interactions between cells, which we hypoth- triggered by cell surface cues that esize help myxobacteria transition from indi- activate T6SS vidual cells into a well-controlled and coherent A. Dey, A. Conklin; multicellular community. University of Wyoming, Laramie, WY. Myxobacteria are known for their relatively n 93 complex social behaviors that involve cell-cell A novel paradigm of interbacterial signaling. One of their interesting behav- communication of antibiotic iors is the ability of Myxococcus xanthus to resistance mediated by conserved transiently fuse their outer membranes and secreted bacterial lipocalins exchange (OME) contents. To understand O. M. El-Halfawy1, M. A. Valvano2; the mechanism of OME, we identified two 1The University of Western Ontario, London, genes, traA and traB, required in donors and ON, CANADA, 2Queen’s University Belfast, recipients for protein transfer. TraA was further Belfast, UNITED KINGDOM. shown to be involved in molecular recognition to distinguish between kin and nonkin cells Communication among bacteria in mixed for OME. Self-recognition is determined by a infections may result in increased resistance polymorphic region (PA14-like domain) con- to antibiotics, thereby leading to therapeutic tained in TraA. One OME-dependent behavior failures. Recently, small secreted molecules

5th ASM Conference on Cell-Cell Communication in Bacteria 87 Poster Abstracts were implicated in communication of transient to hydrophobic moieties, revealing a defined resistance to antibiotics. YceI, a small secreted function for the conserved BCNs in the inter- protein produced by the extremely antibiotic bacterial communication of resistance. This resistant Burkholderia cenocepacia, medi- offers a new avenue for targeting antibiotic ated protection of Pseudomonas aeruginosa in resistance and its spread among bacteria by co-culture from the lethal action of polymyxin developing inhibitors for BCNs. B (PmB). YceI belongs to a large family of conserved bacterial small proteins that share a n 94 common tertiary lipocalin fold, but diverge in NagC repression of TfoX enhances amino acid sequence homology. Recombinant colonization of bacterial lipocalins or “bacteriocalins” (BCNs) by Vibrio fischeri from B. cenocepacia bind PmB; however, until now, there has been no direct demonstration of Y. Sun, S. C. Verma, H. Bogale, T. Miyashiro; BCN function. We hypothesize that BCNs are Pennsylvania State University, University involved in the bacterial response to antimicro- Park, PA. bial stress through binding antibiotics. Here, Gene regulation enables bacteria to adapt to we show by deletion mutagenesis that the B. different and frequently unpredictable environ- cenocepacia BCN BCAL3311 contributes to ments. NagC is a transcriptional repressor resistance against PmB and other amphiphilic conserved in γ-proteobacteria, including Vibrio antibiotics, such as rifampicin and norfloxa- fischeri, that controls the expression of genes cin, but not the hydrophilic gentamicin. The involved in N-acetyl-glucosamine (GlcNAc) expression of BCAL3311, determined by utilization. NagC repression of these genes is chromosomal promoter-luxCDABE transcrip- released by allosteric inhibition with GlcNAc- tional fusion, was stimulated by amphiphilic 6-phosphate, which is the intracellular form of antibiotics and not by gentamicin. Fluoromet- GlcNAc. Previously, we found that a ΔnagC ric binding assays showed that BCAL3311 mutant is unable to compete with wild-type has higher binding affinity to the hydrophobic cells in colonizing juvenile Euprymna scolopes probe Nile Red relative to basic and acidic squid. Our present study seeks to under- dyes. The more hydrophobic antibiotics PmB, stand the mechanism underlying the ΔnagC rifampicin and norfloxacin replaced Nile Red colonization defect. A bioinformatics search from its complex with BCAL3311 whereas for NagC-regulated genes based on potential gentamicin and the deacylated PmB nonapep- NagC binding sites within the ES114 genome tide did not, further highlighting the interaction identified 25 genes within 20 . One of of BCAL3311 with hydrophobic moieties. these genes was tfoX, which encodes a primary Exogenous BCAL3311 protected clinically regulator of natural transformation in Vibriona- relevant pathogens in vitro such as P. aerugi- ceae. Natural transformation is a primary mode nosa, Acinetobacter baumannii, Salmonella of horizontal gene transfer among microbes, typhi and Shigella flexneri from PmB; it also and, in Vibrionaceae members, requires protected P. aeruginosa from the innate im- many TfoX-regulated genes including comEA mune response of Galleria mellonella larvae in and pilA, which encode a periplasmic DNA vivo. Heterologous expression of BCNs from shuttling protein and pseudopilus subunit, P. aeruginosa, Mycobacterium tuberculosis and respectively. Using genetic and biochemical Staphylococcus aureus USA300 in B. ceno- assays, we found that NagC represses tfoX by cepacia ∆BCAL3311 revealed a common role directly binding to two sites upstream of tfoX of these BCNs against PmB and rifampicin as well as to a site upstream of tfoR, which en- based on cfu count and Etest assays respec- codes a small RNA that post-transcriptionally tively. We propose for the first time a model of enhances TfoX. Investigation of several genes antibiotic resistance based on physical binding

