BLAST XI MEETING ASTOR CROWNE PLAZA HOTEL

NEW ORLEANS, LOUSIANA JANUARY 16-21, 2011

Meeting Chairperson:

Dr. Robert Bourret – University of North Carolina, Chapel Hill, NC

Meeting Vice-Chairperson: Dr. Urs Jenal – University of Basel, Basel, Switzerland

Program Committee: Dr. John S. Parkinson (Chairperson) – University of Utah, Salt Lake City, UT

Dr. Joe Falke – University of Colorado, Boulder, CO

Poster Awards Committee: Dr. Kelly Hughes – University of Fribourg, Fribourg, Switzerland

Dr. Karen Ottemann – University of California at Santa Cruz, Santa Cruz, CA Dr. Barry Taylor (Chairperson) – Loma Linda University, Loma Linda, CA

Speaker Award Committee: Dr. Robert Bourret – University of North Carolina, Chapel Hill, NC

Dr. Urs Jenal – University of Basel, Basel, Switzerland

Meeting Review Committee: Dr. Gladys Alexandre (Chairperson) – University of Tennessee, Knoxville, TN

Dr. Sean Crosson – University of Chicago, Chicago, IL Dr. Tarek Msadek – Institut Pasteur, Paris, France

Dr. Thomas Shimizu – FOM Institute for Atomic & Molecular Physics (AMOLF), Amsterdam, The Netherlands

Board of Directors – BLAST, Inc.: Dr. Joe Falke – University of Colorado, Boulder, CO

Dr. Michael Manson – Texas A&M University, College Station, TX Dr. Philip Matsumura (Chairperson) – University of Illinois at Chicago, IL

Dr. John S. Parkinson – University of Utah, Salt Lake City, UT

Conference Coordinators: Ms. Tarra Bollinger – Molecular Biology Consortium, Chicago, IL Ms. Peggy O’Neill – Molecular Biology Consortium, Chicago, IL

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AWARDS INFORMATION

Robert M. Macnab Award for an Outstanding Poster Presentation by a Young Investigator

This award was established and named in memory of the late Robert M. Macnab, Ph.D., who was an integral member of the Bacterial Locomotion and Signal Transduction Community. Dr. Macnab spent his 30 year career studying the assembly, structure and function of the bacterial flagellum. Bob actively participated in the BLAST meetings and served on the Program and Review Committees for BLAST IV. At the time of his death in 2003, Bob was a professor in the Department of Molecular Biophysics and Biochemistry at Yale University.

(This award is sponsored by a generous donation from Mrs. May K. Macnab)

Robert J. Kadner Award for an Outstanding Poster Presentation by a Young Investigator This award was established and named in memory of the late Robert J. Kadner, Ph.D., who was an integral member of the Bacterial Locomotion and Signal Transduction Community. Dr. Kadner spent his career studying microbial physiology of E. coli transport systems. Bob actively participated in the BLAST meetings and served as Chair of the Review Committee for BLAST V, Vice-Chair of BLAST VII and Meeting Chair of BLAST VIII. At the time of his death in 2005, Bob was the Norman J. Knorr Professor of Basic Sciences in the Department of Microbiology at the University of Virginia, School of Medicine.

Nucleic Acids Research Award for an Outstanding Poster Presentation by a Young Investigator

Nucleic Acids Research (NAR), an Oxford University Press Journal, publishes the results of leading edge research into physical, chemical, biochemical and biological aspects of nucleic acids and proteins involved in nucleic acid metabolism and/or interactions. NAR is sponsoring a

poster award to be presented to a young investigator whose research is in the area of transcriptional regulation of gene expression. (http://nar.oxfordjournals.org)

BLAST Board of Directors' Award for an Outstanding Talk The Board of Directors is pleased to announce the establishment of the first BLAST award for speakers. (This award is sponsored by a generous donation from Dr. Philip Matsumura)

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BLAST XI MEETING SCHEDULE TIME EVENT LOCATION

Sunday, January 16, 2011 5:00 pm Poster room available for poster setup St. Charles Ballroom 4:00 pm – 7:00 pm Meeting Registration Grand Ballroom Gallery 7:00 pm – 8:30 pm Dinner Grand Ballroom D 9:00 pm – 11:00 pm Welcome Reception Grand Ballroom Gallery Monday, January 17, 2011 7:30 am – 8:30 am Breakfast Astor Ballroom III 8:45 am – 9:00 am Welcome/ Announcements – Meeting Chair (R. Bourret) Astor Ballrooms I & II 9:00 am – 12:00 pm Meeting Session – “Two-Component Signaling Systems” Astor Ballrooms I & II 10:15 am – 10:30 am Coffee Break Astor Gallery 12:00 pm – 1:30 pm Lunch Astor Ballroom III 2:00 pm – 4:00 pm Poster Session – even numbered posters St. Charles Ballroom 6:00 pm – 7:30 pm Dinner Astor Ballroom III 7:30 pm – 10:00 pm Meeting Session – “Behavior and Bioinformatics” Astor Ballrooms I & II 8:30 pm – 8:45 pm Coffee Break Astor Gallery Tuesday, January 18, 2011 7:30 am – 8:30 am Breakfast Astor Ballroom III 9:00 am – 12:00 pm Meeting Session – “Chemotactic Signaling” Astor Ballrooms I & II 10:15 am – 10:30 am Coffee Break Astor Gallery 12:00 pm – 1:30 pm Lunch Astor Ballroom III 2:00 pm – 4:00 pm Poster Session – odd numbered posters St. Charles Ballroom 6:00 pm – 7:30 pm Dinner Astor Ballroom III 7:30 pm – 10:00 pm Meeting Session – “Receptors” Astor Ballrooms I & II 8:30 pm – 8:45 pm Coffee Break Astor Gallery Wednesday, January 19, 2011 7:30 am – 8:30 am Breakfast Astor Ballroom III 9:00 am – 12:00 pm Meeting Session – “Regulation” Astor Ballrooms I & II 10:15 am – 10:30 am Coffee Break Astor Gallery 12:00 pm – 1:30 pm Lunch Astor Ballroom III Thursday, January 20, 2011 7:30 am – 8:30 am Breakfast Astor Ballroom III 9:00 am – 12:00 pm Meeting Session – “Flagella” Astor Ballrooms I & II 10:15 am – 10:30 am Coffee Break Astor Gallery 12:00 pm – 1:30 pm Lunch Astor Ballroom III 6:00 pm – 7:30 pm Dinner Astor Ballroom III 7:30 pm – 10:00 pm Meeting Session – “Gliding and Swarming” Astor Ballrooms I & II 8:30 pm – 8:45 pm Coffee Break Astor Gallery 10:00 pm – 10:30 pm Awards Presentation Astor Ballrooms I & II 10:30 pm – 12:30 am Reception Astor Ballroom Gallery Friday, January 21, 2011 7:30 am – 8:30 am Breakfast Astor Ballroom III

BLAST XI PROGRAM January 17, 2011 Two-Component Signaling Systems Monday Morning (8:45 am – 12:00 pm) Chair – Linda Kenney

PRESENTER TITLE ABSTRACT PAGE NO.

Huynh, Tu-Anh Conserved mechanism for sensor phosphatase control of two-component signaling: Evidence from the nitrate sensor NarX 2

Petters, Tobias Wiring of two-component signal transduction systems in Myxococcus xanthus 3

Higgs, Penelope Coordination of cell fates is mediated by negative regulatory signaling systems in the Myxococcus xanthus multicellular developmental program 4

BREAK Boll, Joseph Michael

A specificity determinant in a response regulator prevents in vivo crosstalk and phosphorylation by non-cognate phosphodonors 5

Pawelczyk, Sonja Prediction of interspecies cross-talk in two-component systems 6 Wang, Loo Chien Defining the signaling pathway of the osmosensor EnvZ 7 Huangyutitham, Varisa

The response regulator WspR forms subcellular clusters as a possible mechanism to increase activity 8

January 17, 2011 Behavior and Bioinformatics Monday Evening (7:30 pm – 10:00 pm) Chair – Christopher Rao Hobley, Laura

Motility, taxis and predatory behaviour in Bdellovibrio: Multiple mot proteins and GGDEF regulation of motility 9

Li, Guanglai Rotational brownian motion and bacterial near surface swimming 10

Tu, Yuhai Frequency-dependent Escherichia coli chemotaxis behaviors revealed by microfluidics experiments and pathway-based modeling 11

BREAK

Sneddon, Michael Signaling noise in bacteria coordinates flagellar motors to improve chemotactic performance 12

Wuichet, Kristin Computational identification of novel phosphatases for chemotaxis signal transduction 13

Galperin, Michael Visualizing the evolution of signal transduction machinery with signaling protein family profiles 14

January 18, 2011 Chemotactic Signaling Tuesday Morning (9:00 am – 12:00 pm) Chair – Howard Berg

Erbse, Annette Conformational changes in the assembled, membrane-associated chemotatic signaling complex 15

Li, Mingshan The stoichiometry of the minimal core unit of the chemotaxis signaling complex 16

Hall, Benjamin Models of structure and dynamics of a complete Tsr trimer of dimers 17 Briegel, Ariane Electron cryotomography of bacterial chemotaxis arrays 18

BREAK Lertsethtakarn, Paphavee

Helicobacter pylori CheZ cellular localization is independent of other chemotaxis proteins 19

Sukomon, Nattakan

Structural and functional studies of HAMP domain signaling in bacterial chemoreceptors 20

Watts, Kylie PAS-HAMP interactions in the aerotaxis receptor, Aer 21 Fukuoka, Hajime Coordinated regulation of flagellar motors on a single Escherichia coli cell 22

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January 18, 2011 Receptors Tuesday Evening (7:30 pm – 10:00 pm) Chair – Birgit Prüß

PRESENTER TITLE ABSTRACT PAGE NO.

Krell, Tino Identification of McpS as the chemoreceptor for TCA cycle intermediates: Novel structure and conserved ligand binding mode 23

Manson, Michael The general quorum-sensing autoinducer AI-2 is a potent attractant for enteric bacteria 24

Russell, Matthew A chemotaxis-receptor for nitrogenous compounds in Azospirillum brasilense 25

BREAK Pham, Hai How do E.coli chemoreceptors sense phenol? 26

Ottemann, Karen The TlpD chemoreceptor of H. pylori binds zinc and represents a new class of soluble chemoreceptors 27

Haneburger, Ina New insights into the signaling mechanism of the pH-responsive, membrane-integrated transcriptional activator CadC of Escherichia coli 28

January 19, 2011 Regulation Wednesday Morning (9:00 am – 12:00 pm) Chair – Mark McBride Hendrixson, David Polar flagellar biosynthesis influences bacterial cell division 29

Prüß, Birgit FlhC of Escherichia coli O157:H7 regulates genes of cell division, biofilm formation, and virulence, when growing on the surface of meat 30

Msadek, Tarek The ABCs of bacitracin resistance in Staphylococcus aureus 31

Geng, Haifeng Expression of tropodithietic acid (TDA) biosynthesis is controlled by a novel autoinducer 32

BREAK Lan, Ganhui The energy cost of sensory adaptation 33 Wilson, Laurence Quantitative high-speed imaging of motile microorganisms 34

Hu, Linda Protein acetylation modulates phosphorylation-dependent activation of a small RNA gene 35

Walthers, Don Global regulators and anti-silencing control the Salmonella pathogenicity island 2 virulence locus 36

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January 20, 2011 Flagella Thursday Morning (9:00 am – 12:00 pm) Chair – David Blair

PRESENTER TITLE ABSTRACT PAGE NO.

Inoue, Yuichi Torque steps of the bacterial flagellar motor induced by heating 37 Nishiyama, Masayoshi

Reverse rotation in bacterial flagellar motors at high hydrostatic pressures 38

Yuan, Junhua Asymmetry in the clockwise and counter-clockwise rotation of the bacterial flagellar motor 39

Nieto, Vincent The YcgR::c-di-GMP complex acts as a ‘backstop brake’ by first locking the Salmonella flagellar motor in a CCW mode and then braking 40

BREAK Tipping, Murray Light-powering the flagellar motor 41

Li, Na Characterization of the periplasmic region of PomB, a sodium-driven stator component in Vibrio alginolyticus 42

Schniederberend, Maren

The role of FlhF in the regulation of flagella assembly in Pseudomonas aeruginosa 43

Zarbiv, Gabriel FoF1 ATP synthase binds to FliG and is important for proper function of the flagellar motor-switch complex 44

January 20, 2009 Gliding and Swarming Thursday Evening (7:30 pm – 10:00 pm) Chair – Penelope Higgs

Zhang, Haiyang Mechanism and physiological role of predatory ripples in Myxococcus xanthus swarms 45

Nan, Beiyan Myxobacteria gliding motility requires cytoskeleton rotation powered by proton motive force 46

Wall, Dan Protein transfer between myxobacteria cells involves motilty 47 BREAK

McBride, Mark Cell-surface proteins and polysaccharides involved in flavobacterium Johnsoniae gliding 48

Rhodes, Ryan

Flavobacterium johnsoniae sprB encodes a mobile cell-surface gliding motility protein and is part of an operon spanning five additional motility genes 49

Wu, Yilin Microbubbles reveal chiral fluid flows in bacterial swarms 50

POSTERS - BLAST XI

Poster # Lab Presenter Title Page #1 Gladys

Alexandre Amber Bible Modulation of clumping and flocculation behavior by a

chemotaxis-like pathway (Che1) in the alphaproteobacterium, Azospirillum brasilense

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2 Karlheinz Altendorf

Petra Zimmann Nature of the stimulus for the KdpD/KdpE system of Escherichia coli

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3 Judy Armitage

Jennifer Anne de Beyer Specificity of receptor adaptation in Rhodobacter sphaeroides chemotaxis

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4 Judy Armitage

Mostyn Brown Biotinylation of the flagellar hook in E. coli 55

5 Tatsuo Atsumi

Tatsuo Atsumi Phenamil binding site of the Na(+)-driven flagellar motor of alkalophilic Bacillus strain RAB

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6 CancelledPoster

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7 Shannon Au

Shannon AuKwok Ho Lam

Conformational flexibility of FliG provides structural insights for motor switching and coupling mechanism

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8 Carl Bauer

Qian Dong Two open reading frames involved in cGMP secretion and cyst formation in Rhodospirillum centenum

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9 Robert Belas

Yi-Ying Lee FliL, A Gatekeeper of Proteus mirabilis swarming differentiation

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10 Robert Belas

Yi-Ying Lee FlhDC, the flagellar master regulator, regulates its target promoters in a two-stage fashion

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11 Howard Berg

Richard Branch Binding cooperativity in the bacterial flagellar motor 62

12 Howard Berg

Pushkar Lele Particle-wall hydrodynamic interactions in multi-particle ensembles

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13 David Blair

Eun A. Kim Mutational and crosslinking studies of cytoplasmic parts of the flagellar stator

64Blair of the flagellar stator

14 David Blair

Koushik Paul Adjusting the spokes of the flagellar motor with the DNA-binding protein H-NS

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15 David Blair

Mayukh K. Sarkar Flagellar direction switching in Escherichia coli : CheY binds to the rotor protein FliN to induce CW rotation

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16 David Blair

Yang Zhang Systematic mutagenesis of proton binding residues of the flagellar export apparatus

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17 Nyles Charon

Nyles W. Charon CheY3 of Borrelia burgdorferi is the key response regulator essential for chemotaxis and forms a long-lived phosphorylated intermediate

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18 Brian Crane

Xiaoxiao Li Building soluble models of chemoreceptors 69

19 Brian Crane

Ria Sircar Probing the structure of the flagellar switch complex 70

20 Sean Crosson

Sean Crosson A structural model of anti-anti-sigma inhibition by a two-component receiver domain: The PhyR stress response regulator

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21 Rick Dahlquist

Armand S. Vartanian Solution structure of FliG analyzed by NMR 72

22 John Dow

Karen O'Donovan HD-GYP domain proteins regulate virulence and biofilm formation of the human pathogen Pseudomonas aeruginosa

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23 Roger Draheim

Roger Draheim In vivo reconstitution of the EnvZEc/OmpR osmosensing circuit suggests a non-piston mechanism of transmembrane communication

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POSTERS - BLAST XI

Poster # Lab Presenter Title Page #24 Thierry

EmonetMichael Sneddon Overcoming complexity in systems biology modeling

and simulation with NFsim75

25 Joseph Falke

Adam Berlinberg Conformational changes in the assembled, membrane-associated chemotatic signaling complex

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26 Joseph Falke

Peter F. Slivka Investigating the mechanism of ultrastability in chemoreceptor clusters

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27 Joseph Falke

Kalin Swain Testing models for HAMP on-off switching in the E.coli serine chemoreceptor

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28 Rasika Harshey

Rasika M. Harshey Newly identified McpB/McpC chemoreceptors and the adaptation protein CheV function in taxis towards L -cystine in Salmonella enterica

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29 Rasika Harshey

Jaemin Lee FlhE acts as a proton plug in the Salmonell a flagellar Type III secretion system after the switch to late substrate secretion

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30 Caroline Harwood

Claudine Baraquet Mechanism of transcriptional regulation of exopolysaccharide genes by FleQ in response to c-di-GMP in Pseudomonas aeruginosa

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31 Caroline Harwood

Jennifer O'Connor Subcellular localization determinants of the Pseudomonas aeruginosa Wsp sensory transduction complex for biofilm formation

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32 Gerald Hazelbauer

Divyaben Amin Investigating dependence of chemoreceptor structure and function on the lipid environment

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33 Michio Homma

Mizuki Gohara Attempt to investigate dynammic conformational changes in FliG using solution NMR spectroscopy

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34 Michio Homma

Seiji Kojima Interaction between the rotor protein FliG and stator is essential for the functional motor assembly of NA+-driven flagella in Vibrio alginolyticus

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driven flagella in Vibrio alginolyticus35 Christine

JosenhansWiebke Behrens Role of the proposed Helicobacter pylori energy sensor

TlpD in vivo and characterization of protein-protein interactions of TlpD

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36 Kirsten Jung

Kirsten Jung Autoinducer-mediated signaling in Vibrio harveyi 87

37 Barbara Kazmierczak

Ruchi Jain Spatial and temporal regulation of bacterial motility: analysis of the cyclic di-GMP modulating protein FimX

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38 Duncan Krause

Clinton Page Mutant analysis reveals correlation between gliding motility and protein phosphorylation in Mycoplasma pneumoniae

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39 Chunhao Li

Chunhao Li Carbon storage regulator A (CsrABb) is a repressor of Borrelia burgdorferi flagellin protein FlaB

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40 Jan Liphardt

Adam T. Politzer The flagellar motor switch is sensitive to proton motive force

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41 Jun Liu

Jun Liu Cryo-electron tomography of pathogenic and saprophytic Leptospira reveals novel structures of flagellar C-ring and chemotaxis receptor array

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42 Jun Liu

Xiaowei Zhao Molecular architecture of stator assembly in situ revealed by cryo-electron tomography

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43 LuhuaLai

Shuangyu Bi Discovery of novel chemo-effectors for E.coli chemoreceptor Tar

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44 Michael Manson

Christopher Adase The role of the cytoplasmic aromatic anchor of transmembrane helix 2 (TM2) of E. coli Tar in transmembrane signaling

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POSTERS - BLAST XI

Poster # Lab Presenter Title Page #45 Michael

MansonMike David Manson Mechanism of AI-2 chemoreception in Escherichia coli 96

46 Richard Marconi

Jessica Lynn Kostick The Borrelia burgdorferi diguanylate cyclase, Rrp1, controls important steps in the enzootic cycle of Lyme disease spirochetes

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47 Richard Marconi

Lee Thomas Szkotnicki Investigation of the c-di-GMP phosphodiesterase PdeB reveals a critical role in proper motility in the bacteria Borrelia burgdorferi

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48 Jonathan McMurry

Jonathan McMurry Kinetic simulations of interactions among flagellar export apparatus proteins: Is complexity really

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49 Vincent Méjean

Cécile Castelli Chemotactic response to anaerobic electron acceptors involves new types of chemoreceptors in Shewanella oneidensis

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50 Steven Norris

Tao Lin Dissect the mechanism of Borrelia chemotaxis and motility and the relationship between the virulence and chemotaxis/motility

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51 Christopher O'Connor

Christopher O'Connor Binding of CheY to FliM is necessary but not sufficient to switch flagellum rotation

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52 Qi Ouyang

Guangwei Si Chemotaxis behaviors of the Escherichia coli population in spatially and temporally varying enviroments

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53 Chankyu Park

Jihong Kim Role of the mqsRA operon and reactive carbonyl species in flagella expression of Escherichia coli K-12

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54 Chankyu Park

Junghoon Lee Cis - and trans -acting mutations upregulating the flagellar genes

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55 Steven Porter

Steven Porter The GacS phosphorelay of Pseudomonas aeruginosa 106Porter

56 Simon Rainville

Guillaume Paradis Taking control of the bacterial flagellar motor 107

57 Birgit Scharf

Gaurav Dogra The novel Sinorhizobium meliloti chemotaxis protein CheS participates in signal termination

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58 Birgit Scharf

Hardik M. Zatakia Analyzing the role of two Type IVb pili systems in Sinorhizobium meliloti

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59 Tom Shimizu

Milena Lazova Response rescaling in bacterial chemotaxis 110

60 LotteSøgaard-Andersen

Daniela Keilberg Function and interactions of RomR, a response regulator required for A-motility in M. xanthus

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61 Claudia Studdert

Claudia Studdert Tsr constructions with symmetric heptad deletions display full function

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62 Lynmarie Thompson

Lynmarie Thompson Active arrays of bacterial chemoreceptor complexes: Solid-state NMR tests of current models

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63 Kai Thormann

Kai Martin Thormann Analysis of the BarA/UvrY two-component system in Shewanella oneidensis MR-1

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64 Yuhai Tu

Ganhui Lan Mechanical and kinetic principles of bacterial flagellar motor operation

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65 Mandy Ward

Mandy J. Ward Behavioral responses in the metal-reducer Shewanella oneidensis

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66 Zhaomin Yang

Zhaomin Yang DifA, an MCP-like sensory protein, uses a novel signaling mechanism to regulate exopolysaccharide production in Myxococcus xanthus

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POSTERS - BLAST XI

Poster # Lab Presenter Title Page #67 Igor

ZhulinDavi R. Ortega Investigating structural properties of CheW with

molecular dynamics and NMR118

68 David Zusman

Eva M. Campodonico Myxococcus xanthus Frz pathway signaling and the Mgl proteins

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SPEAKER ABSTRACTS

BLAST XI Mon. Morning Session CONSERVED MECHANISM FOR SENSOR PHOSPHATASE CONTROL OF TWO-COMPONENT SIGNALING: EVIDENCE FROM THE NITRATE SENSOR NarX Tu-Anh Huynha, Chris Noriegab, and Valley Stewartb aFood Science Graduate Group, bDepartment of Microbiology, University of California, Davis

Two-component signal transduction mediates a wide range of phenotypes in microbes and plants. The sensor transmitter module controls the phosphorylation state of the cognate response regulator receiver domain. Whereas the two-component autokinase and phosphotransfer reactions are well-understood, the mechanism by which sensors accelerate the rate of phospho-response regulator dephosphorylation, termed transmitter phosphatase activity, is unknown. We identified a conserved DxxxQ motif adjacent to the phospho-accepting His residue in the HisKA_3 subfamily of two-component sensors. We used site-specific mutagenesis to make substitutions for these conserved Gln and Asp residues in the nitrate-responsive NarX sensor, and analyzed function both in vivo and in vitro. Results show that the Gln residue is critical for transmitter phosphatase activity, but is not essential for autokinase or phosphotransfer activities. The documented role of an amide moiety in phosphoryl group hydrolysis suggests an analogous catalytic function for this Gln residue in HisKA_3 members. Results also indicate that the Asp residue is important for both autokinase and transmitter phosphatase activities. Furthermore, we noticed that sensors of the HisKA subfamily exhibit an analogous E/DxxT/N motif, the conserved Thr residue of which is critical for transmitter phosphatase activity of the EnvZ sensor. Thus, two-component sensors likely employ similar mechanisms for receiver domain dephosphorylation. Lab: Valley Stewart

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BLAST XI Mon. Morning Session WIRING OF TWO-COMPONENT SIGNAL TRANSDUCTION SYSTEMS IN MYXOCOCCUS XANTHUS Tobias Petters1, Anke Treuner-Lange1, Michael Hoppert2 & Lotte Søgaard-Andersen1 1Max-Planck Institute for terrestrial Microbiology, Marburg, Germany 2Georg-August-University, Göttingen, Germany Tobias.Petters@mpi-marburg.mpg.de

The Myxococcus xanthus genome encodes a large number of proteins of two-component systems. The genetic organization of the corresponding genes is intriguing with 55% being orphan, 16% being organized in complex gene clusters and only 29% being organized in the standard paired gene configuration. As part of our ongoing efforts to determine the connectivity of proteins of two-component systems, we have analyzed the orphan hybrid histidine protein kinase, which is encoded by MXAN_4640. This gene was previously suggested to be important for S-motility (Youderian et al. 2006) and named sgmT. The structure of SgmT includes an N-terminal GAF domain, a typical histidine protein kinase domain, a receiver domain and a GGDEF domain at the C-terminus. Based on the primary sequence, the GGDEF domain likely has an inactive A(active)-site for c-di-GMP synthesis and an intact I(inhibitory or allosteric)-site for c-di-GMP binding.

We found that an in-frame deletion of sgmT causes a defect in S-motility. The ∆sgmT mutant synthesizes type IV pili and LPS O-antigen whereas synthesis of constituents of the extracellular matrix (ECM) is abnormal. Specifically, exopolysaccharide (EPS) accumulation is increased and accumulation of FibA, a protein component of the ECM, is decreased. To understand the mechanism of SgmT we characterized mutant SgmT proteins in which key residues were substituted or whole domains were deleted. Surprisingly, only inactivation of the kinase domain and deletion of the GAF domain result in defects in ECM accumulation. Substitutions in the receiver domain and the GGDEF domain as well as the deletion of both of these domains did not affect ECM accumulation. These findings suggest that the GAF domain serves as the major sensory input domain to regulate SgmT kinase activity. Our data also suggest that neither the SgmT receiver domain nor the GGDEF domain regulate SgmT kinase activity or give an output response. As a potential interacting partner of SgmT we focussed on the orphan response regulator DigR consisting of an N-terminal receiver domain and a C-terminal DNA-binding domain. A ∆digR mutant phenocopies the ∆sgmT mutant under all conditions tested. To investigate if SgmT and DigR are part of the same signal transduction pathway we performed microarray analysis in which the transcription profile of wild-type cells was compared to that of ∆sgmT mutant cells or ∆digR mutant cells. We found that 210 genes in total were strongly up- or down-regulated. Importantly, 93 of these displayed the same regulation in the two mutants. These data strongly suggest that SgmT and DigR are part of the same signal transduction pathway. Consequently, we performed in vitro phosphotransfer assays with purified SgmT and DigR proteins. The SgmT kinase domain autophosphorylates on the conserved His residue. Subsequently, the phosphate is transferred to the conserved Asp residue in the receiver domain of DigR. Thus, SgmT and DigR are cognate partners in a two-component system. The precise function of the SgmT/DigR will be discussed. Lab: Lotte Søgaard-Andersen

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BLAST XI Mon. Morning Session COORDINATION OF CELL FATES IS MEDIATED BY NEGATIVE REGULATORY SIGNALING SYSTEMS IN THE MYXOCOCCUS XANTHUS MULTICELLULAR DEVELOPMENTAL PROGRAM Bongsoo Lee1, Andreas Schramm1, Vidhi Grover1, Anke Treuner-Lange2, and Penelope I. Higgs1 1Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, 35043 Marburg, Germany 2nstitute for Microbiology and Molecular Biology, University of Giessen, 35392 Giessen, Germany Myxococcus xanthus is a Gram-negative soil dwelling bacterium that is distinguished by a preference for a multicellular life cycle. Under nutrient-replete conditions, cells obtain nutrients cooperatively in predatory swarms. In a nutrient-depleted environment, cells enter a developmental program wherein ~15% of the initial population aggregate into mounds of ~100 000 cells and then within these mounds (fruiting bodies) differentiate into environmentally resistant spores; ~6% of the initial population do not aggregate and remain as peripheral rods; and the remaining 80% of the cells are thought to undergo programmed cell death. Several atypical two-component signal transduction (TCS) systems have been described which act as negative regulators of progression through the developmental program. Mutants in the corresponding genes (espA, espC, todK and red) cause cells to develop earlier than wild type, producing disorganized fruiting bodies and spores outside of the fruiting bodies. A combination of rigorous comparative phenotypic analyses and genetic epistasis experiments suggest these signaling proteins are organized into three distinct signaling pathways that are necessary to coordinate multicellular behavior during development. Our analyses also suggested that in these kinase mutants, the proportion of cells in the different developmental fates was altered. To understand how these kinases influence cell fate, we next rigorously analyzed the proportions of aggregating, non-aggregating, and dying cells throughout the developmental program in the wild type and in strains disrupted in the negative regulator genes. We also examined protein production patterns and gene expression profiles of known key developmental regulators (genes) in each developmental subpopulation in these strains. Our analyses suggest the negative regulatory signaling systems function to promote gradual accumulation of MrpC, a key developmental regulatory protein, specifically in the non-aggregating cell population such that these cells are induced to aggregate and eventually sporulate within fruiting bodies. In the absence of any of the negative regulatory signaling systems, MrpC accumulation is inappropriately rapid giving rise to cells that sporulate before completion of aggregation. Thus, the M. xanthus developmental program is controlled by a balance of positive and negative regulatory systems that coordinate cell fates in the multicellular developmental program. Lab: Penelope Higgs

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BLAST XI Mon. Morning Session A SPECIFICITY DETERMINANT IN A RESPONSE REGULATOR PREVENTS in vivo CROSSTALK AND PHOSPHORYLATION BY NON-COGNATE PHOSPHODONORS Joseph M. Boll and David R. Hendrixson Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX

Members of the NtrC family of response regulators often function in signaling pathways to influence expression of σ54-dependent regulons. The Campylobacter jejuni FlgR NtrC-like response regulator is dependent on phosphorelay from its cognate FlgS histidine kinase to activate σ54-dependent expression of many flagellar genes. Typically, these response regulators possess DNA-binding activity in their C-terminal domain (CTD) that is often essential for these proteins to regulate expression of target genes. We found that the CTD of FlgR binds DNA, but this domain is not required in vivo to activate expression of σ54-dependent flagellar genes. FlgR lacking its CTD (FlgR∆CTD) has equivalent activity as wild-type FlgR in activating expression of flagellar genes. Whereas wild-type FlgR is solely dependent on FlgS for activation via phosphorelay, FlgR∆CTD activates flagellar gene expression in the absence of FlgS. These findings suggest that the CTD of FlgR functions in an unconventional role to limit the specificity of phosphorylation exclusively to FlgS. Transposon mutagenesis revealed that mutations in the pathway for generating acetyl-phosphate severely limited the ability of FlgR∆CTD to positively influence σ54-dependent flagellar gene expression in a FlgS-independent manner. Biochemical, genetic, and physiological experiments verified that acetyl-phosphate promotes phosphorylation-dependent activation of FlgR∆CTD. In contrast, wild-type FlgR does not demonstrate significant activation by acetyl-phosphate in vivo to result in expression of flagellar genes. These findings suggest that the CTD of FlgR has evolved to insulate the response regulator from promoting crosstalk with non-cognate histidine kinases or other phosphodonors in the cell, which allows the protein to maintain its specificity for phosphorelay to FlgS. This study may have implications for how other response regulators of some bacterial signaling networks maintain specificity of activation to cognate sensor kinases. Lab: David Hendrixson

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BLAST XI Mon. Morning Session PREDICTION OF INTERSPECIES CROSS-TALK IN TWO-COMPONENT SYSTEMS Pawelczyk, S.; Scott, K.; Hamer, R.; Blades, G.; Wadhams, G.H. Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU

Protein-protein interactions are a fundamental part of all biological systems. However, the molecular basis underlying the specificity by which one pair of proteins will interact with each other in vivo whilst not interacting with other similar proteins and hence insulating paralogous pathways from unwanted cross-talk is relatively poorly understood. Currently, much use is made of the presence of protein domains and genome context to propose interaction maps, however these approaches do not work well in cases where large numbers of protein paralogues exist in a single organism and these genes are not encoded within specific operons or adjacent to their obvious protein partners. Two Component Systems (TCS) are a good example of this with their prevalence in bacteria and lower eukaryotes and the fact that many bacteria contain tens or hundreds of these signalling proteins, many encoded in operons with their partners but others as orphans in the genome. One of the best characterized TCS is the osmosensing EnvZ/OmpR system from gammaproteobacterium Escherichia coli.

Skerker et al. (2008) recently predicted and functionally verified the residues determining the specificity of the histidine kinase (HK) EnvZ for its response regulator (RR) OmpR. Bioinformatics suggests that the alphaproteobaterium Rhodobacter sphaeroides has an orphan HK (RSP_0203) which contains these specificity residues and has homology to EnvZ in its C-terminal signalling domain but no homology to the N-terminal sensing domain of EnvZ. BLAST searches with RSP_0203 revealed significant homology to other orphan histidine kinases in alphaproteobacteria, which are all annotated as EnvZ like kinases. Analysis of the R. sphaeroides genome also finds a RR with specificity residues similar to those from OmpR.

Phosphotransfer assays confirmed that in vitro RSP_0203 is a kinase for RSP_1138. We also showed that cross-talk between this system and the EnvZ/OmpR system can occur as RSP_0203 phosphorylates OmpR and EnvZ also phosphorylates RSP_1138. To investigate whether this cross-talk also occurs in vivo, we introduced the E.coli promoter of OmpC fused to yfp and OmpR into the genome of R.sphaeroides. High levels of fluorescence in the absence of the native OmpR kinase EnvZ showed that OmpR is phosphorylated in R. sphaeroides. A significant reduction in the fluorescence output from a RSP_0203 deletion strain confirmed that cross-talk occurs in vivo between RSP_0203 and OmpR.

These data not only validate bioinformatic approaches for predicting the interaction of a

class of orphan HK’s with RR’s, but also confirm our ability to predict interspecies cross-talk when introducing TCS components from one bacteria into another species. These results have implications for network predictions, understanding the molecular basis of protein:protein interactions specificity and our ability to utilise non-native components in the pursuit of synthetic biology. Skerker, J. M. et al. Rewiring the Specificity of Two-Component Signal Transduction Systems. Cell 133, 1043-1054 (2008). Lab: George Wadhams

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BLAST XI Mon. Morning Session DEFINING THE SIGNALING PATHWAY OF THE OSMOSENSOR EnvZ Loo Chien Wang1, Linda J. Kenney2,3, Ganesh S. Anand1 1Department of Biological Sciences, National University of Singapore, 2Department of Microbiology and Immunology, University of Illinois-Chicago, 3Mechanobiology Institute, National University of Singapore

In Escherichia coli, the EnvZ/OmpR two-component system regulates the expression of the outer membrane porins OmpF and OmpC in response to osmotic stress. While the intermolecular signaling pathway between EnvZ and OmpR is fairly well established, knowledge of the intramolecular signaling events is limited mostly to mutagenesis studies that identified residues critical for function. In the case of EnvZ, there are conflicting results as to the importance of the periplasmic and transmembrane domains of the protein in coupling stimulus sensing to a downstream response. In light of this, we employed amide hydrogen/deuterium exchange mass spectrometry (HDXMS) combined with the available NMR solution structures of EnvZc to determine the effect of osmolality on EnvZc conformations. Our results indicate that the cytoplasmic domain is itself sufficient to sense and respond to changes in osmolality. The region containing the His243 autophosphorylation site is highly sensitive to osmolality and can distinguish between sucrose and salt. The amphipathic four-helix bundle, which includes His243, appears to be the core region where signals and responses are integrated. Additionally, our results suggest that both the four-helix bundle and the ATP binding domain of EnvZc are closely coupled in a two-way communication. Perturbation of one domain triggers changes in the other. Upon AMP-PNP binding, a specific conformational change occurs at the N-terminus region of EnvZc, which suggests a downward signaling event from the N-terminus effects a change in the cytoplasmic domain or an upward signaling event is communicated from the cytoplasmic domain to the transmembrane/periplasmic domains. Our model of EnvZ signal transduction is: osmolality signals are first integrated at the four-helix bundle, which elicits a response in the ATP-binding domain. Binding of ATP and the subsequent phosphorylation of His243 propagates a signal upstream to the transmembrane/periplasmic domain. The use of HDXMS is a valuable tool for defining signal transduction pathways in other bacteria and with other two-component regulatory systems. This work is supported by NIH GM-058746 to LJK. Lab: Ganesh Anand

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BLAST XI Mon. Morning Session THE RESPONSE REGULATOR WspR FORMS SUBCELLULAR CLUSTERS AS A POSSIBLE MECHANISM TO INCREASE ACTIVITY Varisa Huangyutitham and Caroline S. Harwood Department of Microbiology, University of Washington, Seattle, WA 98195, USA

Pseudomonas aeruginosa encodes about forty proteins that are predicted to produce or degrade cyclic-di-GMP, an intracellular signaling molecule that promotes a biofilm lifestyle. To elucidate the temporal and spatial regulation of c-di-GMP in cells, we are focusing on WspR, a hybrid response-regulator diguanylate cyclase. Phosphorylation of WspR is controlled by the Wsp alternative chemotaxis-like system. WspR-P forms nonpolar, dynamic subcellular cytoplasmic clusters, whereas unphosphorylated WspR is diffuse in cells. Purified WspR protein has increased catalytic activity upon phosphorylation. Our previous results suggest a model in which a surface-associated stimulus activates the Wsp system to phosphorylate WspR, which consequently forms subcellular clusters, assumes an active conformation, and produces c-di-GMP, resulting in exopolysaccharide production and biofilm formation.

