BLAST X MEETING CAMINO REAL SUMIYA HOTEL

CUERNAVACA, MEXICO JANUARY 18-23, 2009

Meeting Chairperson:

Dr. David Zusman –University of California, Berkeley, CA

Meeting Vice-Chairperson: Dr. Robert Bourret – University of North Carolina, Chapel Hill, NC

Local Arrangements Chairperson

Dr. Georges Dreyfus - Universidad Nacional Autonoma de Mexico, Mexico City

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

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

Robert Kadner and Robert Macnab Awards Selection Committee: Dr. Gladys Alexandre – University of Tennessee, Knoxville, TN

Dr. Gerald Hazelbauer (Chairperson) – University of Missouri, Columbia, MO Dr. Ruth Silversmith - University of North Carolina, Chapel Hill, NC

Dr. Alan Wolfe – Loyola University, Maywood, IL

Meeting Review Committee: Dr. Phillip Aldridge – Newcastle University, Newcastle upon Tyne, UK

Dr. Brian Crane (Chairperson) – Cornell University, Ithaca, NY Dr. John Kirby – University of Iowa, Iowa City, IA

Dr. Birgit Scharf – Virginia Tech University, Blacksburg, VA

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

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

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BLAST X MEETING SCHEDULE

TIME EVENT LOCATION

Sunday, January 18, 2009 5:00 pm Poster room available for poster setup Orquideas Room 7:00 pm – 8:00 pm Dinner Garden 9:00 pm – 11:00 pm Welcome Reception Crisantemos Room Monday, January 19, 2009 7:30 am – 8:30 am Breakfast Garden 8:45 am – 8:50 am Welcome -- Local Arrangements Chair (G. Dreyfus) Crisantemos Room 8:50 am – 8:55 am Welcome/ Announcements - Meeting Chair (D. Zusman) Crisantemos Room 9:00 am – 12:00 pm Meeting Session – “Two-Component Systems” Crisantemos Room 10:15 am – 10:30 am Coffee Break 12:00 pm – 1:30 pm Lunch Garden 2:00 pm – 4:00 pm Poster Session – even numbered posters Orquideas Room 6:00 pm – 7:30 pm Dinner Garden 7:30 pm – 10:00 pm Meeting Session – “Gliding Motility” Crisantemos Room 8:30 pm – 8:45 pm Coffee Break Tuesday, January 20, 2009 7:30 am – 8:30 am Breakfast Garden 9:00 am – 12:00 pm Meeting Session – “Flagella” Crisantemos Room 10:15 am – 10:30 am Coffee Break 12:00 pm – 1:30 pm Lunch Garden 2:00 pm – 4:00 pm Poster Session – odd numbered posters Orquideas Room 6:00 pm – 7:30 pm Dinner Garden 7:30 pm – 10:00 pm Meeting Session – “Regulation” Crisantemos Room 8:30 pm – 8:45 pm Coffee Break Wednesday, January 21, 2009 7:30 am – 8:30 am Breakfast Garden 9:00 am – 12:00 pm Meeting Session – “Chemoreceptors” Crisantemos Room 10:15 am – 10:30 am Coffee Break 12:00 pm – 1:30 pm Lunch Garden 1:30 pm Tour bus to Taxco leaves from Hotel Hotel entrance 1:45 pm Tour buses to Xochicalco leave from Hotel Hotel entrance Thursday, January 22, 2009 7:30 am – 8:30 am Breakfast Garden 9:00 am – 12:00 pm Meeting Session – “Biofilms and Host Interactions” Crisantemos Room 10:15 am – 10:30 am Coffee Break 12:00 pm – 1:30 pm Lunch Garden 6:00 pm – 7:30 pm Dinner Garden 7:30 pm – 7:45 pm Robert Kadner and Robert Macnab Awards Presentation Crisantemos Room 7:45 pm – 10:00 pm Meeting Session – “Signaling and Behavior” Crisantemos Room 8:30 pm – 8:45 pm Coffee Break 10:00 pm – 12:00 am Reception Crisantemos Room Friday, January 23, 2009 7:30 am – 8:30 am Breakfast Garden Shuttle buses leave for Mexico City Airport

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BLAST X PROGRAM January 19, 2009 Two-Component Systems Monday Morning – 9:00 am – 12:00 pm Chair – Georges Dreyfus

PRESENTER TITLE ABSTRACT PAGE NO.

Bourret, Bob A structural investigation of response regulator autodephosphorylation 2

Peña-Sandoval, G. Intramolecular autophosphorylation of the Escherichia coli ArcB sensor kinase 3

Porter, Steven A bifunctional kinase-phosphatase in bacterial chemotaxis 4 BREAK

Szurmant, Hendrik Message passing: Protein structure assembly from sequence data for two-component signaling proteins 5

Crosson, Sean Integrated control of Caulobacter cell envelope physiology by a hybrid two-component/ECF sigma factor signaling network 6

Bisicchia, Paola The role of signal transduction in cell wall metabolism in Bacillus subtilis 7

Dubrac, Sarah The WalK/WalR essential signal transduction pathway and cell wall homeostasis in Staphylococcus aureus 8

January 19, 2009 Gliding Motility Monday Evening – 7:30 pm – 9:30 pm Chair – Lotte Søgaard Andersen Bulyha, Iryna

Dynamic assembly and disassembly of the Type IV molecular machine 9

Higgs, Penelope A “four component” signal transduction system regulates developmental progression in Myxococcus xanthus 10

Yang, Zhaomin Independence and interdependence of Dif and Frz chemosensory pathways in Myxococcus xanthus chemotaxis 11

BREAK Berleman, James Predataxis behavior in Myxococcus xanthus 12 Mauriello, Emilia Dynamic localization of FrzCD in Myxococcus xanthus 13

Gitai, Zemer Identifying novel bacterial cytoskeletal elements and cytoskeletal interactors through high-throughput imaging 14

January 20, 2009 Flagella Tuesday Morning – 9:00 am – 12:00 pm Chair – Robert Belas

Rao, Christopher The role of positive feedback in controlling flagella assembly dynamics 15

Imada, Katsumi Crystal structure of FliT, a bacterial flagellar export chaperone for the filament cap protein Hap2 (FliD) 16

Kojima, Seiji Structural insight into active flagellar motor formation through the periplasmic region of MotB 17

Thormann, Kai Stator selection in Shewanella oneidensis 18 BREAK

Gauthier, Mathieu Taking control of the bacterial flagellar motor 19

Branch, Richard Experimental evidence for conformational spread in the bacterial switch complex 20

Reuven, Peter Do individual bacterial flagellar motors use hysteresis to maintain a robust output in a noisy environment? – an experimental study 21

Tu, Yuhai Dynamics of the bacterial flagellar motor with multiple stators 22

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January 20, 2009 Regulation Tuesday Evening – 7:30 pm – 9:30 pm Chair – Linda Kenney

PRESENTER TITLE ABSTRACT PAGE NO.

Kaserer, Alla Effect of osmolytes on regulating the activities of the SSK1 response regulator from Saccharomyces cerevisiae 23

Partridge, John Regulation of Escherichia coli motility by the nitric oxide sensitive transcriptional repressor NsrR 24

Reimann, Sylvia Synthetic lethality uncovers a novel link between the MalT and OmpR regulons 25

BREAK

Tran, Hoa A chemotaxis-like signaling pathway regulates the expression of extracellular materials in Geobacter sulfurreducens 26

Martínez, Luary

The two-component regulatory system BarA/SirA is at the top of a multi-factorial regulatory cascade controlling the expression of the SPI-1 and SPI-2 virulence regulons in Salmonella 27

Lee, Yi-Ying Interaction of the transcriptional regulatory complex, FlhDC, with its target DNA 28

January 21, 2009 Chemoreceptors Wednesday Morning – 9:00 am – 12:00 pm Chair – Sandy Parkinson Glekas, George A novel amino acid binding structure in bacterial chemotaxis 29

Remington, James Structure and function of the Helicobacter pylori chemoreceptor TlpB 30

Wright, Gus The TM2-HAMP connection 31

Khursigara, Cezar

Structure, assembly and conformational changes in chemoreceptors studied in intact bacterial cells using Cryo-electron tomography 32

BREAK

Watts, Kylie Discrete signal-on and -off conformations in the Aer HAMP domain 33

Bhatnagar, Jaya

Investigating the structure of ternary complex of histidine kinase CheA, coupling protein CheW, and chemoreceptor by pulsed dipolar ESR spectroscopy 34

Erbse, Annette The chemotactic core signalling complex is ultrastable 35 Briegel, Ariane Electron cryotomography of bacterial chemotaxis arrays 36

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January 22, 2009 Biofilms & Host Interactions Thursday Morning – 9:00 am – 12:00 pm Chair – Alan Wolfe

PRESENTER TITLE ABSTRACT PAGE NO.