88 ASM Conferences Poster Abstracts whose homologues in other Vibrionaceae fication (service) to investigate how metabolic members are regulated by TfoX revealed that constraints of individual species shape the pilA, but not comEA, is up-regulated in the emergent spatial and functional relationships ΔnagC mutant relative to wild-type expression of species within the community. We find that levels. Using single-strain and co-colonization strong metabolic interdependence drives the experiments, we have found that deletion of emergence of mutualism, robust interspecific tfoX in a ΔnagC mutant restores normal squid mixing, and increased community productiv- colonization profiles. In addition, deletion ity. These emergent community properties are of pilA in a ΔnagC mutant partially rescues driven by demographic feedbacks, such that the ΔnagC colonization defect. At 48 post- aid from neighbouring cells directly enhances inoculation, we also found that V. fischeri focal cell growth, which in turn feeds back represses tfoX transcription and translation positively to neighbouring helpers. In contrast, within the squid light organ. Taken together, we find that weak metabolic interdepen- our results show that NagC repression of TfoX dence drives conflict and greater interspecific is important for V. fischeri cells to colonize E. segregation. Together, these results suggest scolopes. We propose that the pilus involved that species spatial and functional relation- in natural transformation interferes with host ships represent the net balance of metabolic colonization and that NagC repression of TfoX interdependencies. is the underlying mechanism preventing this interference. We also speculate that NagC n 96 repression of TfoX may stabilize the squid- A Novel Application of Nisin in vibrio symbiosis through the suppression of Inhibiting the Formation and natural transformation within host-associated Maintenance of Complex Oral V. fischeri populations. Biofilms by Disrupting the Bacterial Community n 95 J. Shin, Y. Kapila; Metabolic interactions and University of Michigan, Ann Arbor, MI. demographic feedbacks shape the emergent function and The accumulation of oral biofilms on the teeth spatial organisation of microbial and gums can lead to the development of communities caries and periodontal diseases, some of the most common diseases in humans. Nisin is a S. Estrela1, S. P. Brown2; unique antimicrobial peptide that has a broad- 1University of Washington, Seattle, WA, spectrum inhibitory effect on both Gram- 2University of Edinburgh, Edinburgh, UNITED positive and Gram-negative bacteria. Nisin has KINGDOM. been approved for human use by the World Microbes live predominantly in dense and Health Organization (1969) and the Food and spatially-structured polymicrobial communi- Drug Administration (1988). Recently, there ties. Within these communities, microbial cells has been a growing interest in utilizing nisin’s excrete a wide range of metabolites, and this unique bacteriocin properties for clinical use. sets the stage for interspecific metabolic in- Our preliminary data suggest that nisin has an- teractions. The links, however, between meta- tibacterial effects against a periodontal patho- bolic and ecological interactions, and species gen, Treponema denticola and a cariogenic spatial-organisation are still poorly understood. pathogen, . We hypoth- Here we develop an individual-based model esize that nisin may be a novel agent that can of a two-species surface-attached community inhibit the formation and growth of complex where food (resource) is exchanged for detoxi- oral biofilms by disrupting the mutualistic

5th ASM Conference on Cell-Cell Communication in Bacteria 89 Poster Abstracts relationship of the bacterial community. To n 97 determine the effects of nisin on formation and Microwave-Assisted High Resolution growth of oral biofilms, biofilms were cultured Imaging in the Study of Bacterial under both static and flowing conditions. To Colony Organization of Individual emulate the physiological conditions, human and Co-Colonizing Species saliva was used to grow and inoculate multi- species biofilms. In addition, a high throughput D. L. Chance, T. J. Reilly, T. P. Mawhinney; Bioflux microfluidic system was used to grow University of Missouri, Columbia, MO. and treat biofilms. Biofilms were cultured for In the study of chronic polymicrobial infec- different time periods ranging from 24 - 72 tions, such as are common in patients with hours and post-treated with nisin at multiple cystic fibrosis (CF) and of particular interest time points (30 sec - 10 min) and concentra- to our laboratories, one questions whether tions (0.05 - 50 ug/ml). Following the nisin the multiple opportunists isolated from sputa treatment, biofilms were stained with a LIVE/ colonize different zones of the airway or DEAD bacterial viability kit. Biofilms were may co-exist in the same zone or biofilm and visualized through confocal microscopy and thereby affect one another’s persistence in were rendered in 3D to visualize the biofilm the human airway. To learn more about the killing, biofilm biomass, biofilm surface cover- potentially interacting species and contribu- age, average depth of the biofilm, and rough- tions of individual members to the community, ness (quantitative measure of killing). Our we employed a microwave-assisted specimen results demonstrated that nisin has a unique processing method for stabilization of micro- ability to inhibit the formation and growth of bial colonies within their extracellular matrices oral biofilms. Nisin at lower concentrations for transmission electron microscopy imaging were able to disrupt the biofilm formation (TEM). For investigating colony structure and and had a significant dispersive effect. Nisin organization, CF sputum outgrowths as well at higher concentrations had a dual effect of as individual and mixed species colonies of inhibiting formation and killing the bacterial opportunistic pathogens such as Pseudomonas population. Our data showed that nisin was aeruginosa (PA), Staphylococcus aureus (SA), able to penetrate and disrupt the complex and (CA), were covered interactions within the biofilm to prevent its with gelatin or agarose and processed with a formation and maintenance. In conclusion, we microwave-assisted paraformaldehyde with showed that nisin is a novel bacterial peptide ruthenium red fixation strategy. For assess- that can inhibit and disrupt the growth patterns ing preservation of enzymatic activities and of multi-species oral biofilms. In addition, localization within a colony by this mild nisin has a unique mode action on disrupting approach, acid phosphatase activity (AcPase) the symbiotic relationships of oral biofilm spe- of anaerobic pathogen Clostridium perfringens cies based on its concentration and duration of (CP) was evaluated with a modified Gomori treatment. In future studies, we plan on carry- stain. Intact colonies with stabilized extracel- ing out genome sequencing based community lular matrices were imaged at high resolution analyses on biofilms in presence and absence and immunogenicity was preserved. PA and of nisin to further evaluate nisin’s specific ef- SA from CF patient samples differed as to fect within the complex oral microbiome. how they co-existed, in some cases in distinct zones, and in others within the same matrix. Sputum outgrowths revealed interfaces be- tween PA and both CA and SA. For CP, TEM