Understanding how and why WspR forms subcellular clusters will contribute to basic knowledge of the subcellular localization properties of proteins and the molecular mechanisms behind biofilm formation. We asked which of the following properties contribute to WspR clustering: 1) phosphorylation, 2) cyclase activity 3) inhibition by c-di-GMP. We also asked how important clustering is to biofilm formation, a measure of the in vivo function of WspR. To answer these questions we analyzed a set of WspR point mutants. WspR subcellular localization was observed by fluorescence microscopy using YFP-tagged proteins. Biofilm formation was assayed by observing colony morphology. Additionally, we determined the effects of in vivo phosphorylation on WspR cluster formation in three backgrounds: (i) wild type, (ii) a strain where the Wsp system constitutively phosphorylates WspR, and (iii) a strain where WspR will not be phosphorylated by the Wsp system. Finally, we purified the WpsR proteins and assayed their cyclase activity.

Our results suggest that subcellular clustering is an intrinsic property of phosphorylated WspR. In addition, we saw a positive correlation between the percentage of cells that had clusters of mutant WspR proteins, the in vivo activity of these WspR mutants, and their in vitro activity. A straightforward interpretation of our results is that WspR-P is most active when it forms higher order oligomerized structures in cells. Lab: Caroline Harwood

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BLAST XI Mon. Evening Session MOTILITY, TAXIS AND PREDATORY BEHAVIOUR IN BDELLOVIBRIO: MULTIPLE MOT PROTEINS AND GGDEF REGULATION OF MOTILITY Laura Hobley, Karen Morehouse, Michael Capeness, Maximilian Harris, Yoshiko Iida, Kaoru Uchida, Shin-Ichi Aizawa, Liz Sockett

Bdellovibrio bacteriovorus are highly-motile, predatory bacteria that replicate by growing within the periplasm of Gram-negative prey. Their bi-phasic lifecycle consists of a free-swimming, non-replicative ‘attack-phase’, and an intra-periplasmic growth phase. Bdellovibrio swim using a single sheathed polar flagellum, at speeds of up to 160um/s. We have previously shown that a non-motile flagellin mutant has inefficient predation in liquid cultures, but that when placed in close proximity to a prey cell, invasion and replication can occur, and thus disproving the idea that Bdellovibrio used flagella motility to ‘bore’ themselves into the prey cell. The Bdellovibrio genome contains multiple copies of the key proteins involved in flagella synthesis and rotation, and we have recently completed a study of the roles of each of the three pairs of motAB genes. This has revealed that loss of a single pair of motAB genes does not result in immotile cells, that all three pairs are proton-driven and are likely to have been acquired by horizontal gene transfer rather than gene duplication. We have also shown that Bdellovibrio can be seen swimming in the remnants of the dead prey cell immediately prior to lysis, and that all three pairs of mots contribute to the flagellar motor in both Host-Dependent and Host-Independent growth. Current work has revealed that cyclic-di-GMP regulates flagellar synthesis and motility in Bdellovibrio: deletion of an individual GGDEF encoding gene results in non-flagellate Bdellovibrio, which can enter prey but not escape the lysed remnants of the prey cell. Locating and collision with prey is key to Bdellovibrio survival, the genome reveals an extensive number of genes encoding chemotaxis sensors, but the exact nature of the signals which the Bdellovibrio respond to is currently unknown, but is the focus of our forward investigations. Lab: Liz Sockett

BLAST XI Mon. Evening Session ROTATIONAL BROWNIAN MOTION AND BACTERIAL NEAR SURFACE SWIMMING Guanglai Li & Jay X. Tang Physics Department, Brown University, Providence, RI 02912 Brownian motion, the random movement of microscopic objects in fluid caused by incessant thermal agitation, is of fundamental significance in life science, particularly in the microbial world. The trajectory of a bacterium in aqueous environments is constantly altered by the rotational Brownian motion, dictated by its short length. It has been found that swimming bacteria tend to accumulate near a surface, which undoubtedly facilitates their adhesion to the surface [1]. Here we report our study, by experiments and by computer simulations, of the vital roles of rotational Brownian motion on bacterial near surface swimming, accumulation, and adhesion.

We use a double mutant of Caulobacter crescentus as a model bacterium for this study. This mutant has no pili and its flagellar motor turns only clockwise, thus the swarmer cell only swims forward. A three-dimensional tracking technique based on darkfield microscopy is used to measure the near surface swimming trajectory up to 10 µm from the surface. Unlike backward swimming cells that stay near a surface for an extended period of time and follow circular trajectories [2], these forward swimming cells only stay at a close distance for under one second and do not form circular trajectories. Nevertheless, the measured cell density distribution shows that these forward swimming cells strongly accumulate within 1 µm from the surface. We propose a physical model that attributes this accumulative distribution to the collision of swimming cells with the surface, counter-balanced by rotational Brownian motion. The simulation results based on our analytical model confirm the influence of rotational Brownian motion on near surface accumulation of the forward swimming mutant. Future study are planned to include the mutant strain that lacks pili but can switch swimming directions, as well as the wildtype swarmer cells that switch swimming directions and can quickly adhere due to the presence of pili. By dissecting the effects of variously factors separately and then in combination, our ultimate goal is to understand the essential steps and stages of Caulobacter adhesion.

(a) Schematic drawing of the setup. A cell body appears as a ring when out of focus. The cell-surface distance is calculated from the radius of the ring. (b) An atomic force microscopy image of a C. crescentus swarmer cell dried on a glass surface. The image was acquired using a Nanoscope III AFM from Veeco, Inc. (c) An overlay of images of swimming cells showing the swimming trajectories. (d) A three dimensional trajectory (black) and its projection on the X-Y surface (red) of the cell marked by a white arrow in (c).

References: 1. Li, G. and Tang, J.X., 2009, Accumulation of Microswimmers near a Surface Mediated by

Collision and Rotational Brownian Motion. Phys. Rev. Lett. 103: 078101. 2. Li, G., Tam, L.-K., and Tang, J.X., 2008, Amplified effect of Brownian motion in bacterial

near-surface swimming. Proc. Nat. Acad. Sci. 105: 18355-59.

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Lab: Jay Tang ___________

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BLAST XI Mon. Evening Session FREQUENCY-DEPENDENT ESCHERICHIA COLI CHEMOTAXIS BEHAVIORS REVEALED BY MICROFLUIDICS EXPERIMENTS AND PATHWAY-BASED MODELING Xuejun Zhu1, 2*, Guangwei Si1, 2*, Nianpei Deng2, Qi Ouyang1, 2

, Tailin Wu2, Zhuoran He2, Lili jiang2

, Chunxiong Luo1, 2†, Yuhai Tu2, 3† 1Center for Microfluidic and Nanotechnology, The State Key Laboratory for Artificial Microstructures and Mesoscopic Physics, School of Physics, 2Center for the Theoretical Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China. 3 T. J. Watson Research Center, IBM, P.O. Box 218, Yorktown Heights, NY 10598

We have investigated the E. coli chemotaxis behaviors in complex environments with spatio-temporally varying attractant source and different stimulus waveforms by developing a microfluidic system where controlled non-stationary chemical gradients can be established by integrating time-varying perfusion, on-chip mixture, and agarose-filtered diffusion. Measuring the bacterial density profile in response to periodic stimulus of various cycle lengths reveals that the E. coli population response is highly frequency dependent. At low cycle frequency, the E. coli population synchronizes with the attractant waveform, in consistent with the response to quasi-stationary gradient. In contrast, under fast-changing environment, the population response is out of synchrony with the attractant waveform with a phase shift that increases with frequency. A coarse-grained continuum model is presented to describe the dynamic behavior of E. coli at the population level. With the inclusion of the finite receptor methylation rate, our model successfully captures the distinct population behaviors under the experimentally accessed stimulus frequencies, whereas the well-known classical Keller-Segel type chemotaxis-equation fails. Lab: Yuhai Tu

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BLAST XI Mon. Evening Session SIGNALING NOISE IN BACTERIA COORDINATES FLAGELLAR MOTORS TO IMPROVE CHEMOTACTIC PERFORMANCE Michael W. Sneddon1,2, William Pontius2,3 & Thierry Emonet1,2,3 1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520- 8103, USA. 2 Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511, USA. 3 Department of Physics, Yale University, New Haven, CT 06520- 8103, USA

Biochemical noise arising from the stochastic interactions of small numbers of molecules is an inherent aspect of signal processing in cells. In Escherichia coli slow fluctuations arising within the signaling pathway of the bacterial chemotaxis system are particularly pronounced. We investigated theoretically the influence of these slow fluctuations on the response of the flagellar motors and the behavior of single cells. We found that noise in the signaling system can coordinate the timing of flagellar motor switching events and accounts quantitatively for the time-correlations in the switching dynamics of adjacent flagellar motors observed by Berg and coworkers. Furthermore, with a coarse-grained model of the interaction of multiple flagella, we found that this motor coordination extends the run lengths of single cells and improves the search capabilities of single cells in the absence of attractants. Next we investigated the cost of the signaling noise on chemotactic performance. Surprisingly, although the signaling noise does reduces a cell’s ability to detect a signal, the enhanced exploration and diffusion of cells actually enhances, on average, the drift velocity up shallow gradients while only negligibly reducing performance on steep gradients. Together, these results indicate that noise in bacterial chemotaxis can be beneficial to cell behavior. In the broader context of biochemical signal processing, we have identified a novel example of how noise can coordinate the response of multiple downstream effectors. This work raises the intriguing prospect that biochemical noise might be an evolutionarily selectable trait. Lab: Thierry Emonet

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BLAST XI Mon. Evening Session COMPUTATIONAL IDENTIFICATION OF NOVEL PHOSPHATASES FOR CHEMOTAXIS SIGNAL TRANSDUCTION Kristin Wuichet and Igor Zhulin Joint Institute of Computational Sciences, University of Tennessee - Oak Ridge National Laboratory, Oak Ridge, TN 37831

Three families of CheY phosphatases have been characterized in the prokaryotic chemotaxis system. The CheZ phosphatase was first characterized in E. coli, but more recent experimental and computational studies have revealed that CheZ is represented in all classes of Proteobacteria. In Bacillus subtilis and Thermotoga maritima, the CYX family of phosphatases composed of three homologous enzymes, CheC, FliY, and CheX, has been characterized. Most recently, studies in Rhodobacter sphaeroides revealed a phosphatase that is currently considered to be unique, unlike the CheZ and CYX phosphatases that are widespread among diverse organisms and chemotaxis systems. Despite the lack of homology between these three families, all share similar active site motifs at the sequence level, and the co-crystals of CheY-CheX and CheY-CheZ show that both utilize nearly identical mechanisms of action despite large differences in the interface orientations. Although chemotaxis phosphatases are ubiquitous, many systems appear to lack characterized phosphatases and at least some of them are proposed to use alternative methods to regulate CheY phosphorylation, such as the “phosphate sink” described in Sinorhizobium meliloti. We have found that similarity between CheY sequences is primarily mediated by amino acid residues that determine their interactions with phosphatases rather than their cognate CheA kinases. Using this knowledge and the known similarities of characterized phosphatases we identified a large new class of putative chemotaxis phosphatases with surprisingly diverse structures and domain architectures. They are present in many systems that were previously presumed to lack phosphatases, and their distribution supports that the vast majority of chemotaxis systems regulating flagellar motility require phosphatases. Lab: Igor Zhulin ___________

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BLAST XI Mon. Evening Session VISUALIZING THE EVOLUTION OF SIGNAL TRANSDUCTION MACHINERY WITH SIGNALING PROTEIN FAMILY PROFILES Michael Y. Galperin NCBI, NLM, National Institutes of Health, Bethesda, MD 20894, USA

Even in classical model microorganisms, signal transduction systems are so complex that their systematic analysis still presents a problem. The availability of complete genome sequences has finally allowed us to predict all regulatory components in a given organism, compare their organization in various organisms, and evaluate how much do we know – and still do not know – about microbial signal transduction. In the course of the past several years, we have been conducting and updating a census of the key signal transduction proteins encoded in the completely sequenced bacterial and archaeal genomes: sensor histidine kinases, response regulators, methyl-accepting chemotaxis receptors, Ser/Thr/Tyr protein kinases and protein phosphatases, adenylate and diguanylate cyclases and phosphodiesterases. This census (publicly available at http://www.ncbi.nlm.nih.gov/Complete_Genomes/SignalCensus.html) allows one to easily compare the signal protein content of various closely and distantly related organisms. It has been used to develop a quantitative measure of the complexity of signal transduction machinery in any given genome, the so-called “bacterial IQ”. Here, I will introduce a new metric of signaling complexity, signal protein family profiles, which reflect the abundance of each type of signal transduction proteins in a several different organisms. These family profiles are consistent among closely related microorganisms and can be used to trace their evolution in the course of adaptation to their specific ecological niches.

Lab: Eugene Koonin ____

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BLAST XI Tue. Morning Session CONFORMATIONAL CHANGES IN THE ASSEMBLED, MEMBRANE-ASSOCIATED CHEMOTATIC SIGNALING COMPLEX Annette H. Erbse, Adam J. Berlinberg and Joseph J. Falke Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO 80309

The chemosensory pathway of bacterial chemotaxis forms a polar signaling cluster in

which receptor trimers-of-dimers are arrayed in a highly cooperative, hexagonal lattice. Within this lattice, the core signaling complexes are composed of a receptor trimer-of-dimers, the CheA histidine-kinase homodimer, and one or two CheW coupling proteins. The core complex stimulates CheA kinase activity in the absence of attractant, and inhibits kinase activity when attractant binds to the receptors.

Despite the wealth of information available about the chemotactic signaling complex,

there are still many fundamental questions open. The detailed molecular architecture of the membrane associated, active core complex is still largely unclear. Although some aspects of the signal transduction along the receptor are known, the nature of the signal transfer to CheA, the conformational changes in CheA leading to loss of activity and the possible role of CheW in this are still an enigma.

We have previously been able to map out the protein-protein docking surfaces for

receptor and CheW on CheA and were able to construct a working model for the membrane associated, active core complex architecture 1. We have developed a novel One Sample FRET technique (OS-FRET) that allows us to test our model spectroscopically and to monitor conformational changes in the active, regulated, membrane-associated complex under near physiological conditions. We have focused on the relative spatial arrangement between CheA and CheW. Our results support the overall core complex architecture of our published model. Ligand binding to the receptor triggers conformational changes that cause P5 and CheW to move apart without CheW being completely released from the complex. This is the first time that a direct effect of ligand binding to the receptor on CheA/CheW has been observed, allowing a first glimpse into the mechanism of receptor-mediated kinase regulation. Our results further suggest that the catalytic P4 domain and substrate P1 domain of CheA both possess considerable mobility in the assembled complex. P4 mobility is consistent with a recent study of the soluble Thermotoga Maritima complex 2. (This project is supported by NIH R01 GM-040731) 1 A. S. Miller, S. C. Kohout, K. A. Gilman et al., Biochemistry 45 (29), 8699 (2006). 2 J. Bhatnagar, P. P. Borbat, A. M. Pollard et al., Biochemistry 49 (18), 3824 (2010).

Lab: Joseph Falke

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BLAST XI Tue. Morning Session THE STOICHIOMETRY OF THE MINIMAL CORE UNIT OF THE CHEMOTAXIS SIGNALING COMPLEX Mingshan Li and Gerald L. Hazelbauer Department of Biochemistry, University of Missouri, Columbia, MO 65211 Transmembrane chemoreceptors, histidine kinase CheA and coupling protein CheW interact to create signaling complexes that are localized as clusters in the membrane. These clustered complexes enhance kinase activity ~100-fold and put that enhanced activity under receptor control. The notable sensitivity and wide dynamic range of the chemotactic response are thought to reflect extended interactions in clusters among multiple receptors and kinases. Are extended interactions also necessary for formation of signaling complexes or for kinase activation? Specifically, how many receptor dimers are required for effective binding of CheA and CheW; how many are required for full activation of the kinase? We are addressing these questions using Nanodiscs, ~10 nm plugs of lipid bilayer rendered water-soluble by an annulus of amphipathic protein, to manipulate the number of neighboring dimers in a lipid bilayer. Nanodiscs prepared with E. coli chemoreceptor Tar are fractionated by size-exclusion chromatography to enrich for a particular number of receptor dimers/disc. We assay binding of the soluble proteins CheA and CheW to these enriched preparations of intact, membrane-embedded receptors using a 6-His tag on Tar to separate bound from free. By varying the concentration of one soluble component in the presence of a constant concentration of the other, we generate binding curves from which can be derived apparent dissociation constants and the maximal amount bound for the varied component. In addition, we quantify the amount of the constant component that becomes associated with receptors, reflecting formation of signaling complexes. The combined measurements provide values for the stoichiometry of the complexes formed. Complementary experiments utilize a second affinity tag on CheA, allowing isolation of signaling complexes by sequential affinity columns. Quantification of the amounts of receptor, CheA and CheW in these isolated signaling complexes provides independent values for the stoichiometry. Companion experiments determine kinase activity as a function of the number of neighboring receptor dimers and identify the smallest group of receptor dimers that can generate kinase activation equivalent to activation by many neighboring receptors in native membrane. The combined data define the stoichiometry of the minimal core unit of the chemotaxis signaling complex. Lab: Gerald Hazelbauer ____

BLAST XI Tue. Morning Session MODELS OF STRUCTURE AND DYNAMICS OF A COMPLETE Tsr TRIMER OF DIMERS Benjamin A Hall, Judith P Armitage, Mark SP Sansom Oxford Centre for Integrative Systems Biology, Department of Biochemistry, University of Oxford

The bacterial methyl accepting chemoreceptor Tsr controls cellular motility in response to serine, and is representative of the large class of bacterial chemoreceptors. Through its quaternary structure and organisation in the membrane into large, hexagonal arrays it achieves exquisite sensitivity to binding events, whilst maximising the signal to noise ratio. Whilst structures of individual domains have been known for a number of years, high resolution data for the biologically active trimer of dimers has been unavailable. Through iterative rounds of coarse grain modelling of domain pairs, we have built a complete model of the structures of the receptor dimer and the trimer of dimers. Analysis of microsecond dynamics in coarse grain molecular dynamics (CGMD) simulations and of elastic network models of the structure demonstrate that hinging of the whole protein occurs around the HAMP domain. Alongside data from the application of a novel high throughput CGMD technique to characterised mutants of TM2 from homologues, we propose a model for the signalling mechanism through which a swinging piston motion of TM2 changes the trimeric structure and changes downstream effectors.

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Lab: Mark Sansom ____

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BLAST XI Tue. Morning Session ELECTRON CRYOTOMOGRAPHY OF BACTERIAL CHEMOTAXIS ARRAYS Ariane Briegel and Grant J. Jensen California Institute of Technology and Howard Hughes Medical Institute, 1200 East California Blvd., Pasadena, CA 91104

Chemotactic bacteria utilize a highly sensitive and adaptable sensory system to swim towards attractants and away from repellents. Changes in nutrient concentrations are detected by a polar, highly organized sensory patch of transmembrane receptor proteins together with a number of accessory proteins. Attractants and repellents bind to the sensory domains, thereby regulating the activity of a histidine kinase, which phosphorylates a soluble messenger protein. This messenger protein in turn diffuses through the cytoplasm to the flagellar basal body, where it modulates the direction of flagellar motion.

Electron cryotomograhy (ECT) makes it possible to visualize chemoreceptor clusters in bacteria in vivo to macromolecular resolution (4-8 nm). While high-resolution crystal structures of the individual chemotaxis proteins are available, their arrangement and position in the arrays remain unclear. Understanding this "mesoscale" architecture of the clusters is critical, however, since it is vital to the arrays'' emergent properties of cooperative signal amplification and regulation. We have used ECT to investigate a wide range of diverse bacterial species, revealing a universal architecture of the chemoreceptor arrays in hexagonal lattices with a center-to center spacing of 12 nm, suggesting a universal mechanism of transmembrane signaling amongst bacteria. We are now investigating the structural effects on the chemoreceptor arrays upon nutritional changes in their environment. Lab: Grant Jensen ____

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BLAST XI Tue. Morning Session HELICOBACTER PYLORI CheZ CELLULAR LOCALIZATION IS INDEPENDENT OF OTHER CHEMOTAXIS PROTEINS Paphavee Lertsethtakarn and Karen M. Ottemann Department of Microbiology and Environmental Toxicology, University of California, Santa Cruz, Santa Cruz, California 95064

We have recently shown that a remote CheZ orthologue in H. pylori termed CheZHP has phosphatase activity toward phosphoryl-CheY, -CheAY, and -CheV2 using the same mechanism employed by E. coli CheZ. The sequence of CheZHP is not well conserved at the N-terminus and is significantly longer compared to E. coli CheZ. This difference suggests that CheZHP might have an additional function that is different from canonical CheZ proteins. To further characterize CheZHP, we employed immunofluoresence to determine the cellular location of natively-expressed CheZHP using an antibody specific to this protein. We have found that CheZHP localizes to both cellular poles and its localization pattern is not changed in strains lacking any of the known H. pylori chemotaxis genes individually, the flagellar basal body protein, or a strain lacking all four of the chemoreceptors. It thus appears that CheZHP localizes to the poles via an-yet unidentified protein. In addition, we generated several CheZHP mutants in H. pylori strain G27 to further determine the functions and role in localization of the various portions of the protein. All of the mutants have reduced swarming ability in soft agar suggesting partial loss of chemotaxis ability, whereas a complete removal of CheZHP resulted in fully nonchemotactic H. pylori. These results thus demonstrate that the CheZHP plays a role in H. pylori chemotaxis and that its cellular localization mechanism deviates from the E. coli model. Lab: Karen Ottemann ____

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BLAST XI Tue. Morning Session STRUCTURAL AND FUNCTIONAL STUDIES OF HAMP DOMAIN SIGNALING IN BACTERIAL CHEMORECEPTORS Nattakan Sukomon1, Michael V. Airola1, Kylie J. Watts2, Brian R. Crane1 1Department of Chemistry and Chemical Biology, Cornell University, Ithaca NY, 14853 2Division of Microbiology and Molecular Genetics, Loma Linda University, Loma Linda, CA 92350

HAMP domains are signaling motifs found in numerous prokaryotic signaling proteins. Recently, the crystal structure of three consecutive HAMP domains from the soluble Pseudomonas aeruginosa receptor Aer2 was determined. The three HAMP domains show a parallel four-helix-bundle structure with two distinct conformations, which may participate in a conformational switching mechanism. An in vivo chemotaxis assay has been developed to test whether the two conformations play a role in the signaling mechanism of the HAMP domain. Aer2-Tar chimeric receptors (ATCs) containing the Aer2 HAMP domains and the E. coli aspartate receptor Tar were generated and opposite signaling outputs from the different HAMP conformers were observed. Insertion of the DELG motif, but not a conserved Proline, into the HAMP1 domain rescued the response to aspartate. Single amino acid mutations in HAMP1 altered signal output. The crystal structure of HAMP1/L44H, a mutant that shows clockwise (CW)-locked flagellar rotation, displays an altered AS1 helical position, but identical AS2 position to wild-type, which indicates that a HAMP1-like conformation invokes the CW-signaling state. Pulsed-Electron spin resonance (ESR) distance measurements verify the HAMP conformers in solution and assess HAMP domain dynamics. Additionally, effects of residue substitutions on HAMP domain stability have been evaluated with circular dichroism (CD) spectroscopy. In total, this work contributes new insight to our understanding of HAMP domain signaling. Lab: Brian Crane ____

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BLAST XI Tue. Morning Session PAS-HAMP INTERACTIONS IN THE AEROTAXIS RECEPTOR, AER Kylie J. Watts, Darysbel Pérez and Barry L. Taylor Div. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda, CA, USA The PAS domain of the aerotaxis receptor, Aer, signals to downstream HAMP and proximal signaling domains via direct interaction. To identify interacting protein surfaces, residues in the PAS, HAMP, and proximal signaling domains were substituted with cysteine and tested in vivo for accessibility to methoxypolyethylene glycol-maleimide 5000 (PEG-mal). PEGylated Aer migrates more slowly than unmodified Aer and is easily differentiated on Western blots. Using this method, residues were classified as having low, intermediate, or high accessibility. The highest accessibilities were found in the N-terminus of the PAS domain (the N-cap), and in the proximal signaling domain that follows the HAMP domain. Low accessibilities, in contrast, were identified in the interior of the PAS and HAMP domains, but also on one face of HAMP AS-2, and the PAS β-scaffold, even though these two regions were predicted to be accessible. The inaccessible HAMP surface overlapped with a previously identified helical discontinuity in AS-2, and the inaccessible PAS surface overlapped with a cluster of previously identified signal-on lesions. We assessed potential interaction sites between HAMP AS-2 and the PAS β-scaffold by oxidizing PAS-HAMP di-Cys mutants in vivo and analyzing the crosslinked products. Using this method, PAS residues N98 and I114, located on adjacent strands of the β-scaffold, were both found to be proximal to HAMP Q248 on the inaccessible surface of AS-2. Crosslinking between these PAS and HAMP residues occurred in the folded protein and produced dimers that were unaffected by increased expression of Tar. Collectively, these data suggest that PAS-HAMP interactions occur between opposite monomers of a folded dimer, a conclusion also supported by the results of previous heterodimer experiments. We next measured the influence of the kinase-on state on accessibility using an N85S substitution in the PAS FAD-binding cleft, which maintains Aer in the kinase-on state. In the presence of N85S, there was a patch of residues in the HAMP domain that became significantly more accessible. This patch was comprised of residues from AS-1 and AS-2′, and overlapped with residues that have low accessibility in wild-type Aer, but higher accessibility in PAS-less Aer. In contrast, residues in the proximal signaling domain became significantly less accessible in the presence of N85S. This suggests that the kinase-on state may weaken PAS interactions with HAMP AS-2, and strengthen PAS interactions with the proximal signaling domain. These preliminary results may provide insights into how PAS-HAMP interactions change during Aer signaling.

Lab: Barry Taylor ____

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BLAST XI Tue. Morning Session COORDINATED REGULATION OF FLAGELLAR MOTORS ON A SINGLE ESCHERICHIA COLI CELL Hajime Fukuoka1, Shun Terasawa2, Yuichi Inoue1, Takashi Sagawa2, Hiroto Takahashi1, Akihiko Ishijima1. 1Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan 2Graduate School of Life Sciences, Tohoku University, 2-1-1 Katahira, Aoba-ku, Sendai, 980-8577, Japan

Escherichia coli cells swim by rotating flagellar motors and reorient themselves during

swimming by controlling the rotational direction of their flagellar motors in response to extracellular stimuli. E. coli cells swim smoothly when all the flagellar motors rotate counterclockwise (CCW), which causes the flagellar filaments to form a bundle. When one or more of the motors switch to clockwise (CW) rotation, the bundle is disrupted and the cells tumble. When chemotacitc signals are sensed by chemoreceptors, which are localized primarily at one of the cell poles, they modulate the auto-phosphorylation activity of a histidine protein kinase, CheA. The phosphoryl group on CheA is rapidly transferred to a response regulator, CheY. Phosphorylated CheY (CheY-P) binds to the flagellar motor and increases the probability of CW rotation. The functions of proteins involved in chemotaxis and their localization within the cell are relatively well understood. However, it is still uncertain how the cell changes the rotation of its multiple flagella in response to extracellular stimuli or if the switching of their rotational directions is coordinated in some way.

In this study, we simultaneously measured the rotation of multiple flagellar motors of

individual E. coli cell. To monitor the rotation of the motor, a polystyrene bead (φ = 0.5 μm) was attached to each of the sticky flagellar filaments as a probe and their rotation was recorded by a high-speed CCD camera at 1,250 frames / s. These cells express a GFP-fused form of the chemotaxis protein CheW, which represents the position of chemoreceptor patch. Two different motors on the same cell often showed coordinated switching in their rotational direction both from CCW to CW (CCW-to-CW switching) and from CW to CCW (CW-to-CCW switching). In both CCW-to-CW and CW-to-CCW switching, the switching of the motor farther from the chemoreceptor patch delayed compared to that of another motor closer to the patch. The delay was in under a second and was correlated with the distances of two motors from the chemoreceptor patch. Our results suggest that a transient increase and decrease in the concentration of signal protein, which is probably a wave-like change propagated from the chemoreceptor patch in under a second, trigger and regulate the coordinated switching of flagellar motors. Lab: Akihiko Ishijima ____

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BLAST XI Tue. Evening Session IDENTIFICATION OF McpS AS THE CHEMORECEPTOR FOR TCA CYCLE INTERMEDIATES: NOVEL STRUCTURE AND CONSERVED LIGAND BINDING MODE Jesús Lacal1, Estela Pineda2, Jose Antonio Gavira2, Rebecca Parales3, Juan Luis Ramos1 and Tino Krell1* * presenting author (1) Department of Environmental Protection, EEZ (CSIC), Granada-18008, Spain (2) Laboratory of Crystallographic Studies, IACT (CSIC-Granada University), P.T. Ciencias de la Salud, Granada-18100, Spain (3) Department of Microbiology, University of California, Davis, California 95616, USA

Many bacteria show a chemotactic behaviour towards root exudates. Tricarboxylic acid (TCA) intermediates are abundantly present in root exudates and the aim of this study is to identify the molecular basis for taxis towards TCA cycle intermediate using Pseudomonas putida KT2440 as model organism. This strain is predicted to contain 26 chemoreceptors of which only a few have been annotated with a function.

Screening of bacterial mutants deficient in single mcp genes revealed that the mutant

lacking a functional pp4658 (renamed McpS) did not show taxis towards succinate. The ligand-binding region of McpS (McpS-LBR) is un-annotated in all relevant programs and is some 100 amino acids longer than TarH domains. McpS-LBR was produced as a recombinant protein and subjected to isothermal titration calorimetry ligand screening studies to precisely determine the ligand profile of this protein. The screening of a large number of compounds revealed that McpS specifically recognizes the TCA cycle intermediates succinate, fumarate, malate, oxalacetate, citrate and isocitrate with affinities ranging between 8-330 μM (Lacal et al. 2010, J. Biol. Chem 285, 23126). Data will be presented which document that McpS is the only receptor for these compounds. Interestingly, very close structural homologues and derivatives of these compounds like maleate, aspartate, itaconate or tricarballylate did not bind. The chemotactic response towards TCA cycle intermediates varied enormously and did not correlate well with the measured binding affinities. Instead, the increase in thermal stability of McpS-LBR in the presence of different chemoattractants as measured by differential scanning calorimetry did correlate with the magnitude of the chemotactic response. Analytical ultracentrifugation studies revealed that the observed increase in thermal stability can be attributed to the stabilization of the protein dimer.

X-ray crystal structures of the McpS-LBD in complex with malate and succinate have

been solved to 2.1 Å resolution (Pineda et al. manuscript in preparation). The structure is composed of 2 long and 4 small helices, which are arranged as two stacked 4-helical bundles. Although McpS and Tar show no significant sequence similarities, structural similarities between the 4-helical bundles present in their structures are obvious. McpS-LBD forms dimers and, as in the case of Tar, the binding of ligand molecules is accomplished by amino acids from both monomers, which is the structural reason for the ligand-mediated dimer stabilization mentioned above. Site-directed mutagenesis of amino acids involved in ligand binding caused loss of binding activity. The McpS structure corresponds to a novel small molecule sensing domain. Current investigations include an evaluation of the contribution of McpS to the efficient colonization of plant roots. Lab: Tino Krell ____

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BLAST XI Tue. Evening Session THE GENERAL QUORUM-SENSING AUTOINDUCER AI-2 IS A POTENT ATTRACTANT FOR ENTERIC BACTERIA Michael Manson Department of Biology, Texas A&M University

AI-2 is an autoinducer used in quorum sensing by many Gram-negative and Gram-positive bacteria. It is derived from the ribose moiety of S-adenosylhomocysteine, the product left after methyl group donation by S-adenosylmethionine. We have shown that AI-2 strongly attracts Escherichia coli and Salmonella enterica serovar Typhimurium. The periplasmic LsrB binding protein, which is the ligand-recognition component of the ABC transporter for AI-2, is essential for AI-2 chemotaxis, but AI-2 uptake is not. Strains lacking the Tsr chemoreceptor are also almost totally defective for AI-2 chemotaxis. Our hypothesis is that ligand-bound LsrB directly interacts with Tsr in the periplasm to initiate an attractant response, in much the same manner as other binding proteins interact with their cognate receptors. LsrB would thus be the first known binding protein partner for Tsr. We are currently in the process of determining whether LsrB actually does dock onto Tsr and whether it competes with the canonical Tsr ligand L-serine for signaling. Chemotaxis to AI-2 could serve to attract E. coli and S. Typhimurium to high-density populations of a variety of bacterial species and could play an important role in recruitment of planktonic bacteria to biofilms. Lab: Michael Manson ____

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BLAST XI Tue. Evening Session A CHEMOTAXIS-RECEPTOR FOR NITROGENOUS COMPOUNDS IN AZOSPIRILLUM BRASILENSE Matthew Russell and Gladys Alexandre Biochemistry, Cellular and Molecular Biology Department, The University of Tennessee, Knoxville TN 37996

Azospirillum brasilense is a motile soil alpha-proteobacterium that can also colonize the rhizosphere of several crops including corn, rice and wheat. The draft A. brasilense genome sequence suggests that it encodes for four chemotaxis-like signal transduction pathways and 48 chemotaxis receptors. The A. brasilense Che1 chemotaxis-like signal transduction pathway regulates multiple cellular responses including chemotaxis, cell length and flocculation. Most chemoeffectors appear not to be sensed directly but rather by the effect on the energy metabolism and thus, energy taxis was proposed to be a dominant motility behavior in A. brasilense. Two energy taxis receptors, named Tlp1 and AerC, have been previously characterized. Here, we characterize the sensory specificity and function of a third chemotaxis receptor, named Tlp2. Tlp2 encodes for a prototypical chemotaxis receptor with a conserved C-terminal signaling region and a periplasmic domain of unknown function. Similarity searches using the N-terminal domain indicate that this domain is conserved in alpha-proteobacteria that inhabit soil and aquatic environments, suggesting that it has recently evolved and spread within this group of microorganisms. Translational fusion of the putative promoter region of tlp2 to a promoterless gusA gene indicates that the tlp2 promoter is constitutively expressed and it is up-regulated under conditions of nitrogen fixation growth or when the preferred nitrogen source, ammonium, is not present and replaced with nitrate. Using a set of behavioral assays and a mutant deleted for tlp2, we have determined that Tlp2 functions to modulate chemotaxis per se (and not any other cellular behaviors). Further biochemical characterization using intrinsic tyrosine fluorescence indicates specificity of the Tlp2 sensory domain for nitrogenous compounds including nitrate, nitrite, ammonium, and urea with a Kd in the low-to-submicromolar range but amino acids that elicit a weak taxis response in A. brasilense such as alanine and glutamate did not bind to the Tlp2 sensory domain in this assay. Interestingly, the sensory domain of Tlp2 does not show any similarity with other characterized nitrate sensors, including NarX. Therefore, Tlp2 functions as a chemotaxis sensor that allows A. brasilense motile cells to actively seek metabolically favorable nitrogen sources. Lab: Gladys Alexandre ____

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BLAST XI Tue. Evening Session HOW DO E.COLI CHEMORECEPTORS SENSE PHENOL? Hai The Pham, Peter Ames, Sandy Parkinson University of Utah, Biology Department, 257 South 1400 East, Salt Lake City, Utah 84112

In E. coli, the serine chemoreceptor Tsr (taxis to serine and repellents) senses phenol as a repellent whereas the aspartate chemoreceptor Tar (taxis to aspartate and repellents) senses it as an attractant. This study is aimed at revealing how these receptors sense phenol and understanding the relevant signaling process. We employed the “gradient soft agar assay”, which allows the creation of a gradient of phenol on soft agar plates, to study the chemotactic responses of our E.coli hosts that expressed different receptor constructs. By mutagenesis studies, we found that critical amino acid residues of the aspartate binding site in Tar, R73 and Y149, were not crucial for the binding of phenol. Furthermore, F40, a residue assumed to be important for the binding of Tar to phenanthroline (also an aromatic compound), was found to be irrelevant to the binding of Tar to phenol. Hybrid receptors created by replacing the periplasmic domain of Tar with that of Tsr or other foreign receptors exerted good attractant responses to phenol. Even a hybrid formed by replacing the HAMP domain of Tar by that of Tsr could exert an attractant response to phenol. Noticeably, a number of Tsr HAMP mutants (with E248 replaced by other residues) appeared to sense phenol as an attractant, in contrary to the wild-type Tsr. Furthermore, certain Tar mutants that lack the periplasmic domain and have some minor change appeared to sense phenol. These results suggest that the mechanism by which Tar and Tsr sense phenol might not only be restricted to the function of the periplasmic domain of the receptors but also involve the roles of the HAMP domain and the transmembrane domain as well as their interactions to one another and to phenol. Lab: Sandy Parkinson ____

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BLAST XI Tue. Evening Session THE TlpD CHEMORECEPTOR OF H. PYLORI BINDS ZINC AND REPRESENTS A NEW CLASS OF SOLUBLE CHEMORECEPTORS Jenny Draper, Lisa Collison, Susan Williams and Karen M. Ottemann Department of Microbiology and Environmental Toxicology, University of California at Santa Cruz, Santa Cruz, CA 95064 Helicobacter pylori is an epsilon proteobacter that uses chemotaxis and motility to infect stomachs and cause ulcer disease. Our lab studies the advantages that chemotaxis confers for pathogens. We have made and characterized mutants of H. pylori that have general chemotaxis defects or lack specific chemoreceptors. Such mutants are attenuated for stomach infection in a variety of different ways. Mutants lacking general chemotaxis colonize somewhat less well than wild-type H. pylori and cause significantly less inflammation. Mutants lacking either the TlpA or TlpC chemoreceptors have modest colonization defects, while a mutant lacking the TlpD chemoreceptor has a strong colonization defect. TlpD has no transmembrane domains, and has been shown to localize to both the cytoplasm and membrane fractions (J Bacteriol 190:3244, 2008). We have extended those studies to find that membrane localization is dependent on some core siganaling proteins, particularly CheV1 and CheW, but not CheA or the other CheV proteins. These findings suggest that TlpD forms part of a large chemotaxis signaling complex that is dependent on both CheV1 and CheW. We furthermore discovered that TlpD bears a C-terminal conserved motif that is shared amongst many cytoplasmic chemorecetors, but had not been uncharacterized. Using TlpD purified from E. coli or H. pylori, we find that this set of amino acids binds zinc with high affinity. We thus refer to this motif as CZB, for Chemoreceptor Zinc Binding. CZB domains are found throughout the bacterial domain, most frequently in chemoreceptors but often in other signaling proteins as well. Using in vitro assays, we have determined that H. pylori displays a chemotactic response to zinc and iron, but do not yet know whether the CZB domain of TlpD is required for these responses. Lab: Karen Ottemann ____

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BLAST XI Tue. Evening Session NEW INSIGHTS INTO THE SIGNALING MECHANISM OF THE PH-RESPONSIVE, MEMBRANE-INTEGRATED TRANSCRIPTIONAL ACTIVATOR CADC OF ESCHERICHIA COLI Ina Haneburger (1,2), Andreas Eichinger (1,3), Christiane Koller (1,2), Arne Skerra (1,3) and Kirsten Jung (1,2) From the Center of integrated Protein Science Munich (1) and the Department of Microbiology, Ludwig-Maximilians-Universitaet Muenchen (2) and the Lehrstuhl für Biologische Chemie, Technische Universitaet Muenchen (3)

Adaptation of E. coli to acidic stress is mediated by the concerted action of several

proteins, among them the inducible amino acid decarboxylase systems. One of these systems is the Cad system that is induced at low external pH and concomitantly available lysine. The transcriptional activator CadC of the Cad system belongs to the ToxR-like proteins that are characterized by a common topology. These proteins possess a periplasmic sensor domain, a single transmembrane helix and a cytoplasmic DNA-binding domain. Recent data revealed that the periplasmic domain of CadC is responsible for pH sensing, while lysine signaling is mediated by an interaction of CadC with the lysine permease LysP.