Belas, Robert Two regulatory proteins control the swim-or-stick switch in Roseobacters 37

Oropeza, Ricardo Deletion analysis of RcsC reveals a novel signaling-pathway controlling biofilm formation in Escherichia coli 38

Vlamakis, Hera Regulation of cell fate in Bacillus subtilis biofilms 39

Chapman, Matt Protein misfolding done right: The biogenesis of bacterial amyloid fibers 40

BREAK Martinez del Campo, Ana

Rhodobacter sphaeroides, a bacterium with two flagellar systems and multiple chemotaxis gene homologs 41

Motaleb, MD Motility, chemotaxis and virulence of Borrelia burgdorferi, the lyme disease spirochete 42

Prüß, Birgit Pleiotropic phenotypes of a Yersinia enterocolicia flhD mutant include reduced lethality in a chicken embryo model 43

Gonzalez, Juan Regulation of motility by quorum sensing in Sinorhizobium meliloti and its role in symbiosis establishment 44

January 22, 2009 Signaling & Behavior Thursday Evening – 7:30 pm – 9:30 pm Chair – Judy Armitage Goldberg, Shalom Engineered single- and multi-cell chemotaxis in E. coli 45

Jung, Kwang-Hwan Photo-energy conversion and sensory transduction of microbial rhodopsins in photosynthetic microbes

46

Bible, Amber

Function of multiple chemotaxis-like pathways in mediating changes in motility patterns and cellular morphology in Azospirillum brasilense

47

BREAK

Shimizu, Thomas Probing adaptation kinetics in vivo by fluorescence resonance energy transfer

48

Neumann, Silke Minor receptor signalling in E. coli 49

Chastanet, Arnaud A systems biology approach to understanding how Bacillus makes up its mind

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POSTERS - BLAST X

Poster # Lab Presenter Title Page #1 Phillip

AldridgeAldridge, Christine The characterisation of the dynamics of the

FliT:FliD:FlhD4C2 interaction and its role in regulating flagellar assembly

52

2 Phillip Aldridge

Aldridge, Phillip Subunit feedback control of flagellar filament assembly in Caulobacter crescentus

53

3 GladysAlexandre

Alexandre, Gladys Function of unique domains of CheA1 from A. Brasilense in regulating multiple cellular behaviors

54

4 Judith Armitage

Brown, Mostyn How does the Rhodobacter sphaeroides flagellar motor stop – Using a clutch or a brake?

55

5 Judith Armitage

Delalez, Nicolas Jacques Dynamics of the flagellar motor protein FliM 56

6 Judith Armitage

Roberts, Mark A J Using control theory to elucidate connectivity in R. Sphaeroides chemotaxis

57

7 Howard Berg

Yuan, Junhua Behavior of the flagellar rotary motor near zero load 58

8 DavidBolam

Diaz-Mireles, Edith Nutrient sensing by a human gut symbiont 59

9 EdmundoCalva

De la Cruz, Miguel Ángel EnvZ-OmpR and CpxA-CpxR regulate ompS1 by differential promoter expression

60

10 EdmundoCalva

Gallego, Ana The LeuO regulon in Salmonella 60

11 NylesCharon

Miller, Kelly Ann The complex hook basal body structure of the Lyme disease spirochete Borrelia burgdorferi

61

12 Brian Airola Michael How do PAS and HAMP domains communicate? 6212 BrianCrane

Airola, Michael How do PAS and HAMP domains communicate? Insights from Aer2, a heme based sensor for aerotaxis

62

13 BrianCrane

Pollard, Abiola M Structure of soluble chemoreceptor suggests a mechanism for propagating conformational signals

63

14 GeorgesDreyfus

Castillo, David Jaime Functional analysis of a large non-conserved region of FlgK (HAP1) from Rhodobacter sphaeroides

64

15 GeorgesDreyfus

De la Mora, Javier The flagellar muramidase from the photosynthetic bacterium Rhodobacter sphaeroides

65

16 ThierryEmonet

Alexander, Roger Modeling scaffold phosphorylation as an adaptation mechanism in bacterial chemotaxis

66

17 JosephFalke

Swain, Kalin Testing the Yin-Yang model of signal transduction in a bacterial chemoreceptor cytoplasmic domain

67

18 DimitrisGeorgellis

Alvarez, Adrian Fernando Cytochrome d but not cytochrome o rescues the toluidine blue growth sensitivity of arc mutants in E. Coli

68

19 Georgellis, Dimitris

González, Ricardo Searching the physiological signal(s) that regulate the activity of the sensor kinase BarA

69

20 JuanGonzalez

Gurich, Nataliya The role of quorum sensing in the control of motility and plant invasion by Sinorhizobium meliloti

70

21 BerthaGonzález-Pedrajo

García-Gómez, Elizabeth Characterization of etga, a muramidase associated with the type III secretion system of enteropathogenic Escherichia coli

71

22 RasikaHarshey

Lee, Jae-Min FlhE: a periplasmic chaperone of flagellin? 72

23 RasikaHarshey

Nieto, Vincent Michael The cyclic-di-GMP receptor protein YcgR localizes to the flagellar basal body and changes motor bias in Salmonella

73

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POSTERS - BLAST X

Poster # Lab Presenter Title Page #24 Michio

HommaHizukuri, Yohei Analysis of the peptidoglycan-binding domain of the

flagellar stator protein MotB using systematic mutagenesis and chimeric protein in Escherichia coli

75

25 MichioHomma

Koike, Masafumi Attempt to purify the hook-basal body with C-ring from the Na+-driven flagellar motor

76

26 ScottHultgren

Kline, Kimberly Mechanism for sortase localization and role in efficient pilus assembly in Enterococcus faecalis

77

27 AkihikoIshijima

Fukuoka, Hajime Visualization of exchange of rotor component in functioning bacterial flagellar motor

78

28 AkihikoIshijima

Inoue, Yuichi Torque response of the sodium-driven chimeric flagellar motor in E.coli induced by reversible temperature change

79

29 ChristineJosenhans

Josenhans, Christine The Helicobacter pylori flagellar anti-sigma factor FlgM remains bacteria-associated and interacts with FlhAc

80

30 IkuroKawagishi

Inaba, Takehiko The localization patterns of all histidine kinases in Escherichia coli cell

81

31 IkuroKawagishi

Nishiyama, So-ichiro Thermosensing function of Aer, a redox sensor of E. Coli

82

32 KenjiOosawa

Hayashi, Fumio ATPase activity of T3SS specific ATPase InvC 83

33 KenjiOosawa

Hayashi, Fumio Characterizations of the pseudorevertants from Salmonella typhimurium strain SJW1655 and

84Oosawa Salmonella typhimurium strain SJW1655 and

SJW1660 with the R- and the L-type straight flagellar filaments

34 KenjiOosawa

Hayashi, Fumio Raman optical activity and vibrational circular dichroism of flagellar filaments of Salmonella

85

35 Cancelled Poster

86

36 JohnKirby

Willett, Jonathan CrdC negatively regulates CheW3 and CheA3 interaction during signal transduction in Myxococcus xanthus

87

37 JunLiu

Liu, Jun Molecular architecture of intact flagellar motor revealed by Cryo-Electron tomography

88

38 JanineMaddock

Dobkowski, Jason Mining the E. Coli GFP fusion collection 89

39 MichaelManson

Adase, Christopher Understanding the fundamental elements of signaling in the Tar chemoreceptor

90

40 MichaelManson

Crowder, Rachel Leann Linking the TM2 to HAMP—a tough nut to crack? 91

41 MichaelManson

Seely, Andrew Electrostatic effects on signaling mutations in the C-terminal region of the Escherichia coli aspartate chemoreceptor

92

42 PhilipMatsumura

Lee, Yi-Ying Interaction of the transcriptional regulatory complex, FlhDC, with its target DNA

93

43 JonathanMcMurry

McMurry, Jonathan Application of biolayer interferometry to understanding interactions among Salmonella enterica flagellar export apparatus proteins

94

44 PaulMilewski

Simons, Julie A Cross-Species Comparison of Chemotactic Behavior

95

45 MakotoMiyata

Nakane, Daisuke Tethered Mycoplasma 96

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POSTERS - BLAST X

Poster # Lab Presenter Title Page #46 Makoto

MiyataNonaka, Takahiro Molecular shapes of Gli123 and Gli521 involved in

gliding motility of Mycoplasma mobile97

47 TarekMsadek

Delauné, Aurelia The essential nature of the WalK/WalR signal transduction pathway is linked to cell wall hydrolase activity in Staphylococcus aureus

98

48 TarekMsadek

Falord, Mélanie The GraS/GraR two-component system and dermaseptin resistance in Staphylococcus aureus