90 ASM Conferences Poster Abstracts with matrix stabilization revealed that the sig- an outermembrane receptor recently identified nificant cell-wall associated enzymatic activity as FpvU. In contrast, PAO1 lacks a receptor within the colony structure was provided by a capable of recognizing PVDPf-5. We find that relatively small percentage of the organisms. pyoverdine-deficient PAO1 and Pf-5 mutants In conclusion, TEM analysis of bacterial colo- lacking the sigma factor PvdS grow robustly ny biology with matrix stabilization employing and at equal rates when supplemented with microwave-assisted processing permitted high PVDPAO1. However, in co-culture, PAO1 ΔpvdS resolution imaging providing valuable insights has a strong fitness advantage relative to Pf-5 into the status of activities and associations ΔpvdS. This result led us to hypothesize FpvU within individual and mixes species commu- is an inferior receptor for PVDPAO1 compared nities. This “snapshot” tool may assist in the to PAO1’s native receptors, FpvA and FpvB. understanding, and development of new strate- We explored this hypothesis by developing a gies to combat complex infections. mathematical model where receptor affinity can be manipulated. An important prediction n 98 from this model is that receptor affinity is most important when siderophore concentrations are Receptor affinity limits exploitation low. We tested this prediction empirically by of pyoverdine production from measuring fitness at a range of PVD con- interspecies cross-feeding PAO1 centrations. Consistent with the model, we find J. Sexton1, S. Mitri2, F. Hoegy3, J. Loper1, I. PAO1 ΔpvdS has the highest relative fitness 3 2 1 Schalk , K. Foster , M. Schuster ; advantage when PVDPAO1 concentrations are 1Oregon State University, Corvallis, OR, low. Our hypothesis will be further validated 2University of Oxford, Oxford, UNITED KING- by measuring receptor affinities directly. These DOM, 3École Supérieure de Biotechnologie, results indicate that differences in receptor Strasbourg, FRANCE. affinity influence interspecies competition for siderophores. Specifically, cross-feeding may In response to iron limitation, many microor- be successful at high siderophore concentra- ganisms secrete siderophores, a diverse group tions. If limiting however, the superior receptor of secondary metabolites that chelate ferric of the producer confers a distinct fitness iron from the extracellular medium. This be- advantage. This hypothesis provides a simple havior is considered cooperative because sid- explanation for the persistence of siderophore erophores can deliver iron to an individual cell production despite the presence of interspecies other than the producer, creating potential for cross-feeding. exploitation by non-producers. Interestingly, bacteria frequently have systems enabling the uptake of non-native or “xenosiderophores”. n 99 Considering the prevalence of such cross-feed- In vitro assessment of antimicrobial ing, it is unclear how siderophore production efficacy of secondary metabolites remains evolutionarily stable. In this study, we produced by lichen sp. collected address this question using controlled fitness from North West Himalayan Region, studies with two Pseudomonas species, P. India aeruginosa PAO1 and P. protegens Pf-5, which S. Bisht1, S. Sharma1, S. Sharma2, V. kumar3, respectively serve as the siderophore-pro- M. Kumar3, S. Bisht4, K. Sharma1; ducing and cross-feeding strain. The primary 1VCSG college of Horticulture, Uttarakhand siderophores of PAO1 and Pf-5 are the pyover- University of Horticulture & Forestry, Bharsar, dines PVD and PVD . Our strain selec- PAO1 Pf-5 Pauri-2461, Bharsar, Pauri, INDIA, 2De- tion exploits the ability of Pf-5 to utilize the partment of Biotechnology, MLN National non-native PVD through the expression of PAO1 Institute of Technology, Allahabad, U.P. India,

5th ASM Conference on Cell-Cell Communication in Bacteria 91 Poster Abstracts

Allahabad, INDIA, 3Institute of Microbial n 100 Technology, Amity Univeristy, Noida, India, 4 Effects of butyric acid on the Noida, INDIA, Department of Basic Science biofilm formation of A. naeslundii in and Humanities, VCSG college of Horticul- flow cell system ture, Uttarakhand University of Horticulture & Forestry, Bharsar, Pauri-246123, Uttara- T. Arai1, T. Kondoh2, A. Tominaga1, N. Ogura2, khand, India, Bharsar, Pauri, INDIA. K. Ochiai3, H. Senpuku1; 1National Institute of Infectious Diseases, Over the last few year microbial pathogens Tokyo, JAPAN, 2Department of Maxillofacial get mostly resistant to various synthetic drugs, Surgery, Nihon University School of Dentistry available in the global market so, there is a at Matsudo, Chiba, JAPAN, 3Department of vivid interest in developing a novel drug espe- Microbiology, Nihon University School of Den- cially from the natural origin i.e lichens as it tistry, Tokyo, JAPAN. produce a great variety of secondary metabo- lites and most of them are unique. Our investi- Actinomyces naeslundii is an early colonizer gation deal with the assessment of two different and has important roles in the development of lichen Peltigera sp. (Sample A) and Cladomia the oral biofilm. Short Chain Fatty Acids (SC- sp. (Sample B) extracted in two separate FAs) is secreted extracellularly as a product of solvent methanol (ME) and water (AQ). Their metabolism by Gram negative anaerobes e.g., antimicrobial efficacies were assessed against Porphyromonas gingivalis and Fusobacterium various pathogenic bacteria and fungal agents. nucleatum. Recently, we reported that one The MIC value of both these samples varied of SCFAs, butyric acid induced significant within the range of 0.7-27 mg/ml. Quantitative biofilm formation of A.naeslundii in 96 wells and qualitative phytochemical analysis such microtiter plate (Yoneda et al., Molecular Oral as total phenol, polysaccharide concentration, Microbiology, 2013 28:354-65.). As a next TBA, reducing capacity, antioxidant and free issue, to clear biofilm formation in flow condi- radical scavenging ability were also determined. tion and the mechanism of increased biofilm, Higher total phenol concentration was observed we performed A.naeslundii’s biofilm formation in sample A in both the extract ME and AQ i.e. in flow cell system using Tryptic soy broth 15.6 mg GA/g and 14.3 respectively while the without dextrose and with 0.25% sucrose (TSB AQ extract in both the samples exhibited sig- with sucrose) supplemented with butyric acid. nificant inhibitory activity towards lipid peroxi- Various concentrations (0, 6, 30, 40, 50 and 60 dation by TBA method. Consequently, reducing mM) of the butyric acid and other supple- power also increases with the concentration ments {60 mM sodium butyrate (pH7.0) and of extract. Moreover, AQ extract of both the 60 mM sodium butyrate (pH4.9) adjusted with sample were observed with appreciable amount hydrochloric acid} were applied in the flow of polysaccharides comparatively to ME cell culture. The biofilms were stained with extracts. Lichen samples also gave remarkable BacLight LIVE/DEAD stain and examined antioxidant capacity in AQ extract of sample A using a confocal laser scanning microscope i.e.5.4 µg AA/g. In addition to this, in ME ex- (CLSM). Z-stack images were collected via tracts of both samples showed comparable color CLSM and analyzed by COMSTAT. The bio- change in TLC chromatogram. Therefore, our film formation level was significantly higher results clearly gave insight that lichen extract in in the condition containing 60 mM butyric water and organic solvent contain medicinally acid than 30, 40, and 50 mM butyric acid. important bioactive compounds and which justi- Furthermore, the biofilm formation failed in 0 fies their use in the traditional medicine. Key and 6 mM butyric acid. The biofilm formation words: lichens, antimicrobial activity, MIC, failed in 60 mM sodium butyrate (pH7.0), but phytochemical properties. 60 mM sodium butyrate (pH4.9) was similar