We are interested in elucidating how the inner-membrane protein CadC is able to perceive and transduce these signals across the membrane and subsequently activates transcription of the cadBA operon. To gain insights into the pH-dependent activation, the crystal structure of the periplasmic domain of CadC (CadCpd) was solved at 1.8 Å resolution. This is the first structure of a signal input domain of a ToxR-like membrane-integrated transcriptional activator. CadCpd consists of two subdomains with different substructures. The N-terminal subdomain is formed by a beta-sheet that is in contact with several alpha-helices, whereas the C-terminal subdomain is a pure alpha-helical bundle. Within the crystals dimers were observed, that are hold together by polar interactions.

A directed mutagenesis approach identified several amino acids to be involved in the detection of low external pH. Most of these amino acids are part of a negatively charged surface patch. This patch is located at the dimer interface. It is suggested that upon a drop in external pH, protonation of the negatively charged residues reduces the repulsive forces between the two subdomains and thereby enables intramolecular conformational changes and/ or multimerization.

Lab: Kristen Jung ____

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BLAST XI Wed. Morning Session POLAR FLAGELLAR BIOSYNTHESIS INFLUENCES BACTERIAL CELL DIVISION Murat Balaban and David R. Hendrixson* Department of Microbiology, University of Texas Southwestern Medical Center Dallas, TX

Campylobacter jejuni produces a single flagellum at both bacterial poles. Considering this pattern of flagellar biosynthesis, the bacterium must use mechanisms to ensure the production of a single flagellum at the new pole after division. Like other polarly-flagellated bacteria, we found that the FlhF GTPase and the FlhG ATPase control spatial and numerical parameters of flagellar biosynthesis in C. jejuni. FlhF is a positive determinant of flagellar biosynthesis whose activity is negatively influenced by FlhG. In flhG mutants, wild-type FlhF produces multiple flagella at poles. In the presence of wild-type FlhG, FlhF mutant proteins with decreased GTPase activity are able to produce flagella at lateral sites or multiple flagella at a single pole. Considering these observations, we hypothesize that FlhF may function after division to organize construction of the initial cytoplasmic and inner membrane flagellar components at the new pole with FlhG inhibiting additional rounds of polar flagellar biosynthesis. Unexpectedly, we found that FlhF and FlhG are also involved in proper cellular division as respective mutants produce minicells in addition to normal-sized bacteria. We found that Campylobacter species lack the Min system that functions in many bacteria to inhibit polar septation so that division occurs at the midpoint of the cell body to result in symmetrical division. FlhG proteins of polarly-flagellated bacteria share homology to the MinD ATPase of the Min system. As shown in other systems, MinD homologs either solely or in conjunction with other proteins inhibit FtsZ septal ring formation at polar sites. Characterization of C. jejuni FlhG revealed multiple MinD-like characteristics in influencing proper septation, indicating that FlhG has dual functions in numerical control of flagellar biosynthesis and regulating correct cellular division. Strikingly, we found that proper division is also influenced by the basal components of the flagellar organelle. Mutants lacking components of the MS- and C-rings produce minicells at similar levels as flhF and flhG mutants. Polar construction of the initial basal flagellar ring structures, presumably by FlhF, appears to be required for the efficiency of subsequent FlhG-mediated events to limit polar septation and properly form a division site at the midpoint of a cell. Our data suggest a new role for the flagellum in C. jejuni biology, possibly by being a marker of the new pole after division, and indicate that polar flagellar biosynthesis influences proper septal site determination and division in this bacterium and perhaps other polarly-flagellated bacteria. Lab: David Hendrixson ____

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BLAST XI Wed. Morning Session FlhC OF ESCHERICHIA COLI O157:H7 REGULATES GENES OF CELL DIVISION, BIOFILM FORMATION, AND VIRULENCE, WHEN GROWING ON THE SURFACE OF MEAT Preeti Sule, Shelley M. Horne, and Birgit M. Prüß Veterinary & Microbiological Sciences Department, North Dakota State University, Fargo ND 58108

Escherichia coli O157:H7 has emerged over the years, as a major food safety concern. Contaminated meat and meat products have been implicated as primary contributors in transmission. Approximately 70, 000 people are infected annually. Acidic sprays were initially used to control transmission, though development of resistance by the pathogen against such sprays has rendered this method ineffective. Rampant resistance to sprays in the face of decreased but persistent reports of infection called for effective control measures. We hypothesize that sprays or chemicals that will alter the bacterial signal transduction pathways, without posing an immediate threat to bacterial survival will considerably reduced chances of inducing resistance.

To start the development of the spray, we first investigated the role of FlhC mediated gene regulation in E. coli O157:H7 on the surface of meat. FlhC is part of the FlhD/FlhC heterohexameric complex that has been established as a global regulator of gene expression. FlhD/FlhC in turn is regulated by signaling molecules such as acetyl-phosphate and hence serves as a critical link in the signal transduction pathway from the environmental signal to the affected cellular process.

Microarray experiments were carried out using labeled cDNA obtained from RNA extracted from the parental and FlhC mutant strains grown separately on the surface of meat. The putative FlhC targets identified with microarrays were subjected to real-time PCR. The physiological effect of FlhC mediated regulation was also investigated in a series of phenotypic experiments.

The microarray experiment revealed 287 putative FlhC targets genes in E. coli O157:H7. The genes were grouped according to their functions and 15 representative genes encompassing all the groups were confirmed using real-time PCR. 87% of the tested genes were confirmed as regulated by FlhC. These were the metabolic genes mdh, cyoA and sdhA, the transporter genes ompF, ompC and oppB, the cell division genes ftsY, ftsZ, ftsK and minC, the virulence (LEE) genes tir and escN, the biofilm related genes rcsF and wcaB, and a protease encoding gene clpX. The phenotypic experiments indicated that the FlhC mutants divided to 20 times higher cell densities than the parental strain, formed about 5 times more biofilm and were about twice as lethal than the parental strain in the embryo lethality assay.

In conclusion we hereby report for the first time, the role of FlhC in regulating various genes and processes in E. coli O157:H7 on the surface of meat. This study shall provide valuable insight into the complex regulatory network of the pathogen and can be used to manipulate and control bacterial spread, transmission and virulence. Lab: Birgit Prüß ____

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BLAST XI Wed. Morning Session THE ABCS OF BACITRACIN RESISTANCE IN STAPHYLOCOCCUS AUREUS Aurelia Hiron and Tarek Msadek Biology of Gram-positive Pathogens, Department of Microbiology, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France

Bacterial signal transduction pathways often involve two-component systems (TCSs) and ABC transporters. Bacitracin, a cyclic dodecylpeptide antibiotic, binds to undecaprenyl pyrophosphate, the lipid carrier for cell wall precursors, effectively inhibiting peptidoglycan biosynthesis. Gram-positive bacteria have developed several mechanisms of bacitracin resistance. Among these the Bacillus subtilis BceSR TCS /BceAB ABC transporter is the most efficient and well studied, and the prototype for TCS/ABC transporter modules, where the genes are genetically linked; the ABC transporter permease has 10 transmembrane domains and a long extracytoplasmic loop (180 to 300 aa); and the TCS has a so-called intramembrane-sensing HK with an N-terminal domain consisting only of two trans-membrane regions with but a few amino acids in between (Mascher, 2006). In B. subtilis the BceAB ABC transporter was shown to be essential for bacitracin sensing by the BceS/BceR TCS, highlighting a new mechanism for TCS-mediated signal transduction (Bernard et al., 2007; Rietkötter et al., 2008).

Staphylococcus aureus, a major human opportunistic pathogen, is endowed with sixteen

sets of genes encoding TCSs, many of which remain to be characterized. We have constructed a collection of S. aureus mutants inactivated for each of the TCS encoding genes, allowing us to identify a novel and previously uncharacterized system that we show to be essential for bacitracin resistance, the BraS/BraR system (Bacitracin Resistance Associated). The braSR genes are located immediately upstream from genes encoding an ABC transporter, accordingly designated BraDE. We have shown that the BraSR/BraDE module is a key bacitracin resistance determinant in S. aureus. In the presence of low bacitracin concentrations, BraS/BraR activate transcription of two operons encoding ABC transporters: braDE and vraDE. We identified a highly conserved imperfect palindromic sequence upstream from the braDE and vraDE promoter sequences, essential for their transcriptional activation by BraS/BraR, suggesting it is the likely BraR binding site. We demonstrated that the two ABC transporters play distinct and original roles in bacitracin resistance: BraDE is only involved in bacitracin sensing and signaling through BraS/BraR, whereas VraDE acts specifically as a detoxification module and is sufficient to confer bacitracin resistance when produced on its own. This is the first example of a TCS associated with two ABC transporters playing separate roles in signal transduction and antibiotic resistance. Bernard, R. et al. 2007. Resistance to bacitracin in Bacillus subtilis: unexpected requirement of

the BceAB ABC transporter in the control of expression of its own structural genes. J. Bacteriol. 189:8636-42.

Mascher, T. 2006. Intramembrane-sensing histidine kinases: a new family of cell envelope stress sensors in Firmicutes bacteria. FEMS Microbiol. Lett. 264:133-44.

Rietkötter, E. et al. 2008. Bacitracin sensing in Bacillus subtilis. Mol. Microbiol. 68:768-85. Lab: Tarek Msadek ____

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BLAST XI Wed. Morning Session EXPRESSION OF TROPODITHIETIC ACID (TDA) BIOSYNTHESIS IS CONTROLLED BY A NOVEL AUTOINDUCER Haifeng Geng and Robert Belas Department of Marine Biotechnology, University of Maryland Baltimore County, and Institute of Marine and Environmental Technology 701 East Pratt Street, Baltimore, MD 21202 The interactions between marine prokaryotic and eukaryotic microorganisms are crucial to many biological and biogeochemical processes in the oceans. Often the interactions are mutualistic, as in the symbiosis between phytoplankton, e.g., the dinoflagellate Pfiesteria piscicida, and Silicibacter sp. TM1040, a member of the Roseobacter taxonomic lineage. It is hypothesized that an important component of this symbiosis is bacterial production of tropodithietic acid (TDA), a biologically active tropolone compound whose synthesis requires the expression of tdaA-F as well as six additional genes (cysI, malY, paaIJK, and tdaH). The factors controlling tda gene expression are not known, although growth in laboratory standing liquid cultures drastically increases TDA levels. In this report, we measured the transcription of tda genes to gain a greater understanding of the factors controlling their expression. While the expression of tdaAB was constitutive, tdaCDE and tdaF mRNA increased significantly (3.7- and 17.4- fold, respectively) when cells were grown in standing liquid broth compared to shaking liquid culturing. No transcription of tdaC was detected when a tdaCP::lacZ transcriptional fusion was placed in 11 of the 12 Tda- mutant backgrounds, with cysI being the sole exception. Expression of tdaC could be restored to 9 of the remaining 11 Tda- mutants – tdaA and tdaH failed to respond – by placing wild-type (Tda+) strains in close proximity or by adding exogenous TDA to the mutant, suggesting that TDA induces tda gene expression. Results from electrophoretic mobility shift assays (EMSA) showed that TdaA, a LysR type-transcriptional regulatory protein, binds directly to tdaC promoter DNA and thereby regulates tdaC expression. These results indicate that TDA acts as an autoinducer of its own synthesis and suggest that roseobacters may use TDA as a quorum signal. Lab: Robert Belas ____

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BLAST XI Wed. Morning Session THE ENERGY COST OF SENSORY ADAPTATION Ganhui Lan1, Pablo Sartori1, Silke Neumann2, Victor Sourjik2, Yuhai Tu1 1IBM T.J. Watson Research Center, P. O. Box 218, Yorktown Heights, NY 10598, USA 2ZMBH, Im Neuenheimer Feld 282, 69120 Heidelberg,

Cells are highly nonequilibrium systems, consuming energy to carry out different biological functions. While it is intuitive to relate physical functions, such as biomolecule synthesis and cell motility, to energy consumption, the energetics of regulatory functions remains obscure. Sensory adaptation is a basic regulatory function possessed by all living systems: after the initial responses to stimuli, biological sensory systems can readjust themselves to balance prolonged environmental changes and reset themselves back to their optimum “ready-to-sense” states. Robust adaptation is crucial for the survival of organisms, and various molecular feedback networks have evolved to ensure accurate sensory adaptation in different organisms and for different sensory functions, from bacterial chemotaxis to osmotic sensing in yeast to olfactory and light sensing in mammalian sensory neurons. However, despite the accumulating knowledge about the biochemical network structures underlying different sensory adaptations, it remains unclear what drives the adaptation dynamics and what is the energetic cost of the feedback control.

In this work, we address these questions in general by modeling the stochastic dynamics of the core negative feedback motif shared by various adaptation networks. We show rigorously that feedback control systems always operate out of thermodynamic equilibrium and continuous energy consumption is needed to stabilize the adapted state against noise in the system. From our model, we determine the energy dissipation rate, the adaptation speed and its maximum accuracy, which are found to satisfy a universal relation. We then verify this energy-speed-accuracy (ESA) relationship in the specific case of E. coli chemosensory system, in which we identify the energy source, the cost of adaptation, and the key system requirements (design principles) to achieve the maximum accuracy for a given energy budget. In addition, direct monitoring of the response of E. coli cell to chemical stimuli confirms the slowing down of adaptation as cell gradually de-energizes in mediums without nutrient. We believe that the established ESA relationship may provide a unifying description for regulatory and information processing functions in biology. Lab: Yuhai Tu_ ____

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BLAST XI Wed. Morning Session QUANTITATIVE HIGH-SPEED IMAGING OF MOTILE MICROORGANISMS Laurence G. Wilson*1, R. Zhang1, V.A. Martinez2, J. Schwarz-Linek2, M. Li2, J. Arlt2, P.N. Pusey2, G. Bryant3 and W.C.K. Poon2 1The Rowland Institute at Harvard, 100 Edwin H Land Blvd., Cambridge, MA. 02142. 2School of Physics and Astronomy, Kings Buildings, Mayfield Road, Edinburgh, UK, EH9 3JZ. 3Applied Physics, School of Applied Sciences, RMIT University, Melbourne, Victoria 3000, Australia. *Corresponding author.

We have developed two novel microscopy techniques to measure the swimming speed, motile fraction, body rotation rate and flagellar rotation rate of approximately five thousand free-swimming bacteria in a `snapshot’ assay. The first method (Differential Dynamic Microscopy - DDM) is derived from light scattering techniques. Instead of using laser light to produce a scattering pattern, high-speed brightfield microscopy images are taken and the scattering pattern calculated. This method has advantages over particle tracking approaches in that it is insensitive to static background structure, fluctuating target shape, and uneven sample illumination, and because the averaging method is insensitive to motion perpendicular to the focal plane. Our second method (Darkfield Flicker Microscopy – DFM) is used to measure the body rotation rate and flagellar rotation rate, in the same population used for the DDM measurements. The `wiggling’ motion of a swimming bacterium is converted into localized, periodic fluctuations in intensity that have spectral peaks at the body rotation rate (around 10Hz) and the flagellar rotation rate (around 100Hz). Again, this technique is insensitive to static background structure in the sample, as only time-varying signals are studied. Finally, I will present some preliminary results that demonstrate the power of these techniques to quantify time-dependent chemotactic response in E. coli. Lab: Laurence Wilson ____

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BLAST XI Wed. Morning Session PROTEIN ACETYLATION MODULATES PHOSPHORYLATION-DEPENDENT ACTIVATION OF A SMALL RNA GENE Linda I. Hu and Alan J. Wolfe Loyola University Chicago, 2160 South First Avenue, Rm 3822 Bldg 105, Maywood, IL 60153

Phosphorylation-dependent signal transduction via two-component signal transduction

pathways is a common mechanism used by bacteria to respond to environmental stimuli. One such pathway is the Rcs phosphorelay, which includes the signaling proteins RcsC, RcsD, and RcsB. Certain extracytoplasmic stresses stimulate the sensor kinase RcsC to autophosphorylate using the phosphodonor ATP. Phospho-RcsC then donates its phosphoryl group to the histidine phosphotransferase RcsD, which relays the phosphoryl group to a conserved aspartyl residue on the receiver domain of the response regulator RcsB. In response, phospho-RcsB undergoes a conformational change compatible with DNA binding and in that form regulates the transcription of over 100 genes. In the absence of these extracytoplasmic stressors, RcsC/D can function as a net phosphatase, dephosphorylating phospho-RcsB. Under such conditions, acetyl phosphate functions as the phosphoryl donor.

We now present evidence that a novel signaling mechanism, protein acetylation,

modulates phospho-RcsB regulated transcription of the small RNA rprA. Escherichia coli cells growing exponentially in tryptone broth activated rprA transcription in a strictly RcsB-dependent manner. Since RcsC overexpression decreased rprA transcription, while deletion of rcsC increased transcription, we propose that acetyl-phosphate serves as the phosphoryl donor. We further propose that acetylation of RcsB inhibits this activation. We base this conclusion on the following observations. First, a null mutation in the gene that encodes CobB, a member of the sirtuin family of NAD+-dependent deacetylases, inhibited rprA transcription. Exposure to nicotinamide, which inhibits sirtuins, also inhibited rprA transcription in a concentration-dependent manner. The effect of CobB cannot be due its role in activating acetyl-coenzyme A synthetase as an acs mutation did not affect rprA transcription. Second, mass spectrometric analyses of overexpressed RcsB revealed acetylation of two surface-exposed lysines. One lysine was acetylated under all conditions tested, while the other lysine was acetylated only under acetylation-promoting conditions (i.e., in the cobB mutant and in the WT parent exposed to nicotinamide). This latter acetylation occurs within the flexible linker that connects the receiver and effector domains of RcsB. We propose that protein acetylation, historically attributed to eukaryotes, inhibits phosphorylation-dependent RcsB activation of rprA transcription by interfering with the phosphorylation-mediated conformational change required for DNA binding. Lab: Alan Wolfe ____

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BLAST XI Wed. Morning Session GLOBAL REGULATORS AND ANTI-SILENCING CONTROL THE SALMONELLA PATHOGENICITY ISLAND 2 VIRULENCE LOCUS Don Walthers1, You Li2,3, Yingjie Liu2,3, Ganesh Anand4, Jie Yan2,3, and Linda J. Kenney1,3 Department of Microbiology and Immunology1, University of Illinois at Chicago, Chicago, IL 60612, USA Department of Physics2, Mechanobiology Institute, National University of Singapore 3, and Department of Biology4, National University of Singapore, Singapore, 119007 The SsrA/B two-component regulatory system activates genes on Salmonella Pathogenicity Island 2 (SPI-2) that encode a type III secretion system required for replication in macrophages and systemic infection in mice. Activation of the SsrA/SsrB system is governed by a complex interplay of multiple regulators, including the response regulators OmpR and PhoP, the MarR homologue SlyA and the nucleoid protein Fis. SPI-2 and other AT-rich horizontally-acquired genetic elements are selectively silenced by the nucleoid protein H-NS. Counter-silencing of H-NS requires integration of these loci into the host transcriptional control network. Despite their adjacent location within the SPI-2 locus, transcription of ssrA and ssrB is uncoupled, and they display distinct expression patterns in response to environmental signals. OmpR and PhoP are at the top of this regulatory hierarchy; OmpR directly activates expression of ssrA, and PhoP directly activates transcription of ssrB. Once activated, the response regulator SsrB directly activates expression of multiple operons within SPI-2. SsrB also binds upstream of the sifA, sifB and sseJ effector genes located outside of SPI-2 and directly regulates transcription. SsrB relieves gene silencing by the nucleoid protein H-NS. In single molecule experiments with magnetic tweezers, we demonstrated that SsrB displaces H-NS from DNA only when it is bound in a polymerization (stiffening) mode and not when H-NS is bound to DNA in the bridging mode. Thus, in contrast to previous views, the polymerization binding mode of H-NS is the relevant form for counter-silencing by SsrB. Our results reveal that response regulators can directly activate transcription and also relieve H-NS silencing. This study adds to the repertoire of mechanisms by which NarL/FixJ subfamily members regulate transcription. Because SsrB-dependent promoters are diversely organized, additional mechanisms of transcriptional activation at other loci are likely. Supported by VA (1IO1BX000372) and NIH GM-058746 to LJK. Lab: Linda Kenney ____

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BLAST XI Thurs. Morning Session TORQUE STEPS OF THE BACTERIAL FLAGELLAR MOTOR INDUCED BY HEATING Yuichi Inoue (1), Kuniaki Takeda (2), Hajime Fukuoka (1), Hiroto Takahashi (1) and Akihiko Ishijima (1). (1: IMRAM, Tohoku University; 2: Graduate School of Life Sciences, Tohoku University)

We developed a simple method of temperature control for single molecule measurements of motor proteins to understand molecular mechanism of force generation as well as that of thermal response (BLAST X, 2009). In an application for Na+-driven chimeric flagellar motor in E. coli, we found that the transient heating over 40 deg.C can induce the stepwise change of torque in a reversible fashion. To understand the mechanism of the torque change, we measured the torque steps of the chimeric motor and the stator mutants using back-focal-plane interferometry.

In contrast to the fast torque change after transient heating of the chimeric motor, long heating at ~40 deg.C for 10-20 min induced the slower torque change at high load. Analysis with an automatic step-finding algorithm showed that the step size of the torque change decreased with increasing the heating time. However, maximum number of the torque steps, 10-13, did not depend on the heating time up to 20 min. Point mutations in a stator protein at a possible interface to a rotor protein showed similar results in both reduced step size of torque and comparable maximum number of the steps, but the response of the mutants occurred at 5-10 deg.C lower temperature than the original motor.

Since it is reported that maximum number of the torque generation in a motor is > 11 (Reid et al., 2006), each step likely reflects dissociation or incorporation of the stator. Thermally induced stator dynamics during rotational movement and the change in the unitary torque found in this study may be a key to understand the mechanism of torque generation and motor construction. Further test of the fast heating with a new system would be discussed in the meeting. Lab: Akihiko Ishijima ____

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BLAST XI Thurs. Morning Session REVERSE ROTATION IN BACTERIAL FLAGELLAR MOTORS AT HIGH HYDROSTATIC PRESSURES Masayoshi Nishiyama1,2, Yoshiyuki Sowa3, Shigeichi Kumazaki2, Yoshifumi Kimura2, Michio Homma4, Akihiko Ishijima5 & Masahide Terazima2. 1PRESTO, JST, Japan. 2Department of Chemistry, Graduate School of Science, Kyoto University, Japan. 3Hosei University, Japan. 4Nagoya University, Japan. 5Tohoku University, Japan.

The bacterial flagellar motor is a reversible rotary machine that rotates a flagellar filament, allowing bacteria to swim towards a more favourable environment. The chemotactic response switches the rotational direction from counter-clockwise (CCW) to clockwise (CW), and vice versa. The reversal of motor direction is caused by the binding of phosphorylated response regulator CheY (CheY-P) that triggers dynamic conformational changes in the switch complex on the rotor. The detailed mechanism remains unresolved because it is technically difficult to regulate the binding of CheY-P to the switch complex and detect the resulting reactions in vivo. Here, we demonstrate that pressure reverses the rotational direction of flagellar motors, even in the absence of CheY-P. The motility of single motors in Escherichia coli that lacked cheY was monitored at various pressures and temperatures. Application of pressure >1,200 bar induced a switch in the motor direction from CCW to CW at 20°C, although no motor switched its rotational direction at ambient pressure (~1 bar). At lower temperatures, pressure-induced direction changes were found at pressures <1,200 bar. CCW rotation increased with pressure in a sigmoid fashion, which is similar to chemotactic response against a concentration of CheY-P. Application of pressure generally promotes formation of clusters of water on the surface of proteins, which possibly induces structural changes in the switch complex of the flagellar motor, similar to the binding of CheY-P to the switch complex.

Lab: Masayoshi Nishiyama ____

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BLAST XI Thurs. Morning Session ASYMMETRY IN THE CLOCKWISE AND COUNTER-CLOCKWISE ROTATION OF THE BACTERIAL FLAGELLAR MOTOR Junhua Yuan, Karen A. Fahrner, Linda Turner & Howard C. Berg Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, and Rowland Institute for Science, Cambridge, MA 02142 Cells of Escherichia coli are able to swim up gradients of chemical attractants by modulating the direction of rotation of their flagellar motors, which spin alternately clockwise (CW) and counter-clockwise (CCW). Rotation in either direction has been thought to be symmetric and exhibit the same torques and speeds. The relationship between torque and speed is one of the most important measurable characteristics of the motor, used to distinguish specific mechanisms of motor rotation. Previous measurements of the torque-speed relationship have been made with cells lacking the response regulator CheY that spin their motors exclusively CCW. In this case, the torque declines slightly up to an intermediate speed called the “knee speed” after which it falls rapidly to zero. This result is consistent with a “power-stroke” mechanism for torque generation. Here, we measure the torque-speed relationship for cells that express large amounts of CheY and only spin their motors CW. We find that the torque decreases linearly with speed, a result remarkably different from that for CCW rotation. We obtain similar results for wild-type cells by re-examining data collected in previous work. We speculate that CCW rotation might be optimized for runs, with higher speeds increasing the ability of cells to sense spatial gradients, while CW rotation might be optimized for tumbles, where the object is to change cell trajectories. Lab: Howard Berg ____

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BLAST XI Thurs. Morning Session THE YcgR::c-di-GMP COMPLEX ACTS AS A ‘BACKSTOP BRAKE’ BY FIRST LOCKING THE SALMONELLA FLAGELLAR MOTOR IN A CCW MODE AND THEN BRAKING Vincent Nieto and Rasika Harshey Section of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712 Cyclic-di-GMP (c-di-GMP) is a bacterial second-messenger that affects surface-associated cellular functions. Often, c-di-GMP levels control the transition between motility and sessility. Our model organisms E. coli and Salmonella have multiple c-di-GMP cyclases and phosphodiesterases, yet absence of a specific phosphodiesterase YhjH impairs motility in both bacteria. yhjH mutants have elevated c-di-GMP levels and require YcgR, a c-di-GMP-binding protein, for motility inhibition. Our published work demonstrates that YcgR interacts with the flagellar switch-complex proteins FliM and FliG, most strongly in the presence of c-di-GMP. This interaction reduces the efficiency of torque generation and induces a CCW motor bias, effectively slowing bacterial speed and inhibiting chemotaxis (1). We have proposed a ‘‘backstop brake’’ model showing how both effects can result from disrupting the organization of the FliG C-terminal domain, which interacts with the stator protein MotA to generate torque. In follow-up experiments to test this model, we have used fliM, fliG and che mutants locked in CW or CCW modes of rotation to ask which comes first – motor switching or braking. Our data show that upon increase of YcgR::c-di-GMP, cells first display CCW rotation, followed by a reduction in rotation speed. Cells eventually become completely immobilized. These data support our suggested sequence of events where YcgR::c-di-GMP first interacts with FliM. However, they suggest that a CCW conformation of the switch is induced before the proposed flipping of the FliG C-terminal domain to disrupt the FliG/MotA interface. 1. The c-di-GMP Binding Protein YcgR Controls Flagellar Motor Direction and Speed to Affect Chemotaxis by a “Backstop Brake” Mechanism. Koushik Paul, Vincent Nieto, William C. Carlquist, David F. Blair and Rasika M. Harshey. March 2010. Molecular Cell 38 (1) 128-139

Lab: Rasika Harshey ____

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BLAST XI Thurs. Morning Session LIGHT-POWERING THE FLAGELLAR MOTOR Murray Tipping and Judith P. Armitage Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU

The bacterial flagellar motor is one of the best-known examples of a molecular nanomachine, and one of the few rotary motors found in nature. Powered by an ion-motive force, the flagellar motor generates torque through the interaction between membrane-bound stators and a cytoplasmic rotor complex. Using a proteorhodopsin-based p.m.f. recovery system, we have developed a new way to control and monitor wild-type E. coli motors in vivo.

Recent work into motor dynamics has shown that the motor itself is in constant flux. Turnover studies using fluorescently-tagged stators indicate that individual stator subunits are free to diffuse in and out of functioning motors. Additionally, resurrection studies show that stators can bind sequentially to empty motors. The role of the ion-motive force in motor assembly and maintenance of motor integrity is still not fully understood.

By using the light-driven proton pump proteorhodopsin we are able to disrupt and restore a p.m.f. in wild-type E. coli cells. Light-powered E. coli have been used to observe resurrection of a wild-type motor in vivo for the first time. The mechanics of individual steps in rotation have been explored by manipulating speed through p.m.f. control. The system is in active development, and promises to be a powerful tool for investigating the relationship between the motor and the bioenergetics of the cell. Lab: Judy Armitage ____

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BLAST XI Thurs. Morning Session CHARACTERIZATION OF THE PERIPLASMIC REGION OF PomB, A SODIUM-DRIVEN STATOR COMPONENT IN VIBRIO ALGINOLYTICUS Li Na1,2, Seiji Kojima1 and Michio Homma1 1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; 2Division of Microbiology, Graduate School of Life Science, Northwest A&F University, Yanglin, Shaanxi, Yanglin 712100, China

Stator protein PomA and PomB form a complex that functions as a Na+ channel and couples Na+ influx to torque generation in the polar flagellar motor of Vibrio alginolyticus. This stator complex must be anchored to an appropriate place around the rotor through a putative peptidoglycan-binding (PGB) domain in the periplasmic region of PomB (PomBC). Crystal structure of the corresponding domain of H+-driven Salamonella MotB (MotBC) indicated that this domain must dimerize to form the H+-conducting channel and conformational changes in its N-terminal portion are required both for PG binding and the H+-channel activation. To understand the role of PomBC in the Na+-driven PomA/PomB stator complex, series of N-terminally truncated variants and in-frame deletions in the linker region between the transmembrane (TM) segment and the PGB domain of PomB were constructed. A PomBC fragment containing residues 135 to 315 (PomBC5) forms a stable homodimer and significantly inhibited motility of wild type cells when overexpressed in the periplasm. An in-frame deletion variant, PomB(∆41-120) is functional and its overexpression impaired cell growth. This growth inhibition was suppressed by the mutation at the functionally critical Asp (D24N) in the TM segment of PomB. A mutation L168P on the putative N-terminal α-helix connecting to the PGB domain improved the motile fraction of the cells expressing PomB(∆41-120) and still exhibited growth inhibition when overproduced, suggesting that the conformational change in the periplasmic region of PomB promote assembly of the PomA/PomB(∆41-120) complex around the rotor. Thus we conclude that the periplamic region of PomB plays an important role in targeting and stable anchoring of PomA/B complex around the rotor and in the control of ion flux. Lab: Michio Homma ____

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BLAST XI Thurs. Morning Session THE ROLE OF FLHF IN THE REGULATION OF FLAGELLA ASSEMBLY IN PSEUDOMONAS AERUGINOSA Maren Schniederberend1, Kholis Abdurachim1, Thomas S. Murray2, and Barbara I. Kazmierczak1,3 1Department of Internal Medicine (Infectious Diseases), 2Department of Pediatrics, 3Section of Microbial Pathogenesis, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520

Pseudomonas aeruginosa is a motile, opportunistic pathogen responsible for numerous acute and chronic infections in humans. Two surface organelles of P. aeruginosa - flagella and type IV pili (TFP) - appear to play important roles in host-pathogen and pathogen-pathogen interactions. The single, unipolar flagellum is required for swimming while type IV pili are necessary for twitching across solid surfaces. A SRP-like protein, FlhF, is required for efficient polar assembly of flagella; in its absence, flagellar assembly occurs but is no longer restricted to the pole. ∆flhF bacteria exhibit altered swimming and swarming motility. These phenotypes may in part be due to inappropriate TFP assembly when DflhF bacteria are not on a surface, which we have observed, and/or the consequence of non-polar flagellar placement. Several point mutants of FlhF were constructed and assayed phenotypically and biochemically. The positions for the mutations were selected based on the sequence alignment of P. aeruginosa FlhF with previously crystallized Bacillus subtilis FlhF and Escherichia coli Ffh and FtsY GTPases. Amino acids predicted to be involved in either catalytic activity and/or GTP binding were targeted. All tested FlhF point mutants (expressed under the control of an inducible promoter, located in single copy on the chromosome) were targeted to the bacterial pole and supported polar flagellar assembly. Nonetheless, some point mutants exhibited defective swimming and swarming motility. We could show that wild-type FlhF hydrolyses GTP; however the catalytic reaction occurs slowly which suggests that the main role of this GTPase is not to generate energy but to regulate the flagellar machinery. Enzymatic activity and nucleotide binding characteristics of purified mutant proteins of FlhF will be presented. Lab: Barbara Kazmierczak ____

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BLAST XI Thurs. Morning Session FoF1 ATP SYNTHASE BINDS TO FliG AND IS IMPORTANT FOR PROPER FUNCTION OF THE FLAGELLAR MOTOR-SWITCH COMPLEX Gabriel Zarbiv1, Hui Li2, Amnon Wolf1, Gary Cecchini3, Victor Sourjik2 and Michael Eisenbach1 1Department of Biological Chemistry, The Weizmann Institute of Science, 76100 Rehovot, Israel. 2Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Alliance, Heidelberg, Germany. 3Molecular Biology Division, Veterans Administration Medical Center, San Francisco, California 94121, and Department of Biochemistry and Biophysics, University of California, San Francisco, California 94158

Flagellar rotation and switching between counterclockwise and clockwise directions are done in many bacteria at the motor-switch complex. The energy required for rotation of the flagella originates from the protonmotive force across the cytoplasmic membrane, generated by membrane-bound enzyme complexes and used by the FoF1 ATP synthase for ATP synthesis. Here we provide evidence for the association of this enzyme with the flagellar motor-switch complex. Pull-down assays of the switch protein FliG with an E. coli extract demonstrated that the β subunit of the F1 part of FoF1 ATP synthase (F1-β) interacts with the switch protein FliG. In vivo FRET assays confirmed the binding of F1-β to FliG. Furthermore, a mutant deleted for the gene encoding F1-β exhibited decreased counterclockwise bias, increased switching frequency, and a reduced response to a repellent. In line with this interaction, we found that membrane vesicles made of membrane areas adjacent to the flagellar motor had higher ATPase activities than vesicles made of other membrane areas. Overall, these results suggest that FoF1 ATP synthase, and specifically the β subunit in its F1 part, binds to FliG. The function of this interaction still remains to be elucidated.