99

49 KeiichiNamba

Che, Yong-Suk Characterization of suppressors of the MotB(D33E) mutation, a putative proton-binding residue of the bacterial flagellar motor

100

50 KeiichiNamba

Ibuki, Tatsuya Structure of FliJ, a cytoplasmic component of the flagellar type III; protein export apparatus of Salmonella

101

51 KeiichiNamba

Makino, Fumiaki CryoEM structure of the hook-filament junction of Salmonella

102

52 KeiichiNamba

Nakamura, Shuichi Effect of intracellular pH on the torque-speed relationship of bacterial proton-driven flagellar motor

103

53 KeiichiNamba

Yoshimura, Shinsuke Fluorescence imaging of assembly and disassembly of the bacterial flagellar protein export ATPase FliL to the flagellar basal body

104

54 Karen Ottemann

Ottemann, Karen M Analysis of Helicobacter pylori lacking all four chemoreceptors

105Ottemann chemoreceptors

55 RebeccaParales

Parales, Rebecca E. Chemotaxis to pyrimidines and identification of a cytosine chemoreceptor in Pseudomonas putida

106

56 ChankyuPark

Lee, Changhan Reactive aldehydes and motility in Escherichia coli K12

107

57 SimonRainville

Rainville, Simon Taking control of the bacterial flagellar motor 108

58 TracyRaivio

Malpica, Roxana Characterization of the periplasmic domain of the sensor kinase CpxA: Role of conserved residues in CpxA activity

109

59 ChristopherRao

Saini, Supreet Characterization of FliZ as an activator of flagellar genes in Salmonella enterica serovar typhimurium

110

60 ChristopherRao

Wu, Kang Localization of the chemotaxis proteins in Bacillus subtilis

111

61 KathleenRyan

Ryan, Kathleen R RcdA structure and function in regulated CtrA proteolysis

112

62 MarkSansom

Hall, Benjamin Modelling MCP signalling mechanisms with high-throughput simulation of Tar TM2

113

63 FlorianSchubot

Schubot, Florian D. Structural evidence suggests that antiactivator ExsD from Pseudomonas aeruginosa is a DNA binding protein

114

64 VictoriaShingler

Herrera Seitz, María Karina A novel PAS-GGDEF-EAL protein involved in regulation of motility in Pseudomonas putida

115

65 VictorSourjik

Sommer, Erik In vivo study of the two-component signalling network in E. coli

116

66 AnnStock

Han, Hua Gene regulation by Escherichia coli response regulator PhoB

117

67 ClaudiaStuddert

Massazza, Diego Ariel New reporter residues of trimer formation by Escherichia coli MCPs

118

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POSTERS - BLAST X

Poster # Lab Presenter Title Page #68 Motohide

TakahashiKenri, Tsuyoshi Systematic detection of proteins that localize at the

attachment organelle regions of Mycoplasma119

Takahashi attachment organelle regions of Mycoplasma pneumoniae by fluorescent-protein tagging

69 BarryTaylor

Campbell, Asharie Johnson Mapping the signal transduction pathway within the PAS domain of the Aer receptor

120

70 Gunnar von Heijne

Draheim, Roger Modulating two-component signal output with protein-membrane interactions

121

71 Cancelled Poster

122Poster

72 DouglasWeibel

Copeland, Matthew Francis A mechanical and genetic study of Escherichia coli swarming motility

123

73 DouglasWeibel

Muralimohan, Abishek Probing hydrodynamic interactrions between swimming bacteria using microfulidics

124

74 ZhaominYang

Black, Wesley P. Examination of phosphorylation in the Dif chemotaxis-like system in Myxococcus xanthus

125

75 Igor Cantwell, Brian Evolution of chemotaxis proteins on a micro scale 126g(Zhulin) Jouline

, p

76 Igor(Zhulin) Jouline

Shanafield, Harold Evolution of signal transduction in a bacterial genus 127

77 DavidZusman

Nan, Beiyan The chemosensory receptor FrzCD interacts with two A-motility proteins, AglZ and AgmU

128

78 DimitrisGeorgellis

Barba Ostria, Carlos Arturo Phenotypic characterization of all single mutants of two component system proteins in Neurospora

129

crassa79 Makoto

Miyata Miyata, Makoto AFM study of Mycoplasma mobile's gliding motility 129b

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SPEAKER ABSTRACTS

BLAST X Mon. Morning Session A STRUCTURAL INVESTIGATION OF RESPONSE REGULATOR AUTODEPHOSPHORYLATION Yael Pazy1, Amy C. Wollish1, Stephanie A. Thomas1, Peter J. Miller2, Edward J. Collins1,2, Robert B. Bourret1, and Ruth E. Silversmith1 Departments of Microbiology & Immunology1 and Biochemistry & Biophysics2, University of North Carolina, Chapel Hill, NC 27599

Assorted two-component regulatory systems are used to control a wide variety of biological processes, which occur over a broad range of time scales. To appropriately synchronize the adaptive responses implemented by response regulators with the environmental stimuli detected by sensor kinases, the kinetics of biochemical signaling reactions must be at least as fast as the biological process that they regulate. The fraction of response regulators in the phosphorylated state is determined by the net result of phosphorylation and dephosphorylation. Autodephosphorylation rates reported for various wildtype response regulators span a range of at least 40,000x, consistent with timescales ranging from about one second to one day. Furthermore, the range of rates observed suggests that autodephosphorylation rates are an important contributor to setting the overall timescales of two-component signaling systems.

We previously found that changing the amino acids at the variable active site positions

corresponding to residues 59 and 89 of Escherichia coli CheY could alter response regulator autodephosphorylation rates about 100x (1). Thus, the particular amino acids found at positions '59' and '89' in response regulators can account for two orders of magnitude in autodephosphorylation rate, but other factors to account for an additional two to three orders of magnitude in reaction rate must also exist and remain to be identified. To begin to characterize the structural basis of response regulator autodephosphorylation rate, we determined high-resolution X-ray crystal structures for five CheY mutants that bear amino acid substitutions at positions 14, 59, and 89 and consequently exhibit autodephosphorylation rates six to 40 times slower than wildtype CheY. Each structure was determined in the presence of the phosphoryl group analog BeF3

- and thus represents the starting point of the autodephosphorylation reaction. Comparison of mutant and wildtype CheY structures, which are matched at all but three residues and yet support different reaction rates, can potentially provide insight into the mechanistic basis by which positions '59' and '89' influence autodephosphorylation rate. Similarly, comparison of each mutant CheY structure with the structure of a wildtype response regulator that is matched at eight (three variable and five conserved) active site residues and yet catalyzes autodephosphorylation at rates 10-80x slower than the CheY mutants can potentially suggest candidates for additional factors that might contribute to autodephosphorylation rate.

Response regulator autodephosphorylation likely involves an inline attack on the

phosphoryl group by a nucleophilic water molecule. The structural comparisons outlined above clearly suggest that autodephosphorylation rate is influenced by the extent to which amino acid sidechains at positions '59' or '89' sterically occlude access to the phosphoryl group. Additional factors potentially affecting autodephosphorylation rate will also be discussed. Reference 1. Thomas, S.A., Brewster, J.A., & Bourret, R.B. (2008) Two nonconserved active site residues

affect response regulator phosphoryl group stability. Molecular Microbiology 69, 453-465.

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BLAST X Mon. Morning Session INTRAMOLECULAR AUTOPHOSPHORYLATION OF THE ESCHERICHIA COLI ArcB SENSOR KINASE Gabriela R. Peña-Sandoval, Luis A. Nuñez Oreza and Dimitris Georgellis Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México City, México. The Arc two-component system is a complex signal transduction system that plays a key role in regulating energy metabolism at the level of transcription in bacteria. This system comprises the ArcB protein, a tripartite membrane-associated sensor kinase, and the ArcA protein, a typical response regulator. Under anoxic growth conditions, ArcB autophosphorylates and transphosphorylates ArcA, which in turn represses or activates the expression of its target operons. Under aerobic conditions, the kinase activity of ArcB is silenced by the oxidation of two cytosol-located redox-active cysteine residues that participate in intermolecular disulfide bond formation, a reaction in which the quinones provide the source of oxidative power.

Here we present results demonstrating that the putative leucine-zipper near the second

transmembrane segment of ArcB is functional and necessary for proper ArcB signaling. Moreover, we provide data demonstrating that in contrast to the proposed model of intermolecular autophosphorylation, ArcB autophosphorylation is an intra-molecular reaction.