92 ASM Conferences Poster Abstracts level to 60 mM butyric acid. The pH was 7.0, semi-synthetic antimicrobials. The Gram- 6.7, 5.5, 5.0, 4.8 and 4.7 in TSB with sucrose negative opportunistic pathogen Pseudomonas containing 0, 6, 30, 40, 50 and 60 mM butyric aeruginosa causes various acute and chronic acid respectively. Same pH conditions adjusted infections in immunocompromised individu- using hydrochloric acid were also tested in als and accounts for 15% of all nosocomial the assay but didn’t induce significant biofilm infections. P. aeruginosa is both intrinsically formation. Therefore, 60 mM of butyric acid resistant and can acquire resistance to multiple condition involving low pH are required for antimicrobial classes. Here, we employed significant biofilm formation in the flow -sys two genome-wide sequencing methods to: 1) tem. In the previous report, we found that the elucidate the transcriptional response to sub- GroEL played as an adhesion of A.naeslundii inhibitory levels of 17 natural, synthetic, and in the biofilm formation assay condition with- semi-synthetic antimicrobials; and 2) identify out flow. As a next issue, we investigated how genes required for resistance to these antimi- GroEL would contribute to the biofilm forma- crobials. We hypothesized that P. aeruginosa tion in the flow condition. The initial coloniza- has evolved an adaptive response to antibiotics tion of A.naeslundii was analyzed utilizing commonly produced by soil microbes whereas CLSM after incubation for 3 hours + 1hour synthetic or semi-synthetic antimicrobials after washing in TSB with sucrose. Initial will not elicit an adaptive response. Using colonization increased in 60 mM butyric acid RNA-sequencing, we found that the transcrip- condition. To observe contribution of GroEL tional response varied from having as little as to the initial colonization, anti-GroEL antibody 0 genes to as many as 500 genes differentially was applied. As a result, anti-GroEL antibody expressed upon exposure to sub-MIC levels inhibited clearly the initial colonization. In of an antimicrobial. In general, P. aeruginosa conclusion, the metabolic products such as had a more robust transcriptional response butyric acid from Gram negative anaerobes to natural antimicrobials than semi-synthetic stimulate the GroEL-dependent initial coloni- antimicrobials. A closer look at the genes zation of A.naeslundii resulting in significant identified by RNA-seq indicated that oxidative biofilm formation under the low pH condition stress-related genes are induced upon exposure in the flow system. to several different classes of antimicrobials. To identify genes required for resistance to n 101 these antimicrobials, we profiled the fitness of ~300,000 P. aeruginosa transposon mutants Comparing the response of during growth in the presence of sub-inhibitory Pseudomonas aeruginosa to levels of antimicrobials using Transposon- natural, synthetic, and semi- sequencing (Tn-Seq). Tn-seq revealed that synthetic antimicrobials oxidative stress-related genes are also critical J. L. Murray; fitness determinants for multiple classes of University of Texas at Austin, Austin, TX. antimicrobials. Our results reveal that P. aeru- ginosa possesses unique responses to natural, Opportunistic pathogens encounter antimicro- synthetic, and semi-synthetic antimicrobials bials during infection as well as outside the and we anticipate this work will continue to host. At sub-inhibitory levels, antimicrobi- yield important insights in the involvement of als induce subtle changes in expression of antimicrobials in growth inhibition as well as stress-related and metabolic genes, which has communication. led to the idea that antimicrobials not only function to inhibit bacterial growth but also act as cues. However, little is known about how bacteria respond to natural versus synthetic or

5th ASM Conference on Cell-Cell Communication in Bacteria 93 Poster Abstracts n 102 n 103 Scanning Electrochemical A FRET based sensor for quantitative Microscopy (SECM): A toolbox to in vivo measurements of PhrA- study microbial metabolic exchange peptide signaling in Bacillus subtilis K. Nguyen, G. Ummadi, D. Koley; H. Babel1, V. Sourjik2, I. Bischofs1; Oregon State University, Corvallis, OR. 1University of Heidelberg, Heidelberg, GER- MANY, 2MPI for Terrestrial Microbiology, Scanning electrochemical microcopy (SECM) Marburg, GERMANY. has proved to be a powerful tool to study microbial metabolic exchanges at high spatial Extracellular signaling systems of the Rap-Phr resolution (koley et. al). This technique has type control a variety of cellular differentia- been used (koley et. al) to detect and quantify tion pathways in the Gram-positive model the reduced pyocyanin layer produced by organism Bacillus subtilis. Rap-Phr systems pseudomonas aeruginosa biofilm. SECM is a are export-import systems where Phr-peptides technique that allows precise positioning of are exported, processed, reimported and sensed the electrochemical probe over the substrate intracellularly by Rap-proteins. Often several of interest (e.g., biofilm) without touching or Rap-Phr systems act on the same pathway and destroying it, thus making it a noninvasive are genetically interconnected. The resulting method. The SECM probe or ultramicrosen- complexity makes it difficult to investigate sor is positioned above the substrate at a these systems. Here we developed a FRET known distance of 1- 10 μm with the aid of based PhrA-biosensor that consists of a pair an approach curve (plot of probe current vs. of stable and functional fluorescent protein z-direction distance). Later the probe can be fusions. The sensor responds specifically and moved in the x, y, and z direction over a range reproducibly to extracellular PhrA, as deter- of 1-1,000 μm, a perfect working range for mined by acceptor photobleaching measure- biofilm studies. The surface pattern of a single ments and shows a high sensitivity in the range or multispecies biofilm fabricated by a new of low nanomolar concentrations. We show alginate-based electroaddressing technique that PhrA can be detected in the supernatant is being used as a SECM substrate to study of wild-type cells. Hence, the new biosensor the polymicrobial metabolic exchanges. Time might be used to measure extracellular concen- dependent 3D distribution of phenazine and tration profiles and help to elucidate dynamical its derivatives produced by the pseudomonas features of PhrA signaling during develop- biofilm grown on these substrates will be ment. It might also facilitate the determination addressed. Reference: 1. Koley, D., Ramsey, of biochemical rate constants by combining M. M., Bard, A. J. & Whiteley, M. Discovery quantitative measurements with mathematical of a biofilm electrocline using real-time 3D modeling. As a first step, we characterized the metabolite analysis. Proc. Natl. Acad. Sci. U. oligopeptide permease Opp, a shared network S. A. 108, 19996-20001 (2011). component of all Rap-Phr-systems. Overall, our data suggests that FRET-biosensors show a promising potential to serve in the quantitative analysis of complex signaling networks.