Lab: Michael Eisenbach ____

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BLAST XI Thurs. Evening Session MECHANISM AND PHYSIOLOGICAL ROLE OF PREDATORY RIPPLES IN MYXOCOCCUS XANTHUS SWARMS H. Zhang1, Z. Vaksman2, H. Kaplan2 and O.A. Igoshin1

1Department of Bioengineering, Rice University, Houston, TX 2Department of Microbiology and Molecular Genetics, UT Medical School, Houston, TX

Myxococcus xanthus cells are known to self-organize into periodic bands of traveling waves, termed ripples, when exhibiting two types of multicellular behaviors: starvation-induced fruiting body development and predation on other bacteria. Developmental ripples were hypothesized to form due to reversal-inducing C-signaling occurring as a result of head-to-head collisions of M. xanthus cells [1,2]. However, recent observations [3,4] of the widespread presence of predatory ripples occurring without C-signaling raise questions concerning the mechanism of the intercellular signaling leading to this self-organization phenomenon. We have constructed a mathematical agent-based model that demonstrates that three ingredients are sufficient to generate rippling behavior: (1) side-to-side signaling between two cells that causes one or both of cells to reverse, (2) a minimal refractory time-period after each reversal during which cells can not reverse and therefore are not sensitive to signaling, and (3) physical interactions that cause the cells to locally align. Furthermore, we hypothesize that the presence of prey induces ripples by stimulating side-by-side signaling and limits rippling behavior to occur on top of prey. This model leads to experimentally confirmed predictions of the relation between the wavelength and reversal time and suggests several physiological benefits to rippling when prey is present. First, when swarming over prey M. xanthus cells will spread faster when subject to side-by-side signaling. Second, when prey is covered individual M. xanthus cells within waves are subject to more periodic motion patterns and as a result drift will be reduced. Thus, rippling behavior allows M. xanthus cells to cover its prey faster and stay over it for longer. These modeling predictions were tested experimentally by observing ripples over E. coli prey with fluorescence microscopy.

1. Sager, B., D. Kaiser. 1994. Intercellular C-signaling and the traveling waves of Myxococcus. Genes Dev. 8:2793-2804

2. Igoshin, O.A., A. Mogilner, R. Welch, D. Kaiser, G. Oster. 2001. Pattern formation and traveling waves in myxobacteria. PNAS 98:14913-14918

3. Berleman, J. E., T. Chumley, P. Cheung, and J. R. Kirby. 2006. Rippling is a predatory behavior in Myxococcus xanthus. J. Bacteriol. 188:5888-5895

4. Berleman, J. E., J. Scott, T. Chumley, and J. R. Kirby. 2008. Predataxis behavior in Myxococcus xanthus. PNAS 105:17127-17132

Lab: Oleg Ioshin ____

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BLAST XI Thurs. Evening Session MYXOBACTERIA GLIDING MOTILITY REQUIRES CYTOSKELETON ROTATION POWERED BY PROTON MOTIVE FORCE Beiyan Nan1, Jing Chen2, John C. Neu3, Richard M. Berry4, George Oster1* and David R. Zusman1* 1Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA 2Biophysics Graduate Group, University of California, Berkeley, CA 94720, USA 3Department of Mathematics, University of California, Berkeley, CA 94720, USA 4Department of Physics, University of Oxford, Oxford OX1 3PU, UK

Myxococcus xanthus is a Gram-negative bacterium that can glide over surfaces without

the aid of flagella. Two motility systems are used for locomotion: social (S)-motility, powered by the retraction of Type IV pili, and adventurous (A)-motility, powered by unknown mechanism(s). In this study, we present evidence that, an A-motility protein, AgmU, decorates a looped continuous helix that rotates as the cell glides and reverses its rotation when the cell reverses its direction. The helix rotation is driven by proton motive force (PMF) and depends on actin-like MreB cytoskeletal filaments. A mechanochemical model explains our observations: PMF-driven motors, similar to bacterial flagella stator complexes, run along a looped helical track, driving rotation of the track and translocation of the cell.

Lab: David Zusman ____

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BLAST XI Thurs. Evening Session PROTEIN TRANSFER BETWEEN MYXOBACTERIA CELLS INVOLVES MOTILTY Xueming Wei, Darshan Pathak and Daniel Wall* Department of Molecular Biology, University of Wyoming, Laramie, WY 82071.

Myxococcus xanthus is a Gram-negative soil bacteria that utilizes cell-cell interactions to direct its multicellular behaviors. In response to starvation thousands of cells swarm in a coordinated manner to build fruiting body structures in which vegetative cells differentiate into environmentally resistant spores. Two distinct systems control gliding motility; adventurous (A) motility propels single and groups of cells, while social (S) motility only involves group cell movements and is powered by type IV pili. To investigate the role cell-cell interactions in motility we characterized a subset of mutants. These mutants are unique in that their motility defects can be complemented extracellularly (stimulation); a process by which the mutants motility is transiently restored by physical contact with donor cells that contains the corresponding wild type gene. In the A-motility system there are five stimulatable cgl loci; cglB, cglC, cglD, cglE and cglF. Analogously, the S-motility system contains a single mutant locus, tgl, which can be complemented extracellularly. In prior work we established that the Tgl and CglB lipoproteins are efficiently transferred between cells during stimulation. Recently we identified the remaining four cgl genes and found that cglC and cglD also encode type II signal sequences for lipoproteins, while cglE and cglF genes contain type I signal sequences. Fusion proteins were constructed to visualize protein transfer and to identify cis elements required for transfer. These studies revealed that a type II signal sequence fused to the fluorescent mCherry (Lipo-mCherry) protein was necessary and sufficient for transfer. Protein transfer was found to occur on a solid surface, not in liquid, and was dependent on gliding motility. To help elucidate the transfer mechanism a donor defective (dodA) mutant was isolated. The dodA mutation blocked Lipo-mCherry transfer when placed either in donor or recipient cells. The dodA gene encodes a unique protein rich in cysteine amino acids. Current studies are further investigating the mechanism of protein transfer and its biological role. Lab: Daniel Wall ____

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BLAST XI Thurs. Evening Session CELL-SURFACE PROTEINS AND POLYSACCHARIDES INVOLVED IN FLAVOBACTERIUM JOHNSONIAE GLIDING Mark J. McBride, Sreelekha Bollampalli, Ryan G. Rhodes, and Soumya Pochiraju Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, WI 53201

Cells of the gliding bacterium Flavobacterium johnsoniae move rapidly over surfaces. These cells do not have well-studied motility organelles such as flagella or pili. Instead they rely on a novel motility apparatus composed of proteins that are unique to the phylum Bacteroidetes. 12 Gld proteins are required for motility, and some of these (GldA, GldB, GldD, GldF, GldG, GldH, GldI, GldJ) likely comprise the 'motor'. SprB is a cell surface protein that appears to be propelled rapidly by this motility machine. GldK, GldL, GldM, GldN, SprA, SprE, and SprT are thought to comprise a protein secretion system, the PorSS, that is needed for secretion of SprB to the cell surface.

Disruption of F. johnsoniae motility genes results not only in motility defects, but also in

resistance to bacteriophages. Completely nonmotile gld mutants are resistant to infection by all bacteriophages whereas sprB mutants exhibit partial motility defects and are resistant to some but not all phages. SprB may be a phage receptor and other cell-surface components of the motility machinery may also interact with specific phages. A novel bacteriophage resistance screen was used to identify genes involved in motility. We performed transposon mutagenesis on an sprB mutant and identified eight mutants with increased resistance to bacteriophages. Cells of the mutants exhibited more severe motility defects than did the parent strain, suggesting that the disrupted genes encode proteins involved in cell movement. Four of the mutants had insertions in an sprB paralog that we named remA (redundant motility gene A). RemA is a large repetitive protein that has a lectin domain and may function as a polysaccharide-binding cell-surface adhesin. Three of the remaining mutants had insertions in remC, wzc, and wza, which are predicted to be involved in polysaccharide synthesis and secretion. RemC is similar in sequence to glycosyltransferases, and Wzc and Wza are similar in sequence to E. coli outer membrane (Wza) and cytoplasmic membrane (Wzc) proteins involved in polysaccharide secretion. Polysaccharides may aid motility by coating the surface and interacting with the lectin domain of RemA.

A model of F. johnsoniae gliding is presented in which Gld proteins constitute the ‘motor’

in the cell envelope that converts chemical energy into movement. The motor propels adhesins such as SprB and RemA along the cell surface. Cells may express different SprB-like adhesins to allow movement on different types of surfaces. Exopolysaccharides might enhance motility on some surfaces by coating the substratum and interacting with the adhesins.

Lab: Mark McBride ____

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BLAST XI Thurs. Evening Session FLAVOBACTERIUM JOHNSONIAE sprB ENCODES A MOBILE CELL-SURFACE GLIDING MOTILITY PROTEIN AND IS PART OF AN OPERON SPANNING FIVE ADDITIONAL MOTILITY GENES Ryan G. Rhodes, Shawn S. Nelson, Soumya Pochiraju, Halley Pucker, and Mark J. McBride Department of Biological Sciences, University of WI-Milwaukee, Milwaukee, WI 53201

Cells of Flavobacterium johnsoniae move rapidly over surfaces by a process known as

gliding motility. Gld proteins are thought to comprise the gliding motor that propels cell surface adhesins, such as the 669 kDa SprB. A novel protein secretion apparatus called the Por secretion system (PorSS) is required for assembly of SprB on the cell surface. Genetic and molecular analyses revealed that sprB is part of an operon spanning 28.7 kbp of DNA. In addition to sprB, five other genes of this operon (sprC, sprD, sprF, remF, and remG) are involved in gliding. Mutations in sprB, sprC, sprD, and sprF resulted in cells that failed to form spreading colonies on agar but that exhibited some motility on glass in wet mounts. Cells with nonpolar mutations in remF and remG formed spreading colonies and exhibited wild-type motility. Paralogs of remF and remG (remH and remI respectively) are found elsewhere on the genome raising the possibility of redundancy. Cells carrying mutations in remF and remH exhibited motility defects and formed nonspreading colonies, as did cells carrying mutations in remG and remI suggesting that the proteins encoded by these genes perform redundant roles in motility. SprF exhibits similarity to Porphyromonas gingivalis PorP, which is required for secretion of gingipain protease virulence factors via the P. gingivalis PorSS. F. johnsoniae sprF mutants produced SprB protein but were defective in localization of SprB to the cell surface, suggesting a role for SprF in secretion of SprB. The F. johnsoniae PorSS is involved in secretion of extracellular chitinase in addition to its role in secretion of SprB. SprF was not needed for chitinase secretion and may be specifically required for SprB secretion by the PorSS. Cells with nonpolar mutations in sprC or sprD produced and secreted SprB, and propelled it rapidly along the cell surface. Multiple paralogs of sprB, sprC, sprD, and sprF are present in the genome, which may explain why mutations in sprB, sprC, sprD, and sprF do not result in complete loss of motility, and suggests the possibility that semi-redundant SprB-like adhesins may allow movement of cells over different surfaces. Lab: Mark McBride ____

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BLAST XI Thurs. Evening Session MICROBUBBLES REVEAL CHIRAL FLUID FLOWS IN BACTERIAL SWARMS Yilin Wu, Basarab G. Hosu, and Howard C. Berg Rowland Institute at Harvard, Cambridge, MA 02142 and Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Flagellated bacteria colonize surfaces by spreading within a thin film of fluid, a process known as swarming. An interesting model system is the bacterium Escherichia coli that swarms over nutrient agar. The fluid film provides a medium in which flagella can operate and chemical signals can propagate, thus critical for the expansion and physiology of swarms. Much has been learned about the biochemistry and genetics of bacterial swarming, and some things have been learned about its biophysics. However, little is known about the swarm fluid. We found that micron-sized bubbles, that form spontaneously when droplets of a water-insoluble surfactant are exposed to an air/water interface, make excellent tracers for the motion of this micron-thick fluid. Using these bubbles, we have discovered an extensive stream (or river) flowing clockwise along the leading edge of an Escherichia coli swarm, at rates of order 10 micron/s. The river provides an avenue for long-range communication in the swarming colony. Lab: Howard Berg ____

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POSTER ABSTRACTS

BLAST XI Poster #1 MODULATION OF CLUMPING AND FLOCCULATION BEHAVIOR BY A CHEMOTAXIS-LIKE PATHWAY (Che1) IN THE ALPHAPROTEOBACTERIUM, AZOSPIRILLUM BRASILENSE Amber Bible and Gladys Alexandre Department of Biochemistry, Cellular, and Molecular Biology at the University of Tennessee, Knoxville

The Che1chemotaxis-like pathway of Azospirillum brasilense was recently shown to contribute to chemotaxis behavior as well as clumping (cell-to-cell aggregation) and flocculation (Bible et al, 2008). Che1 comprises prototypical components found in similar chemotaxis pathways, and the molecular basis underlying the modulation of multiple cellular responses by this pathway remains to be elucidated. Clumping and flocculation are differentiation processes of A. brasilense that take place under specific conditions of growth thought to correspond to nutritional stress in this microaerophilic diazotroph. Mutants lacking cheA1 (AB101), cheY1 (AB102), or the entire che1 cluster (AB103) flocculate more than the wild type. However, a double mutant lacking cheB1 and cheR1 (BS104) flocculates very little, if at all. Here, we characterize the function of Che1 proteins in clumping and flocculation.

The process of flocculation in wild type A. brasilense takes place through a series of steps with clumping preceding “mini-floc” formation (an intermediate step between clumping and flocculation) and flocculation (formation of cell aggregates visible to the naked eye). The formation of mini-flocs takes place earlier than the wild type in mutants that also flocculate more than the wild type and may be due to an effect on the pattern of clumping. Phenotypes associated with clumping were not consistent with the prototypical pattern of signal transduction in chemotaxis pathways, suggesting that the effect of Che1 on clumping behavior may be indirect. We are examining the possibility that differences in clumping behavior may instead be due to differences in the sensitivity of cells to external cues. Consistent with this hypothesis, clumping and flocculating cells of A. brasilense have the ability to express nitrogenase, the oxygen-sensitive enzyme involved in nitrogen fixation, where oxygen diffusion is limited, allowing oxygen-sensitive processes to take place. Bible AN, Stephens BB, Ortega DR, Xie A, Alexandre G. “Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense.” J. Bacteriology. 2008. 190 (19): 6365-6375. Lab: Gladys Alexandre

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BLAST XI Poster #2 NATURE OF THE STIMULUS FOR THE KdpD/KdpE SYSTEM OF ESCHERICHIA COLI Petra Zimmann and Karlheinz Altendorf Universität Osnabrück, Fachbereich Biologie/Chemie, D-49069 Osnabrück, Germany

For survival, bacteria have evolved surface-exposed signal transduction systems. The predominant systems in bacteria are the two-component systems, which allow adaptational responses to a huge variety of environmental stimuli. The KdpD/KdpE system, one of the most existent sensor kinase/response regulator system in bacteria, including many pathogens, controls the expression of the kdpFABC operon, coding for the K+-dependent P-type ATPase KdpFABC, which serves as an emergency system to scavenge K+ when the other transporters cannot keep up with the cell’s requirement for K+.

In general, little is known how membrane-bound sensor kinases sense their extra- or

intracellular stimuli and propagate information across the cytoplasmic membrane. The stimulus that is perceived by KdpD remains also enigmatic. The determination of the kdpFABC mRNA using the method of qRT-PCR revealed that the system responds very effectively and permanently to K+-limiting conditions in the medium, but hardly and only transiently to osmotic upshifts. Furthermore, based on different experimental approaches, it was shown that changes in turgor, membrane strain, the K+-gradient across the cytoplasmic membrane, external and internal K+-concentrations, intracellular ATP-concentration, membrane lipid composition or changes in the concentration of different cytoplasmic solutes cannot be the stimulus for KdpD (1, 2). The cells’ need for K+ must therefore be recognized by so-far-unaddressed processes that heavily depend not only on the presence of K+ but also on the continuing uptake of K+ to allow cell growth. Novel parameters, which might be considered as stimuli for KdpD, will be discussed. (1) Hamann, K., Zimmann, P., and Altendorf, K. (2008) The role of turgor in stimulus perception by the

sensor kinase KdpD of Escherichia coli. J. Bacteriol. 190, 2360-2367 (2) Schniederberend, M., Zimmann, P., Bogdanov, M., Dowhan, W., and Altendorf, K. (2010) Influence

of membrane lipid composition on the expression of the kdpFABC operon in Escherichia coli. Biochim. Biophys. Acta 1798, 32-39

Lab: Karlheinz Altendorf

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BLAST XI Poster #3 SPECIFICITY OF RECEPTOR ADAPTATION IN RHODOBACTER SPHAEROIDES CHEMOTAXIS Jennifer A. de Beyer, Mark A. J. Roberts, Kathryn Scott, David Staunton and Judith P. Armitage Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK

Rhodobacter sphaeroides has two distinct chemotaxis protein clusters, one located at the pole and the other in the cytoplasm. The polar cluster contains membrane-bound MCPs, as in other chemotaxis bacteria, while the cytoplasmic cluster contains transducer-like proteins (Tlps), analogous to the MCPs but without a membrane-spanning region. Two CheR and two CheB homologues are expressed under laboratory conditions. Fluorescent tagging has shown that the CheRs are localised, one to each chemotaxis cluster, while the CheBs are diffuse in the cytoplasm.

Thus far, evidence for methylation-based adaptation in R. sphaeroides chemotaxis is

unspecific. Both clusters appear to have the components required for adaptation: methyltransferase CheR, methylesterase CheB and conserved glutamate-containing regions in both the MCPs and Tlps. Methanol release has been observed in vivo during the addition and removal of certain attractants. This suggests that adaptation occurs in R. sphaeroides, but which cluster and which receptors are involved is unknown.

Receptor adaptation was investigated in vitro using mass spectrometry to track receptor mass

changes due to changing methylation state. Escherichia coli chemotaxis proteins were used as controls.

Results will be presented suggesting which of the two clusters and which receptors are involved

in adaptation and whether post-translational deamidation by a CheB is required before methylation can occur. Further, CheB specificity for receptor deamidation and demethylation and whether CheR specificity for receptor methylation is determined solely by localisation will be discussed. Lab: Judy Armitage

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BLAST XI Poster #4 BIOTINYLATION OF THE FLAGELLAR HOOK IN E. COLI Claudio Silvestrin & Mostyn Brown, Bradley Steel, Richard Berry, Judy Armitage Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU

We engineered a strain to test the long-standing assumption that a flexible hook is essential for bundle formation in swimming E. coli.

The chromosomal copy of the flgE gene was modified by the addition of a 45bp insert that

encoded a 15 amino acid ‘biotin-accepting peptide’, which meant the strain could be externally biotinylated. When exposed to streptavidin-conjugated Alexa-532, several fluorescent spots per cell were observed proving the biotinylation was successful.

We then found that the binding of free streptavidin prevented normal swimming (cells swam

slowly end-over-end or tumbled continuously). Fluorescent labelling of filaments and preliminary biophysical data revealed that this was because (i) bundles could not form, (ii) due to a significant reduction in hook flexibility.

Lab: Judy Armitage

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BLAST XI Poster #5 PHENAMIL BINDING SITE OF THE Na+-DRIVEN FLAGELLAR MOTOR OF ALKALOPHILIC BACILLUS STRAIN RAB Tomiyama, M.(1), Ishida, K.(1), Souma, N.(1), Abbas, E. M. (2), Kato, M.(2), Kido, N.(3), Atsumi, T.(1,4) (1)Dept. Med. Technol., Gifu Univ. Med. Sci., Nagamine, Ichihiraga, Seki, Gifu 501-3892, JAPAN (2) Dept. Biol. Sci., Grad. Sch. Sci., Osaka Pref. Univ., Gakuen-cho, Naka, Sakai, Osaka 599-8531, JAPAN, (3) Div. Biol. Sci., Grad. Sch. Sci., Nagoya Univ., Furo-cho, Chikusa, Nagoya, Aichi 464-8602, JAPAN, (4) Present address: Dept. Radiol. Technol.

Flagellar motors convert electrochemical potential energy into mechanical work of rotation and are interesting for their mechanism of mechanochemical coupling. MotAB and their homologs are thought to interact with coupling ions, and from this interaction the flagellar motors accept electrochemical energy from the coupling ions. The use of specific inhibitors is crucial to investigating the interaction between MotAB and coupling ions. Amiloride analog phenamil strongly inhibits Na+-driven flagellar motors and phenamil resistant mutants have been reported in one type of alkalophilic Bacillus (strain RAB) and two types of Vibrio. Only in the latter, however, have the mutation sites been analyzed.

In this study, we sequenced the homologs of motAB in the Na+-driven flagellar motor of

alkalophilic Bacillus strain RAB and also identified the mutation sites in the phenamil resistant mutants. At first, we tried to sequence the 16S rRNA gene in the strain RAB in order to find related

strains. We were able to amplify the regions coding for 16S rRNA by PCR with a set of universal eubacterial primers, and the sequences were determined. Similarity search in international databases has shown that the 16S rRNA gene of RAB was identical to that of Bacillus firmus OF4, in which mot genes were reported as motPS.

Secondarily, we have amplified the genes of motAB homologs in the strain RAB by PCR with

the primers designed from motPS sequence of OF4. The PCR products were cloned into pUC19 and sequenced. The results have shown that the homologs of motAB in the strain RAB were also identical to motPS of OF4.

Thirdly, we have sequenced the homologs of motAB (motPS) of the phenamil resistant mutants.

We found substitutions in the homolog of motB (motS) (Pro13(cca) to Ser(tca), or to Thr(aca)), which corresponded to the mutation site reported in the phenamil resistant mutant of Vibrio (Pro16(ccg) to Ser(tcg)), and also found another substitution (Thr18(aca) to Ala(gca)). Both mutation sites existed close to the cytoplasmic ends of the putative transmembrane region. We could not find any substitution in the homolog of motA, which was reported to have been observed in Vibrio. Lab: Tatsuo Atsumi

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BLAST XI Poster #6

Poster Cancelled

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BLAST XI Poster #7 CONFORMATIONAL FLEXIBILITY OF FliG PROVIDES STRUCTURAL INSIGHTS FOR MOTOR SWITCHING AND COUPLING MECHANISM Lam KH2, Ling TKW3, and Au SWN1

1Biochemistry Programme and 2Molecular Biotechnology Programme, School of Life Sciences, The Chinese University of Hong Kong, HKSAR, China. 3Department of Microbiology, The Chinese University of Hong Kong, HKSAR, China.

Flagellar rotation is controlled by a reversible rotary motor. Motor switching is regulated by the binding of phosphorylated CheY to FliM that triggers the conformational change of FliG and alter its binding with stator MotA4B2. Binding of CheY-P is highly cooperative to the switch response. A recent hallmark study explains the switch mechanism by a conformational spread model and shows that the presence of conformational heterogeneity among subunits is critical to both allosteric mechanism and to the cooperativity of the motor. However, the structural information to understand the basis of conformational heterogeneity remains limited. Here we report FliG structures from H. pylori with distinct conformations from previously reported FliG. We uncovered at least three key flexible regions that allow FliG to exist as multiple conformations. We argue that the local conformational changes conferred by the flexible loops are independent, thus intensifying the possible conformations of FliG states. The “dominant” conformation is instead stabilized by stochastic interactions with FliM which depends of the concentration of CheY-P. In the presentation, the switching mechanism based on the structural information of chemotaxis and switch proteins from H. pylori will be discussed. Lab: Shannon Au

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BLAST XI Poster #8 TWO OPEN READING FRAMES INVOLVED IN cGMP SECRETION AND CYST FORMATION IN RHODOSPIRILLUM CENTENUM Qian Dong1, Jeremiah Marden2 and Carl Bauer1 1Molecular and Cellular Biochemistry Department, Indiana University- Bloomington, IN, 47405 2Department of Microbiology and Immunology, University of North Carolina, Chapel Hill, NC, 27599

Rhodospirillum centenum is a purple photosynthetic α-proteobacterium with a complex developmental life-cycle featuring three morphologically distinct cell types; swim cells, swarm cells and resting cysts. Cysts are metabolically dormant as a means of surviving environmental stresses such as desiccation. Wild-type R. centenum excretes large amounts of cGMP when transitioning from vegetative growth into encystment, whereas a strain deleted for recently characterized guanylyl cyclase fails to synthesize cGMP, is severely disrupted in cyst-cell maturation, and can be complemented by exogenously added cGMP.

There are two open reading frames, rc1_3786 and rc1_3787 linked to the R. centenum guanylyl

cyclase with this gene linkage also present in several plant associated soil bacteria that also synthesize cGMP. To explore the function of these two genes we constructed in frame deletions of ORFs rc1_3786 and rc1_3787 in R. centenum. Deletions of either rc1_3786 or rc1_3787 results in a phenotype that is virtually indistinguishable from the strain deleted for the cyclase. Specifically, these additional deletion strains are defective in synthesis of cGMP and in the forming cysts. The defect in synthesis of cysts can be complemented by the exogenous addition of cGMP. Our working model for cGMP signaling in R. centenum involves the synthesis of cGMP in response to an unknown signal that activates the activity of the guanylyl cyclase which interacts or forms a complex with products of ORF’s rc1_3786 and rc1_3787. Lab: Carl Bauer

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BLAST XI Poster #9 FliL, A GATEKEEPER OF PROTEUS MIRABILIS SWARMING DIFFERENTIATION Yi-Ying Lee and Robert Belas Department of Marine Biotechnology, University of Maryland, Baltimore County, and Institute of Marine and Environmental Technology, 701 East Pratt Street, Baltimore, Maryland 21202

Proteus mirabilis is a dimorphic Gram-negative enterobacterium. In liquid culture, P. mirabilis cells are uniformly 1.5-2.0 μm rods, called swimmer cells; while on a solid surface, swimmer cells differentiate into elongated (25 μm or longer) swarmer cells bearing many flagella. The swarmer cell phenotype is required for movement over solid surfaces, which is known as swarming behavior. P. mirabilis swarming is a multicellular behavior and requires flagellar rotation. Conditions that prevent rotation of swimmer cell flagella trigger production of swarmer cells, suggesting that inhibition of flagellar rotation is the signal cuing cells that they are on a surface. However, it remains a mystery as to how information from the stalled flagellum motor is transduced to affect swarmer cell gene expression. We have previously shown that fliL, a gene encoding a ubiquitous flagellar basal body-associated protein, is involved in sensing a surface, and that a strain (BB2204) bearing a transposon insertion in fliL inappropriately produces swarmer cells when grown in liquid cultures. We therefore hypothesize that FliL is involved in transducing information from the stalled motor to control gene expression. To gain an understanding of the molecular mechanism involved in surface sensing, we have characterized of a set of spontaneous motile revertants of BB2204, which is non-motile due to polar effects on essential downstream genes in the fliL operon, that regain both swimming and swarming motility. Analysis of the fliL sequence in these motile revertants revealed that the transposon has partially excised from the original insertion point leaving a 68 bp ‘scar’. The excision allows transcription to proceed through the fliL operon, but importantly preserves the original constitutive swarmer cell phenotype, as seen in BB2204. Real-time PCR results reveal that, in contrast to little increase of the transcriptional level of the flhD gene, the transcriptional level of flagellar Class II genes (fliA and fliL genes) increases evidently in BB2204 and the motile revertants. It suggests that disruptions of FliL up-regulate the expression and the function of the master regulator. Bioinformatic analyses suggest that, in both BB2204 and the motile revertants, the transposon insertion created a chimeric FliL whose C-terminal 14 amino acids are replace by an amino acid sequence with 4 arginines and 4 prolines. We hypothesize that this modification of FliL may cause the constitutive swarmer phenotype. If correct, this suggests a function for FliL in bacterial surface sensing.

Lab: Robert Belas ___________

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BLAST XI Poster #10 FlhDC, THE FLAGELLAR MASTER REGULATOR, REGULATES ITS TARGET PROMOTERS IN A TWO-STAGE FASHION Yi-Ying Lee1,2, Robert Belas2 and Philip Matsumura1 1Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, 835 South Wolcott Avenue, M/ C 790, Chicago, Illinois 60612 2 Department of Marine Biotechnology, University of Maryland, Baltimore County, and Institute of Marine and Environmental Technology, 701 East Pratt Street, Baltimore, Maryland 21202

The bacterial flagellum is a suborganelle that is required for motility. It is a complex structure and requires over 50 genes for flagellar function and composition. The flagellar genes in Escherichia coli are organized in a regulatory hierarchy of 3 levels. The flhDC operon, encoding the flagellar master regulator FlhDC, is at the top of the hierarchy (Class I promoter). FlhDC directly activates the transcription of 7 Class II promoters whose genes include fliA encoding a flagellum specific sigma factor (σ28) that activates the transcription of Class III promoters. Genetic and microarray data suggest that FlhDC also regulates non-flagellar genes as a global regulator. While many genes are known to be regulated by FlhDC, one major question is what factors determine the specificity of FlhDC action on different promoters.

FlhD and FlhC form an unusual heterohexameric complex, FlhD4C2. This complex protects an

unusually large ‘footprint’ region of about 50 base pairs in its target Class II promoters (e.g., fliAp, flhBp and fliLp). Biochemical and bioinformatic studies suggest FlhDC binds a large imperfect inverted repeat with 16-17 base pairs half-site in the protected region. We have determined a refined FlhDC binding consensus sequence (AN2AN18TN2TN7T) in the FlhDC-regulated promoters. Site-specific changes were introduced at the single base pair “As” and “Ts” in the consensus sequence in fliAp, flhBp and fliLp. We found the sequence specificity in the proximal end of the consensus sequence is high, while the specificity in the distal end is lower. Mutations in the 9-10 base pairs in the distal and proximal ends of the FlhDC footprint region in the fliAp, flhBp and fliLp showed that FlhDC uses different binding mechanism at the different promoters. In the fliA promoter, for example, the proximal end but not the distal end of the footprint region is important for the function of FlhDC; while in the flhB and fliL promoters, both ends of the footprint region are important. We constructed fliA-fliL hybrid promoters containing portions of the proximal fragments from the fliA (or fliL) promoters and distal fragments from the other. The hybrid promoters revealed two stages of regulation occur during FlhDC-mediated promoter activation. These results suggest that the FlhDC consensus sequence determines the ‘on/off’ action of FlhDC, while the flanking regions around the footprint act as a rheostat to control the level of promoter activity.

Lab: Robert Belas ___________

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BLAST XI Poster #11 BINDING COOPERATIVITY IN THE BACTERIAL FLAGELLAR MOTOR Richard Branch, Puskar Lele, Gabriel Hosu, Vedavalli Nathan & Howard Berg Department of Molecular and Cellular Biology, Harvard University

E. coli can respond to small changes in stimuli concentration (1), partly due to signal amplification in the bacterial flagellar motor (2). Amplification in motors is understood in terms of allosteric cooperativity models (3). An indispensable assumption of these models is that the clockwise and counterclockwise states of the motor have different affinities for the phosphorylated response regulator CheY. Constrained models indicate that approximately 20% more CheY-P should bind the motor in the clockwise state. Here we describe the preliminaries of an experiment designed to measure this binding difference in single motors.

(1) SEGALL, J. E., BLOCK, S. M. & BERG, H. C. (1986). Temporal comparisons in bacterial

chemotaxis. Proc Natl Acad Sci USA, 83(23), 8987‐8991. (2) CLUZEL, P., SURETTE, M. & LEIBLER, S. (2000). An ultrasensitive bacterial motor revealed by

monitoring signaling proteins in single cells. Science, 287(5458), 1652‐1655. (3) BAI, F., BRANCH, R. W., NICOLAU, D. V., PILIZOTA, T., STEEL, B. C., MAINI, P. K., BERRY, R.

M. (2010). Conformational spread as a mechanism for cooperativity in the bacterial flagellar switch. Science, 327(5966), 685-689.

Lab: Howard Berg

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BLAST XI Poster #12 PARTICLE-WALL HYDRODYNAMIC INTERACTIONS IN MULTI-PARTICLE ENSEMBLES Pushkar P. Lele, Norman J. Wagner and Eric M. Furst Harvard University, Department of Molecular and Cellular Biology, 16 Divinity Ave., Rm. 3063, Cambridge, MA 02138

The dynamics of microscopic objects suspended in a liquid close to surfaces, is a fairly complex problem due to hydrodynamic screening. Even for isotropic particles this phenomenon is not well understood and, research groups disagree on the nature of the interactions. To investigate the decays of the fluid velocities due to a cluster of point forces over varying distances (r), we use blinking holographic optical tweezers and measure the wall-induced hydrodynamic screening phenomenon for spherical-particle clusters. Using eigen-decompositions of the diffusion tensors, we show that the particle interactions decay as 1/r, 1/r2 or 1/r3, depending on the distance (h) from the no-slip surface1. We also show that Blake’s point-particle model2 captures the experimentally observed relative and collective modes successfully, although corrections to the self-diffusivities of particles can not be predicted3. Such studies can form the basis for understanding the more complicated behavior of motile bacteria near surfaces. References [1] P. P. Lele, PhD Thesis, University of Delaware (2010). [2] J. R. Blake, Proc. Camb. Phil. Soc., 1971, 70, 303-310. [3] P. P. Lele, J. W. Swan, J. F. Brady, N. J. Wagner, E. M. Furst, manuscript submitted for publication.

Lab: Howard Berg

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BLAST XI Poster #13 MUTATIONAL AND CROSSLINKING STUDIES OF CYTOPLASMIC PARTS OF THE FLAGELLAR STATOR Eun A. Kim and David F. Blair University of Utah Salt Lake City, UT 84112

The stator of the bacterial flagellar motor is formed from the membrane proteins Mot A and MotB. Mot A has four transmembrane segments and a large domain in the cytoplasm; Mot B has a single transmembrane segment and a large domain in the periplasm. The motor contains ten or more independently functioning stator complexes, each with subunit composition MotA4MotB2. These complexes function to conduct protons across the membrane, and harness proton flow to rotation by a mechanism that appears to involve conformational changes in the cytoplasmic domain. Previous mutational and crosslinking studies revealed the organization of the membrane segments of MotA and Mot B and gave evidence that the conserved residue Asp 32 of Mot B is critical for rotation and might function directly in proton association / dissociation. The crosslinking-based structural model suggests that residue Tyr 217 of MotA might also be critical, possibly regulating the hypothesized conformational changes. Several mutational replacements of Tyr 217 were made and were found to disrupt motility. Genetic-suppression analysis identified several mutations in the cytoplasmic domain that can largely rescue the motility defect of the Tyr 217 mutants. Prompted by these suppression data, we are attempting to extend the cross-linking studies to develop a structural model encompassing the cytoplasmic domain of MotA.