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BLAST X Mon. Morning Session A BIFUNCTIONAL KINASE-PHOSPHATASE IN BACTERIAL CHEMOTAXIS Steven L. Porter, Mark A.J. Roberts, Cerys S. Manning and Judith P. Armitage1

Oxford Centre for Integrative Systems Biology (OCISB), Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU

Phosphorylation based signaling pathways employ dephosphorylation mechanisms for signal termination. Histidine to aspartate phosphosignaling in the two-component system controlling bacterial chemotaxis has been studied extensively. Rhodobacter sphaeroides has a complex chemosensory pathway with multiple homologues of the Escherichia coli chemosensory proteins, although it lacks homologues of known signal terminating CheY-P phosphatases such as CheZ, CheC, FliY or CheX. Here we demonstrate that an unusual CheA homologue, CheA3, is not only a phosphodonor for the principal CheY protein, CheY6, but is also is a specific phosphatase for CheY6-P. This phosphatase activity accelerates CheY6-P dephosphorylation to a rate that is comparable with the measured stimulus response time of ~1 s. CheA3 possesses only two of the five domains found in classical CheAs, the Hpt (P1) and regulatory (P5) domains, which are joined by a novel 794 amino acid sequence that is required for phosphatase activity. The P1 domain of CheA3 is phosphorylated by CheA4 and it subsequently acts as a phosphodonor for the response regulators. A CheA3 mutant protein deleted for the 794 amino acid region lacked phosphatase activity, retained phosphotransfer function but did not support chemotaxis, suggesting that the phosphatase activity may be required for chemotaxis. Using a nested deletion approach we show that a 200 amino acid segment of CheA3 is required for phosphatase activity. The phosphatase activity of previously identified non-hybrid histidine protein kinases depends upon the dimerization and histidine phosphorylation (DHp) domains. CheA3, however, lacks a DHp domain, suggesting that CheA3 is a novel phosphatase.

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BLAST X Mon. Morning Session MESSAGE PASSING: PROTEIN STRUCTURE ASSEMBLY FROM SEQUENCE DATA FOR TWO-COMPONENT SIGNALING PROTEINS Hendrik Szurmant1, Martin Weigt2,3, Robert White1,3, Terry Hwa3 and James A. Hoch1

1 Division of Cellular Biology, Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, CA 92037

2 Institute for Scientific Interchange, I-10133 Torino, Italy 3 Center for Theoretical Biological Physics and Department of Physics, University of California at

San Diego, La Jolla, CA 92093 The crystal structure of the Bacillus subtilis response regulator Spo0F in complex with the histidine kinase structural homologue Spo0B defined the active site of phosphotransfer and the spatial interactions of two-component systems of microbes and plants. The limited bioinformatic data available at the time was sufficient to deduce and understand the molecular basis for recognition specificity between histidine kinases and response regulators (J. A. Hoch and K. I. Varughese. 2001. J. Bacteriol. 183:4941-4949). Today, the availability of large protein databases generated from sequences of hundreds of bacterial genomes enables more sophisticated statistical approaches to extract interacting and specificity determining positions between proteins from protein databases. The goal of such studies is to identify protein interaction surfaces from sequencing data alone, without previous structural knowledge, i.e. co-crystal data. A number of co-variance based approaches producing nearly identical results have been applied to the highly amplified two-component systems as a means to verify mathematical data with structural knowledge of sensor kinase/response regulator interaction; including one presented by us at the BLAST IX in 2007 (R. A. White et al. 2007. Methods Enzymol. 422:75-101). While producing potential specificity determining position information, these methods have an important shortcoming in predicting spatial proximity. They cannot distinguish between directly correlated (interacting) and indirectly correlated (non-interacting) residue positions. To address this issue we developed a novel method that combines co-variance analysis with global inference analysis, adopted from use in statistical physics. When applied to a set of over 2500 representatives of the bacterial two-component signal transduction system, the combination of covariance with global inference methods successfully and robustly identified residue pairs that are proximal in space without resorting to ad hoc tuning parameters, both for hetero-interactions between sensor kinase (SK) and response regulator (RR) proteins and for homo-interactions between RR proteins. The spectacular success of this approach illustrates the effectiveness of this combination approach in identifying direct interaction positions based on sequence information alone. We expect this method to be applicable for predicting interaction surfaces between proteins present in only one copy per genome as the number of sequenced genomes continues to expand, and for assembling multi-protein structures.

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BLAST X Mon. Morning Session INTEGRATED CONTROL OF CAULOBACTER CELL ENVELOPE PHYSIOLOGY BY A HYBRID TWO-COMPONENT/ECF SIGMA FACTOR SIGNALING NETWORK Robert Foreman, Erin Purcell, Aretha Fiebig, Dan Siegal-Gaskins & Sean Crosson Department of Biochemistry and Molecular Biology, University of Chicago, 929 E. 57th St., Chicago, IL 60637

We present evidence that Caulobacter crescentus encodes a regulatory network that integrates information about two different signals, visible light and oxidative/osmotic stress, to regulate the cell envelope and cell adhesion. In this hybrid signaling system, light signals via the LovK histidine kinase and oxidative/osmotic stress signals via the ECF sigma factor σT are integrated to regulate cell envelope physiology. Caulobacter LovK, exhibits light-controlled autokinase activity and forms a two-component signaling system with the single-domain receiver protein, LovR. We have shown that the LovK/LovR system can function to modulate cell adhesion in response to blue light. LovK/LovR is a negative regulator of σT, an envelope stress sigma factor that is critical for cell survival under osmotic and oxidative stress. σT, in turn, is a positive transcriptional regulator of the lovK/lovR two-component system. This feedback-regulated signaling network can serve as a model to probe how bacterial cells integrate and coordinate their responses to multiple environmental queues.

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BLAST X Mon. Morning Session THE ROLE OF SIGNAL TRANSDUCTION IN CELL WALL METABOLISM IN BACILLUS SUBTILIS Paola Bisicchia, David, Noone, Efthimia Lioliou and Kevin M Devine. Smurfit Institute of Genetics, Trinity College Dublin, Dublin 2. Ireland.

The cell wall of Gram positive bacteria is an extracellular structure, physically removed from the biosynthesis of the precursors used in its synthesis. Peptidoglycan and teichoic acid precursors are synthesized within the cytoplasm and transported across the cytoplasmic membrane where they are incorporated into the cell wall during growth and cell division. The spatial separation of these processes implies bidirectional signaling between the cell wall and cytoplasmic compartments, that recent work has begun to elucidate. We have shown that the essential YycFG two-component signal transduction system of Bacillus subtilis controls cell wall metabolism – during exponential growth, it activates expression of the YocH, YvcE and LytE autolysins and represses expression of YoeB (IseA), an inhibitor of autolysin activity, and YjeA a peptidoglycan deacetylase whose activity on peptidoglycan modulates its susceptibility to autolysin digestion (Howell et al., 2003; Bisicchia et al., 2007; Salzberg and Helmann, 2007; Yamomoto et al., 2008). Thus we propose that YycG senses some aspect(s) of the cell wall externally, perhaps the Lipid II intermediate, and transduces this information into the cytoplasm so that the cell wall synthetic activities in these two compartments are coordinated (Dubrac et al., 2008). We have also demonstrated a close connection between YycFG and the PhoPR two-component system that controls one of the phosphate limitation responses in B. subtilis (Hulett, 2002). YycFG and PhoPR are closely related phylogenetically - hybrid YycF’-‘PhoP and PhoP’-‘YycF response regulators are functional and there are similarities in the YycF and PhoP DNA binding sequences. We have also shown (i) that while YycG can phosphorylate only its cognate response regulator YycF, PhoR can phosphorylate both PhoP and YycF and (ii) that cells depleted for YycFG cannot mount a normal PhoPR-mediated phosphate limitation response. From these observations, we postulated that the roles of YycFG and PhoPR might be linked during cell wall metabolism and phosphate limitation.

In this talk we will present the results of further analysis on the relationships between the

YycFG and PhoPR two-component systems and their roles in cell wall metabolism during growth and phosphate limitation. References: Bisicchia et al., (2007) Molecular Microbiology 65: 180-200. Dubrac et al., (2008) Molecular Microbiology In Press Howell et al., (2003) Molecular Microbiology 49: 1639-1655. Howell et al., (2006) Molecular Microbiology 59:1199-1215. Hulett, (2002) The Pho regulon. in ‘B. subtilis and is closest relatives’ ASM Press. Salzberg and Helmann (2007) J. Bacteriology 189: 4671-4680. Yamamoto et al., (2008) Molecular Microbiology 70: 168-182.