94 ASM Conferences Poster Abstracts

n 104 from a frequency change (ΔF2), which was the Bioaffinity Analysis of Quorum index of the adsorbed mass on the electrode Sensing Receptor SpnR with spn avoiding the effects on the viscosity change of box in Serratia marcescens Using the solution around the oscillating electrode. a Quartz Crystal Microbalance Technique n 105 E. Nasuno, T. Umemura, Y. Takayama, H. Genetic determinants of fitness Sasa, K. Iimura, T. Ikeda, N. Kato; in polymicrobial chronic wound Utsunomiya University, Utsunomiya, JAPAN. infections K. H. Turner1, J. Everett2, D. Fleming2, K. P. Intracellular interaction of SpnR, a quorum Rumbaugh2, M. Whiteley1; sensing (QS) receptor, with the regulatory 1The University of Texas at Austin, Austin, TX, target DNA sequence in Serratia marcescens 2Texas Tech University Health Sciences Center, AS-1 was successfully analyzed by quartz Lubbock, TX. crystal microbalance based on admittance analysis (QCM-A). S. marcescens is one of the Most naturally occurring infections are poly- opportunistic human pathogens and activation microbial. Clinical and experimental observa- of the QS-related gene expression depends tions suggest that when infections contain on a concentration of N-acyl-L-homoserine more than one bacterium, they are often worse. lactone (AHL). The QS-dependent production One particularly significant example of this is of an antibacterial red pigment prodigiosin infections in chronic wounds, such as pressure is controlled by N-hexanoyl-L-homoserine ulcers (bedsores), surgical wounds, and dia- lactone and N-(3-oxo-hexanoyl)-L-homoserine betic ulcers. These wounds contribute to high lactone. It is considered that SpnR stably healthcare costs, and are rapidly increasing in binds to approximately 20 bp sequence of prevalence in the US. These chronic infections the QS-related DNA, spn box, possessing lux are often polymicrobial, and the opportunis- box-like sequence at low concentration of the tic pathogens Pseudomonas aeruginosa and AHL before the QS activation. To evaluate the Staphylococcus aureus are among the most bioaffinity using the QCM-A technique, the commonly isolated bacteria from infected target promoter spn box was immobilized on chronic wounds. Infections containing both of a gold electrode of the 27 MHz QCM sensor. these bacteria take longer to heal and are more By using approximately 40 bp of double- resistant to antibiotic therapy. Yet specific mo- stranded DNA fragment labelled at the 5’-end lecular mechanisms potentiating this synergy with biotin, the spn box sequence could be are not well understood. To investigate the introduced to NeutrAvidin-immobilized self- precise bases for synergy between these two assembly membrane formed on the Au-surface. organisms, we used genome-wide insertion The aqueous solution of maltose-binding mutant fitness profiling (Tn-seq) to character- protein (MBP) tagged SpnR produced by gene ize fitness determinants in monomicrobial and engineering technique was prepared as desired polymicrobial murine model chronic wound concentration. The aliquot of the MBP-SpnR infections. Previous work has shown that P. solution was gently mixed to the spn box-mod- aeruginosa must biosynthesize numerous me- ified electrode immersed in TE buffer solution tabolites in single-species infections, including (pH 7.8) after reaching the steady state output. riboflavin and lipoic acid, while it is able to A decrease of the resonant frequency observed harvest many others from the wound environ- just after adding MBP-SpnR clearly showed ment. Our polymicrobial Tn-seq analysis sug- the effective interaction with the spn box. The gests that co-infection with S. aureus changes apparent stability constant could be evaluated

5th ASM Conference on Cell-Cell Communication in Bacteria 95 Poster Abstracts the metabolic requirements of P. aeruginosa in activity of receptor proteins belonging to vivo, requiring it to synthesize some metabo- the Rgg family of transcription factors. In lites previously available from wound environ- S. pyogenes, four Rgg paralogs exist, each ment and relieving biosynthetic requirements serving as transcriptional regulators of genes seen in monomicrobial infection. Our results associated with pathogenesis (RopB), biofilm also show that the MexEF-OprN multidrug development (Rgg2 and Rgg3), or a cryptic efflux pump is required of P. aeruginosa competence regulon (ComR). Our ongoing specifically in co-infection, suggesting that aims are to elucidate the mechanisms by which efflux of small, possibly toxic molecules from communication is propagated and regulated the cytoplasm is a determinant of fitness in during the course of bacterial colonization and polymicrobial infections. Finally, we will de- infection and to identify small molecules that tail advances made using genomic approaches disrupt signaling as a therapeutic strategy to to dissect the genetic determinants of S. aureus treat or prevent disease. We have conducted required for polymicrobial synergy in chronic genetic, biochemical and mass spectrometry wounds. Our results provide novel insight into analyses to identify several components of the genetic requirements for P. aeruginosa Rgg signaling pathways, that includes defining and S. aureus polymicrobial chronic wound the mature pheromones. We have also begun infections and demonstrate the power of using identifying environmental signals and bacterial fitness profiling for probing bacterial virulence enzymes that control the expression and activ- and persistence in infections. ity of signaling pathways. Finally, we have identified inhibitory compounds that specifi- n 106 cally block individual Rgg pathways across several species. Cell-cell communication among the Streptococci: Rgg pheromones, inhibitors and regulated behaviors n 107 Activation of Vibrio cholerae C. Aggarwal1, J. Chang1, J. C. Jimenez2, quorum sensing via small molecules K.Ratia3, M. J. Federle1 1Department of Medicinal Chemistry and A. Hurley, B. Bassler; Pharmacognosy, Center for Pharmaceutical Department of Molecular Biology, Princeton Biotechnology, College of Pharmacy; 2Dept. University of Microbiology and Immunology, College of Quorum sensing is a form of bacterial cell-cell Medicine, 3High-throughput Screening Facil- communication that uses extracellular signal- ity, Research Resources Center; University of ing molecules called autoinducers to control Illinois at Chicago, Chicago, IL. individual vs. group behavior. In the pathogen , a human pathogen, Vibrio cholerae, the quorum-sensing response causes a variety of diseases including, but not is initiated by the binding of the autoinducer limited to, pharyngitis, rheumatic fever and CAI-1 to its cognate receptor CqsS. The necrotizing fasciitis. It accounts for substan- histidine kinase CqsS controls the activity tial mortality related to infections worldwide. of the response regulator LuxO via phos- Recent studies indicate that streptococci pro- phorylation. In the absence of autoinducer, duce and respond to several secreted peptide the CqsS-LuxO cascade leads to virulence signaling molecules (pheromones), including factor expression while binding and response those known as SHPs (short hydrophobic to CAI-1 terminates the virulence program. peptides). Upon transport into the bacterial Thus, compounds that activate the circuit cell, the pheromones bind to and modulate (CqsS agonists or LuxO antagonists) could