Lab: David F. Blair

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BLAST XI Poster #14 ADJUSTING THE SPOKES OF THE FLAGELLAR MOTOR WITH THE DNA-BINDING PROTEIN H-NS Koushik Paul, William C. Carlquist and David F. Blair Department of Biology, University of Utah, Salt Lake City, UT 84112

The Heat-stable Nucleoid Structuring (H-NS) protein of bacteria is a global regulator that stimulates transcription of flagellar genes and also acts directly to modulate flagellar motor function. H-NS binds to FliG, a rotor protein that interacts directly with the stator to produce torque. Using a GST-pull down assay we found that the DNA binding domain of H-NS binds to a well-conserved EHPQR motif of FliG. Strong binding was shown to require that H-NS be a dimer. The binding of H-NS restricts mobility of a helix joining the middle- and C-terminal domains of FliG, and stabilizes FliG in its normal relationship with the stator. Thus, H-NS serves to tighten the spokes of the motor, improving motor function under conditions requiring a strengthened rotor-stator interface. The effects of H-NS are roughly opposite those of the recently characterized motility regulator YcgR. A structurally grounded model is proposed that can account for the opposing effects of these regulators. Lab: David F. Blair

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BLAST XI Poster #15 FLAGELLAR DIRECTION SWITCHING IN ESCHERICHIA COLI: CheY BINDS TO THE ROTOR PROTEIN FliN TO INDUCE CW ROTATION Mayukh K Sarkar, Koushik Paul and David F Blair University of Utah, Department of Biology, Salt Lake City, UT 84112

The direction of rotation of the Escherichia coli flagellum is controlled by a large assembly on the rotor called the switch complex, formed from the proteins FliG, FliM, and FliN. The switch complex contains about 25 FliG, 35 FliM, and 140 FliN subunits, and corresponds structurally to the basal body C-ring. Flagellar direction reversals are the basis of chemotaxis: In E.coli, the motors turn counterclockwise (CCW) in their default state, resulting in smooth swimming, but switch to clockwise (CW) rotation, and rapid tumbling of the cell, in response to the signaling molecule phospho-CheY (CheYP). CheYP has previously been shown to bind to a conserved segment near the N-terminus of FliM. Here, we show that this FliM-CheY interaction serves to capture CheYP and that the switch to CW rotation involves subsequent interaction of CheYP with FliN. Targeted crosslinking experiments showed that FliN is organized in donut-shaped tetramers at the bottom of the C-ring. The C-terminal domain of FliM (FliMC) is inserted between adjacent FliN tetramers. The switch to CW rotation is associated with a movement in FliN relative to FliMC, which is predicted to increase the accessibility of the CheY binding site identified here. This would provide a simple mechanism for linking CheY binding to the conformational switch from CCW to CW rotation.

Lab: David F. Blair

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BLAST XI Poster #16 SYSTEMATIC MUTAGENESIS OF PROTON BINDING RESIDUES OF THE FLAGELLAR EXPORT APPARATUS Yang Zhang, Mayukh K Sarkar and David F Blair University of Utah, Department of Biology, Salt Lake City, UT 84112

Bacterial flagella contain a specialized secretion apparatus that functions to deliver the protein subunits that form the filament and other structures to outside the membrane. This export by the flagellum is closely related to the export process occurring in injectisome systems of pathogens, and is termed ‘type III’ secretion. Flagellar type III secretion obtaining energy for export from the membrane proton gradient, allows efficient assembly of the exterior part of the complex macromolecular of the apparatus machine. To obtain more detailed information on functional roles of machine proteins, we undertook the systematic mutagenesis of conserved titratable residues that might participate in proton binding/dissociation reactions. The mutational data suggests that FlhA might contribute to the H+ translocation pathway of the export apparatus, while FliP might form channel for translocated protein subunit b.

Lab: David F. Blair

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BLAST XI Poster #17 CheY3 OF BORRELIA BURGDORFERI IS THE KEY RESPONSE REGULATOR ESSENTIAL FOR CHEMOTAXIS AND FORMS A LONG-LIVED PHOSPHORYLATED INTERMEDIATE Md. A. Motaleb1,2,*, Syed Z. Sultan1, Michael R. Miller3, Chunhao Li2,4, and Nyles W. Charon2* 1Department of Microbiology and Immunology, East Carolina University School of Medicine, Greenville, NC 27834. 2Departments of Microbiology, Immunology, and Cell biology, and Biochemistry3, Health Sciences Center, West Virginia University, Morgantown, WV 26506-9177. 4Current address: Department of Oral Biology, State University of New York at Buffalo, Buffalo, NY 14214.

Spirochetes have a unique cell structure, as these organisms have internal periplasmic flagella subterminally attached at each cell end that are involved in motility. How spirochetes coordinate the rotation of the periplasmic flagella for chemotaxis is poorly understood. In other bacteria, modulating flagellar rotation is essential for chemotaxis, and phosphorylation–dephosphorylation of the response regulator CheY plays a key role in regulating this rotary motion. The genome of the Lyme disease spirochete Borrelia burgdorferi contains multiple homologues of chemotaxis genes, including three copies of cheY, referred to as cheY1, cheY2, and cheY3. To investigate the function of these genes, each was targeted separately or in combination by allelic exchange mutagenesis. Only those mutants containing inactivated cheY3 had an altered phenotype. Whereas wild-type cells ran, paused (flexed), and reversed, cells from all single, double, and triple mutants that contained an inactivated cheY3 constantly ran. Swarm plate and capillary tube chemotaxis assays indicated that only those strains with a mutation in cheY3 were deficient in chemotaxis, and cheY3 complementation resulted in regaining the wild-type phenotype. In vitro phosphorylation assays indicated that CheY3 was more efficiently phosphorylated by CheA2 than CheA1, and the CheY3-P intermediate generated was considerably more stable than most CheY-P found in other bacteria. The results point towards CheY3 being the key response regulator essential for chemotaxis in B. burgdorferi, and that its stability may be critical for the coordination of the periplasmic flagella rotation.

Lab: Nyles Charon

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BLAST XI Poster #18 BUILDING SOLUBLE MODELS OF CHEMORECEPTORS Xiaoxiao Li, Brian R. Crane Cornell University, Ithaca, NY, 14850

In bacterial chemotaxis, the signal transduction machinery involves a sensory complex that is composed of transmembrane chemoreceptors, the auto-kinase CheA and coupling protein CheW. CheA phosphorylates the response regulator CheY, which controls the direction bias of the flagella rotation. The activity of CheA depends on the state of the chemoreceptors.

Our understanding is increasing with regards to interactions within the ternary signaling

complex, the assembly process of single complex into large arrays, and the conformational changes associated with kinase regulation. However, many issues remain unresolved.

A stable, soluble trimer of dimer chemoreceptors would be useful to probe these problems in

vitro. Here we report on our ongoing effort to build trimer of dimeric chemoreceptors. Two identical

truncated cytoplasmic regions of the Tar chemoreceptor were tethered together by a short amino acid linker, thereby generating a single-chain “homodimer”. Then we fused an outside ultra-stable trimeric motif onto the single-chain homodimer. Design and characterization of the fusion proteins are described.

Lab: Brian Crane ____

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BLAST XI Poster #19 PROBING THE STRUCTURE OF THE FLAGELLAR SWITCH COMPLEX Ria Sircar, Brian R. Crane Cornell University, Cornell University, Ithaca, NY

The bacterial flagellum is a complex machine consisting of the export apparatus, the reversible rotary motor, the joint and the filament. The rotary motor efficiently converts chemical energy into torque directed in either the clockwise or counterclockwise direction. The rotor proteins FliG, FliM and FliN together form the switch complex of the cytoplasmic C-ring which is essential for binding phosphorylated CheY and switching. In some bacteria, the N-terminus of FliN has an additional phosphatase domain and is known as FliY. FliY is a multidomain protein that shares conserved residues with the CheC/X phosphatase family. We are pursuing crystal structures of the switch protein components, and complexes to understand the architecture of the rotor and how the proteins interact to produce a functional switch. To complement our crystallographic work we apply Pulsed Dipolar Electron Spin Resonance Spectroscopy (PDS) to study changes in the interactions among components in the presence of phosphorylated CheY. Progress towards the understanding of flagellar structure and switching will be presented.

Lab: Brian Crane ____

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BLAST XI Poster #20 A STRUCTURAL MODEL OF ANTI-ANTI-SIGMA INHIBITION BY A TWO-COMPONENT RECEIVER DOMAIN: THE PhyR STRESS RESPONSE REGULATOR Julien Herrou, Robert Foreman, Aretha Fiebig and Sean Crosson Department of Biochemistry and Molecular Biology, The University of Chicago, 929 E. 57th St., Chicago, IL, USA

PhyR is a hybrid stress regulator conserved in α-proteobacteria that contains an N-terminal σ-like (SL) domain and a C-terminal receiver domain. Phosphorylation of the receiver domain is known to promote binding of the SL domain to an anti-σ factor. PhyR thus functions as an anti-anti-σ factor in its phosphorylated state. We present genetic evidence that Caulobacter crescentus PhyR is a phosphorylation-dependent stress regulator that functions in the same pathway as σT and its anti-σ factor, NepR. Additionally, we report the X-ray crystal structure of PhyR at 1.25 Å resolution, which provides insight into the mechanism of anti-anti-σ regulation. Direct intramolecular contact between the PhyR receiver and SL domains spans regions σ2 and σ4, likely serving to stabilize the SL domain in a closed conformation. The molecular surface of the receiver domain contacting the SL domain is the structural equivalent of α4-β5-α5, which is known to undergo dynamic conformational change upon phosphorylation in a diverse range of receiver proteins. We propose a structural model of PhyR regulation in which receiver phosphorylation destabilizes the intramolecular interaction between SL and receiver domains, thereby permitting regions σ2 and σ4 in the SL domain to open about a flexible connector loop and bind anti-σ factor.

Lab: Sean Crosson ____

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BLAST XI Poster #21 SOLUTION STRUCTURE OF FliG ANALYZED BY NMR Armand S. Vartanian, Hongjun Zhou, and Frederick W. Dahlquist Biomolecular Science and Engineering Program, University of California, Santa Barbara, Santa Barbara, CA 93106

Often the crystal structure and solution structure of a protein will have minor to extreme differences. These differences are commonly the result of crystal formation but do not preclude the value of information gained from the structure. The RCSB data bank contains three structures of the flagellar motor protein FliG: the C-terminal domain from T. maritima (1QC7), the middle and C-terminal domains from T. maritima (1LKV) and most recently the full-length structure from A. aeolicus (3HJL). Through the use of residual dipolar couplings (RDCs) and dynamic analysis of FliG, the solution structure and orientation can be compared to the crystal structures. Here we report NMR analysis of the middle and C-terminal domains of FliG from T. maritima. We have determined that FliG in solution is comprised of multiple domains and motifs that individually fit the crystal structures very well, correlation factors >0.8. When compared as a whole the solution and crystal structures fit poorly, correlation factor 0.576. This data implies that in solution the individual domains of FliG are in their crystal configurations but are oriented differently than as observed in the crystal. The dynamic analysis of FliG by NMR relaxation methods suggests that in solution FliG either has a single conformation or relative positions of the domains convert rapidly (τ < 1 ms) among their various orientations. This data in conjunction with the RDCs suggests that in solution FliG as a whole takes a different arrangement of its domains than as seen in the crystal structures.

Lab: Frederick Dahlquist ____

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BLAST XI Poster #22 HD-GYP DOMAIN PROTEINS REGULATE VIRULENCE AND BIOFILM FORMATION OF THE HUMAN PATHOGEN PSEUDOMONAS AERUGINOSA Karen O’Donovan, Yvonne McCarthy, Robert P. Ryan and J. Maxwell Dow BIOMERIT Research Centre, Department of Microbiology, University College Cork, Ireland

HD-GYP is a protein domain involved in the hydrolysis of the bacterial second messenger cyclic-di-GMP. The genome of P. aeruginosa PAO1 encodes two proteins (PA4108, PA4781) with an HD-GYP domain and a third protein, PA2572, which contains the variant YN-GYP domain. Our work addresses the role of these three HD-GYP domain proteins in virulence and biofilm formation in P. aeruginosa. Mutation of PA4108 and PA4781 led to an increase in the level of cyclic-di-GMP, consistent with the activity of these proteins as cyclic-di-GMP phosphodiesterases. Mutation was also associated with reduced swarming motility and production of the virulence determinants ExoS, pyocyanin and pyoverdin and on biofilm architecture. The PA2572 mutant had altered biolfilm architecture and increased rhamnolipid production, but had no effect on cyclic-di-GMP levels, suggesting it was enzymatically inactive. However PA2572 had a negative influence on swarming that was cryptic and was revealed only after removal of an uncharacterised C-terminal domain. Importantly all three proteins contributed to the virulence of P. aeruginosa to larvae of Galleria mellonella. We are currently investigating the role of protein-protein interactions in the regulatory activities of the HD-GYP domain proteins. We are using yeast two-hybrid analysis to identify possible interacting proteins.

Lab: John Dow

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BLAST XI Poster #23 IN VIVO RECONSTITUTION OF THE EnvZEc/OmpR OSMOSENSING CIRCUIT SUGGESTS A NON-PISTON MECHANISM OF TRANSMEMBRANE COMMUNICATION Roger R. Draheim*1,2, Morten H. H. Nørholm1, Salomé C. Botelho1, Karl Enquist1 and Gunnar von Heijne*1

1Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University, Stockholm, Sweden; 2Institute of Biochemistry, Biocenter, Goethe University Frankfurt, Frankfurt, Germany

Two-component signaling systems are modular and several discrete periplasmic and cytoplasmic domains have been evolutionarily conserved and shuffled to yield different functional combinations. Because of this diversity, we designed a strategy that targeted the membrane-spanning domain that is common to all SHKs. Our “aromatic tuning” strategy targets the second transmembrane helix (TM2) because, in most cases, it serves as the sole covalent connection between the periplasmic and cytoplasmic domains. This strategy is based on previous work with Trp-flanking (WALP) and Tyr-flanking (YALP) poly-Ala-Leu a-helical peptides that demonstrated the preference of amphipathic aromatic residues for the polar/hydrophobic interfacial regions of synthetic lipid bilayers.

One of our long-term goals is to establish a universal method for stimulus-independent

modulation of the receptors within these systems. Based on these pervious results, we sought to harness this intrinsic affinity to force displacements of TM2 within the cytoplasmic membrane. We began by subjecting the TM2s of E. coli TarEc and EnvZEc to glycosylation-positioning analyses, which, in both cases, demonstrated that moving aromatic residues that normally reside at the cytoplasmic polar-hydrophobic interface within the intact receptor, is sufficient to reposition TM2 within a biological membrane. However, unlike TarEc, which shows a direct correlation between the vertical position of TM2 of steady-state signal output, with EnvZEc we observed a different pattern of activity that did not correlate directly with vertical position. Instead, the helical face of TM2 that the aromatic residues reside upon was found to be the dominant factor rather than their vertical position along the helix. Therefore, the first two receptors that have been subjected to aromatic tuning resulted in two different classes of results: one where the vertical position of TM2 directly correlates with the signal output (TarEc) and one where the helical face of TM2 dominates over the vertical displacement (EnvZEc).

Based on these results, we propose that aromatic tuning may be used to classify the functional

mechanisms of various SHKs. Finally, we suggest that aromatic tuning could be used to circumvent the necessity of ligand-specific modulation of SHK output and therefore could serve as a valuable tool for unraveling complex multicellular developmental and physiological processes governed by two-component signaling systems. Lab: Roger Draheim

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BLAST XI Poster #24 OVERCOMING COMPLEXITY IN SYSTEMS BIOLOGY MODELING AND SIMULATION WITH NFsim Michael W Sneddon1,2, James R Faeder3 & Thierry Emonet1,2,4

1 Department of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT 06520- 8103, USA. 2 Interdepartmental Program in Computational Biology and Bioinformatics, Yale University, New Haven, CT 06511, USA. 3Department of Computational and Systems Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15260, USA. 4Department of Physics, Yale University, New Haven, CT 06520- 8103, USA

Standard methods for modeling biochemical reaction systems require the enumeration of every

possible molecular species and reaction, which is tedious and can be impossible for many complex processes. For example, enumerating all the possible methylation states of chemoreceptor signaling teams cannot be accomplished with a differential equation or standard stochastic approach. Here we present the Network-Free Stochastic Simulator (NFsim), a new software platform that allows efficient, yet exact, stochastic simulation of large or even infinite reaction networks. By using an agent and rule-based approach, the performance of NFsim is independent of the size of the reaction network providing orders of magnitude speedup in many situations. Additionally, reaction rates can be defined and arbitrary mathematical or conditional functions to facilitate coarse-graining of reaction mechanisms such as long-range cooperativity. We demonstrate the capabilities and performance of NFsim with models of multi-site phosphorylation, receptor aggregation in the immune system, actin filament assembly, and bacterial chemotaxis signaling.

Lab: Thierry Emonet ____

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BLAST XI Poster #25 CONFORMATIONAL CHANGES IN THE ASSEMBLED, MEMBRANE-ASSOCIATED CHEMOTATIC SIGNALING COMPLEX Adam J. Berlinberg, Annette H. Erbse and Joseph J. Falke Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO 80309

Receptor-kinase complexes organize in ultrastable, ordered signal sensing arrays at the cell poles, sense chemical attractants and transfer the signal via phospho-transfer to an effector protein that regulates the flagella motor. The simplest array retaining ultrastability and receptor-regulated kinase activity is composed of receptor trimer-of-dimers, the homodimeric histidine-kinase CheA, and the CheW coupling protein. Complex formation enhances the latent kinase activity of CheA strongly while attractant binding to the receptor causes inactivation of CheA. Although our knowledge of the structure of the core complex is steadily increasing, we do not have a detailed accurate complex structure and more importantly, the mechanism of signal transduction between receptor and CheA is still an enigma.

FRET is a powerful method for the analysis of large, multi-component soluble or membrane associated protein complexes in their native environment. It makes use of the radiationless transfer of energy from a fluorescent donor chromophore to a nearby acceptor molecule and exhibits a characteristic 1/r6 distance dependence. In order to elucidate the core complex and the nature of the communication between its elements in more detail, we have developed a new One Sample FRET (OS-FRET) method. The ability to make all the necessary measurements to calculate FRET efficies in just one sample abolishes the need to precisely know the concentration of multiple samples, while it maintains accuracy. As added benefit, it increases sample efficiency and decreases overall time needed for experiment1.

These advantages allow us to apply OS-FRET to probe possible conformational changes in

CheA and CheW triggered by ligand binding to the receptor. For the study presented here we focused on the P5 domain of CheA, which is known to interact with CheW, and is believed to also provide direct interaction with the receptor and CheW. Results have shown that Ser binding induces a conformational change between the P5 domain of CheA and CheW, and that the domain motions are not the result of CheW falling off the complex. Data also shows that the FRET efficiencies are relevant for our current model of the complex2. Preliminary data for the P1 domain of CheA interacting with CheW also demonstrates this domain is likely mobile and has a high degree of flexibility in solution. (This project is supported by NIH R01 GM-040731) 1 Annette H. Erbse, Adam J. Berlinberg, Ching-Ying Cheung, Wai-Yee Leung, and Joseph J. Falke

(2010) OS-FRET: A new one-sample method for improved FRET measurements (Accepted). 2 A. S. Miller, S. C. Kohout, K. A. Gilman et al., Biochemistry 45 (29), 8699 (2006).

Lab: Joseph Falke

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BLAST XI Poster #26 INVESTIGATING THE MECHANISM OF ULTRASTABILITY IN CHEMORECEPTOR CLUSTERS Peter F. Slivka and Joseph J. Falke Department of Chemistry and Biochemistry and the Molecular Biophysics Program University of Colorado, Boulder, CO 80309-0125

Chemosensory receptors in bacteria and archea form highly cooperative and ultrastable arrays. In the simplest core array, receptor trimers-of-dimers are arranged in a well-ordered hexagonal lattice with two additional proteins, CheA kinase and coupling protein CheW. Recent literature suggests that a possible mechanism for ultrastability could arise from multiple contacts between receptors, CheA and CheW within the ordered lattice, much like the stability exhibited by a completed jigsaw puzzle. In this model, ultrastability requires a highly ordered array with a low density of packing defects. To test this model we are investigating the cooperative array formed by the serine receptor (Tsr), CheA and CheW. Our strategy is to systematically introduce defects into the array in a controlled fashion by labeling a population of mutant Cys-containing receptors with increasing densities of a bulky fluorescein probe known to disrupt receptor/CheA/CheW association. Subsequently the mixture of functional and non-functional receptors is reconstituted with CheA and CheW. The cumulative effect of receptor defects may manifest themselves in at least two ways. First, labeled receptors may be completely excluded from the lattice, so that the remaining functional lattice exhibits normal ultrastability although its area and kinase activity are decreased. Alternatively, the labeled receptors may intercalate into the lattice and disrupt ultrastability by reducing CheA and CheW binding, thereby causing lattice defects and decreasing the lattice lifetime. By studying the lattice lifetime as a function of defect type and density we hope to better understand the mechanism of lattice ultrastability. This poster will present our current results.

Support Provided by: NIH R01 GM-040731

Lab: Joseph Falke

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BLAST XI Poster #27 TESTING MODELS FOR HAMP ON-OFF SWITCHING IN THE E.COLI SERINE CHEMORECEPTOR

Ka Lin E. Swain and Joseph J. Falke Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO, 80309

The bacterial transmembrane serine receptor (Tsr) of E. coli and S. typhimurium chemotaxis is a homodimer that assembles to form larger oligomers, yielding a trimer-of dimers that in turn assembles into hexagonal arrays. The homodimer can be divided into three modules: (i) the transmembrane signaling module comprised of the periplasmic ligand binding domain and the transmembrane helices, (ii) the cytoplasmic HAMP domain which serves as a signal conversion module, and (iii) the cytoplasmic kinase control module possessing the adaptation sites and a kinase docking region. Previous structural studies show the HAMP domain folds into a parallel 4-helix bundle, consisting two α-helices (“HD1” and “HD2”). This HAMP motif is an essential signal transduction element believed to convert its input signal (the attractant-triggered piston transmembrane signal) into a different conformational output signal (that weakens packing between the adaptation helices of the kinase control module). There are at least four current models in the field for HAMP on-off switching. The piston-triggered scissors model proposes that the input piston displacement of TM2 triggers a scissors-displacement of the HD2 and HD2’ helices of the homodimeric HAMP, thereby perturbing the packing between the adaptation helices. The dynamic bundle model proposes that Tsr-HAMP switches between a dynamic on-state and stable off-state, such that attractant binding imposes a structural force that results in the HD2/HD2’ helices to pack out-of-phase with that of the adjoining MH1/MH1’ helices, thereby destabilizing the four-helix methylation bundle and shifting HAMP to the kinase-off state (Ames et al., submitted, 2010). The gearbox model proposes that the attractant signal is a rotation of TM2 that triggers rotations of all four helices in the bundle about their long axes, thereby generating the off-state (Hulko et al., Cell, 2006). Finally the “gymnastics” model proposes that HAMP undergoes large movements during signaling in which the entire HAMP domains in a trimer-of-dimers get closer and further apart (Khursigara et al., PNAS, 2008). We are using disulfide mapping and disulfide trapping approaches to test these models for HAMP on-off switching. To test the gymnastics model we have targeted single cysteines at surface-exposed positions on the 4-helix bundle and are measuring the attractant-triggered disulfide formation rates of Tsr-HAMP in the active, ternary signaling core complexes. If one HAMP domain interacts with other HAMPs between neighboring trimer-of-dimer receptors, it is predicted that an increase in disulfide formation rates will be observed for one signaling state in the Serine chemoreceptor. The latest findings, which are expected to have a broad relevance to HAMP-containing receptors, will be reported at the meeting.

Lab: Joseph Falke

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BLAST XI Poster #28 NEWLY IDENTIFIED McpB/McpC CHEMORECEPTORS AND THE ADAPTATION PROTEIN CheV FUNCTION IN TAXIS TOWARDS L-CYSTINE IN SALMONELLA ENTERICA Mitchell T. Butler1, Manjunath Hegde2, Susana Mariconda1, Arul Jayaraman2 and Rasika M. Harshey1,* 1Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, TX 78712. 2Departments of Chemical Engineering and Biomedical Engineering, Texas A&M University, College Station, TX 77843

The newly identified chemoreceptors McpB and McpC in Salmonella enterica serovar Typhimurium promote positive chemotaxis in LB or tryptone media. Of the amino acids/sugars/vitamins/nucleosides tested as potential attractants sensed by these chemoreceptors, the only consistent response was towards cystine. The response was optimal when both McpB and McpC were present, but McpC was clearly the dominant receptor for cystine chemotaxis. McpB and McpC are homologous to other chemoreceptors that have a periplasmic sensory domain, a HAMP domain, a methylation module and receptor-trimer contact sites. The C-terminal pentapeptide sequence NWET/SF is important for methylation-dependent adaptation to ligand stimuli in the high-abundance chemoreceptors Tsr and Tar. The pentapeptide EWVSF at the C-terminus of McpB resembles NWET/SF, but DTQPA at the C-terminus of McpC is very different. The McpBC response could not be improved by substitution of NWETF on the C-terminus of McpC, or by providing Tsr or Tar in trans. McpBC-dependent chemotaxis occurred only in the presence of the newly discovered methylation-independent adaptation protein CheV, but chemosensing by Tsr or Tar was unaffected by the absence of CheV. Thus, CheV appears be dedicated to chemotaxis mediated by McpBC. Lab: Rasika M. Harshey

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BLAST XI Poster #29 FlhE ACTS AS A PROTON PLUG IN THE SALMONELLA FLAGELLAR TYPE III SECRETION SYSTEM AFTER THE SWITCH TO LATE SUBSTRATE SECRETION Jaemin Lee and Rasika M. Harshey* Section of Molecular Genetics and Microbiology, University of Texas at Austin, TX 78712

flhE is the last gene in the flhBAE flagellar operon whose first two members encode components of the Type III secretion system (T3SS) in Salmonella enterica. flhE is found only in Enterobacteria, and its role has been a mystery, since it is not essential for swimming motility. However, absence of FlhE reduces or even abolishes swarming, which requires inclusion of glucose in the motility media. We have localized FlhE to the periplasm and within the basal body. Based on a chance observation of a ‘green’ colony phenotype of flhE mutants on pH indicator plates containing glucose, we have established that this phenotype is associated with a lowered cytoplasmic pH and cell lysis in an acidic environment created by glucose metabolism. The lowered cytoplasmic pH is dependent on the switch to late flagellar secretion, while the cell lysis phenotype is dependent on filament assembly (but not rotation), irrespective of whether flagella grow outside the cell or within the periplasm. We conclude that FlhE regulates proton flow during the late phase of flagellar biogenesis. In addition, our experiments reveal that a long flagellar filament causes membrane stress.

Lab: Rasika M. Harshey

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BLAST XI Poster #30 MECHANISM OF TRANSCRIPTIONAL REGULATION OF EXOPOLYSACCHARIDE GENES BY FleQ IN RESPONSE TO c-di-GMP IN PSEUDOMONAS AERUGINOSA Claudine Baraquet and Caroline Harwood Department of Microbiology, University of Washington, SEATTLE, WA98195 Key words: biofilm, c-di-GMP, transcription factor

The intracellular concentration of c-di-GMP varies depending on the activities of diguanylate cyclases or phosphodiesterases. These activities are modulated by environmental signals that are largely unknown. Binding of c-di-GMP to downstream proteins leads to the regulation of various cellular functions. One of these c-di-GMP binding proteins is the transcription factor FleQ of Pseudomonas aeruginosa. FleQ regulates the expression of Pel and Psl exopolysaccharide genes required for biofilm formation. Previous data showed that FleQ represses pel expression and that this repression is relieved in response to c-di-GMP. However, full expression of pel genes in the presence of c-di-GMP occurs only when another protein, FleN, is present. It was suggested that the binding of c-di-GMP to FleQ induces a dissociation of FleQ from the pel promoter allowing the RNA polymerase to access DNA and form an active transcription complex. To test this hypothesis and to further probe mechanism we analyzed pel promoter DNA footprinting patterns with various combinations of FleQ, FleN and c-di-GMP. We identified two FleQ binding sites of 19 base pairs, one on each side of the -10 and -35 region of the pel promoter. Mutations in the FleQ binding sites of the pel promoter showed that the binding of FleQ on one site is independent of protein occupancy at the other site and that only the occupancy of the site close to the -10 region is required for repression. Surprisingly, it appears that the presence of c-di-GMP neither promotes the dissociation of FleQ from the pel promoter nor changes the binding sites of FleQ. We also found that when FleN is present, FleQ binds to the same sites on the pel promoter, but FleN induces a bending of the pel DNA. This bending requires the presence of ATP but not its hydrolysis. When c-di-GMP is added, FleQ stays bound to the promoter, but the bending mediated by FleN is relieved. Limited proteolysis and bacterial two-hybrid experiments indicate that FleQ and FleN interact in the presence or in absence of ATP or c-di-GMP. The binding of c-di-GMP to FleQ appears to induce a conformational change of the FleQ/FleN complex.

Our data indicate that FleQ and FleN form a complex on the pel promoter at a position that

prevents binding of RNA polymerase, resulting in repression of gene expression. Addition of c-di-GMP seems to promote a change in the FleQ-FleN complex such that repression is relieved and pel transcription can occur. The mechanism by which FleQ-FleN complex apparently still stays bound to the pel promoter while allowing RNA polymerase access remains to be determined. Lab: Caroline Harwood ____

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BLAST XI Poster #31 SUBCELLULAR LOCALIZATION DETERMINANTS OF THE PSEUDOMONAS AERUGINOSA Wsp SENSORY TRANSDUCTION COMPLEX FOR BIOFILM FORMATION Jennifer R. O’Connor and Caroline S. Harwood Department of Microbiology, University of Washington, Seattle WA 98195

The P. aeruginosa Wsp sensory transduction complex is homologous to the paradigm E. coli chemotaxis signal transduction system. In response to a signal associated with surface culture, the WspA receptor stimulates phosphorylation of WspR, a composite CheY-GGDEF domain protein that catalyzes cyclic-di-GMP (c-di-GMP) synthesis. This intracellular messenger stimulates P. aeruginosa to switch to a biofilm lifestyle. A previous study in our lab1 showed that P. aeruginosa chemoreceptors and other chemotaxis proteins localize to the old cell pole. Conversely, another study2 showed that the WspA receptor, which is bioinformatically indistinguishable from the chemoreceptors, localizes laterally along the length of the cell. Phosphorylated WspR forms subcellular clusters that are not necessarily located at cell poles. The localization of WspA and WspR is dynamic and these two proteins only co-localize occasionally. To further investigate the mechanisms that determine the unusual subcellular localization properties of the Wsp complex we are studying the localization of the WspA receptor in more depth. Initially, linescan analysis of the subcellular distribution of WspA-YFP was performed. The results showed that WspA-YFP adopts a helical arrangement along the length of the cell. This suggested that the bacterial cytoskeleton protein MreB, which also forms a helix3, might determine WspA localization. However when we treated cells with a drug (A22) that disrupted the localization of MreB, there was no effect on the helical arrangement of WspA-YFP. To identify the region of WspA that determines its subcellular location we replaced the WspA periplasmic and HAMP domains with those of the P. aeruginosa chemoreceptors PctA, PctB, PctC and PA2652. These chimeric proteins localized laterally in cells, similar to wild type WspA. The functionality of the chimeric chemoreceptors was analyzed by counting the number of cells that contained WspR-YFP clusters – a surrogate measure of WspR phosphorylation. The chimeric chemoreceptors were functional, but not as efficient as wild type WspA. Interestingly, the chimeric receptors were all capable of responding to the WspA activation signal: surface culture. We are currently constructing a WspA chimera that contains the WspA periplasmic and HAMP regions fused to the cytoplasmic domain of PctA to determine whether this chimera will adopt polar or helical subcellular arrangement. Our results suggest that the WspA periplasmic and HAMP domains are not essential for Wsp function or correct subcellular localization of WspA. We continue to test the idea that the unusual localization properties of the Wsp system are important for its function.

1Güvener ZT, Tifrea DF, Harwood CS. (2006) Mol Microbiol. 61:106-18. 2Güvener ZT, Harwood CS. (2007) Mol Microbiol. 66:1459-73. 3Cowles KN, Gitai Z. (2010) Mol Microbiol. 76:1411-26.

Lab: Caroline Harwood ____

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BLAST XI Poster #32 INVESTIGATING DEPENDENCE OF CHEMORECEPTOR STRUCTURE AND FUNCTION ON THE LIPID ENVIRONMENT Divya N. Amin and Gerald L. Hazelbauer Department of Biochemistry, University of Missouri, Columbia, MO 65211

A significant majority of bacterial chemoreceptors are transmembrane proteins but there is little information about the influence of specific lipid environments on receptor structure and function. It has long been known that detergent-solubilized receptors do not perform several crucial functions, presumably reflecting structural disruption, but the degree to which receptor structure and activity is dependent on particular features of the membrane into which they are inserted has not been defined. We are investigating these issues by assaying chemoreceptor Tar from E. coli incorporated into Nanodiscs made with different lipids and in different lipid ratios. Nanodiscs are small (~10 nm) plugs of lipid bilayer rendered water-soluble by an annulus of “membrane scaffold protein”, structures that can be made with many combinations of lipids. We have found that Tar is tolerant of significant variability in its lipid environment but functions best in membranes that approximate the composition of native E. coli cytoplasmic membrane. To determine which features of the native lipid environment are important for receptor structure and activity, we are using synthetic lipids to vary fatty acid chains or head groups while holding other variables constant. In this way we are investigating the effects of membrane fluidity, propensity for bilayer formation and head group properties on chemoreceptors. Lab: Gerald Hazelbauer ____

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BLAST XI Poster #33 ATTEMPT TO INVESTIGATE DYNAMIC COMFORMATIONL CHANGES IN FliG USING SOLUTION NMR SPECTROSCOPY Mizuki Gohara1, Rei Abe-Yoshizumi1, Yoshikazu Hattori2,3, Chojiro Kojima2,3, Michio Homma1 1Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan; 2Insititute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka, 565-0871, Japan; 3Graduate School of Biological Sciences, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

The flagellar motor in Vibrio species is a molecular machine powered by an electrochemical potential gradient of sodium ions. This Na+-driven motor is composed of a stator and a rotor. Stator is composed of four PomA and two PomB. FliG is a rotor component which forms C ring with FliM and FliN. The C ring is attached to the cytoplasmic side of MS ring of basal body. FliG is believed to involve directly in torque generation and in switching of the rotation direction. Recently, crystal structure of full-length FliG, which is divided into three domains, N-terminal domain (N), Middle-domain (M) and C-terminal domain (C), from A. aeolicus was published. In this report, it was suggested that dynamic conformational changes are carried out in switching and torque generation. To investigate the physical character of FliG, we established the over production system and constructed three recombinant proteins, full-length FliG (FliG), G122-FliG (FliG-MC) and G214-FliG (FliG-C). We carried out DSC (Differential scanning calorimetry) on these proteins. Furthermore we measured 1H-15N HSQC spectrum of these proteins using solution NMR. FliG-C gave the promising profiles of peaks for the assignments. NMR is ongoing to detect structural changes of FliG-C in which mutations affect the motor function.

Lab: Michio Homma____

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BLAST XI Poster #34 INTERACTION BETWEEN THE ROTOR PROTEIN FliG AND STATOR IS ESSENTIAL FOR THE FUNCTIONAL MOTOR ASSEMBLY OF NA+-DRIVEN FLAGELLA IN VIBRIO ALGINOLYTICUS Seiji Kojima1, Natsumi Nonoyama1, Norihiro Takekawa1, Hajime Fukuoka2 and Michio Homma1 1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan; 2Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, 980-8577, Japan.

Torque of the bacterial flagellar motor is generated by the rotor-stator interaction coupled with

specific ion translocation through the stator channel. To produce a fully functional motor, multiple stator units must be properly incorporated around the rotor to engage rotor-stator interactions. However, the mechanism of stator assembly remains unknown. Here, we approached this question by using the Na+-driven polar flagellar motor of Vibrio alginolyticus, whose assembly of the PomA/PomB stator complex can be easily evaluated by its polar localization. We mutated a rotor protein FliG, which is located at the C ring of the basal body and closely participated in torque generation, and found that point mutations L259Q, L270R and L271P completely abolished both motility and polar localization of stator without affecting flagellation. Likewise, mutations V274E and L279P severely affected motility and stator assembly. These residues are localized at the core of globular C-terminal domain of FliG when mapped onto the crystal structure of FliG from Thermotoga maritima, suggesting that mutations induce a quite large structural alteration at the interface responsible for rotor-stator interaction. To test this idea from the stator side, mutations that abolished motility were introduced into the cytoplasmic region of PomA, and their effects on motility and stator assembly were investigated. Results showed that mutations H136Y, R215E and D220K abolished motility and reduced polar localization of stator, further demonstrating the importance of the rotor-stator interaction for stator assembly into the motor. Our study suggests that FliG functions as the target of the stator at the rotor side.