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BLAST X Mon. Morning Session THE WalK/WalR ESSENTIAL SIGNAL TRANSDUCTION PATHWAY AND CELL WALL HOMEOSTASIS IN STAPHYLOCOCCUS AUREUS Sarah Dubrac 1, Aurélia Delauné 1, Olivier Poupel 1, Adeline Mallet 2, Tarek Msadek 1 Biology of Gram-positive Pathogens 1, Department of Microbiology, Plateforme de Microscopie Ultrastructurale 2, Imagopole, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France The highly conserved WalK/WalR (aka YycG/YycF) two-component system is specific to low G+C % Gram-positive bacteria. While this system is essential for cell viability, both the nature of its regulon and its physiological role remained mostly uncharacterized. We have recently shown that the S. aureus WalKR system positively controls autolytic activity, in particular that of the major autolysins, AtlA, LytM and Sle1, and identified at least ten genes belonging to the WalKR regulon that are known or thought to be involved in S. aureus cell wall degradation. While none of these genes appears to be essential, we have shown that their global regulation by the WalKR system results in a drastic down regulation of cell wall dynamics, with a complete arrest of both cell wall biosynthesis and turn over under WalKR depletion. As a consequence of these molecular disorders TEM observations revealed that the cell wall of WalKR depleted cells was significantly thicker and division septa were abnormally distributed. Recent advances presented here have shown that this global regulation is directly linked to WalKR essentiality. While the walRK genes are essential, the WalKR system is inducible since it is assumed that the WalR response regulator is only active when phosphorylated. While the activation signal is still unknown, several recent results suggest that it could be related to cell wall homeostasis. As cell wall metabolism is a major parameter influencing virulence and particularly the innate immune response, we are now interested in characterizing the impact of WalKR on S. aureus virulence. Beyond the regulation of genes involved in cell wall metabolism, we have also shown that the WalKR system activates expression of at least two genes involved in interactions with the extracellular host matrix and influences the capacity of S. aureus to adhere to the host matrix.

References:

1. Dubrac, S., I. G. Boneca, O. Poupel, and T. Msadek. 2007. New insights into the WalK/WalR (YycG/YycF) essential signal transduction pathway reveal a major role in controlling cell wall metabolism and biofilm formation in Staphylococcus aureus. J. Bacteriol. 189:8257-69.

2. Dubrac, S., and T. Msadek. 2008. Tearing down the wall: peptidoglycan metabolism and the WalK/WalR (YycG/YycF) essential two-component system. Adv. Exp. Med. Biol. 631:214-28.

3. Dubrac, S., P. Bisicchia, K. M. Devine and T. Msadek. 2008. A matter of life and death: cell wall homeostasis and the WalKR (YycGF) essential signal transduction pathway. Mol. Microbiol. 70: (in press)

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BLAST X Mon. Evening Session DYNAMIC ASSEMBLY AND DISASSEMBLY OF THE TYPE IV MOLECULAR MACHINE Iryna Bulyha and Lotte Søgaard-Andersen Max Planck Institute for Terrestrial Microbiology, D-35043 Marburg, Germany

Myxococcus xanthus harbors two gliding motility systems, A and S. The S(ocial)-system

depends on type IV pili (T4P) and is generally active only when the cells are within contact distance of each other. T4P undergo cycles of assembly and retraction. The force for S-motility is generated by retraction of T4P. T4P are localized in a unipolar pattern and are present only at the leading pole of the rod-shaped cells. M. xanthus cells periodically undergo cellular reversals in which the old leading pole (which harbors T4P) becomes the new lagging pole (which does not harbor T4P). These observations suggest that in parallel with a cellular reversal, the pole at which T4P assemble switches. The molecular mechanisms regulating the T4P extension/retraction cycle and underlying T4P pole switching remain unknown.

To investigate these mechanisms, we focused on the cellular localization of five highly

conserved T4P biogenesis proteins (PilB, PilT, PilM, PilC and PilQ), which are present in all known T4P systems. PilB and PilT are cytoplasmic proteins and members of the secretion ATPase superfamily of proteins; PilB is required for assembly of T4P, while PilT is necessary for T4P retraction. PilM shows similarity to MreB/FtsA and is indispensable for T4P assembly. PilC is an inner membrane protein and is necessary for T4P assembly. The PilQ secretin forms a gated oligomeric channel for the pilus in the outer membrane. Using immuno-fluorescence microscopy, and time-lapse fluorescence microscopy with functional YFP-fusions, we show that PilQ, PilC and PilM are localized in clusters at both cell poles. These clusters have equal intensities and they do not oscillate between the two poles during reversals. The analysis of PilB and PilT localization revealed that both proteins are localized in polar clusters. PilB is predominantly localized in a cluster at the leading pole and PilT is predominantly localized in a cluster at the lagging cell pole. This localization is dynamic and the two proteins oscillate between the poles during reversals. These observations show that T4P function depends on two sets of proteins: one set is statically localized at both cell poles, and the other set is dynamically localized. Based on these findings we suggest that during cellular reversals, the T4P machinery is disassembled at the old leading pole and reassembled at the new leading pole.

Moreover, we will present the data which suggest that the T4P assembly/retraction cycle

relies on a PilB/PilT competition-based mechanism. According to this model, PilB at the leading cell pole stimulates T4P assembly, and the occasional accumulation of PilT at the leading cell pole results in retraction. Therefore, the two dynamically localized ATPases determine whether assembly or disassembly of pilus takes place.

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BLAST X Mon. Evening Session A "FOUR COMPONENT" SIGNAL TRANSDUCTION SYSTEM REGULATES DEVELOPMENTAL PROGRESSION IN MYXOCOCCUS XANTHUS Sakthimala Jagadeesan, Bongsoo Lee, and Penelope I. Higgs Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, D35043 Marburg, Germany

Myxococcus xanthus responds to starvation by entering a multicellular developmental program in which 105 cells first aggregate into mounds and then within these mounds, differentiate into environmentally resistant spores. Under standard laboratory conditions, formation of spores within the mounds (fruiting bodies) takes approximately 72 hours. We have previously demonstrated that progression through the developmental program appears to be regulated by an atypical two component signal (TCS) transduction system consisting of four TCS homologs (RedC, RedD, RedE, and RedF). While RedC appears to be a typical membrane bound histidine kinase, RedD consists solely of two receiver domains. RedE is a soluble histidine kinase-like protein, and RedF is a single receiver domain response regulator. Based on a combination of genetic and biochemical analyses, we propose a model for how these four Red proteins function together to regulate progression through the developmental program. Our data suggests that development is repressed when the RedC histidine kinase phosphorylates RedF, a single domain response regulator. Developmental repression is relieved when, in response to an unknown signal(s), RedC is instead induced to phosphorylate the response regulator RedD. Surprisingly, the phosphoryl group is then transferred from RedD to the histidine kinase-like protein, RedE. RedE is then likely made accessible to RedF-P, whereupon it removes RedF’s phosphoryl group. We present the data that supports this model. Furthermore, we will address how progression through the developmental program is modulated by the Red system.

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BLAST X Mon. Evening Session INDEPENDENCE AND INTERDEPENDENCE OF Dif AND Frz CHEMOSENSORY PATHWAYS IN MYXOCOCCUS XANTHUS CHEMOTAXIS Qian Xu, Wesley P. Black, Christena L. Cadieux and Zhaomin Yang Biological Sciences, Virginia Tech, Blacksburg, Virginia 24061-0910, USA

Dif and Frz, two Myxococcus xanthus chemosensory pathways, are required in phosphatidylethanolamine (PE) chemotaxis for excitation and adaptation, respectively. DifA and FrzCD, the homologs of methyl-accepting chemoreceptors in the two pathways, were examined for methylation in the context of chemotaxis and inter-pathway interactions. Evidence indicates that DifA may not undergo methylation but signals transmitting through DifA do modulate FrzCD methylation. Results also revealed that M. xanthus possesses Dif-dependent and Dif-independent PE sensing mechanisms. Previous studies showed that FrzCD methylation is decreased by negative chemostimuli but increased by attractants such as PE. Results here demonstrate that the Dif-dependent sensory mechanism suppresses the increase in FrzCD methylation in attractant response and elevates FrzCD methylation upon negative stimulation. In other words, FrzCD methylation is governed by opposing forces from Dif-dependent and Dif-independent sensing mechanisms. We propose that the Dif-independent but Frz-dependent PE sensing leads to increases in FrzCD methylation and subsequent adaptation, while the Dif-dependent PE signaling suppresses or diminishes the increase in FrzCD methylation to decelerate or delay adaptation. We contend that these antagonistic interactions are crucial for effective chemotaxis in this gliding bacterium to ensure that adaptation does not occur too quickly relative to the slow speed of M. xanthus movement.