96 ASM Conferences Poster Abstracts lead to the development of medicinal mol- of the organism. The LeDSF3-regulated HSAF ecules. A chemical screen was performed that production is dependent on the two-component yielded both types of compounds. Isolating regulatory system RpfC/RpfG. Moreover, overexpressed CqsS membranes revealed the LeDSF3 up-regulated the expression of the synthetic CqsS agonists function by blocking global regulator Clp (cAMP receptor-like His194 autophosphorylation, similar to CAI-1. protein). The disruption of clp led to no HSAF Titration assays revealed that several agonists production. Together, the results show that are more potent than the native autoinducer. LeDSF3 is a fatty acid-derived, diffusible sig- While these agonists are structurally unrelated naling factor positively regulating HSAF bio- to the natural ligand, mutagenesis of CqsS synthesis and that the signaling is mediated by suggests their binding sites overlap. Among the RfpC/RpfG-Clp pathway. These findings the LuxO antagonists, both uncompetitive may facilitate the antibiotic production through and competitive inhibitors of LuxO ATPase applied genetics and molecular biotechnol- activity were identified. Characterizing these ogy in Lysobacter, a group of ubiquitous yet pro-QS molecules will provide tools for prob- underexplored microorganisms. ing the quorum-sensing signaling mechanism and have the potential to be developed into n 109 therapeutics. Regulation of violacein production in Chromobacterium violaceum n 108 G. Devescovi1, M. Kojic2, M. Cámara3, P. Identification of a Small Molecule Williams3, S. Subramoni1, I. Bertani1 and V. Signaling Factor That Regulates Venturi1; the Biosynthesis of the Antifungal 1International Centre for Genetic Engineering Polycyclic Tetramate Macrolactam and Biotechnology, Trieste, Italy, 2Institute of HSAF in Lysobacter enzymogenes Molecular Genetics and Genetic Engineer- L. Du; University of Nebraska-Lincoln, Lin- ing, University of Belgrade, Belgrade, Serbia, coln, NE. 3School of Life Sciences, University of Notting- ham, Nottingham, UK. Lysobacter are emerging as a new source of antibiotics. The regulation of these antibiot- The biosynthesis of the violacein purple ics is not well understood. Here, we identified pigment by Chromobacterium violaceum is a small molecule metabolite (LeDSF3) that known to be positively regulated by an N-acyl regulates the biosynthesis of the antifungal homoserine lactone (AHL)-driven quorum antibiotic HSAF, a polycyclic tetramate mac- sensing (QS) system. Wild type C. violaceum rolactam with a structure and mode of action strain ATCC31532 produces low amounts distinct from the existing antifungal drugs. of violacein whereas a transposon mutant of LeDSF3 was isolated from the culture broth ATCC31532 has been reported to produce very of L. enzymogenes, and its chemical structure large amounts of violacein. This suggests that was established by NMR and MS. The purified violacein production in under negative regula- compound induced green fluorescence in a tion. We have studied this regulation show- reporter strain of Xanthomonas campestris, ing that the violacein promoter is positively which contained gfp gene under the control of regulated by AHL-mediated QS and negatively a DSF (diffusible signaling factor)-inducible regulated by a novel protein which we named promoter. Exogenous addition of LeDSF3 in L. VioS. VioS neither affected AHL levels nor enzymogenes cultures significantly increased the expression of luxI/R genes. However, it in- the HSAF yield, the transcription of HSAF terfered with the positive regulation of the vio biosynthetic gene, and the antifungal activity operon by the AHL QS system since in a vioS

5th ASM Conference on Cell-Cell Communication in Bacteria 97 Poster Abstracts mutants much higher levels of transcription of the vio operon could be detected. Interestingly the sequenced C. violaceum strain ATCC12472 naturally produces high amounts of viola- cein and introduction of vioS from strain ATCC31532 resulted in much lower levels of violacein production. We have also sequenced C. violaceum ATCC31532 and performed com- parative genomics speculating possible DNA rearrangements in these strains. It is known that some AHL QS systems undergo nega- tive regulation resulting in QS homeostasis; violacein production on the other hand is an example of strong competition between nega- tive regulation and AHL QS at the target locus in order to carefully control the production levels of a QS regulated phenotype.