Lab: Michio Homma____

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BLAST XI Poster #35 ROLE OF THE PROPOSED HELICOBACTER PYLORI ENERGY SENSOR TLPD IN VIVO AND CHARACTERIZATION OF PROTEIN-PROTEIN INTERACTIONS OF TlpD Wiebke Behrens1*, Tobias Schweinitzer1*, Daniela Goeppel1, Peter Loewen2, Jonathan McMurrry3, Christine Josenhans1 1Department for Medical Microbiology and Hospital Epidemiology, Medical School Hannover, Carl-Neuberg-Straße 1, 30625 Hannover, Germany 2 Department of Microbiology, University of Manitoba, Canada 3 Kennesaw State University, Kennesaw, U.S.A.

The human gastric colonizer Helicobacter pylori requires motility and taxis in infection and

persistence. In particular, energy taxis or pH-taxis were shown to be essential for the colonization. H. pylori does not appear to possess Aer-like sensors which could act as energy sensors, however we identified a novel type of soluble cytoplasmic taxis sensor, TlpD, which has a proposed role in energy taxis. We suggested that energy taxis is dominant over other modes of taxis in H. pylori in vitro and in vivo (Schweinitzer et al., 2008).

Previous in vivo colonization data of a H. pylori TlpD mutant in a mouse model (Williams et al.,

2007) indicated that TlpD is not essential in vivo. However, gastric conditions and in the mouse are quite different from humans. Therefore, the role of TlpD in vivo remained unclear. We have now investigated the role of TlpD in a Mongolian gerbil infection model, which more closely mimicks the human gastric physiology, including a very low pH in the gastric lumen. The results indicate that TlpD has a decisive function during initial colonization, but also during bacterial persistence.

One hypothesis how H. pylori TlpD mediates energy sensing would be by interaction with

enzymes which enable metabolism. This dual role of proposed partner proteins would enable energy sensing to be closely coordinated with the activity or inhibition of metabolic properties. We have tested this hypothesis by performing protein-protein interaction studies including TlpD as a partner and by testing the energy sensing abilities of H. pylori mutants in proposed partner proteins. We identified and confirmed protein interaction partners of TlpD. These results and the taxis phenotype of mutants in TlpD partner proteins will be discussed. Williams SM, Chen YT, Andermann TM, Carter JE, McGee DJ, Ottemann KM, 2007. Infect Immun. 75:3747-57 Schweinitzer T, Mizote T, Ishikawa N, Dudnik A, Inatsu S, Schreiber S, Suerbaum S, Aizawa S, Josenhans C, 2008. J. Bacteriol. 190:3244-55. Lab: Christine Josenhans____

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BLAST XI Poster #36 AUTOINDUCER-MEDIATED SIGNALING IN VIBRIO HARVEYI Claudia Anetzberger, Nina Kramer and Kirsten Jung Munich Center for integrated Protein Science (CiPSM) at the Department of Biology I, Microbiology, Ludwig-Maximilians-Universitaet Muenchen, Grosshaderner Straße 2-4, 82152 Martinsried

Bacteria produce and excrete signaling molecules, so called autoinducers (AIs), which allow them to monitor their population density and/or their environment in a process best known as Quorum Sensing. In Vibrio harveyi AIs regulate type III secretion, siderophore production, biofilm formation, exoprotease activity and bioluminescence. This bacterium responds to three different classes of AIs, HAI-1, an N-(3-hydroxybutyryl)-D-homoserine lactone, AI-2, a furanosylborate diester and CAI-1, a 3-hydroxytridecan-4-one. In order to understand how single cells behave within an AIs-activated community, AIs-induced processes in V. harveyi were investigated in a homogeneous environment over time. Analysis of wild-type single cells with respect to AIs-induced bioluminescence revealed that even at high cell densities only 69% of the cells of a population produced bioluminescence, 25% were non-luminescent, and 6% were dead. Moreover, fractionation of the population was found for other AIs-controlled promoters. These results indicated phenotypic heterogeneity of a genetic homogeneous population. An artificial increase of the AIs concentration in the wild-type resulted in an all-bright cell population similar as observed for a luxO mutant. Both wild-type and mutant switched to biofilm formation at high cell density. However, the capability of the luxO mutant to produce biofilm was significantly reduced in comparison to wild-type. These data suggest that a high-dense population of the non-differentiating bacterium V. harveyi takes advantage of division of work. In addition, evidence is provided for the temporal variation of the AIs concentration within a growing population. Moreover, in vitro phosphorylation experiments of the complete signaling cascade indicate a differentiated response to various ratios of the three AIs. Our results demonstrate that not the cell density is important, but availability and concentration of the AIs at certain growth phases influence AIs-dependent gene expression. Mechanisms are discussed how V. harveyi adjusts the externally available AIs.

Lab: Kirsten Jung ____

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BLAST XI Poster #37 SPATIAL AND TEMPORAL REGULATION OF BACTERIAL MOTILITY: ANALYSIS OF THE cyclic di-GMP MODULATING PROTEIN FimX Ruchi Jain1, Barbara I. Kazmierczak1,2 1Department of Internal Medicine (Infectious Diseases), 2Section of Microbial Pathogenesis, Yale University School of Medicine, New Haven, CT 06520

Pseudomonas aeruginosa, a Gram-negative bacterium causes opportunistic tissue destructive infections chronically colonizes many patients with cystic fibrosis. Two surface organelles, type IV pili and flagella, have been shown to be important for bacterial infection and colonization of the host. These organelles are required for bacterial adherence to biotic and abiotic surfaces, and subsequent biofilm formation. Type IV pilus assembly and twitching motility require FimX, a dual domain GGDEF/EAL protein that binds and hydrolyzes cyclic-di-GMP. FimX shows a predominantly unipolar localization in actively twitching cells.

In this study, an extragenic suppressor screen was carried out in fimX deletion strain in order to

identify regulators of pilus assembly working in concert with FimX. A random transposon insertion library was generated in ΔfimX and the transformants were screened visually for restoration of twitching motility. The suppressor mutations were mapped to several genes associated with small colony variant (SCV) phenotypes. Whole cell c-di-GMP levels were elevated in these mutants as compared to the ΔfimX strain. Detailed analysis showed that the suppressor mutations restored surface pilus assembly, but pili originated from both polar and non-polar sites on the bacteria surface. These strains were able to plaque a Type IV pilus specific phage with wild type efficiency, suggesting that pili are functional.

We propose a model in which high affinity cyclic-di-GMP binding by FimX facilitates type IV pilus

assembly at the bacterial pole at the relatively low whole cell concentrations of cyclic-di-GMP present in P. aeruginosa cells upon surface attachment. Elevated whole cell cyclic-di-GMP levels in our suppressors circumvent the requirement for FimX, but Type IV pilus assembly now occurs at random sites on the cell surface. Further work is focused on identifying these cyclic-di-GMP responsive proteins in the ΔfimX suppressors, and understanding how they and FimX work in concert to lead to pilus assembly at a single pole.

Lab: Barbara Kazmierczak ____

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BLAST XI Poster #38 MUTANT ANALYSIS REVEALS CORRELATION BETWEEN GLIDING MOTILITY AND PROTEIN PHOSPHORYLATION IN MYCOPLASMA PNEUMONIAE Clinton A. Page and Duncan C. Krause Department of Microbiology, University of Georgia, Athens, GA, USA

Mycoplasma pneumoniae is a human pathogen and etiologic agent of primary atypical pneumonia and tracheobronchitis. While severely limited in biosynthetic capabilities, M. pneumoniae and several related species exhibit a novel form of gliding motility along the liquid/solid interfaces of host respiratory epithelium. In vitro studies reveal that gliding occurs only in the direction of a polar terminal structure also involved in cell division as well as adhesion to host cell receptors and the gliding substrate.

Given that genes known to be involved in gliding in other organisms are absent in the M.

pneumoniae genome, random transposon mutagenesis was employed to generate mutants with gliding-deficient phenotypes. Transposon insertions in the only annotated serine/threonine kinase (prkC; MPN248) and its cognate phosphatase (prpC; MPN247) in M. pneumoniae resulted in significant and contrasting effects on gliding frequencies. prkC mutants glided at approximately one-third the frequency of wild type cells, while prpC mutants glided more than twice as frequently as wild type cells. Furthermore, the removal of a phosphate source from gliding media resulted in a reduced gliding frequency in wild type cells but not in either mutant strain, suggesting that phosphorylation by PrkC has a role in regulation of gliding but is not a power source for gliding. The combined application of western immunoblotting and Pro Q Diamond phosphoprotein staining identified several high molecular weight proteins as apparent targets for PrkC phosphorylation, including HMW2, which is known to localize to the M. pneumoniae terminal structure and has been previously shown to contain phosphoserine and phosphothreonine.

In order to confirm the correlation between phosphorylation / dephosphorylation and gliding

frequency, the prkC and prpC mutants were complemented with wild type copies of their respective disrupted alleles by transposon delivery. We present evidence that wild type gliding frequencies and phosphorylation levels are returned to the wild type standard in the complemented prpC mutant. Attempts to detect the corresponding gene product in M. pneumoniae and enriched fractions thereof were unsuccessful, despite the availability of antibodies that react strongly with synthetic PrpC peptides, suggesting that the phosphatase is a low-abundance protein in M. pneumoniae.

Cumulatively, these data contribute to an eventual testable model to define the precise

mechanism of gliding in the M. pneumoniae group of mycoplasmas. Additionally, a proven link between motility and phosphorylation opens the discussion of a possible signaling system associated with M. pneumoniae gliding motility, which is not predicted from the genome as currently annotated.

Lab: Duncan Krause ____

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BLAST XI Poster #39 CARBON STORAGE REGULATOR A (CsrABb) IS A REPRESSOR OF BORRELIA BURGDORFERI FLAGELLIN PROTEIN FlaB ChingWooen Sze1, Dustin Reed Morado2, HongBin Xu1, Jun Liu2, and Chunhao Li1* 1. Department of Oral Biology, the State University of New York at Buffalo, New York 14214 2. Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Texas 77030

CsrA, a key component of the Escherichia coli Csr system, regulates gene expressions post-transcriptionally via affecting either the mRNA stability or the translation initiation. A recent study describes that the over-expression of BB0184 (CsrABb), a homolog of CsrA, significantly repressed the level of FlaB, a major flagellin protein of the Lyme disease spirochete Borrelia burgdorferi. However, the mechanism involved and its overall impact on the other flagellar proteins of B. burgdorferi have not been studied yet. In this report, we attempt to decipher the regulatory role of CsrABb on FlaB and the potential mechanism involved by studying csrABb

-, a deletion mutant of csrABb, and csrABb+, a mutant

that over-expresses csrABb. Genetic and biochemical studies demonstrated that the level of FlaB was significantly repressed in csrABb

+ but was substantially increased in csrABb-, whereas the level of other

flagellar proteins remains unchanged in these two mutants. Consistently, cryo-electron tomography and immune fluorescence microscopic analyses revealed that the altered CsrABb in these two mutants only affects the flagellar filament, but not the other parts of a flagellum such as the flagellar basal body and hook. Further studies revealed that there are two well conserved CsrA binding sites within the leader sequence of the flaB transcript with one of them overlapping with the Shine-Dalgarno sequence, and CsrABb binds to the flaB transcript via these two binding sites and affects the level of FlaB at the translational level. The results taken together demonstrate that CsrABb is a repressor of FlaB via blocking of the translation initiation of the flaB transcript. Lab: Chunhao Li

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BLAST XI Poster #40 THE FLAGELLAR MOTOR SWITCH IS SENSITIVE TO PROTON MOTIVE FORCE Adam T Politzer, Junhua Yuan, Will Draper, Howard C Berg, Carlos Bustamante, Jan Liphardt Biophysics Graduate Group, UC Berkeley, QB3 Institute, Berkeley, CA 94720-3220

The switch complex of the bacterial flagellar motor controls the direction of motor rotation in response to varying concentrations of the signaling molecule CheY-P. We have found that the switch is also sensitive to changes in proton motive force (PMF), even when [CheY-P] is constant. We studied the E. coli switch’s response to a range of PMF levels by controlling the PMF with light using proteorhodopsin, a light-powered proton pump. Switching events in the high and medium load regimes were observed by monitoring rotation of tethered cells and beads. As the PMF decreases the counterclockwise bias of the motor increases. Interestingly, this result is in contrast to previous experiments where motor speed was reduced by increasing load, which results in a decrease in counterclockwise bias. Consequently, the PMF-dependent change in bias is not simply a product of reduced speed or proton flux. We have also observed PMF-dependent discrete steps in rotation velocity that likely correspond to changes in the number of torque-generating stators; stators disengage at reduced PMF, and reengage at full PMF. The observed change in motor bias is not correlated with these changes in stator number. These experiments demonstrate how chemotaxis behavior is altered as cells run out of energy. Lab: Jan Liphardt

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BLAST XI Poster #41 CRYO-ELECTRON TOMOGRAPHY OF PATHOGENIC AND SAPROPHYTIC LEPTOSPIRA REVEALS NOVEL STRUCTURES OF FLAGELLAR C-RING AND CHEMOTAXIS RECEPTOR ARRAY Gianmarco Raddi, Feng Xue, Frank Yang and Jun Liu Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77030, USA Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202, USA

Leptospira interrogans is the primary causative agent of the most widespread zoonotic disease, leptospirosis. Motility and Chemotaxis are generally recognized to be important virulence factors, while they are poorly understood, largely due to the lack of robust tools for genetic manipulation and in-depth structural insight of this pathogen. In this study, cryo-electron tomography (cryo-ET) was utilized to study the intact bacteria from two Leptospira species with the focus on flagellar motor and chemotaxis receptor array. Cryo-ET provided an unprecedented view of the flagellar machine of Leptospira, which appears to differ considerably from that of B. burgdorferi and Treponema. In particular, there are two FliN and three FliG genes in Leptospira. The presence of multiple copies of these genes may contribute to the high degree of complexity in the C-ring of the leptospiral flagellar motor. Cryo-ET allows direct visualization of methyl-accepting chemotaxis proteins (MCPs) arrays, and confirms that Leptospiral MCPs arrays were mostly located near the poles in close proximity to the flagellar motor. They not only formed a characteristically organized lattice, but also formed a novel pattern. The structural and genetic diversities of MCPs arrays may contribute to the bacterial unique strength to survive in variable natural environments. Lab: Jun Liu____

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BLAST XI Poster #42 MOLECULAR ARCHITECTURE OF STATOR ASSEMBLY IN SITU REVEALED BY CRYO-ELECTRON TOMOGRAPHY Xiaowei Zhao1, Joshua E. Pitzer2, Md A. Motaleb2, Jun Liu1 1 Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Texas 77030 2 Department of Microbiology and Immunology, Brody School of Medicine, East Carolina University, Greenville, NC 27834

Bacterial flagella are helical filaments propelled by rotary motors embedded in the bacterial cell envelope. Flagellar motion is powered by transmembrane ion flux through the stator complexes that push on a central rotor. Flagellar motor structure has been the subject of extensive analysis by X-ray crystallography and electron microscopy, yet the mechanism of flagellar rotation remains elusive. This is partly because the lack of structural information about the torque generating unit – stator and its interaction with the rotor during flagellar rotation. In this study, targeted mutagenesis and high-throughput cryo-electron tomography (cryo-ET) methodologies were utilized to determine in situ flagellar motor structures by using Borrelia burgdorferi (Lyme disease pathogen) as the model system. A motB mutant (motB-) was successfully constructed using a newly developed non-polar gene inactivation system that does not affect downstream gene expression. The non-polar motB- cells synthesize periplasmic flagella but were paralyzed. The defect was corrected when the mutant was complemented (motB+) in trans. Three-dimensional (3-D) structures of wild-type, motB-, and motB+ flagella motors were reconstructed by cryo-ET and 3-D subvolume averaging. The comparative analysis of motor structures from wild-type and motB- cells indicates that a distinct stator ring composed by 16 subunits is embedded in the cytoplasmic membrane of wild-type cell. Our results provide a better understanding of the stator structure and the stator–rotor interaction in the in situ motor, which underlies the fundamental mechanism of flagella rotation. Lab: Jun Liu____

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BLAST XI Poster #43 DISCOVERY OF NOVEL CHEMO-EFFECTORS FOR E. COLI CHEMORECEPTOR Tar Shuangyu Bi1, Daqi Yu1,2, Guangwei Si1, Chunxiong Luo1, Yuhai Tu1,3 Luhua Lai1,2* 1Center for Theoretical Biology, Peking University, Beijing 100871, China 2BNLMS, State Key Laboratory for Structural Chemistry of Unstable and Stable Species, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China 3IBM T. J. Watson Research Center, Yorktown Heights, New York 10598 *Email: lhlai@pku.edu.cn

E.Coli cells have the ability to mediate chemotaxis to various chemical signals in the environment. They can swim towards or away from a number of ligands by temporal comparing ligand concentrations. Chemoreceptors, which locate on the upstream of chemotaxis signal transduction pathway, have the function of sensing, adaptation and signal transduction. Tar, the major chemoreceptor in E.Coli, senses native ligands like aspartate, maltose, nickel, and a few other chemical compounds. In order to understand the relationship between Tar ligand binding and signal transduction, we have tried to screen for novel chemicals that can bind to Tar and give different biological responses. Virtual screen by molecular docking was done using the Available Chemical Directory database and the 3-dimensional structure of the periplasmic domain of Tar to search for compounds that might bind to Tar. The periplasmic domain of Tar was expressed and purified in vitro, and its secondary structures, aggregation state, and concentration dependent oligomeric structure were characterized. Isothermal Titration Calorimetry (ITC) was used to measure the binding affinity of selected compounds with the protein. Among the eighty compounds tested, sixteen showed significant binding in the ITC study. The responses of E. Coli cells to the selected compounds were also investigated using microfluidic assay. Five compounds were found to function as novel attractants for Tar receptor. The concentration response ranges of cells to those novel attractants were quantitatively measured, and the distribution profile in each attractant gradient was analyzed. The relationship between chemoreceptor ligand binding and downstream signal transduction is under investigation. Lab: Luhua Lai____

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BLAST XI Poster #44 THE ROLE OF THE CYTOPLASMIC AROMATIC ANCHOR OF TRANSMEMBRANE HELIX 2 (TM2) OF E. COLI Tar IN TRANSMEMBRANE SIGNALING Christopher Adase and Michael Manson Department of Biology, Texas A&M University, College Station, TX 77843

The Tar chemoreceptor of Escherichia coli mediates responses to two different attractants: L-aspartate, which binds directly to the receptor; and maltose, which interacts with the receptor indirectly through maltose-binding protein. Ni2+, a repellent sensed by Tar, also interacts directly with the periplasmic domain. The most likely mechanism for transmembrane signaling in response to attractant binding involves a downward, piston-like displacement of TM2 vertical to the plane of the membrane. Draheim et al. (2005 & 2006) showed that the aromatic anchor of E. coli Tar, composed of the residues Trp-209 and Tyr-210, is essential for maintaining a normal signaling state. They also showed that moving the aromatic anchor in single-residue steps influences the signaling state of the receptor in a predictable fashion. My research involves substituting the native residues in the aromatic anchor of Tar with different combinations of aromatic and non-aromatic residues to determine which properties of the aromatic anchor are essential to its normal function. Defects in receptor function caused by certain combinations of residues at the anchor positions were alleviated by moving the defective anchors in single-residue steps. The results reported here identify the structural constraints that operate on the aromatic anchor E. coli Tar. A TM2 aromatic anchor is present in the other chemoreceptors and all of the transmembrane sensor kinases of E. coli, but the anchors of individual proteins differ widely in their residue composition and spacing. This diversity suggests that the aromatic anchor of each TM2 may be “tuned” to the transmembrane signal it propagates. Lab: Michael Manson____

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BLAST XI Poster #45 THE MECHANISM OF AI-2 CHEMORECEPTION IN ESCHERICHIA COLI Manjunath Hegde*, Derek Englert*, William Cohn#, Sneha Jani#, Arul Jayaraman*, Michael Manson# Departments of Chemical Engineering* and Biology#, Texas A&M University, College Station, TX 77843

The general quorum-sensing autoinducer AI-2 is a 5-carbon compound derived from the ribose moiety of S-adenosylhomocysteine, the product remaining after methyl group donation by S-adenosylmethionine. AI-2 can exist in two interchangeable enantiomeric forms, one of which is complexed with borate and the other of which is borate-free. The borate derivative binds to the periplasmic LuxP protein of Vibrio harveyi and induces bioluminescence in that organism. The borate-free enantiomer binds to the periplasmic LsrB protein of Salmonella enterica serovar Typhimurium. LsrB is the ligand-recognition component of an ABC transporter for AI-2. We have used two microfluidic chemotaxis assays and the traditional capillary assay to show that AI-2 is a potent chemoattractant for E. coli strain RP437. Mutational analysis shows that LsrB is essential for AI-2 chemotaxis, but uptake of AI-2 into the cell is not. A strain lacking the Tsr chemoreceptor for L-serine also is defective for AI-2 chemotaxis. Our hypothesis is that AI-2-bound LsrB interacts directly with the periplasmic domain of Tsr to initiate an attractant response, in much the same manner as the maltose-binding protein (MBP) interacts with the E. coli L-aspartate chemoreceptor Tar. We previously showed that aspartate-saturated Tar can still mediate a chemotaxis response to maltose, indicating that aspartate and MBP can signal simultaneously through opposing subunits of the Tar chemoreceptor. We are conducting similar experiments to see whether saturating levels of L-serine block chemotaxis to AI-2. We previously used the SPOCK software to derive an energy-minimized MBP-Tar docking model. We have now used the SPOCK software to simulate the LsrB-Tsr interaction, using the MBP-Tar docked complex as a template. We are carrying out alanine-scanning mutagenesis on the regions of the LsrB and Tsr proteins that come into closest contact in the simulated LsrB-Tsr complex to identify residues that are important for the interaction of the two proteins. We hope to have preliminary results of the competition experiments and the mutational analysis to present at BLAST XI. Lab: Michael Manson____

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BLAST XI Poster #46 THE BORRELIA BURGDORFERI DIGUANYLATE CYCLASE, Rrp1, CONTROLS IMPORTANT STEPS IN THE ENZOOTIC CYCLE OF LYME DISEASE SPIROCHETES Jessica L. Kostick1, Lee T. Szkotnicki1, Elizabeth A. Rogers1, & Richard T. Marconi1,2 1Department of Microbiology & Immunology, 2Center for the Study of Biological Complexity, Medical College of Virginia at Virginia Commonwealth University, Richmond, VA

Response regulator protein 1 (Rrp1) is a diguanylate cyclase that regulates approximately 10% of the Borrelia burgdorferi genome. Diguanylate cyclases produce c-di-GMP, an important bacterial secondary messenger. C-di-GMP plays a critical role in the pathogenesis of numerous bacteria. In other bacteria the study of diguanylate cyclases is complicated by significant pathway redundancy. In contrast, Rrp1 is the sole diguanylate cyclase of B. burgdorferi, making the Borrelia a simplified and ideal model for c-di-GMP signaling. In this study, an infectious B. burgdorferi B31-∆rrp1 mutant (B31-∆rrp1) and an rrp1 overexpressing mutant (B31-OV) were constructed and evaluated for their ability to progress through the Lyme enzootic cycle. Murine and Ixodes tick infection analyses revealed Rrp1 to be essential for spirochete acquisition by ticks but not for transmission from ticks to mammals. Interestingly, overexpression of rrp1 abolished murine infection but the strain maintained tick colonization capabilities, demonstrating Rrp1 to be a unique and critical regulator of the B. burgdorferi enzootic cycle. Examination of motility and chemotactic responses of in vitro cultivated spirochetes demonstrated rrp1 to influence motility and/or chemotaxis. These results provide clear evidence that Rrp1 and, by extension c-di-GMP, have significant roles in Lyme spirochete enzootic cycle, potentially via its regulation of motility and chemotactic responses. Lab: Richard Marconi____

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BLAST XI Poster #47 INVESTIGATION OF THE c-di-GMP PHOSPHODIESTERASE PdeB REVEALS A CRITICAL ROLE IN PROPER MOTILITY IN THE BACTERIA BORRELIA BURGDORFERI Lee T. Szkotnicki, Jessica L. Kostick, John C. Freedman & Richard T. Marconi Department of Microbiology & Immunology, Center for the Study of Biological Complexity, Medical college of Virginia at Virginia Commonwealth University, Richmond, VA

Cyclic di-GMP is a bacterial second messenger molecule that regulates several important cellular functions associated with pathogenicity. Borrelia burgdorferi, the causative agent of Lyme disease, provides an ideal system in which to study cyclic di-GMP mediated regulation, as its genome contains only a single copy of the genes required to synthesize and degrade cyclic di-GMP. Cyclic di-GMP is generated from two molecules of GTP by diguanylate cyclases (DGCs) and subsequently metabolized into two molecules of GMP by the activity of phosphodiesterases (PDEs). There are two families of cyclic di-GMP phosphodiesterases, those which harbor EAL and HD-GYP domains. While the EAL domain containing phosphodiesterase (PdeA) has been studied in B. burgdorferi, the role of the HD-GYP domain containing phosphodiesterase(PdeB) remains unexplored. To investigate the role of PdeB in Borrelia pathogenesis, a pdeB deletion mutant (B31ΔpdeB) was generated using allelic exchange mutagenesis. B31ΔpdeB strain displayed drastically altered motility patterns. We have shown that these cells, rather than exhibiting the normal run-flex-reverse-run motion of wild type cells are in a state of constant flexing. Consistent with this, B31ΔpdeB displayed a greatly reduced ability to migrate using in vitro swarm assays. Despite changes in motility and swarming, B31ΔpdeB retained its ability to establish an infection in mice. Analyses of tissues and organs from infected mice revealed that B31ΔpdeB disseminated efficiently to distal sites. Finally, B31ΔpdeB stain was able to transit from mice into ticks. Taken together, these data suggest PdeB plays an important role in controlling motility of B. burgdorferi cells while being dispensable for infection of both ticks and mammals.

Lab: Richard Marconi____

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BLAST XI Poster #48 KINETIC SIMULATIONS OF INTERACTIONS AMONG FLAGELLAR EXPORT APPARATUS PROTEINS: IS COMPLEXITY REALLY COMPLEX? Joshua W. Francis,1 John D. Salerno2 and Jonathan L. McMurry1

1Department of Chemistry & Biochemistry and 2Department of Biology Kennesaw State University, Kennesaw GA 30144

The bacterial flagellum contains its own type III secretion apparatus that allows for self-assembly by effecting export of more than 20,000 proteins. At a defined point in flagellar morphogenesis, a switch in specificity of substrates secreted by the apparatus occurs (from rod and hook to filament proteins). The switch involved binding of FliK to FlhB and likely other interactions. Our recent study (Morris, et al (2010) Biochemistry 49(30):6386-93) described analysis by optical biosensing of the K-B interaction using Salmonella enterica proteins. Binding was found to be complex, with a faster-than-measurable association state and amplitude differences between association and dissociation states. Overall affinities among wild-type and variant FlhBs were similar. Those results suggested a number of complexities including possible oligomerization of FlhB and a conformational change upon binding as well as the involvement of other apparatus proteins in switching. Here we report on kinetic simulations of finer K-B data and other pairwise interactions. In allowing for degradation of the immobilized ligand on the sensor surface, the biologically relevant binding was found to be less complex than observed. We are developing a general model for accommodating sensor-specific anomalies in binding data. We also report results from our continuing kinetic survey of flagellar protein interactions.

Lab: Jonathan McMurry____

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BLAST XI Poster #49 CHEMOTACTIC RESPONSE TO ANAEROBIC ELECTRON ACCEPTORS INVOLVES NEW TYPES OF CHEMORECEPTORS IN SHEWANELLA ONEIDENSIS

Claudine Baraquet, Chantal Iobbi-Nivol, Vincent Méjean and Cécile Jourlin-Castelli Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, CNRS, Marseille, France. Aix-Marseille Université, France.

Shewanella oneidensis is known to use a wide range of terminal electron acceptors for respiration. Moreover S. oneidensis shows a chemotactic behaviour towards anaerobic electron acceptors such as nitrate, nitrite, fumarate, dimethylsulfoxide (DMSO) and trimethylamine N-oxide (TMAO)1,2. We demonstrated that this response is governed by an energy taxis mechanism1. Indeed deletion of the torA and dmsA genes, respectively encoding the TMAO and the DMSO reductase enzymes, abolished the tactic response towards TMAO and DMSO. Moreover inactivation of the molybdoenzymes (TMAO, DMSO and nitrate reductases), by addition of tungstate, an antagonist of molybdate, abolished the response of S. oneidensis towards TMAO, DMSO and nitrate. The activity of the terminal oxydoreductases is therefore required for exogenous electron acceptor taxis1. We have then shown that addition of nigericin, described to collapse the ∆pH, significantly reduces the tactic response of S. oneidensis towards the different electron acceptors. This result not only confirms that the chemotactic behaviour of S. oneidensis towards the different electron acceptors is governed by energy taxis, but also indicates that the signal is mainly the ∆pH component of the proton motive force generated by respiration1. Surprisingly, a strain deleted of the four PAS-containing chemoreceptors still responds to electron acceptors. We identified one major and four minor chemoreceptors involved in this energy taxis behaviour1. Interestingly the major energy taxis chemoreceptor (SO2240) contains a Cache domain. These results indicate that energy taxis can be mediated by new types of chemoreceptors1,3,4. References : 1. Baraquet, C., Théraulaz, L., Iobbi-Nivol, C., Méjean V., Jourlin-Castelli, C., 2009. Unexpected chemoreceptors mediate energy taxis towards electron acceptors in Shewanella oneidensis. Mol. Microbiol. 73, 278-290. 2. Bencharit, S., Ward, M.J., 2005. Chemotactic responses to metals and anaerobic electron acceptors in Shewanella oneidensis MR-1. J. Bacteriol. 187, 5049-5053. 3. Alexandre, G., 2010. Coupling metabolism and chemotaxis-dependent behaviours by energy taxis receptors. Microbiol. 156, 2283-2293. 4. Schweinitzer, T., Josenhans, C., 2010. Bacterial energy taxis: a global strategy? Arch. Microbiol. 192, 507-520. Lab: Vincent Méjean____

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BLAST XI Poster #50 DISSECT THE MECHANISM OF BORRELIA CHEMOTAXIS AND MOTILITY AND THE RELATIONSHIP BETWEEN THE VIRULENCE AND CHEMOTAXIS/MOTILITY Tao Lin, Lihui Gao, and Steven J. Norris Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston

The mechanisms of pathogenesis in Borrelia burgdorferi are largely unknown. The ability of this spirochete to migrate to distant sites in the tick and mammalian host is likely dependent on a robust chemotaxis response and motility. B. burgdorferi contains multiple copies of the chemotaxis genes cheR, cheW, cheB, cheY, cheA, and cheX. Multiple chemotaxis proteins may provide diversity in terms of function and/or structural location, or may be differentially expressed under varied physiological conditions. Proteins involved in motility are encoded by 39 genes including 37 flagellar & motor genes and 2 flagellin genes, whereas chemotaxis proteins are encoded by 18 genes including 13 chemotaxis genes and 5 chemotaxis receptor genes. To dissect the mechanism of Borrelia chemotaxis and motility and the relationship between the virulence and chemotaxis/motility, we examined transposon mutants in 24 chemotaxis/motility genes including 9 motility genes, 11 chemotaxis genes and 4 chemotaxis receptor genes in the transformable and infectious B. burgdorferi strain 5A18NP1. The infectivity of these mutants was determined using a signature-tagged mutagenesis (STM) procedure in C3H/HeN mice and a newly developed, high throughput Luminex procedure. Among the mutants in 21 genes tested, mutations in flgI and flgJ exhibited reduced infectivity. Mutants in the 18 genes (fliG-1, fliH, fliI, flaA, flbA, cheA-1, cheA-2, cheB-1, cheB-2, cheR-2, cheW-2, cheW-3, cheX, cheY-2, mcp-1, mcp-3, mcp-4, and mcp-5) showed no infectivity, indicating that these genes are required for full infectivity in Borrelia. Examination of infectivity of 3 mutants (fliW-1, fliZ, and cheY-1) is in progress. In examining morphology under dark-field microscopy, 18 mutants did not exhibit obvious morphology defects. Seven mutants (fliH, fliI, flbA, flaA, cheA-2, cheB-2, and cheR-2) exhibited elongated, string-like and/or rod-shaped morphology. In terms of motility, mutations in 13 genes did not induce obvious motility defects, 7 mutants (flaA, flgI, fliG-1, fliW-1, cheA-2, cheR-2, and mcp-5) showed a reduced motility, and string-like mutants (flbA, fliH, and fliI) were nearly non-motile, ‘trembling’ in a few sites of the cell. The string-shaped or rod-shaped mutants often bend at the cell center. The swimming ability of these mutants was evaluated by measuring their velocity in highly viscous media such as 1% methylcellulose. Seven mutants (fliH, fliI, flaA, flbA, flgI, cheX, and mcp-4) appeared to be incapable of translating motion, 7 mutants (fliW-1, cheA-2, cheR-2, cheW-2, cheW-3, cheY-2, and mcp-5) exhibited reduced motility, and inactivation of 6 genes (flgJ, fliZ, cheA-1, cheB-1, cheB-2, mcp-1, and mcp-3) did not induce decreased motility in 1% methycellulose medium. Based on the genetic, structural, morphology, motility, and infectivity information we obtained, twenty-seven chemotaxis and motility mutants are being examined by Cryo-EM to characterize associated structural defects.

Lab: Steven Norris ____

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BLAST XI Poster #51 BINDING OF CheY TO FliM IS NECESSARY BUT NOT SUFFICIENT TO SWITCH FLAGELLUM ROTATION Andres Campos, Philip Matsumura and Christopher O’Connor (1) University of Illinois at Chicago Department of Microbiology and Immunology (M/C 790) 835 S.

Wolcott, Chicago IL 60612 (2) North Central College, Department of Biology. 30 N. Brainard St. Naperville IL, 60540

We have obtained CheY mutants that show higher activity than wild type CheY (WT CheY): CheYL24Q/E27G/A103V and CheYL24Q/K26N/E27G/E35V (Triple [TM] and Quadruple [QM] mutants respectively). However, these mutants are not constitutive, meaning they still require phosphorylation to activate the change of direction of the flagella. The biochemical characterization of these mutants suggests that the activation occurs in two steps and not in one step. Our mutants (TM and QM CheY) are able to bind to FliM with higher affinity via in vitro binding assays, as well as in vivo Fluorescence Resonance Energy Transfer (FRET) experiments. We were unable to observe any significant change in the reversal of flagellar rotation due to TM and QM CheY when compared to WT CheY. Therefore, we believe that binding of CheY to FliM is necessary but not sufficient to switch flagellum rotation. Lab: Christopher O’Connor ____

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BLAST XI Poster #52 CHEMOTAXIS BEHAVIORS OF THE ESCHERICHIA COLI POPULATION IN SPATIALLY AND TEMPORALLY VARYING ENVIRONMENTS Xuejun Zhu1, Guangwei Si1, Qi Ouyang1, Chunxiong Luo1, Yuhai Tu1, 2 1Center for the Theoretical Biology, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing, 100871, China.2 T. J. Watson Research Center, IBM, P.O. Box 218, Yorktown Heights, NY 10598

Bacteria have evolved the ability to process diversified environmental signals and to respond

accordingly to improve their chances of survival. Escherichia coli implement a robust chemotaxis pathway to guide its motion towards favorable chemical conditions. Here, we study how E. coli behaves in presence of spatio-temporally varying attractant source and different stimulus waveforms. We develop a unique microfluidic system in which a controlled sub-mm chemical gradient with tunable frequency is established by integrating time-varying perfusion, on-chip mixture, and agarose-filtered diffusion. Measuring the bacterial density profile in response to periodic stimuli of various cycle lengths reveals that the E. coli population response is highly frequency dependent. At low cycle frequency, the E. coli population synchronizes with the attractant waveform, consistent with the response to quasi-stationary gradient. In contrast, under fast-changing environment, the population response is out of synchrony with the attractant waveform.