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BLAST X Mon. Evening Session PREDATAXIS BEHAVIOR IN MYXOCOCCUS XANTHUS Jeb Berleman, Jodie Scott, Tatiana Chumley, and John R. Kirby* University of Iowa, Department of Microbiology, Iowa City, IA 52246, USA

Spatial organization of cells is important for both multicellular development and tactic responses to a changing environment. We find that the slow-moving, gliding bacterium, Myxococcus xanthus, utilizes a Che-like pathway to regulate multicellular rippling during predation of other microbial species. Tracking of GFP-labeled cells indicates directed movement of M. xanthus cells during the formation of rippling wave structures. Quantitative analysis of rippling indicates that ripple wavelength is adaptable and dependent on prey cell availability. Methylation of the receptor, FrzCD, is required for this adaptation: a frzF methyltransferase mutant is unable to construct ripples, whereas a frzG methylesterase mutant forms numerous, tightly packed ripples. Both the frzF and frzG mutant strains are defective in directing cell movement through prey colonies. These data indicate that the transition to an organized multicellular state during predation in M. xanthus relies on the tactic behavior of individual cells, mediated by a Che-like signal transduction pathway. Predataxis behavior differs from chemotaxis behavior in that it seems to depend heavily on tactile-stimulation, as opposed to chemical-stimulation.

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BLAST X Mon. Evening Session DYNAMIC LOCALIZATION OF FrzCD IN MYXOCOCCUS XANTHUS Emilia M.F. Mauriello1, David P. Astling1, Oleksii Sliusarenko2 and David R. Zusman1

University of California, Department of Molecular and Cell Biology, Berkeley, CA 94720, USA1; Yale University, Department of Molecular, Cell and Developmental Biology New Haven, CT 06520, USA2

Directional motility in the gliding bacterium Myxococcus xanthus requires controlled cell

reversals mediated by the Frz chemosensory system. FrzCD, a cytoplasmic chemoreceptor, does not form membrane bound polar clusters typical for most bacteria, but rather cytoplasmic clusters that are helically arranged and span the cell length. This unusual localization is maintained in the absence of the CheA homologs FrzE or CheA4, and the CheW homologs FrzA or FrzB. In contrast, MCPs lose their respective polar or cytoplasmic localization in Escherichia coli and Rhodobacter spheroides strains lacking CheA and/or CheW (1, 2). The distribution of FrzCD in living cells was found to be dynamic: FrzCD was localized in clusters that continuously changed their size, number, and position. The number of FrzCD clusters was correlated with cellular reversal frequency: fewer clusters were observed in hypo-reversing mutants and additional clusters observed in hyper-reversing mutants. When moving cells made side-to-side contacts, FrzCD clusters in adjacent cells showed transient alignments. These events were frequently followed by one of the interacting cells reversing. These observations suggest that FrzCD detects signals from a cell-contact sensitive signaling system and then re-localizes as it directs reversals to distributed motility engines. References: 1. Maddock, J.R., and Shapiro, L. (1993) Science 259: 1717-1723. 2. Wadhams, G.H., Martin, A.C., Warren, A.V., and Armitage, J.P. (2005) Mol Microbiol 58(3): 895-902. 3. Bustamante, V.H., Martínez-Flores, I., Vlamakis H.C., and Zusman, D. (2004) Mol Microbiol 58(5): 1501-1513.

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BLAST X Mon. Evening Session IDENTIFYING NOVEL BACTERIAL CYTOSKELETAL ELEMENTS AND CYTOSKELETAL INTERACTORS THROUGH HIGH-THROUGHPUT IMAGING John Werner, Michael Ingerson-Mahar, Jonathan Guberman, and Zemer Gitai Princeton University, Department of Molecular Biology, LTL-355 Washington Rd., Princeton, NJ 08540 Bacterial cytoskeletal proteins polymerize into filamentous structures that represent key regulators of a wide array of cellular processes, including cell shape determination, cell division, and cell polarity. While bacterial homologs of all three major eukaryotic cytoskeletal families have already been described, the upstream regulators of bacterial cytoskeletal assembly and downstream effectors of cytoskeletal function remain poorly understood. In addition, recent studies have suggested that additional filament-forming proteins remain uncharacterized. All known cytoskeletal elements in both bacteria and eukaryotes have distinct non-uniform subcellular distributions. Focusing on the asymmetric bacterium, Caulobacter crescentus, we thus employed a directed high-throughput imaging approach to identify both novel bacterial cytoskeletal proteins and proteins that act upstream or downstream of the previously-characterized cytoskeletons. We developed a pipeline of high-throughput methods for generating fluorescent protein fusions, expressing them in Caulobacter, imaging their subcellular distribution at high resolution, and quantitating the resulting imaging data. By using this pipeline to analyze over 2,800 Caulobacter proteins as both N-and C-terminal mCherry fusions, we identified a set of ~300 localized Caulobacter proteins. This set included the three known Caulobacter cytoskeletons (the MreB actin homolog, FtsZ tubulin homolog, and Crescentin intermediate-filament). We also identified a novel protein that localizes to a tight line that hugs a short region of the inner curvature of Caulobacter cells. This protein may polymerize on its own, as it is capable of forming linear structures when expressed in heterologous systems such as E. coli or S. pombe. Preliminary studies suggests that this previously-uncharacterized cytoskeletal element plays a role in cell shape determination and may exhibit crosstalk with other cytoskeletal proteins. To identify upstream regulators of cytoskeletal assembly, we modified our pipeline to allow us to assay the effects of overexpressing genes of interest on the localization patterns of MreB, FtsZ, and Crescentin. A pilot screen of ~200 conserved proteins with no known function identified four proteins that perturbed FtsZ, one that perturbed MreB, one that perturbed both MreB and FtsZ, and one that perturbed Crescentin. The functions and mechanisms of action of these candidate cytoskeletal regulators are currently being examined. Finally, to identify downstream cytoskeletal effectors, we imaged our library of localized Caulobacter proteins in the presence of the MreB-delocalizing compound, A22. We found a large number of proteins that are either delocalized or mislocalized by A22 and are currently determining the cellular functions of these proteins as well as the nature of their direct or indirect associations with MreB.

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BLAST X Tue. Morning Session THE ROLE OF POSITIVE FEEDBACK IN CONTROLLING FLAGELLA ASSEMBLY DYNAMICS Supreet Saini1, Christy Aldridge2, Jonathan Brown2, Philip Aldridge2, Christopher Rao1 1Department of Chemical and Biomolecular Engineering, University of Illinois, Urbana, IL 61801, United States 2Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom

Flagellar assembly in Salmonella enterica serovar Typhimurium (S. typhimurium) proceeds in a sequential manner, starting from the base of the flagella and concluding at the filament tip. A key regulatory step in the assembly process is the σ28-FlgM checkpoint, which prevents the activation of σ28-dependent Pclass3 promoters prior to completion of the hook basal body. This regulatory checkpoint is typically assumed to involve a binary decision process: either proceed with Pclass3 gene expression or not, depending on the state of assembly. However, mathematical modeling suggests that this binary checkpoint may in fact result in more subtle, rheostat-like control. σ28 is involved in a positive feedback loop, as ιτ positively regulates its own expression along with the expression of FliZ, an activator of Pclass2 gene expression. In addition, the ability of σ28 to regulate gene expression depends on the concentration of FlgM in the cell. This suggests that the response of the σ28 positive feedback loop is tuned by late protein secretion. In fact, our modeling and experimental results suggests that σ28 and FlgM are not only involved in establishing the binary checkpoint between Pclass2 and Pclass3 gene expression but are also involved in fine tuning the relative timing of expression. In particular, the positive feedback loop involving σ28 and FliZ establishes the delay between Pclass2 and Pclass3 gene expression. In this talk, we will discuss our recent work investigating the positive feedback loops involving σ28 and FliZ. We have recently shown that FliZ is an FlhD4C2-dependent activator of Pclass2 gene expression. In addition, our results indicate the FliZ speeds up the induction of Pclass2 genes in a secretion-dependent manner. With regards to σ28, we have found that autoregulation controls the relative timing of gene expression. In cells lacking FlgM, both Pclass2 and Pclass3 genes are induced at the same times. Conversely, when the rate of FlgM secretion is reduced, the delay between Pclass2 and Pclass3 gene expression is exaggerated. These results suggest that timing is responsive to the rate of late protein secretion. Moreover, mathematical modeling predicts that this control is due to autoregulation by σ28. To test this model, we have rewired the flagellar gene circuit by replacing the native PfliA promoter with Pclass1/Pclass2/Pclass3 promoters. Consistent with the model predictions, these promoter replacement experiments show that autoregulation plays a key role in enforcing the timing of flagellar gene expression. Last, we also investigated gene expression dynamics at single-cell resolution, and our preliminary results suggest that the dynamics may exhibit bistability, consistent with control involving feedback. Collectively, our results suggest that the regulation of flagellar gene expression is complex and involves multiple layers of control.