98 ASM Conferences Index

A Belmont, J. 58 Chandler, L. S7:2 Fleming, D. 105, 50 Adlung, L. 85 Chatterjee, S. S4:3 Florez, V. 78 Aliyu, A. B. 70 Chen, S. 84 Förstner, K. U. 22 Amador, C. I. 45 Chen, Y. S5:2 Foster, K. 98 An, J. 19, 27 Chenia, H. Y. 67, 70, Fueki, S. 80 Arai, T. 100 S2:6 Fujita, M. 11, 12 Armstrong, A. 50 Choi, S. 48, 65 Futamata, H. 82 Asfahl, K. L. 41 Clemmer, K. M. S1:3 Garcia-Contreras, R. 58, 59 Babel, H. 103 Clinton, A. 72 García Fontana, C. 1 Baltrus, D. A. 49 Coenye, T. S2:2 Garge, S. 66 Bandyopadhyay, A. A. Cong, J.-P. 22 Gart, E. 76 S5:2 Conklin, A. 92 Gee, M. L. 20 Bang, Y.-J. 48, 65 Connell, J. L. S5:5 Ghosh, S. 66 Bard, A. J. S5:5 Corander, J. 52 Gibbs, K. A. 28, S3:5 Barnes, A. S5:2 Coria-Jiménez, R. 59 Gil, S. 48 Bassler, B. L. 22, 75, 79, Cornforth, D. S2:4 Gilbert, K. 41 83, OS:1, Corral Lugo, A. 1 Gilley, R. 56 S1:2 Cueva, A. R. 50 Gilmore, M. S. S4:1 Bentley, W. E. S5:1 Damkiær, S. 46 Givskov, M. C. S2:1 Ben-Zion, I. 38 Dandekar, A. A. 32, 33 Gloag, E. S. 20, S3:2 Bergeest, J.-P. 24 Dandekar, A. A. 35 Gomez-Santos, N. 25 Bhattacharjee, A. S2:3 Darch, S. E. 52 González-Pedrajo, B. 73 Bischofs, I. B. 24, 85, Darkoh, C. 87 Goo, E. 19, 27 103, S5:3 Deng, Y. 84 Greenberg, E. P. 33, 35, 37, Bisht, S. 99 Devi, S. N. 11, 12 S7:1 Bisht, S. 99 Dey, A. 92 Greenberg, P. 86 Bitzer, A. S. S6:2 Diep, D. S4:2 Gruber, C. 68 Blackwell, H. E. S2:5 Diggle, S. P. 39, 42, 52, Gurney, J. S2:4, S7:4 Blanchette, K. 56 S2:4, S7:4 Hadjifrangiskou, M. S4:7 Bogale, H. 94 Dogsa, I. 40 Haggett, L. 11, 12 Bojer, M. S. S3:6 Dorrestein, P. C. S5:4 Hammer, B. K. 23 Bonocora, R. P. 69 Drees, B. S5:3 Harder, N. 24 Bowers, A. 4 Dunny, G. M. S5:2 Harrison, F. 39, 52 Brackman, G. S2:2 Du Plooy, N. 67 Haruta, S. 88 Brede, D. S4:2 DuPont, H. L. 87 Hasegawa, Y. 82 Brown, S. 39 Duy Duong, A. 21 Hashimoto, Y. 16 Brown, S. P. 95, S2:4, Eldar, A. 34, 38, Hawver, L. A. 14 S7:2 S7:5 Henriques Normark, B. Cady, N. C. 69 Elgamoudi, B. A. 10 47, 54, 55 Callan, C. G. S1:2 El-Halfawy, O. M. 93 Hense, B. A. S5:6 Cao, P. 6 Eslava, C. A. 73 Hentrich, K. 55 Cardarelli, L. S3:5 Espinosa Urgel, M. 1 Hinshaw, K. 36 Chakraborty, B. 71 Estrela, S. 95 Hirakawa, H. 80 Chan, K. 51 Even-Tov, E. 34 Hochbaum, A. S2:3 Chance, D. L. 97 Everett, J. 105, 50, Ho-Ching, T. S1:4 Chandler, J. 86 57 Hoegy, F. 98 Chandler, J. R. 32, 36 Everett, J. A. S4:6 Hong, K. 51

5th ASM Conference on Cell-Cell Communication in Bacteria 99 Index

Hoover, S. S1:4 Kondoh, T. 100 Morohoshi, T. 61 How, K. 51 Konovalova, A. 5 Müller, D. 33 Hu, W.-S. S5:2 Koorbanally, N. A. 70 Murray, J. L. 101 Huang, A. S3:2 Kraigher, B. 26 Musah, R. A. 69 Hung, D. 44 Krell, T. 1 Mutlu, A. 24 Hunsur Rajendra, V. 68 Kroos, L. 29 Myers, S. 76 Hwang, I. 19, 27 Kumar, M. 99 Nadal-Jimenez, P. 90 Iimura, K.-I. 104 kumar, V. 99 Nakayama, J. 74 Ikeda, T. 104, 61 Kurabayashi, K. 80 NAKAZAWA, F. 77 Inaba, H. 16 Kurosawa, M. 8 Nasuno, E. 104 Ingmer, H. S3:6 K Wood, T. 58 Neiditch, M. B. 81 Irie, Y. 42 Lawhon, S. 76 Nerurkar, A. 66 Ismail, A. S. 83 Lazazzera, B. S1:4 Nes, I. S4:2 Ito, S. 63 Leanti La Rosa, S. S4:2 Ng, W.-L. 14, S4:4 Ivens, A. S2:4 Lee, E. 76 Nguyen, K. 102 Jang, K. 48 Lee, J. 91 Nielsen-LeRoux, C. S7:3 Jasso-Chávez, R. 58 Lemy, C. 89 Nishioka, S. 80 Javed, A. 20 Lereclus, D. 89, S7:3 Nomura, N. 16, 62, 63, javvadi, S. S4:3 Levy, S. B. S6:2 8 Jelsbak, L. 45, 46, Lim, J. 48 Normark, S. 54, 55 S3:6 Little, K. 28 Norris, S. J. 87 Jensen, R. V. 21 Löfling, J. 55 Norsworthy, A. S6:1 Jia, A. 7 Loper, J. 98 Nuñez-López, L. 58 Joon-Hee, L. 17, 18 Lopez, D. 2, 3 Ochiai, K. 100 Jorth, P. 57 Lyons, N. 26 Ochiai, S. 64 Jung, K. S5:3 Maeda, T. 58, 59 Ogura, N. 100 Jung, S. A. S4:4 Mandic Mulec, I. 26 Ohtani, K. 74 Kajee, Z. S2:6 Mandic-Mulec, I. 40 Oliveira, A. S6:3 Kakihara, K. 63 Markussen, T. 45 Oluremi, A. S. 43 Kang, Y. 27 Martínez-Vázquez, M. Omer, S. 34 Kapila, Y. 96 59 Orihuela, C. J. 56 Kaplan, H. B. 87 Marvig, R. 46 Oslizlo, A. 40 Kasper, S. H. 69 MASHIMA, I. 77 Papenfort, K. 22, 79 Kato, N. 104 Matsumoto, N. 80 Parashar, V. K. 81 Ke, X. 75 Matsuura, K. 88 Park, W. 60 Kernell Burke, A. 21 Matsuzawa, T. 80 Paszkiewicz, K. 52 Ketley, J. 10 Mawhinney, T. P. 97 Pathak, A. 47, 54 Khademi, S. 46 McCully, L. M. S6:2 Peng, Q. 15 Kiehler, B. 11, 12 McNally, A. 52 Peng, X. 7 Kim, B. 65 McNally, L. 39, S2:4, Peréz-Eretza, B. 59 Kim, B. 65 S7:2 Planinc, J. 26 Kim, J. 60 Michelsen, C. F. S3:6 Polatynska, M. A. 5 Kim, J. S5:5 Miller, L. C. 83 Popat, R. 39, S2:4 Kimbara, K. 82 Min, A. 79 Porat, Z. 31 Koch, G. 2, 3 Mitri, S. 98 Powers, M. J. 4 Kolb, P. 85 Miyashiro, T. 94 Prabhakar, R. S3:2 Koley, D. 102 Mobarec, J. 85 Qin, N. 7 Kolodkin-Gal, I. 31 Monson, R. E. S3:3 Quiñones-Peña, M. A. 73 Kolter, R. 26 Moodley, B. 70 Raffl, S. 68 Morohoshi, S. 88 Rahman, M. 30