Lab: Qi Ouyang ____

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BLAST XI Poster #53 ROLE OF THE mqsRA OPERON AND REACTIVE CARBONYL SPECIES IN FLAGELLA EXPRESSION OF ESCHERICHIA COLI K-12 Jihong Kim, Changhan Lee, and Chankyu Park Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea

The reactive carbonyl compound, e.g. glyoxal (GO), is accumulated in vivo from sugars by oxidative stress. GO is detoxified by glutathione-dependent glyoxalases and other aldehyde reductases. GO modifies proteins and nucleotides, causing cellular malfunctions. Previously, we screened for GO sensitive mutants by random insertions of transposon, TnphoA-132, to search for GO related genes. Among such mutants, the mqsA gene was found, which constitute an operon with mqsR, recently characterized as toxin-antitoxin (TA) system. MqsA functions not only as an antitoxin against toxic MqsR but also regulates its own expression as well as other genes. MqsRA regulates flhDC, the master operon of flagellar expression, via QseBC, a two-component system associated with quorum sensing. In this study, we observed that the mqsA mutant shows decreased motility, so does the expression of flhDC. Furthermore, the expression of mqsRA operon is affected by GO, implying the role of MqsRA system as a redox sensor, regulating the downstream genes including flhDC.

Lab: Chankyu Park ___

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BLAST XI Poster #54 Cis- AND trans-ACTING MUTATIONS UPREGULATING THE FLAGELLAR GENES Junghoon Lee1, Changhan Lee1, Kwang-Hee Baek2, and Chankyu Park1 1Department of Biological Sciences, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea 2Department of Life Science and Biotechnology, Kyung Hee University, Yongin 446-701, Republic of Korea

Bacteria motility is regulated by the flhDC master operon whose expression is subjected to the control of transcription factors such as OmpR, LrhA, HdfR, and HNS. Previously, motile derivatives of the poorly motile MG1655 strain of E. coli K-12 were found to contain the insertion sequences (ISs) in the regulatory region of flhDC gene. However, the relationships between the trans-acting factors and the cis-acting regulatory sequences associated with flhDC have not been clearly established. We report here that not only an integration of insertion sequences, IS1 or IS5, in the regulatory region of flhDC operon but also a mutation in lrhA gene enhances motility by relieving the transcriptional repression of flhDC operon. Furthermore, it was found that effects of the cis- and trans-acting mutations were additive, suggesting that there are more than one independent pathways for regulating flagellar expression. The molecular mechanisms involving such factors as well as their upstream effectors will be discussed.

Lab: Chankyu Park ___

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BLAST XI Poster #55 THE GacS PHOSPHORELAY OF PSEUDOMONAS AERUGINOSA Sutharsan E. Finton-James, David Young and Steven L. Porter Biosciences, College of Life and Environmental Sciences, University of Exeter, Stocker Road, Exeter, EX4 4QD, UK.

The decision between acute and chronic virulence states in the opportunistic pathogen

Pseudomonas aeruginosa is controlled by the GacS/GacA phosphorelay. Unusually this phosphorelay is modulated by input from two additional sensor kinases RetS and LadS. RetS lacks the conserved residues of the ATP binding site and is unable to autophosphorylate. However, RetS has previously been shown to bind and inhibit GacS via an unknown mechanism1. In this study, we biochemically analyse the mechanism by which RetS affects the activity of GacS and demonstrate that RetS promotes the dephosphorylation of GacS. 1.Goodman, A. L., et al. Direct interaction between sensor kinase proteins mediates acute and chronic disease phenotypes in a bacterial pathogen. Genes Dev. 23, 249-259 (2009). Lab: Steven Porter ___

BLAST XI Poster #56 TAKING CONTROL OF THE BACTERIAL FLAGELLAR MOTOR Guillaume Paradis, Mathieu Gauthier and Simon Rainville Department of Physics, Engineering Physics and Optics and Centre of Optics, Photonics and Lasers, Laval University, Québec, Québec, CANADA

The bacterial flagellar motor is a fairly complex machine embedded in the multiple layers of the bacterial membrane. That explains why, unlike many other molecular motors, it has not yet been studied in vitro. As spectacular studies of linear motors (like kinesin, myosin and dynein) have clearly demonstrated, an in vitro system provides the essential control over experimental parameters to achieve the precise study of the motor’s physical and chemical characteristics. Thus the focus of our laboratory for the past 5 years has been the development of a unique in vitro assay to study the bacterial flagellar motor.

Our setup consists of a filamentous Escherichia coli bacterium partly introduced inside a

micropipette (as illustrated below). Femtosecond laser pulses (60 fs and ~ 15 nJ/pulse) are then tightly-focused on the part of the bacterium that is located inside the micropipette. This vaporizes a small portion of the membrane, leaving an essentially permanent hole in the wall of the bacterium. Using a patch-clamp amplifier, we then apply an external voltage between the inside and the outside of the micropipette. That voltage then directly contributes to the proton-motive force that powers the flagellar motor. As we change the applied potential, variations in the motor's rotation speed are observed. The rotation speed was measured using high-speed video microscopy of fluorescently labeled filaments: image sequences from a fast EMCCD camera were analyzed with custom MatLab code. We are also investigating different ways to monitor the rotation using various nanoparticules.

In addition to granting us direct control over the proton-motive force, this in vitro assay give us

full access to the inside of the cell. We can then directly control the pH (hence the other component of the pmf) or the concentration of various proteins that the motor is exposed to. That system therefore opens a world of new possibilities. For example, we have started to study the rotation speed vs applied voltage (linear) relationship and we found that our data directly probes the dynamics of the torque generating units in the motor. It should also be possible to shine a new light on the switching mechanism. This poster will present our first quantitative results using this in vitro assay and our plans for the future.

The in vitro assay. a) Diagram our in vitro assay showing the tip of the micropipette with a filamentous bacterium squeezed in the constriction. To artificially power the motor, an electrical voltage is applied between one electrode back-inserted in the micropipette and a second electrode placed in the bath. b) Brightfield image of a typical micropipette with a bacterium in the constriction. c) Still frame from a movie showing fluorescently labeled filaments whose rotation is under the control of an external voltage. The bright spot at the tip is from all the fluorescent filaments that were stripped as the cell was pulled into the micropipette. Scale bars are 10μm.

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Lab: Simon Rainville ___

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BLAST XI Poster #57 THE NOVEL SINORHIZOBIUM MELILOTI CHEMOTAXIS PROTEIN CheS PARTICIPATES IN SIGNAL TERMINATION Gaurav Dogra1, Frauke G. Purschke2, Verena Wagner2, Martin Haslbeck3, Melanie Niemeyer1, Jonathan G. Hughes1, W. Keith Ray1, Richard F. Helm1, and Birgit E. Scharf1,2 1Virginia Polytechnic Institute and State University, Biological Sciences, Life Sciences I, Blacksburg, VA 24061, USA 2Lehrstuhl für Genetik, Universität Regensburg, D-93040 Regensburg, Germany 3Lehrstuhl für Biotechnologie, Technische Universität München, D-85747 Garching,Germany

Retrophosphorylation of the histidine kinase CheA in the chemosensory transduction chain, first discovered in Sinorhizobium meliloti, is a widespread mechanism for efficient dephosphorylation of the activated response regulator, CheY2-P. A second response regulator, CheY1, serves as a sink for surplus phosphoryl groups from CheA (1). We have identified a new component in this phospho-relay system, a small 97-aa protein, named CheS. CheS has no counterpart in enteric bacteria, but revealed distinct similarities to unassigned genes in other members of the α-subgroup of proteobacteria. Deletion of cheS causes a phenotype similar to that of a cheY1 deletion strain. Fluorescence microscopy revealed that CheS is part of the polar chemosensory cluster and that its cellular localization is dependent on the presence of CheA. In-vitro binding analysis, as well as coexpression and copurification studies give evidence of a high affinity between CheS and CheA. We also showed that the response regulator binding domain of CheA is sufficient for the formation of a stable complex. Using limited proteolysis coupled with mass spectrometric analyses we defined CheA163-256 to be the CheS binding domain, which overlaps with the N-terminal part of the previously defined CheY2 binding domain (CheA174-316) (2). The phenotype of the cheS deletion strain and its tight interaction with CheA indicate that CheS participates in signal termination. We therefore analyzed individual steps of the phosphotransfer reactions between CheA, CheY1, and CheY2 in the presence and absence of CheS. CheS has not influence on the ATP-dependent autophosphorylation of CheA or on the dephosphorylation of CheY1-P or CheY2-P. However, the phosphotransfer from CheA-P to CheY1 and CheY2 is enhanced in the presence of CheS. Our results also suggest that the retro-phosphorylation reaction from CheY2-P to CheA is facilitated by CheS. In conclusion, CheS promotes binding of CheY1 and CheY2 to CheA, thereby enabling efficient phosphotransfer from CheA-P, and more importantly, efficient retrophosphorylation from CheY2-P to CheA.

1. Sourjik, V., and Schmitt, R. (1998) Phosphotransfer between CheA, CheY1, and CheY2 in the

chemotaxis signal transduction chain of Rhizobium meliloti, Biochemistry 37, 2327-2335. 2. Riepl, H., Maurer, T., Kalbitzer, H. R., Meier, V. M., Haslbeck, M., Schmitt, R., and Scharf, B.

(2008) Interaction of CheY2 and CheY2-P with the cognate CheA kinase in the chemosensory-signalling chain of Sinorhizobium meliloti, Mol Microbiol 69, 1373-1384.

Lab: Birgit Sharf ____

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BLAST XI Poster #58 ANALYZING THE ROLE OF TWO TYPE IVb PILI SYSTEMS IN SINORHIZOBIUM MELILOTI Hardik M. Zatakia1, Cassandra E. Nelson1, Veronica M. Meier2, Birgit E. Scharf1,2 1) Department of Biological Sciences, Virginia Polytechnic Institute & State Univeristy, Blacksburg,

USA. 2) Department of Biology, University of Regensburg, Germany. The gram-negative alpha-proteobacterium Sinorhizobium meliloti fixes atmospheric nitrogen after establishing a symbiotic relationship with leguminous plants. Several factors like motility, quorum sensing, biofilm formation, and exopolysaccharide production (EPS) are important for this bacteria-plant interaction. The S. meliloti genome encodes two Type IVb pili systems, one each on the chromosome and the pSymA plasmid. We hypothesize that these pili play a role in adhesion and attachment to biotic and abiotic surfaces, and therefore support interaction with the plant host. We created mutant strains with in-frame deletions of both pilin genes (pilA1 and pilA2). Nodulation experiments with alfalfa showed that the ∆pilA1 strain is less competitive while the ∆pilA2 showed no significant phenotype. Thus, it can be concluded that pilA1 is important for adhesion to plant roots and therefore nodulation. Using transcriptional fusions with lacZ, we observed that pilA1 expression peaks during late-log to early-stationary phase in liquid cultures, while pilA2 was not expressed under the same conditions. We observed differences in pilA1 expression in two strains which also vary in their EPS production. Sm1021, the sequenced S. meliloti strain, has a mutated copy of expR which codes for the transcriptional regulator of EPS II synthesis and therefore produces dry colonies on plates. Sm1021expR+ has a functional copy of expR and produces mucoid colonies. The pilA1 expression in this strain is downregulated to basal levels. In conclusion, pilA1 expression is either directly or indirectly controlled by ExpR. Lab: Birgit Sharf ____

BLAST XI Poster #59 RESPONSE RESCALING IN BACTERIAL CHEMOTAXIS Milena D. Lazova1, Tanvir Ahmed2, Domenico Bellomo3, Roman Stocker2, Thomas S. Shimizu1 1FOM Institute for Atomic and Molecular Physics (AMOLF), Amsterdam, The Netherlands. 2Ralph M. Parsons Laboratory, Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA. 3VU University Amsterdam, Amsterdam, The Netherlands.

Adaptation of sensory systems entails two complementary processes: restoring the output activity to the prestimulus level, and rescaling of the sensitivity to subsequent stimuli. Rescaling of bacterial chemotaxis response thresholds with the background concentration (Weber’s law) was characterized in classic experiments by Adler and colleagues1. However, Weber’s law does not address the time-dependence of the response, which is crucial for gradient sensing. Recent theoretical work has sought to distinguish response-rescaling that applies only to the instantaneous responses to step stimuli (Weber’s law), and response rescaling that applies to the entire time series of response during complex stimulus waveforms2. The latter, more general type of response rescaling has been called fold-change detection (FCD), and is predicted to be a desirable property for sensory systems controlling spatial searches2

. To test whether the E. coli chemotaxis system exhibits this FCD property, we carried out in vivo

fluorescence resonance energy transfer (FRET) measurements3 with time-varying stimuli, and follow the entire time series of the response. Using α-methylaspartate as chemoattractant, we show that under rescaling of stimulus with background, not only the instantaneous response following a step, but also the entire time series of the response to arbitrary stimuli is invariant. Thus, we confirm that response-rescaling in E. coli chemotaxis exhibits FCD2. By systematically varying the background concentration, we identify the ambient concentration range over which FCD holds. Moreover, we test the chemotactic performance of swimming populations in spatial gradients using a microfluidic device4, and confirmed that the FCD property extends to the level of behavioural responses. Finally, we use a simple theoretical model5 to identify a set of requirements for this dynamic response rescaling behavior in the bacterial chemotaxis response. 1 Mesibov R, Ordal GW, & Adler J (1973) The range of attractant concentrations for bacterial chemotaxis and the threshold and size of response over this range. Weber law and related phenomena. J Gen Physiol 62(2):203-223. 2 Shoval O, Goentoro L, Hart Y, Mayo A, Sontag E, Alon U. (2010) Fold-change detection and scalar symmetry of sensory input fields Proc Natl Acad Sci U S A 107(36):15995 16000. 3 Sourjik V & Berg HC (2002) Receptor sensitivity in bacterial chemotaxis. Proc Natl Acad Sci U S A 99(1):123-127. 4 Ahmed T, Shimizu TS, & Stocker R (2010) Bacterial chemotaxis in linear and nonlinear steady microfluidic gradients. Nano Lett 10(9):3379-3385. 5 Tu Y, Shimizu TS, & Berg HC (2008) Modeling the chemotactic response of Escherichia coli to time-varying stimuli. Proc Natl Acad Sci U S A 105(39):14855-14860.

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BLAST XI Poster #60 FUNCTION AND INTERACTIONS OF RomR, A RESPONSE REGULATOR REQUIRED FOR A-MOTILITY IN M. XANTHUS Keilberg, Daniela, Leonardy, Simone, Søgaard-Andersen, Lotte Max Planck Institute for Terrestrial Microbiology, Marburg, 35035 Germany daniela.keilberg@mpi-marburg.mpg.de

M. xanthus cells possess two independent gliding machines: the adventurous (A) system and the social (S) system. A-motility allows movement of single cells, while S-motility is cell-cell contact dependent. Mutations which abolish both of these systems lead to non-motile cells, while mutations in only one of them allow bacterial cells to move by means of the intact system. While S-motility depends on extension and retraction of Type-4-pili, it is still not clear how the A-motility motor works. Current data suggest that A-motility depends on focal adhesion complexes.

RomR is a response regulator, which is required for single cell movement and has a conserved receiver domain and a unique output domain. The subcellular localizations of A-motility proteins have defined two patterns of localization for these proteins. Two proteins required for A-motility localize in a cluster at the leading pole and in focal adhesion complexes, whereas RomR localizes in an asymmetric bipolar pattern with a large cluster at the lagging cell pole. During cell reversals the polar clusters relocate between the poles. The distinct localization patterns of A-motility proteins suggest that the A-motility system consists of distinct functional units. In the case of RomR, a RomRD52E mutant, in which the protein is likely locked in the phosphorylated state, causes a hyper-reversing phenotype; while a RomRD52N, in which the protein is likely locked in the non-phosphorylated state, hypo-reverses. These results suggest that the phosphorylation of RomR plays a direct role in the regulation of reversal frequency and that RomR has a central role in the regulation of polarity of the A-motility system. To further understand how RomR functions in setting-up the polarity of the A-motility system, we systematically mapped cis-acting polar targeting determinants of RomR. We found that the output domain contains two polar targeting determinants, a Pro-rich- and a Glu-rich region that function independently of each other. Moreover, using a genetic approach we have identified trans-acting determinants required for polar-targeting of RomR. We identified seven A-motility genes that are required for RomR localization. In all of the seven mutants, RomR localizes more symmetrically. Interestingly, a romR mutation also causes the abnormal localization of AglZ, which is one of the A-motility proteins located at the leading cell pole and in adhesion complexes. In conclusion, these observations suggest that the two functional units of the A-motility system, although localized to different subcellular addresses, are functionally interdependent. Lab: Lotte Søgaard-Andersen

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BLAST XI Poster #61 Tsr CONSTRUCTIONS WITH SYMMETRIC HEPTAD DELETIONS DISPLAY FULL FUNCTION Massazza D.A., Izzo S.A., Studdert C.A. Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Mar del Plata, Argentina

Methyl-accepting chemotaxis proteins (MCPs) share a highly conserved cytoplasmic domain. It consists in a long alpha-helical hairpin that forms, in the dimer, a coiled coil four-helix bundle.

A comparison between MCPs from different microorganisms has identified the existence of

insertions/deletions of seven aminoacids (heptads) in a symmetric arrangement, that is, situated at similar distances from the hairpin tip. This observation suggests that specific interactions between the helices in the coiled coil arrangement are essential for function. The whole number of heptads (ranging from 28 to 44) in this specific domain, plus certain sequence conservation, have been used to define seven classes of MCPs. Interestingly, some microorganisms possess MCPs belonging to different classes, raising the question of whether they cluster together and form mixed arrangements, or if they need to be segregated in the cell for proper function.

We reasoned that making a shortened Tsr derivative, intended to mimic an MCP from a shorter class, could provide an experimental system for exploring these issues. Accordingly, we introduced heptad deletions into one, the other or both helices of Tsr, the E.coli chemoreceptor for serine (36-heptad class). The location of the deletions was chosen based in an alignment with a chemoreceptor from R. sphaeroides, that naturally lacks a heptad in each helix and thus belongs to the 34-heptad class of MCPs.

We analyzed the effect of the deletions on several functional/structural aspects of Tsr:

- chemotaxis to serine in soft agar plates - kinase activation (pseudotaxis and tethering assays) - response to serine by changing the direction of flagellar rotation in a CW-biased strain - adaptation to serine in free-swimming assays - methylation response after attractant stimulus in the absence of CheA and CheW - trimer formation ability by in vivo crosslinking assays

While single asymmetric deletions abolished serine chemotaxis and caused serious alterations

in several wild type Tsr abilities, the double symmetric deletion generated a receptor with partial function and apparently normal higher order interactions. Moreover, point mutations in this doubly deleted construction, but not in the singly deleted ones, rendered fully functional Tsr derivatives. Interestingly, one of such mutations replaces an isoleucine, conserved in the 36H class to which Tsr belongs, for a valine, a residue that is highly conserved at that position in the 34H class, to which the shortened version mimics.

These results highlight the importance of the coiled coil arrangement for a proper transmission

of the signal from the periplasmic domain of Tsr to the tip of the cytoplasmic domain. The location and effect of mutations that restore function to the shortened version of the receptor might help to identify key features required for this transmission. Lab: Claudia Studdert

BLAST XI Poster #62 ACTIVE ARRAYS OF BACTERIAL CHEMORECEPTOR COMPLEXES: SOLID-STATE NMR TESTS OF CURRENT MODELS Daniel J. Fowler, Robert M. Weis, Lynmarie K. Thompson Department of Chemistry, University of Massachusetts Amherst

The receptor dimers that mediate bacterial chemotaxis form signaling complexes with CheW and the kinase CheA that cluster into large arrays at the poles of the E. coli cell. We have reconstituted active signaling complexes for solid-state NMR studies of receptor packing interactions in the array. A site-directed solid-state NMR distance measurement on complexes formed with soluble receptor fragments demonstrates that the receptors do not adopt either of the two proposed signaling array models, at least in the kinase-active signaling state. Comparisons of simulated and observed 19F-13C REDOR dephasing were used to deduce a closest-approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies in the kinase-active signaling state. Further NMR studies of these active assemblies are in progress to investigate protein-protein contacts in the array; other methods are being used to probe the role of receptor clustering and of protein dynamics in the transmembrane signaling mechanism. This research was supported by U.S. Public Health Service Grants GM47601 and GM085288; DJF was partially supported by National Research Service Award T32 GM08515.

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Lab: Lynmarie Thompson

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BLAST XI Poster #63 ANALYSIS OF THE BarA/UvrY TWO-COMPONENT SYSTEM IN SHEWANELLA ONEIDENSIS MR-1 Lucas Binnenkade, Jürgen Lassak, Kai M. Thormann Max-Planck-Institut für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse 10, 35043 Marburg, Germany

The BarA/UvrY two-component system is well conserved in species of the γ-proteobacteria and regulates numerous processes predominantly by controlling the expression of noncoding small RNAs. In this study, we characterized the BarA/UvrY two-component system in Shewanella oneidensis MR-1. Sensor kinase BarA and the cognate response regulator UvrY were identified by in vivo interaction and phosphotransfer studies. The expression of two predicted small regulatory RNAs (sRNAs), CsrB1 and CsrB2, was demonstrated to be dependent on UvrY. Transcriptomic analysis by microarrays revealed that UvrY is a global regulator and directly or indirectly affects transcription of more than 200 genes in S. oneidensis. Among these are genes encoding key enzymes of central carbon metabolism such as ackA, aceAB, and pflAB. Mutants lacking UvrY have a significant growth advantage over the wild type during aerobic growth on N-acetylglucosamine while under under anaerobic conditions the mutant grew more slowly. A positive effect on growth also occurred with lactate as carbon source but was absent in complex medium. We propose that, in S.oneidensis MR-1, the global BarA/UvrY regulatory system is involved in central carbon metabolism processes. Lab: Kai Thormann

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BLAST XI Poster #64 MECHANICAL AND KINETIC PRINCIPLES OF BACTERIAL FLAGELLAR MOTOR OPERATION Giovanni Meacci1, Ganhui Lan1, Yuhai Tu1 1IBM T.J. Watson Research Center, P. O. Box 218, Yorktown Heights, NY 10598, USA

Bacterial flagellar motor is powered by proton-motive-force (pmf). It contains multiple stator units that tether to the cell wall and step along the rotor ring to generate rotary motion against load. Due to the complexity in its structure, the operating mechanism of bacterial flagellar motor is under debating. Recent measurements largely enriched the knowledge of flagellar motor operation from the physiological conditions to various other mechanical and physical conditions, such as dynamics under near-zero or super-stall loads and the behavior at different temperatures or in different proton-isotope environments, which have brought new insight for understanding the operating mechanism of the motor. In this work, we develop a comprehensive model based on the previously proposed “power-stroke” modeling framework to incorporate more structural and kinetic details. Our model suggests that flagellar motor operates via a torque-dependent stepping mechanism and the mechanical flexibility of motor components must be strongly confined based on the choice of stepping kinetic functions. Our model successfully reproduces the characteristic torque-speed relation and explains the motor dynamics under near-zero and super-stall loads. In addition, our model provides quantitative explanation for the temperature and proton-isotope dependent motor behaviors. Moreover, after largely exploring the parameter dependence of the model prediction, we believe that the developed model can be applied to describe general operating principles of other rotary and linear molecular motor proteins. Lab: Yuhai Tu

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BLAST XI Poster #65 BEHAVIORAL RESPONSES IN THE METAL-REDUCER SHEWANELLA ONEIDENSIS H. Wayne Harris, Kenneth H. Nealson and Mandy J. Ward Dept. of Earth Sciences, University of Southern California, 3651 Trousdale Parkway, Los Angeles, CA 90089 Shewanella oneidensis MR-1 is a facultative anaerobe capable of respiring a wide range of anaerobic electron acceptors, including a number of metals. We have previously reported that MR-1 shows behavioral responses to soluble iron [Fe(III) citrate] and insoluble manganese oxides (MnO2), but not to soluble manganese [Mn(III) pyrophosphate], or insoluble iron (hydr)oxides [Fe(OH)3]. Recently, we have studied behavioral responses of wild-type MR-1 and a number of chemotaxis mutants to a range of manganese minerals including pyrolucite (MnO2), cryptomelane [K(Mn4+Mn2+)8O16], hausmannite (Mn2+Mn3+

2O4), and birnessite [Na0.3Ca0.1K0.1)(Mn4+,Mn3+)2O4 · 1.5 H2O]. We will report the results of these analyses. Lab: Mandy Ward

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BLAST XI Poster #66 DifA, AN MCP-LIKE SENSORY PROTEIN, USES A NOVEL SIGNALING MECHANISM TO REGULATE EXOPOLYSACCHARIDE PRODUCTION IN MYXOCOCCUS XANTHUS Qian Xu, Wesley P. Black, Heidi M. Nascimi and Zhaomin Yang Department of Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061

DifA is an MCP-like sensory transducer that regulates exopolysaccharide (EPS) production in Myxococcus xanthus. Here mutational analysis and molecular biology were used to probe the signaling mechanisms of DifA in EPS regulation. We first identified the start codon of DifA experimentally; this identification extended the N-terminus of DifA for 45 amino acids (AA) from the previous bioinformatics prediction. This extension helped to address the outstanding question of how DifA receives input signals from type-4 pili without a prominent periplasmic domain. The results suggest that DifA uses its N-terminus extension to sense an upstream signal in EPS regulation. We suggest that the perception of the input signal by DifA is mediated by protein-protein interactions with upstream components. Subsequent signal transmission likely involves transmembrane signaling instead of direct intramolecular interactions between the input and the output modules in the cytoplasm. The basic functional unit of DifA for signal transduction is likely dimeric as mutational alteration of the predicted dimeric interface of DifA significantly affected EPS production. Deletions of 14-AA segments in the C-terminus suggest that the newly defined flexible bundle subdomain in MCPs is likely critical for DifA function because shortening of this bundle can lead to constitutively active mutations. Lab: Zhaomin Yang

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BLAST XI Poster #67 INVESTIGATING STRUCTURAL PROPERTIES OF CheW WITH MOLECULAR DYNAMICS AND NMR Davi Ortega (1), Guoya Mo (2), Kwangwoon Lee(2), Hongjun Zhou(2), Jerome Baudry (1), Frederick Dahlquist (2), Igor Zhulin (1) (1) Joint Institute for Computational Sciences, University of Tennessee - Oak Ridge National

Laboratory Oak Ridge, TN 37831 (2) Department of Chemistry and Biochemistry, University of California Santa Barbara, Santa Barbara,

CA 93106-9510

The prokaryotic chemotaxis system is one of the best studied signal transduction pathways in nature. Recently, a comprehensive evolutionary study revealed that the birth of a single component, CheW, led to the divergence of the chemotaxis system from simpler two component systems. CheW increases the binding affinity between the receptors and the kinase in the chemotaxis complex, which supports its role as scaffold and possibly the main promoter of chemotaxis lattice formation. However, in vitro and in vivo experiments targeting a highly conserved position R62 in E. coli suggest that CheW may play a more complex and dynamic role in chemotaxis given its null phenotype despite showing only small changes in binding affinity with the kinase and the receptor. Preliminary Molecular Dynamics simulations of CheW from E. coli show that residue R62 forms a salt bridge with residue E38. The salt bridge formed by these residues appears to be important for the integrity of sub-domain 1 and is present in approximately 76% of non-redundant CheW sequences. Here we report our ongoing efforts to study the structural properties of CheW and the role of the conserved salt bridge using Molecular Dynamics and NMR methods. We have prepared three in silico mutants targeting positions 62 and 38, which underwent a series of 90 ns molecular dynamics simulations along with the wild type structure in order to identify structural perturbations introduced by the mutations. The simulations reveal changes in the flexibility of several parts of CheW upon disruption of the salt bridge. In order to validate the simulation results, we performed measurements of the N15 relaxation parameters with the wild type protein experimentally with NMR. Groundwork results show that the molecular dynamics simulations agree with NMR measurements in identifying flexible and non-flexible regions of the protein. Overall, the molecular dynamics simulations support that the disruption of the salt bridge affects the relative stability of the CheW interfaces with the kinase and receptor. Lab: Igor Zhulin

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BLAST XI Poster #68 MYXOCOCCUS XANTHUS Frz PATHWAY SIGNALING AND THE Mgl PROTEINS Eva M. Campodonico and David R. Zusman Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720 USA

The Frz chemosensory system coordinates directed motility in Myxococcus xanthus by regulating the frequency of cellular reversals. The tandem CheY domain protein FrzZ serves as the output of this signaling pathway; upon phosphorylation by FrzE, FrzZ is proposed to modulate downstream targets to induce cell reversals. The mgl operon encodes both the Ras-like GTPase MglA, and MglB, which serves as a GTPase activating protein (GAP) for MglA. MglA and MglB localize the leading and lagging cell poles, respectively, and are essential for effective M. xanthus movement. It has been shown that the ability of MglB to exert its GAP activity on MglA is required to exclude MglA from the lagging pole. However, the means by which MglB is localized remains to be determined. Here, we seek to understand how the Frz pathway might communicate with MglB. To study the role of FrzZ phosphorylation, we examined the localization of MglB-YFP fusion protein in strains expressing FrzZ phosphorylation site mutants. In the presence of single and double FrzZ point mutants, MglB-YFP still localized at the lagging pole, but showed increased localization at the leading pole as well as clusters throughout the cell. Based upon the canonical Che system, this suggests that phosphorylation may lead to an enhanced binding affinity of FrzZ for MglB. GST pull-down and cross-linking experiments did demonstrate binding between FrzZ and MglB. However, FrzZ phosphorylation was not required for binding to occur. Taken together, these data indicate a role for Frz signaling in MglB targeting. The mechanism by which FrzZ binding and phosphorylation combine to modulation of MglB localization is the subject of ongoing study. Lab: David Zusman

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BLAST XI PARTICIPANT LIST

Christopher Adase Texas A&M University Biochemistry 3258 TAMU BSBE Room 303 College Station, TX 77843 Phone: (979) 845-1249 xpage@neo.tamu.edu

Gladys Alexandre The University of Tennessee Biochemistry, Cellular and Molecular Biology 1414 W. Cumberland Avenue M407 Walters Life Sciences Building Knoxville, TN 37996 Phone: (865) 974-0866 Fax: 865974-6306 galexan2@utk.edu

Divyaben Amin University of Missouri Department of Biochemistry 117 Schweitzer Hall Columbia, MO 65211 Phone: (573) 884-6334 Amind@missouri.edu

Tatsuo Atsumi Gifu University of Medical Science Radiological Technology 795-1 Nagamine Ichihiraga Seki, Gifu 501-3892 Japan Phone: +81-575-22-9401 Fax: +81-575-23-0884 atsumi@u-gifu-ms.ac.jp

Shannon Au The Chinese University of Hong Kong School of Life Sciences The Chinese University of Hong Kong Shatin, NT Hong Kong Phone: +852-3163-4170 shannon-au@cuhk.edu.hk

Claudine Baraquet University of Washington Microbiology 1705 NE Pacific St. Seattle, WA 98195 Phone: (206) 221-2798 baraquet@uw.edu

Wiebke Behrens Hannover Medical School Institute for Medical Microbiology Carl-Neuberg-Strasse 1 Hannover 30625 Germany Phone: +49-5115-328-028 Behrens.wiebke@mh-hannover.de

Howard Berg Harvard University MCB, Physics Harvard Biological Laboratories 16 Divinity Ave., Room 3063 Cambridge, MA 2138 Phone: (617) 495-0924 Fax: (617) 496-1114 hberg@mcb.harvard.edu

Adam Berlinberg University of Colorado at Boulder Campus Box 215 Boulder, CO 80309 Phone: (303) 492-3597 adam.berlinberg@gmail.com

Shuangyu Bi Peking University College of Chemistry and Molecular Engineering Beijing, Beijing 100871 China Phone: +86-01-6275-7520 sybi@mdl.ipc.pku.edu.cn

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Amber Bible University of Tennessee, Knoxville Biochemistry, Cellular, and Molecular Biology M407 Walters Life Sciences 1414 Cumberland Avenue Knoxville, TN 37996 Phone: (865) 974-2364 abible@utk.edu

David Blair University of Utah Biology 257 S 1400 East Salt lake City, UT 84112 Phone: (801) 585-3709 blair@bioscience.utah.edu

Joseph Boll UT Southwestern Medical Center Microbiology 5323 Harry Hines Blvd. Dept. of Microbiology Mail Code 9048 Dallas, TX 75390 Phone: (214) 648-5952 joseph.boll@utsouthwestern.edu

Bob Bourret University of North Carolina at Chapel Hill Microbiology & Immunology Chapel Hill, NC 27599-7290 Phone: (919) 966-2679 Fax: (919) 962-8103 bourret@med.unc.edu

Richard Branch Harvard University Molecular and Cellular Biology Harvard Biological Laboratories 16 Divinity Avenue, Rm. 3063 Cambridge, MA 2138 Phone: (617) 495-4217 rbranch@mcb.harvard.edu

Ariane Briegel California Institute of Technology 1200 E. California Blvd. Mail code 114-96 Pasadena, CA 91125 Phone: (626) 395-8848 briegel@caltech.edu

Mostyn Brown University of Oxford Department of Biochemistry South Parks Road Oxford, UK OX1 3QU United Kingdom Phone: +44-1865-613315 mostyn.brown@bioch.ox.ac.uk

Eva Campodonico University of California, Berkeley Molecular and Cell Biology 31 Koshland Hall Berkeley, CA 94720 Phone: (510) 643-5457 eva.campodonico@berkeley.edu

Cécile Castelli Aix-Marseille University / CNRS 31 Chemin Joseph Aiguier Marseille, Bouches du Rhône 13402 France Phone: +33-4-91-16-40-32 Fax: +33-4-91-71-89-14 jourlin@ifr88.cnrs-mrs.fr

Nyles Charon West Virginia University Microbiology, Immunology, and Cell Biology Health Sciences Center North Box 9177 Morgantown, WV 26506-9177 Phone: (304) 293-4170 Fax: (304) 293-7823 ncharon@hsc.wvu.edu

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Brian Crane Cornell University Chemistry and Chemical Biology G75 Chemistry Research Building Ithaca, NY 14853 Phone: (607) 254-8634 Fax: (607) 255-1248 bc69@cornell.edu

Rachel Creager-Allen University of North Carolina at Chapel Hill 2002 Quaker Creek Dr Mebane, NC 27302 Phone: (919) 966-2679 rachel.creager@gmail.com

Sean Crosson University of Chicago Biochemistry and Molecular Biology 929 E. 57th St. GCIS-W138 Chicago, IL 60637 Phone: (773) 834-1926 scrosson@uchicago.edu

Rick Dahlquist University of California Santa Barbara Department of Chemistry & Biochemistry Santa Barbara, CA 93106 Phone: (805) 893-5326 dahlquist@chem.ucsb.edu

Jennifer De Beyer University of Oxford Department of Biochemistry Merton College Merton Street Oxford, Oxfordshire OX14JD United Kingdom Phone: +44-1865-613317 jennifer.de-beyer@merton.ox.ac.uk

Gaurav Dogra Virginia Polytechnic Institute and State University Biological Sciences Life Sciences 1, Scharf Lab Washington Street Blacksburg, VA 24061 Phone: (540) 231-0772 gauravdogra@vt.edu

Qian Dong Indiana University-Bloomington Molecular and Cellular Biochemistry Department 212 Howthorne Drive Simon Hall Bloomington, IN 47405 Phone: (812) 855-5443 dong3@indiana.edu

Roger Draheim Goethe University Frankfurt Institute of Biochemistry Biozentrum N210, 1.08 Max-von-Laue-Str 9 Frankfurt, Hessen 60438 Germany Phone: +49-6979-829-468 draheim@biochem.uni-frankfurt.de

William Draper University of California, Berkeley 2817 8th St Berkeley, CA 94710 Phone: (510) 666-2786 draper@berkeley.edu

Michael Eisenbach Weizmann Institute of Science Biological Chemistry PO Box 26 Rehovot, Israel 76100 Israel Phone: +972-8-934-3923 Fax: +972-8-947-2722 m.eisenbach@weizmann.ac.il

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Annette Erbse University of Colorado at Boulder Chemistry/Biochemistry 76 Chemistry Boulder, CO 80309 Phone: (303) 492-3597 erbse@colorado.edu

Joseph Falke University of Colorado at Boulder Dept. of Chem. & Biochem. UCB 215 Univ. Colorado Boulder, CO 80309-0215 Phone: (303) 492-3503 falke@colorado.edu

Milana Fraiberg Weizmann Institute of Science Biological Chemistry P.O. Box 26 Rehovot 76100 Israel Rehovot, israel 76100 Israel Phone: +972-8-934-2710 Fax: +972-8-947-2722 milana.fraiberg@weizmann.ac.il

Hajime Fukuoka Tohoku University IMRAM Katahira 2-1-1 Aoba-Ku Sendai, Miyagi 980-8577 Japan Phone: +81-22-217-5804 Fax: +81-22-217-5804 f-hajime@tagen.tohoku.ac.jp

Michael Galperin National Institutes of Health NCBI, NLM 8600 Rockville Pike, MSC3830 Bldg. 38A, Rm. 507 Bethesda, MD 20894 Phone: (301) 435-5910 Fax: (301) 435-7793 galperin@ncbi.nlm.nih.gov