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BLAST X Tue. Morning Session CRYSTAL STRUCTURE OF FLIT, A BACTERIAL FLAGELLAR EXPORT CHAPERONE FOR THE FILAMENT CAP PROTREN HAP2 (FliD) Katsumi Imada, Tohru Minamino, Miki Kinoshita and Keiichi Namba Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan The bacterial flagellum is a filamentous organelle responsible for motility. Since the flagellum extends from the cytoplasm to the cell exterior, the external component proteins have to be exported from the cytoplasm. The protein subunits are exported by the flagellar specific export apparatus, which is a member of the type III secretion system. The export apparatus is believed to be located within the C-ring of the flagellar basal body and consists of at least six integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, FliR) and three soluble proteins (FliH, FliI, FliJ). In addition to these proteins, other cytoplasmic proteins (FlgN, FliA, FliS, FliT) act as substrate-specific chaperons that facilitate the export of their substrates.

FliT is a flagellar export chaperone for FliD (HAP2), which forms a capping complex at

the distal end of the flagellar filament and promotes incorporation of flagellin subunits into the growing filament, and prevents FliD from premature aggregation in the cytoplasm. FliT is not only involved in protein export but also in regulation of flagellar gene expression. FliT negatively regulates transcription of the flagellar class 2 operons by binding to FlhD4C2 complex, which is a transcriptional activator. We have determined a crystal structure of FliT at 3.2 Å resolution. The structure and following genetic and biochemical studies have revealed that the C-terminal region of FliT regulates its interactions with other flagellar proteins. We will discuss the molecular mechanisms of protein export and gene expression based on the FliT structure.

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BLAST X Tue. Morning Session STRUCTURAL INSIGHT INTO ACTIVE FLAGELLAR MOTOR FORMATION THROUGH THE PERIPLASMIC REGION OF MOTB Seiji Kojima1, Mayuko Sakuma1, Yuki Sudo1, Chojiro Kojima2, Tohru Minamino3, Keiichi Namba3, Michio Homma1 and Katsumi Imada3* 1Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-Ku, Nagoya 464-8602, Japan; 2Laboratory of Biophysics, Graduate School of Biological Sciences, Nara Institute of Science and Technology (NAIST), 8916-5 Takayama, Ikoma, Nara, 630-0192 Japan; 3Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan

Bacterial flagellar motor is a supramolecular nano-machine powered by the transmembrane gradient of protons or sodium ions, and spins flagellar filaments to drive cell motility. Motor torque is generated by the rotor-stator interaction that is coupled with ion flow through the ion-channel in the stator unit, which is composed of four MotA and two MotB subunits. About ten stators are assembled around the perimeter of the rotor and anchored to peptidoglycan layer by the peptidoglycan-binding (PGB) domain of MotB. The rotor-stator assembly is not a rigid complex, so each stator unit can dynamically be exchanged in the functional motor, and is activated only when assembled around the rotor. However, the mechanisms of the stator assembly and the activation of the proton flow remain unclear. Here, we report the crystal structure of a C-terminal fragment of MotB (MotBC), which includes the PGB domain and covers the whole periplasmic region essential for cell motility (PEM), at 1.75 Å resolution. The structure, and subsequent mutational and biochemical analyses indicate that dimer formation by the PGB domains is required for motility through the regulation of the arrangement of the transmembrane segment. Moreover, we show that large structural changes in the N-terminal helices should be coupled with both peptidoglycan binding and activation of the stator. This work provides novel structural insight into the dynamic behavior of the ion-channel complex regulated by the periplasmic domain, and the activation mechanism of the complex coupled with completion of the assembly where it works.

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BLAST X Tue. Morning Session STATOR SELECTION IN SHEWANELLA ONEIDENSIS MR-1 Anja Paulick, Andrea Koerdt, Kai M. Thormann Department of Ecophysiology, MPI für terrestrische Mikrobiologie, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany

The Gram-negative metal-ion reducing bacterium Shewanella oneidensis MR-1 is motile by means of a single polar flagellum. We identified two potential stator systems, PomAB and MotAB, each sufficient as a force generator to drive flagellar rotation. Physiological studies demonstrate that PomAB is sodium-dependent while MotAB is powered by proton motive force. Homology comparisons strongly indicate that the MotAB system has been acquired by horizontal gene transfer, probably as a consequence of long-term adaptation from a marine to a low-sodium freshwater environment. As in S. oneidensis MR-1, a number of bacterial species possess more than one stator system to power a single flagellar system but it is yet unclear, how selection of the stators is achieved.

Expression analysis at the single cell level showed that both stator systems of S.

oneidensis MR-1 are expressed simultaneously, and functional fusions of PomB and MotB to mCherry revealed that both stator systems are present in the cell at the same time. While the Pom system is efficiently localizing to the flagellated cell pole under all conditions, the Mot stator is located in the cell membrane and only found at the cell pole at high abundance in media with low sodium content. At low sodium, both stator systems are localizing to the flagellated cell pole in the majority of the cell population, thus indicating that under such conditions a hybrid motor may be formed. We conclude that stator selection occurs at the level of protein localization by alterations in the localization efficiency in response to sodium levels.

In Vibrio species, two additional proteins, MotX and MotY, are involved in stator

recruitment and sodium-dependent swimming. We therefore analyzed whether S. oneidensis MR-1 orthologs to MotX and MotY play a role in stator selection. Mutant and localization analyses demonstrated that both proteins are required for function of the Pom as well as the Mot stator system. As opposed to the Vibrio system, in S. oneidensis MR-1, MotX and MotY are not required for stator recruitment and also do not play a role in stator selection in response to sodium conditions.

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BLAST X Tue. Morning Session TAKING CONTROL OF THE BACTERIAL FLAGELLAR MOTOR Mathieu Gauthier, Dany Truchon and Simon Rainville Department of Physics, Engineering Physics and Optics and Centre d'optique photonique et laser, Laval University, Québec, Québec, CANADA

The bacterial flagellar motor is a fairly complex machine, requiring 40-50 genes for its expression, assembly and control. Furthermore, it is 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. Here, we report significant progress towards the development of a unique in vitro system to study quantitatively the bacterial flagellar motor.

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

a micropipette. 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 submicrometer-sized hole in the wall of the bacterium, thereby granting us access to the inside of the cell and the control over the proton-motive force that powers the motor. Using a patch-clamp amplifier, we applied an external voltage between the inside and the outside of the micropipette. If the hole in the bacterium is open, that voltage should translate into a membrane potential powering the motors outside of the micropipette. As we varied the applied potential, variations in the motor's rotation speed were observed. For these preliminary results, the rotation speed was observed directly using video microscopy of fluorescently labeled filaments. That system opens numerous possibilities to study the flagellar motor and other membrane components.

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BLAST X Tue. Morning Session EXPERIMENTAL EVIDENCE FOR CONFORMATIONAL SPREAD IN THE BACTERIAL SWITCH COMPLEX Richard W. Branch1, Fan Bai1, Dan V. Nicolau2, Teuta Pilizota1, Bradley Steel1, Philip K. Maini2, Richard M. Berry1

1Clarendon Laboratory, Department of Physics, University of Oxford, Parks Road, Oxford OX1 3PU, UK. 2Centre for Mathematical Biology, Mathematical Institute, University of Oxford, St.Giles Road, Oxford OX1 3LB, UK.

The bacterial switch complex in E. coli controls the direction of rotation of the bacterial flagellar motor between clockwise and counterclockwise modes. The complex takes the form of a ring composed of about 110 FliN, 34 FliM and 26 FliG protein subunits. Regulation is through binding of the signaling molecule CheY-P to FliM. FliG interfaces with the torque-generating stator units of the motor. The precise mechanism by which the complex executes a switch is unclear.

The complex displays the ultrasensitive nature typical of allosteric proteins, with a steep

sigmoidal relationship existing between [CheY-P] and motor rotational bias. Allosteric regulation of proteins has classically been understood in terms of the Monod-Wyman-Changeux (MWC) or Koshland-Nemethy-Filmer (KNF) models. However, it is unrealistic that MWC-type concerted transitions could be responsible for quaternary conformational changes of such a large complex, and cooperative binding studies in vitro and in vivo have precluded a KNF-type induced-fit mechanism.

The MWC and KNF models are recognized as limiting cases of a general allosteric

scheme that has recently been described in a model of conformational spread. The model has been shown to be capable of reproducing motor switching kinetics. A directly observable consequence of conformational spread in the switch complex would be the variation of motor speed associated with the conformational spread of ring subunit state. In particular, the duration of switch events should be finite and broadly distributed due to the diffusive random walk of conformational spread, and incomplete switches should be observed due to incomplete growth and shrinkage of subunit state domains.