100 ASM Conferences Index

Rai, R. S4:3 Smith, B. A. 49 Wall, D. 6 Rajagopalan, R. 29 Soberón-Chávez, G. 59 Walsh, J. 41 Ramachandran, R. 21 Søgaard-Andersen, L. Wand, M. P. S3:2 Ramsay, J. S3:3 25, 5 Wang, H. 20 Ranadive, I. 66 Someya, N. 61 Wang, J.-F. 53 Rather, P. N. S1:3 Song, F. 15 Wang, M. 35 Raymond, B. S7:3 Sonomoto, K. 74 Wang, Q. 13 Reiger, M. S5:3 Soo-Kyoung, K. 17, 18 Watve, S. S. 23 Reilly, T. J. 97 Sourjik, V. 103 West, S. 42 Rippa, V. 85 Stacy, A. 57 Whitchurch, C. B. 20, S3:2 Roberts, A. E. 42 Stefanic, P. 26, 40 Whiteley, M. 105, 50, Rohr, K. 24 Steinberg, N. 31 52, 57, Rumbaugh, K. P. 105, 50 Stengel, S. T. 3 S5:5 Rumbaugh, K. P. 57 Stevens, A. M. 21 Williams, P. 39, S4:5 Rumbaugh, K. P. 72, S4:6 Suárez-Moreno, Z. 78 Winans, S. S1:1 Rutherford, S. T. S1:2 Subramoni, S. 78 Wingreen, N. S. S1:2 Saak, C. S3:5 Sumana, A. 71 Winkler, M. S1:4 Sakai, R. 62 Sun, Y. 94 W Kwan, B. 58 Salmond, G. P. S3:3 Taillefumier, T. O. S1:2 Wolf, D. 85 Sanabria-Valentín, E. 4 Takayama, Y. 104 Wood, T. K. 59, 9 Sasa, H. 104 Tan, X. 7 Wu, Q. 53 Sato, R. 61 Tashiro, Y. 82, S3:3 Xavier, K. B. 90, S6:3 Sauer, P. 85 Tateda, K. 62 Xi-Hui, L. 17, 18 Schaefer, A. L. 33 Tavizon, G. 73 Yamaguchi, T. 61 Schaefer, A. L. 35 Thomas, J. 23 Yang, J. 62 Schalk, I. 98 Thompson, J. S6:3 Yang, M. 15 Scholz, R. L. 37 Thrikawala, S. 11, 12 Yokohata, R. 74 Schrank, E. M. 68 Tipping, M. J. 28 Zangger, K. 68 Schuster, M. 41, 98 Tominaga, A. 100 Zechner, E. L. 68 Scott-Phillips, T. C. S2:4 Toyofuku, M. 16, 62, 63, Zhang, J. 15 Seaton, S. C. S6:2 8 Zhang, L.-H. 84 Semmelhack, M. F. 83 Trauth, S. 24, 85 Zhang, L. 91 Sender, V. 47, 54 Trivedi, U. 57 Zhao, J. 13 Sengupta, T. 71 Truong, T. 86 Zhou, L. S7:3 Senpuku, H. 100 Turnbull, L. 20, S3:2 Zhu, Y.-H. 53 Sexton, J. 98 Turner, K. 50 Ziesack, M. 24 Shah, P. 44 Turner, K. H. 105, 52 Shank, E. A. 4, S3:4 Umemura, T. 104 Sharma, C. M. 22 Ummadi, G. 102 Sharma, K. 99 Valastyan, J. S. 83, S1:2 Sharma, S. 99, 99 Valente, R. 90 Shimizu, T. 74 Vallotton, P. S3:2 Shin, J. 96 Valvano, M. A. 93 Shintani, M. 82 Van den Driessche, F. S2:2 Shu, C.-C. S5:2 Verma, S. C. 94 Silby, M. W. S6:2 Vishnoi, M. 11 Silva, P. M. 86 Visick, K. L. S6:1 Singh, R. P. 74 Wade, J. T. 69 Slamti, L. 89, S7:3 Wade, S. A. 20 Smalley, N. E. 32 Wall, D. S3:1

5th ASM Conference on Cell-Cell Communication in Bacteria 101 American Society for Microbiology 1752 N Street, N.W. Washington, DC 20036-2904