Haifeng Geng University of Maryland, Baltimore County 701 East Pratt Street Baltimore, MD 21202 Phone: (410) 234-8877 geng1@umbc.edu

Mizuki Gohara Nagoya University Division of Biological Science Furo-cho, Chikusa-ku Nagoya, Aichi 464-8602 Japan Phone: +81-52-789-3543 Fax: +81-52-789-3001 migomigo16@yahoo.co.jp

Shukui Guo University of Utah Biology 257 South 1400 East Salt Lake city, UT 84112 Phone: (801) 585-3961 guoshk2001@yahoo.com

Ronit Gutman Pasvolsky Weizmann Institute of Science Biological Chemistry P.O.Box 26 Rehovot 76100 Israel Rehovot, Israel 76100 Israel Phone: +972-8-934-2710 Fax: +972-8-947-2722 ronit.pasvolsky@weizmann.ac.il

Benjamin Hall University of Oxford Department of Biochemistry South Parks Road Oxford, Oxfordshire OX1 3QU United Kingdom Phone: +44-1865-275380 Fax: +44-1865-275273 benjamin.hall@bioch.ox.ac.uk

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Ina Haneburger Ludwig-Maximilians-Universität München Grosshaderner Str. 2-4 Munich, Bavaria 82152 Germany Phone: +49-8921-807-4508 Ina.Haneburger@lrz.uni-muenchen.de

Rasika Harshey University of Texas at Austin Mol. Gen. & Microbiology 1 University Station A1000 Austin, TX 78712 Phone: (512) 471-6881 Fax: (512) 471-7088 rasika@uts.cc.utexas.edu

Caroline Harwood University of Washington 1910 25th Ave. E Seattle, WA 98112 Phone: (206) 221-2848 csh5@uw.edu

Gerald Hazelbauer University of Missouri Biochemistry 117 Schweitzer Hall Columbia, MO 65211 Phone: (573) 882-4845 Fax: (573) 882-5635 hazelbauerg@missouri.edu

David Hendrixson University of Texas Southwestern Medical Center Microbiology 5323 Harry Hines Boulevard Dept of Microbiology, Room NA 6.512 Dallas, TX 75390-9048 Phone: (214) 648-5949 Fax: (214) 648-5907 david.hendrixson@utsouthwestern.edu

Penelope Higgs Max-Planck-Institut für terrestrische Mikrobiologie Ecophysiology Karl-von-Frisch Strasse 10 Marburg, Hessen D35043 Germany Phone: +49-6421-178-301 Fax: +49-6421-178-309 higgs@mpi-marburg.mpg.de

Laura Hobley University of Nottingham Centre for Genetics and Genomics Lab C15, Medical School Queen's Medical Centre Nottingham, Nottinghamshire NG7 2UH United Kingdom Phone: +44-1158-230317 Fax: +44-1158-230338 laura.hobley@nottingham.ac.uk

Basarab Hosu Harvard University Molecular and Cellular Biology 16 Divinity Avenue Bio Labs Buliding Rom 3068 Cambridge, MA 2138 Phone: (617) 495-6127 Fax: (617) 496-1114 ghosu@mcb.harvard.edu

Linda Hu Loyola University Chicago Microbiology/Immunology 38 Oakton Drive Lombard, IL 60148 Phone: (708) 216-0845 lihu@lumc.edu

Varisa Huangyutitham University of Washington Microbiology 1705 NE Pacific St HSB K327 Seattle, WA 98195 Phone: (206) 221-2798 varisah@u.washington.edu

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Kelly Hughes University of Fribourg Biology-Microbiology Chemin du Musée 10 Fribourg, Fribourg CH1700 Switzerland Phone: +41-26-300-94-36 kelly.hughes@unifr.ch

F. Marion Hulett University of Illinois at Chicago Bios Lab for Mol Biol 900 South Ashland Rm 4150 MBRB M/C567 Chicago, IL 60614 Phone: (312) 996-5460 hulett@uic.edu

Tu-Anh Huynh University of California, Davis Microbiology 330 J St, Apt 28 Davis, CA 95616 Phone: (530) 754-7995 tnahuynh@ucdavis.edu

Yuichi Inoue Tohoku University Katahira 2-1-1, Aoba-ku Sendai, Miyagi 980-8577 Japan Phone: +81-22-217-5804 inoue@tagen.tohoku.ac.jp

Chantal Iobbi-Nivol CNRS Laboratoire de Chimie Bactérienne 31 chemin Joseph Aiguier Marseille, Bouches du Rhône 13402 France Phone: +33-4-91-16-44-27 Fax: +33-4-91-71-89-14 iobbi@ifr88.cnrs-mrs.fr

Akihiko Ishijima Tohoku University Katahira 2-1-1,Aoba-ku Sendai, Japan 980-8577 Japan Phone: +81-22-217-5802 ishijima@tagen.tohoku.ac.jp

Ruchi Jain Yale University Internal Medicine TAC, 300 Cedar Street New Haven, CT 6520 Phone: (203) 737-1197 ruchi.jain@yale.edu

Urs Jenal Biozentrum, University of Basel Infection Biology Biozentrum der Universität Basel Klingelbergstrasse 70 Basel, BS 4056 Switzerland Phone: +41-61-267-2135 Fax: +41-61-267-21-18 urs.jenal@unibas.ch

Mark Johnson Loma Linda University Biochemistry and Microbiology AHBS 120 Loma Linda, CA 92350 Phone: (909) 558-4480 Fax: (909) 558-4035 mjohnson@llu.edu

Christine Josenhans Medical University Hannover Carl-Neuberg-Strasse 1 Hannover, Germany 30625 Germany Phone: +49-5115-324-354 Fax: +49-5115-324-354 josenhans.christine@mh-hannover.de

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Kirsten Jung Ludwig-Maximilians-Universitaet Muenchen Microbiology Biocenter Grosshaderner Str. 2-4 Martinsried, Bavaria 82152 Germany Phone: +49-89-2180-74500 jung@lmu.de

Christine Kaimer UC Berkeley 31 Koshland Hall Berkeley, CA 94720 Phone: (510) 643-5457 christine.kaimer@berkeley.edu

Barbara Kazmierczak Yale University 333 Cedar St., Box 208022 New Haven, CT 06520-8022 Phone: (203) 737-5062 Fax: (203) 785-3864 barbara.kazmierczak@yale.edu

Daniela Keilberg Max-Planck-Institut für terrestrische Mikrobiologie Karl-von-Frisch-Straße 10 Marburg, Hessen D-35043 Germany Phone: +49-6421-178-221 daniela.keilberg@mpi-marburg.mpg.de

Linda Kenney University of Illinois at Chicago Micro. & Immunol. 835 S. Wolcott M/C 790 Chicago, IL 60612 Phone: (312) 413-0576 Fax: (312) 996-6415 kenneyl@uic.edu

Eun A Kim The University of Utah Salt lake city, UT 84112 Phone: (801) 585-3961 nlcfkea@yahoo.com

Jihong Kim Korea Advanced Institute of Science and Technology Department of Biological Sciences BMRC(E7) Room 4101 373-1 Guseong-dong, Yuseong-gu Daejeon, Daejeon 305-701 Korea, Republic of Phone: +82-42-350-2669 kckacl@kaist.ac.kr

Seiji Kojima Nagoya University Division of Biological Science Furo-cho, Chikusa-ku Nagoya, Aichi 464-8602 Japan Phone: +81-52-789-2992 Fax: +81-52-789-3001 z47616a@cc.nagoya-u.ac.jp

Jessica Kostick Virginia Commonwealth University Microbiology & Immunology McGuire Hall, 1112 E. Clay St. Room 101 Richmond, VA 23298 Phone: (804) 628-2718 kostickjl@vcu.edu

Tino Krell Estación Experimental del Zaidín Albareda 1 Granada, Granada 18008 Spain Phone: +34-958-18-16-00 tino.krell@eez.csic.es

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Kwok Ho Lam The Chinese University of Hong Kong Molecular Biotechnology RM 314, BMSB, CUHK Hong Kong SAR Hong Kong Phone: +852-2609-6118 Fax: +852-2603-5123 s0702001@mailserv.cuhk.edu.hk

Ganhui Lan IBM T.J. Watson Research Center Physics and Quantitative Biology Room 27-133 P. O. Box 218 Yorktown Heights, NY 10598 Phone: (914) 945-2334 glan@us.ibm.com

Milena Lazova FOM Institute for Atomic and Molecular Physics (AMOLF) Systems Biology Sciencepark 104 Amsterdam, Noord Holland 1098 XG Netherlands Phone: +31-20-754-7302 lazova@amolf.nl

Jaemin Lee University of Texas at Austin Molecular Genetics and Microbiology 1 University Station A5000 Austin, TX 78712-0162 Phone: (512) 471-6799 greenjaemin@gmail.com

Junghoon Lee KAIST Biological science BMRC room 4101, 373-1 Guseong-dong, Yuseong-gu Daejeon, Daejeon 305-701 Korea, Republic of Phone: +82-42-350-2669 mydreams83@kaist.ac.kr

Yi-Ying Lee University of Maryland, Baltimore County Department of Marine Biotechnology 701 E Pratt Street Baltimore, MD 21202 Phone: (410) 234-8877 yyinglee@umbc.edu

Pushkar Lele Harvard University Molecular and Cellular Biology Harvard Biological Laboratories 16 Divinity Avenue, Room 3063 Cambridge, MA 2138 Phone: (617) 495-4217 lele@fas.harvard.edu

Paphavee Lertsethtakarn University of California, Santa Cruz Microbiology and Environmental Toxicology 1156 High St. METX Santa Cruz, CA 95064 Phone: (831) 459-4780 plertset@slugmail.ucsc.edu

Robert Levenson University of California, Santa Barbara Chemistry & Biochemistry 761 Birch Walk Apt J Goleta, CA 93117 Phone: (858) 342-5006 rlevenson@chem.ucsb.edu

Chunhao Li SUNY at Buffalo Oral Biology 3435 Main St. Buffalo, NY 14214 Phone: (716)829-6014 Fax: (716)829-3942 cli9@buffalo.edu

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Guanglai Li Brown University Physics Department 182 Hope Street Providence, RI 2912 Phone: (401) 863-2140 guanglai_li@brown.edu

Mingshan Li University of Missouri Biochemistry 117 Schweitzer Hall Columbia, MO 65211 Phone: (573) 884-6334 limin@missouri.edu

Na Li Nagoya University Division of Biological Science Furo-cho, Chikusa-ku Nagoya, Aichi 464-8602 Japan Phone: (81-52-789-3543 Fax: +81-52-789-3001 li.na@i.mbox.nagoya-u.ac.jp

Xiaoxiao Li Cornell University Chemistry G-64 S. T. Olin Lab, Cornell Univeristy Ithaca, NY 14850 Phone: (607) 255-4970 xl97@cornell.edu

Tao Lin UT Medical School at Houston Pathology and Laboratory Medicine PO Box 20708, Room MSB 2.124 Houston, TX 77025 Phone: (713) 500-5350 Fax: (713) 500-0730 tao.lin@uth.tmc.edu

Jun Liu UT Houston Medical School Department of Pathology 6431 Fannin, MSB 2.228 Houston, TX 77030 Phone: (713) 500-5342 jun.liu.1@uth.tmc.edu

Mike Manson Texas A&M University Biology MS 3258 BSBE 303 College Station, TX 77843 Phone: (979) 845-5158 Fax: (979) 845-2891 mike@mail.bio.tamu.edu

Richard Marconi Virginia Commonwealth University Microbiology & Immunology PO BOX 980678 Richmond, VA 23298 Phone: (804) 828-3888 rmarconi@vcu.edu

Philip Matsumura University of Illinois at Chicago Microbiology/Immuno 835 S. Wolcott Ave. M/C 790 Chicago, IL 60612-7344 Phone: (312) 996-2286 Fax: (312) 413-2952 Philip.matsumura@uic.edu

Mark McBride University of Wisconsin-Milwaukee Biological Sciences P. O. Box 413 Milwaukee, WI 53201 Phone: (414) 229-5844 mcbride@uwm.edu

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Jonathan McMurry Kennesaw State University Chemistry & Biochemistry 1000 Chastain Rd. MB #1203 Kennesaw, GA 30144 Phone: (770) 499-3238 jmcmurr1@kennesaw.edu

Guoya Mo University of California, Santa Barbara Biochemistry 708 Bolton Walk Apt 201 Goleta, CA 93117 Phone: (805) 284-2326 gmo@chem.ucsb.edu

Tarek Msadek Institut Pasteur Biology of Gram-Positive Pathogens, Department of Microbiology, Biology of Gram-Positive Pathogens 25 Rue du Dr. Roux Paris, France 75015 France Phone: +33-1-45-68-88-09 Fax: +33-1-45-68-89-38 tmsadek@pasteur.fr

Beiyan Nan UC Berkeley Department of Molecular and Cell Biology University of California, Berkeley Berkeley, CA 94720 Phone: (510) 643-5457 nanbeiyan@gmail.com

Vincent Nieto University of Texas at Austin Molecular Genetics & Microbiology 2506 Speedway Austin, TX 78712 Phone: (512) 471-6799 vince.nieto@gmail.com

Masayoshi Nishiyama Kyoto University Department of Chemistry Oiwake cho, Kitashirakawa, Sakyo-ku Kyoto, Kyoto 606-8502 Japan Phone: +81-75-753-4023 m-nishi@kuchem.kyoto-u.ac.jp

Christopher O'Connor North Central College Biology 30 N. Brainard St. Naperville, IL 60540 Phone: (630) 637-5175 coconnor@noctrl.edu

Jennifer O'Connor University of Washington Microbiology 1705 NE Pacific St. Box 357242 Seattle, WA 98195 Phone: (206) 234-8859 jroc@uw.edu

Karen O'Donovan University College Cork Microbiology BIOMERIT Research Centre Department of Microbiology, UCC Cork Ireland Phone: +353-21-490-1370 kod@umail.ucc.ie

Davi Ortega University of Tennessee / ORNL Physics 1414 W. Cumberland Av. WLS F437 Knoxville, TN 37919 Phone: (865) 384-9507 dortega@utk.edu

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Karen Ottemann University of California, Santa Cruz Microbiology/Env Toxicology 1156 High Street METX Santa Cruz, CA 95064 Phone: (831) 459-3482 ottemann@ucsc.edu

Clinton Page University of Georgia Microbiology 176 Cross Creek Place Apartment 2C Athens, GA 30605 Phone: (706) 542-2670 Fax: (706) 542-2671 capage@uga.edu

Stephani Page University of North Carolina at Chapel Hill Microbology and Immunology 804 Mary Ellen Jones Bulding Chapel Hill, NC 27599 Phone: (919) 966-2679 cheme05bio07@gmail.com

Guillaume Paradis Laval University Physics, Engineering Physics and Optics 2610 d'Oviedo Quebec, QC G2B0G6 Canada Phone: (418) 656-2131 guillaume.paradis.5@ulaval.ca

Rebecca Parales University of California, Davis Department of Microbiology 226 Briggs Hall 1 Shields Ave. Davis, CA 95616 Phone: (530) 754-5233 Fax: (530) 752-9014 reparales@ucdavis.edu

Chankyu Park KAIST Biological Sciences BMRC(E7) room 4106, 373-1 Guseong-dong, Yuseonggu Daejeon, Daejeon 305-701 Korea, Republic of Phone: +82-42-350-2629 ckpark@kaist.ac.kr

John Parkinson University of Utah Biology 257 South 1400 East Salt Lake City, UT 84112-0840 Phone: (801) 581-7639 parkinson@biology.utah.edu

Koushik Paul University of Utah Biology 126 university village Salt lake city, UT 84108 Phone: (801) 585-3961 Koushikpaul20@gmail.com

Sonja Pawelczyk University of Oxford Department of Biochemistry South Parks Road Oxford, Oxfordshire OX1 3QU United Kingdom Phone: +44-1865-613315 sonja.pawelczyk@bioch.ox.ac.uk

Tobias Petters Max-Planck-Institut für terrestrische Mikrobiologie Karl-von-Frisch-Str. 10 Marburg, Hesse 35043 Germany Phone: +49-6421-178-212 tobias.petters@mpi-marburg.mpg.de

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Hai Pham University of Utah Department of Biology 257 South 1400 East Rm. 320 624 Medical Plaza Salt Lake city, UT 84112 Phone: (801) 581-6307 hai.pham@utah.edu

Adam Politzer UC Berkeley Biophysics QB3 Institute 456 Stanley Hall Berkeley, CA 94720-3220 Phone: (510) 666-2786 politzer@berkeley.edu

Steven Porter University of Exeter Biosciences Biosciences, Geoffrey Pope Building Stocker Road Exeter, Devon EX4 4QD United Kingdom Phone: +44-1392-722172 s.porter@exeter.ac.uk

Birgit Prüß North Dakota State University Veterinary and Microbiological Sciences 1523 Centennial Blvd. Fargo, ND 58108 Phone: (701) 231-7848 Birgit.Pruess@ndsu.edu

Simon Rainville Laval University Physics Pavillon optique photonique 2375, rue de la Terrasse Quebec, QC G1V 0A6 Canada Phone: (418) 656-2131 Fax: (418) 656-2623 simon.rainville@phy.ulaval.ca

Christopher Rao University of Illinois Chemical Engineering 211 Roger Adams Lab 600 S Mathews Ave Urbana, IL 61801 Phone: (217) 244-2247 chris@scs.uiuc.edu

Ryan Rhodes University of Wisconsin-Milwaukee Biological Sciences 3209 N Maryland Ave Lapham Hall Rm 181 Milwaukee, WI 53211 Phone: (414) 229-2910 Fax: (414) 229-3926 rhodesrg@uwm.edu

Matthew Russell University of Tennessee, Knoxville M407 Walters Life Science 1414 W. Cumberland Ave. Knoxville, TN 37996 Phone: (865) 974-2364 mhrussel@utk.edu

Mayukh Sarkar University of Utah Biology 201 Biology Building Salt Lake City, UT 84108 Phone: (801) 585-3961 m.sarkar@utah.edu

Birgit Scharf Virginia Tech Biological Sciences Life Sciences I Blacksburg, VA 24061 Phone: (540) 231-0757 Fax: (540) 231-4043 bscharf@vt.edu

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Maren Schniederberend Yale University Infectious Diseases 333 Cedar Street TAC S140 New Haven, CT 6520 Phone: (203) 737-1197 Fax: (203) 785-6815 maren.schniederberend@yale.edu

Thomas Shimizu FOM Institute for Atomic and Molecular Physics (AMOLF) Science Park 104 Amsterdam, North Holland 1098 XG Netherlands Phone: +31-20-754-7242 t.shimizu@amolf.nl

Guangwei Si Peking University Room 454,Physics Building, Peking University Haidian District Beijing, Beijing 100871 China Phone: (914) 945-2132 siguangwei@gmail.com

Ruth Silversmith University of North Carolina at Chapel Hill Microbiology and Immunology Room 804 Mary Ellen Jones Chapel Hill, NC 27599 Phone: (919) 966-2679 Fax: (919) 962-8103 silversr@med.unc.edu

Ria Sircar Cornell University Chemistry & Chemical Biology G 64 S.T. Olin Lab Cornell University Ithaca, NY 14853 Phone: (607) 255-4970 rs693@cornell.edu

Peter Slivka University of Colorado at Boulder Chemistry and Biochemistry CU Boulder Dept. of Chemistry and Biochemistry 215 UCB Boulder, CO 80309 Phone: (303) 492-3597 slivkap@colorado.edu

Michael Sneddon Yale University 600 Orange St #2 New Haven, CT 6511 Phone: (203) 432-3517 michael.sneddon@yale.edu

Lotte Sogaard-Andersen Max-Planck-Institut für terrestrische Mikrobiologie Karl-von-Frisch. Str. Marburg, Germany 35043 Germany Phone: +49-6421-178-201 sogaard@mpi-marburg.mpg.de

Claudia Studdert Universidad Nacional de Mar del Plata Instituto de Investigaciones Biológicas Funes 3250 Mar del Plata, Buenos Aires 7600 Argentina Phone: +54-223-4753030 Fax: +54-223-4753150 studdert@mdp.edu.ar

Nattakan Sukomon Cornell University Chemistry and Chemical Biology 709 Hasbrouck Apartment Ithaca, NY 14850 Phone: (607) 280-8698 ns528@cornell.edu

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Kalin Swain University of Colorado at Boulder Chem & Biochem Univ of Colorado Campus Box 215 Boulder, CO 80020 Phone: (303) 492-3597 kalin.swain@colorado.edu

Lee Szkotnicki Virginia Commonwealth University Microbiology & Immuno McGuire Hall, 1112 E. Clay St. Room 101 Richmond, VA 23298 Phone: (804) 628-2718 ltszkotnicki@vcu.edu

Barry Taylor Loma Linda University Micro & Molec Genetics Alumni Hall Loma Linda, CA 92350 Phone: (909) 558-4881 Fax: (909) 558-4035 bltaylor@llu.edu

Lynmarie Thompson University of Massachusetts Chemistry LGRT 104 Amherst, MA 1003 Phone: (413) 545-0827 thompson@chem.umass.edu

Kai Thormann Max-Planck-Institut für terrestrische Mikrobiologie Ecophysiology Karl-von-Frisch-Strasse Marburg, Hessia D-35043 Germany Phone: +49-6421-178-302 Fax: +49-6421-178-309 thormann@mpi-marburg.mpg.de

Murray Tipping University of Oxford Department of Biochemistry South Parks Road Oxford, Oxon OX1 3QU United Kingdom Phone: +44-1865-613315 murray.tipping@bioch.ox.ac.uk

Yuhai Tu IBM T.J. Watson Research Center Tu 1101 Kitchawan Rd./Rt. 134 Yorktown Heights, NY 10598 Phone: (914) 945-2762 yuhai@us.ibm.com

Armand Vartanian University of California, Santa Barbara BMSE Dahlquist Lab Santa Barbara, CA 93106 Phone: (805) 893-5468 armandsv@gmail.com

Anh Vu University of California, Santa Barbara Chemistry and Biochemistry 706 Bolton Walk Apt. 101 Goleta, CA 93117 Phone: (805) 893-5468 avu@chem.ucsb.edu

George Wadhams Oxford University Biochemistry South Parks Road Oxford, Oxfordshire OX1 3QU United Kingdom Phone: +44-1865-613329 george.wadhams@bioch.ox.ac.uk

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Dan Wall University of Wyoming Molecular Biology 1000 E. University Ave Laramie, WY 82071 Phone: (307) 766-3542 dwall2@uwyo.edu

Don Walthers University of Illinois at Chicago Micro & Immuno 835 S. Wolcott M/C 790 Chicago, IL 60611 Phone: (312) 413-2014 Fax: (312) 996-6415 dwalther@uic.edu

Loo Chien Wang National University of Singapore Department of Biological Sciences S3 Level 1, Lab 1 Science Drive 4 Singapore, Singapore 117543 Singapore Phone: +65-6516-8093 A0066554@nus.edu.sg

Mandy Ward UNiversity of Southern California Earth Sciences 651 Andalusia St Los Angeles, CA 90065 Phone: (443) 854-9289 mandywar@usc.edu

Kylie Watts Loma Linda University Microbiology and Molecular Genetics Div. of Microbiology and Molecular Genetics AHBS 120 Loma Linda, CA 92350 Phone: (909558-1000 Fax: 909558-4035 kwatts@llu.edu

Ben Webb Virginia Polytechnic Institute and State University life sciences 1 washington st. blacksburg, VA 24060 Phone: (571) 426-9433 mercury1@vt.edu

Laurence Wilson The Rowland Institute at Harvard 100 Edwin H Land Boulevard Cambridge, MA 2142 Phone: (617) 497-4643 wilson@rowland.harvard.edu

Alan Wolfe Loyola University Chicago Microbiology and Immunology 2160 South First Avenue Maywood, IL 60153 Phone: (708) 216-5814 awolfe@lumc.edu

Yilin Wu Harvard University Molecular and Cellular Biology / Rowland Institute 100 Edwin H. Land Boulevard Cambridge, MA 2142 Phone: (617) 497-4606 ywu@rowland.harvard.edu

Kristin Wuichet University of Tennessee Department of Microbiology M409 Walters Life Sciences Building Knoxville, TN 37996 Phone: (865) 974-7687 kwuichet@utk.edu

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Zhaomin Yang Virginia Tech Biological Sciences 103 LS1 (Mailcode: 0910) Blacksburg, VA 24061 Phone: (540) 231-1350 zmyang@vt.edu

Junhua Yuan Harvard University Molecular and Cellular Biology 16 Divinity Ave, BioLabs 3063 Cambridge, MA 2138 Phone: (617) 495-4217 jyuan@mcb.harvard.edu

Gabriel Zarbiv Weizmann Institute of Science Biological Chemistry 26 Ben-Yehuda st. Rehovot, Israel 76301 Israel Phone: +972-8-934-2710 gabriel.zarbiv@weizmann.ac.il

Hardik Zatakia Virginia Tech Biological Sciences Life Sciences 1 Washington Street Blacksburg, VA 24060 Phone: (540) 231-0772 hzatakia@vt.edu

Haiyang Zhang Rice University 6100 Main St. MS 142 Houston, TX 77005 Phone: (713) 348-3066 hyzhang@rice.edu

Yang Zhang University of Utah Biology 257 South 1400 East Salt Lake City, UT 84112 Phone: (801) 585-3961 yang.zhang@utah.edu

Xiaowei Zhao University of Texas Medical School at Houston department of pathology and laboratory of medicine 6431 Fannin Houston, TX 77030 Phone: (713) 500-5254 Xiaowei.Zhao@uth.tmc.edu

Petra Zimmann University of Osnabrueck Department of Biology/Chemistry Barbarastr. 11 Osnabrueck, Germany 49078 Germany Phone: +49-5419-692-855 Fax: +49-5419-692-870 zimmann@biologie.uni-osnabrueck.de

David Zusman University of California, Berkeley Molecular and Cell Biology 16 Barker Hall #3204 Berkeley, CA 94720-3204 Phone: (510) 642-2293 Fax: (510) 642-7038 zusman@berkeley.edu

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BLAST STAFF Tarra Bollinger Molecular Biology Consortium 835 S. Wolcott Ave. (M/C 790) Chicago, IL 60612 Phone: (312) 996-1216 Fax: (312) 413-2952 tbolli1@uic.edu 

    Peggy O’Neill Molecular Biology Consortium 835 S. Wolcott Ave. (M/C 790) Chicago, IL 60612 Phone: (312) 996-1216 Fax: (312) 413-2952 oneill@uic.edu 

 

137

INDEX

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab 

138

A Adase, Christopher, 95, 121 - Manson, Michael Alexandre, Gladys, ii, 121 - Alexandre, Gladys Amin, Divyaben, 83, 121 - Gerald , Hazelbauer Atsumi, Tatsuo, 56, 121 - Atsumi, Tatsuo Au, Shannon, 58, 121 - Au, Shannon

B Baraquet, Claudine, 81, 121 – Harwood, Caroline Behrens, Wiebke, 86, 121 - Josenhans, Christine Berg, Howard, v, 121 - Berg, Howard Berlinberg, Adam, 76, 121 - Falke, Joseph Bi, Shuangyu, 94, 121 - Luhua , Lai Bible, Amber, 52, 122 - Alexandre, Gladys Blair, David, vii,122 - Blair, David Boll, Joseph, 5, 122 - Hendrixson, David Bollinger, Tarra, ii, 137 - Matsumura, Philip Bourret, Bob, ii, 122 - Bourret, Bob Branch, Richard, 62, 122 - Berg, Howard Briegel, Ariane, 18, 122 - Jensen, Grant Brown, Mostyn, 55, 122 - Armitage, Judy

C Campodonico, Eva, 119, 122 - Zusman, David Castelli, Cécile, 100, 122 - Méjean, Vincent Charon, Nyles, 68, 122 - Charon, Nyles Crane, Brian, 123 - Crane, Brian Creager-Allen, Rachel, 123 - Bourret, Robert Crosson, Sean, ii, 71, 123 - Crosson, Sean

D Dahlquist, Rick, 123 - Dahlquist, Rick De Beyer, Jennifer, 54, 123 - Armitage, Judith Dogra, Gaurav, 108, 123 - Scharf, Birgit Dong, Qian, 59, 123 - Bauer, Carl Draheim, Roger, 74, 123 - Draheim, Roger Draper, William, 123 - Liphardt, Jan

E Eisenbach, Michael, 123 - Eisenbach, Michael Erbse, Annette, 15, 124 - Falke, Joseph

F Falke, Joseph, ii, 124 - Falke, Joseph Fraiberg, Milana, 124 - Eisenbach, Michael Fukuoka, Hajime, 22, 124 - Ishijima, Akihiko

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab 

139

G Galperin, Michael, 14, 124 - Koonin, Eugene Geng, Haifeng, 32, 124 - Belas, Robert Gohara, Mizuki, 84, 124 - Homma, Michio Guo, Shukui, 124 - Blair, David Gutman Pasvolsky, Ronit, 124 - Eisenbach, Michael

H Hall, Benjamin, 17, 124 - Sansom, Mark Haneburger, Ina, 28, 125 - Jung, Kirsten Harshey, Rasika, 79, 125 - Harshey, Rasika Harwood, Caroline, 125 - Harwood, Caroline Hazelbauer, Gerald, 125 - Hazelbauer, Gerald Hendrixson, David, 29, 125 - Hendrixson, David Higgs, Penelope, vii, 4, 125 - Higgs, Penelope Hobley, Laura, 9, 125 - Sockett, R. Elizabeth Hosu, Basarab, 125 - Berg, Howard Hu, Linda, 35, 125 - Wolfe, Alan Huangyutitham, Varisa, 8, 125 - Harwood, Caroline Hughes, Kelly, ii, 126 - Hughes, Kelly Hulett, F. Marion, 126 - Hulett, F. Marion Huynh, Tu-Anh, 2, 126 - Stewart, Valley

I Inoue, Yuichi, 37, 126 - Ishijima, Akihiko Iobbi-Nivol, Chantal, 126 - Méjean, Vincent Ishijima, Akihiko, 126 - Ishijima, Akihiko

J Jain, Ruchi, 88, 126 - Kazmierczak, Barbara Jenal, Urs, ii, 126 - Jenal, Urs Johnson, Mark, 126 - Taylor, Barry Josenhans, Christine, 126 - Josenhans, Christine Jung, Kirsten, 87, 127 - Jung, Kirsten

K Kaimer, Christine, 127 - Zusman, David Kazmierczak, Barbara, 127 - Kazmierczak, Barbara Keilberg, Daniela, 111, 127 - Søgaard-Andersen, Lotte Kenney, Linda, v, 127 - Kenney, Linda Kim, Eun A, 64, 127 - Blair, David Kim, Jihong, 104, 127 - Park, Chankyu Kojima, Seiji, 85, 127 - Homma, Michio Kostick, Jessica, 97, 127 - Marconi, Richard Krell, Tino, 23, 127 - Krell, Tino

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab 

140

L Lam, Kwok Ho, 58, 128 - Au, Wing Ngor Shannon Lan, Ganhui, 33, 115, 128 - Tu, Yuhai Lazova, Milena, 110, 128 - Shimizu, Tom Lee, Jaemin, 80, 128 - Harshey, Rasika Lee, Junghoon, 105, 128 - Park, Chankyu Lee, Yi-Ying, 60, 61, 128 - Belas, Robert Lele, Pushkar, 63, 128 - Berg, Howard Lertsethtakarn, Paphavee, 19, 128 - Ottemann, Karen Levenson, Robert, 128 - Dahlquist, Frederick Li, Chunhao, 90, 128 - Li, Chunhao Li, Guanglai, 10, 129 - Tang, Jay Li, Mingshan, 16, 129 - Hazelbauer, Gerald Li, Na, 42, 129 - Homma, Michio Li, Xiaoxiao, 69, 129 - Crane, Brian Lin, Tao, 101, 129 - Norris, Steven Liu, Jun, 92, 129 - Liu, Jun

M Manson, Mike, ii, 24, 96, 129 - Manson, Michael Marconi, Richard, 129 - Marconi, Richard Matsumura, Philip, 129 - Matsumura, Philip McBride, Mark, vi, 48, 129 - McBride, Mark McMurry, Jonathan, 99, 130 - McMurry, Jonathan Mo, Guoya, 130 - Dahlquist, Rick Msadek, Tarek, ii, 31, 130 - Msadek, Tarek

N Nan, Beiyan, 46, 130 - Zusman, David Nieto, Vincent, 40, 130 - Harshey, Rasika Nishiyama, Masayoshi, 38, 130 - Nishiyama, Masayoshi

O O'Connor, Christopher, 102, 130 - O'Connor, Christopher O'Connor, Jennifer, 82, 130 - Harwood, Caroline O'Donovan, Karen, 73, 130 - Dow, John Maxwell O'Neill, Peggy, ii, 137 - Matsumura, Philip Ortega, Davi, 118, 130 - Zhulin, Igor Ottemann, Karen, ii, 27, 131 - Ottemann, Karen

P Page, Clinton, 89, 131 - Krause, Duncan Page, Stephani, 131 - Bourret, Robert Paradis, Guillaume, 107, 131 - Rainville, Simon Parales, Rebecca, 131 - Parales, Rebecca Park, Chankyu, 131 - Park, Chankyu

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab 

141

Parkinson, John, 131 - Parkinson, J. S. Paul, Koushik, 64, 131 - Blair, David Pawelczyk, Sonja, 6, 131 - Wadhams, George Petters, Tobias, 3, 131 - Søgaard-Andersen, Lotte Pham, Hai, 26, 132 - Parkinson, John Politzer, Adam, 90, 132 - Liphardt, Jan Porter, Steven, 105, 132 - Porter, Steven Prüß, Birgit, vi, 30, 132 - Prüß, Birgit

R Rainville, Simon, 132 - Simon, Rainville Rao, Christopher, v, 132 - Rao, Christopher Rhodes, Ryan, 49, 132 - McBride, Mark Russell, Matthew, 25, 132 - Alexandre, Gladys

S Sarkar, Mayukh, 66, 132 - Blair, David Scharf, Birgit, 132 - Scharf, Birgit Schniederberend, Maren, 43, 133 - Kazmierczak, Barbara Shimizu, Thomas, ii, 133 - Shimizu, Tom Si, Guangwei, 103, 133 - Ouyang, Qi Silversmith, Ruth, 133 - Bourret, Robert Sircar, Ria, 70, 133 - Crane, Brian Slivka, Peter, 77, 133 - Falke, Joseph Sneddon, Michael, 12, 75, 133 - Emonet, Thierry Søgaard-Andersen, Lotte, 133 - Søgaard-Andersen, Lotte Studdert, Claudia, 112, 133 - Studdert, Claudia Sukomon, Nattakan, 20, 133 - Crane, Brian Swain, Kalin, 78, 134 - Falke, Joseph Szkotnicki, Lee, 98, 134 - Marconi, Richard

T Taylor, Barry, ii, 134 - Taylor, Barry Thompson, Lynmarie, 113, 134 - Thompson, Lynmarie Thormann, Kai, 114, 134 - Thormann, Kai Tipping, Murray, 41, 134 - Armitage, Judith Tu, Yuhai, 11, 134 - Tu, Yuhai

V Vartanian, Armand, 72, 134 - Dahlquist, Rick Vu, Anh, 134 - Dahlquist, Rick

W Wadhams, George, 134 - Wadhams, George Wall, Dan, 47, 135 - Wall, Dan Walthers, Don, 36, 135 - Kenney, Linda Wang, Loo Chien, 7, 135 - Anand, Ganesh Srinivasan

Participant’s Name, Abstract page(s), Contact Information Page – PI Lab 

142

Ward, Mandy, 116, 135 - Ward, Mandy Watts, Kylie, 21, 135 - Taylor, Barry Webb, Ben, 135 - Scharf, Birgit Wilson, Laurence, 34, 135 - Wilson, Laurence Wolfe, Alan, 135 - Wolfe, Alan Wu, Yilin, 50, 135 - Berg, Howard Wuichet, Kristin, 13, 135 - Zhulin, Igor

Y Yang, Zhaomin, 117, 136 - Yang, Zhaomin Yuan, Junhua, 39, 136 - Berg, Howard

Z Zarbiv, Gabriel, 44, 136 - Eisenbach, Michael Zatakia, Hardik, 109, 136 - Scharf, Birgit Zhang, Haiyang, 45, 136 - Igoshin, Oleg Zhang, Yang, 67, 136 - Blair, David Zhao, Xiaowei, 93, 136 - Liu, Jun Zimmann, Petra, 53, 136 - Altendorf, Karlheinz Zusman, David, 136 - Zusman, David