We have used high-resolution back-focal-plane interferometry of polystyrene beads

attached to truncated WT E. coli flagella to resolve intermediate states of the motor predicted by conformational spread, and demonstrate detailed quantitative agreement between our measurements and conformational spread simulations. Individual switch events are not instantaneous, but follow a broad distribution of switch times with mean ~ 20 ms. The shortest switch events are observed to last less than 1ms, while the longest require over 100ms and take several revolutions to complete. Intervals between switches are exponentially distributed at all values of bias. Incomplete switches reaching a range of intermediate speeds are observed. The events are Poisson distributed in time with a bias-dependent frequency.

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BLAST X Tue. Morning Session DO INDIVIDUAL BACTERIAL FLAGELLAR MOTORS USE HYSTERESIS TO MAINTAIN A ROBUST OUTPUT IN A NOISY ENVIRONMENT? – AN EXPERIMENTAL STUDY Peter Reuven1, Oleg Krichevsky2, Michael Eisenbach1 1 Department of Biological Chemistry, Weizmann Institute of Science, 76 100, Israel 2 Department of Physics, Ben-Gurion University, Beer Sheva, 84 105, Israel

It is known that despite imperfections of intracellular environment, flagellar motor outputs are robust against stochastic fluctuations of CheY signal. Indirect evidence from our lab suggests that, to maintain stability, the motor complex might damp out fluctuations in the intracellular level of CheY by having a hysteresis feature - two different thresholds for switching. In this case, hysteresis means that the default-state motor switches at a higher threshold from counterclockwise to clockwise state compared to a lower threshold when switching back. Such behavior will produce hysteretic loop in input/output characteristics of flagellar motor.

Our aim is to pin-point the level at which the noise-filtering (via hysteresis) occurs in the

chemotactic network. We are studying flagellar rotation of single cells as a function of their intracellular CheY-P concentration, changing the concentration up and down in order to cover both - counterclockwise to clockwise and clockwise to counterclockwise - switching routes. Since it is impossible to distinguish CheY from CheY-P in vivo, one has to work under conditions that maintain CheY constantly fully phosphorylated. The challenge was how to effectively decrease the concentration of the phosphorylated signal.

Knowing that we cannot play with the level of phosphorylation we opt to play with the

level of the CheY-P protein instead. While to increase protein concentration is a routine task, to decrease it is less obvious. We cloned a bi-modal, inducible plasmid expressing a CheY fused to yellow fluorescent protein (YFP) and ssrA degradation tag. YFP is used for quantifying the signal whereas the degradation tag makes it possible to shorten the lifetime of CheY-YFP. The core assumption of our approach is that heat-shock-induced proteases accelerate the degradation of the ssrA-targeted CheY-YFP protein. We have verified this assumption experimentally.

We monitor the degradation of CheY-YFP by a decrease in fluorescence intensity and

this decrease is correlated with the change of the direction of motor rotation. Input/output characteristics of individual flagellar motors is build from these correlations.

Advancing this study will, hopefully, enable us to deeper understand how the

mechanisms of intracellular interactions affect the logic of cell's behavior.

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BLAST X Tue. Morning Session DYNAMICS OF THE BACTERIAL FLAGELLAR MOTOR WITH MULTIPLE STATORS Giovanni Meacci and Yuhai Tu IBM T. J. Watson Research Center

The bacterial flagellar motor drives the rotation of flagellar filaments and enables many species of bacteria to swim. Torque is generated by interaction of stator units, anchored to the peptidoglycan cell wall, with the rotor. Recent experiments [Yuan, J. & Berg, H. C. (2008) PNAS 105, 1182-1185] show that near zero load the speed of the motor is independent of the number of stators. Here, we introduce a mathematical model of the motor dynamics that explains this behavior based on a general assumption that the stepping rate of a stator depends on the torque exerted by the stator on the rotor. We find that the motor dynamics can be characterized by two time scales: the moving-time interval for the mechanical rotation of the rotor and the waiting-time interval determined by the chemical transitions of the stators. We show that these two time scales depend differently on the load, and that their crossover provides the microscopic explanation for the existence of two regimes in the torque-speed curves observed experimentally. We also analyze the speed fluctuation for a single motor using our model. We show that the motion is smoothed by having more stator units. However, the mechanism for such fluctuation reduction is different depending on the load. We predict that the speed fluctuation is determined by the number of steps per revolution only at low load and is controlled by external noise for high load. Our model can be generalized to study other molecular motor systems with multiple power-generating units.

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BLAST X Tue. Evening Session EFFECT OF OSMOLYTES ON REGULATING THE ACTIVITIES OF THE SSK1 RESPONSE REGULATOR FROM SACCHAROMYCES CEREVISIAE Alla O. Kaserer, Paul F. Cook and Ann H. West Department of Chemistry and Biochemistry, The University of Oklahoma, 620 Parrington Oval, Norman, OK 73019

The multi-step His-Asp phosphorelay system in Saccharomyces cerevisiae allows cells to adapt to osmotic, oxidative and other environmental stresses. The pathway consists of a hybrid histidine kinase SLN1, a histidine-containing phosphotransfer (HPt) protein YPD1 and two response regulator proteins, SSK1 and SKN7. Under non-osmotic stress conditions, the SLN1 kinase is active and phosphoryl groups are shuttled to SSK1 via YPD1. We have previously demonstrated that YPD1 stabilizes the phosphorylated form of SSK1. The cellular response to hyperosmotic stress involves rapid efflux of water and change in intracellular ion and osmolyte concentration. It is our hypothesis that these changes may affect rates of phosphotransfer within the SLN1-YPD1-SSK1 phosphorelay system and the phosphorylated lifetime of response regulators. Therefore, we examined the effect of different solute concentrations on dephosphorylation of SSK1 and phosphotransfer rates within the phosphorelay system using half-life studies and rapid quench kinetics, respectively. These studies provide new insight and offer a better understanding of how this His-Asp multi-step phosphorelay is environmentally regulated.

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BLAST X Tue. Evening Session REGULATION OF Escherichia coli MOTILITY BY THE NITRIC OXIDE SENSITIVE TRANSCRIPTIONAL REPRESSOR NsrR Jonathan D. Partridge and Stephen Spiro Department of Molecular and Cell Biology, The University of Texas at Dallas, 800 W. Campbell Road, Richardson, Texas 75080, USA

There is circumstantial evidence implicating the water-soluble free radical nitric oxide

(NO) as a regulator of motility, chemotaxis and biofilm development. Heme-containing NO-binding domains of methyl accepting chemotaxis proteins from Clostridium botulinum and Thermoanaerobacter tengcongensis have been characterized, though the prediction that these proteins mediate taxis towards or away from NO has not been tested. In transcriptomics experiments, the expression of some motility genes has been observed to be perturbed by exposure of cultures to sources of NO or nitrosative stress (imposed by S-nitrosothiols), although both positive and negative responses have been reported, and the regulators involved were not identified. In the non-pathogenic organism Nitrosomonas europaea, NO stimulates biofilm formation. In Pseudomonas aeruginosa and Staphylococcus aureus, there is evidence that NO inhibits biofilm formation, or stimulates dispersal, and NO stimulates swimming and swarming motility in P. aeruginosa. In no case has a molecular mechanism been described which accounts for the effects of NO on biofilm development or motility.

NO is made in bacteria either as a by-product of nitrite reduction to ammonia, or as an

intermediate of denitrification, and is made by the inducible NO synthase of host phagocytes. Thus pathogenic bacteria can be exposed both to endogenously-generated NO, and to the NO made by host cells. In Escerichia coli, the regulatory proteins NorR and NsrR mediate adaptive responses to NO, by controlling the expression of genes encoding enzymes that reduce or oxidize NO to less toxic species. The key NO detoxifying activities are the flavohemoglobin (encoded by the hmp gene) and the flavorubredoxin (encoded by norVW), the expression of which is regulated by NsrR and NorR, respectively. As far as is known, the norVW promoter is the sole target for regulation by NorR, while NsrR appears to control a large regulon of genes and operons.

The extent of the NsrR regulon of E. coli has been assessed computationally, and by a

transcriptomics analysis of a strain in which NsrR was titrated by the presence of multiple copies of a cloned NsrR binding site. We believe that neither approach has provided a comprehensive inventory of all of the genes regulated by NsrR. Therefore, we used chromatin immunoprecipitation and microarray analysis (ChIP-chip) to identify NsrR binding sites in the E. coli genome. Surprisingly, we found NsrR binding sites associated with the promoter regions of three transcription units (mqsR-ygiT, fliAZY and fliLMNOPQR) containing genes with well-established or suspected roles in motility and/or biofilm development. We have confirmed that the fliA and fliL promoters are subject to regulation by NsrR and NO, and have identified an NsrR binding site in the fliA promoter. We have shown that NsrR is a negative regulator of motility in both K12 and UPEC strains of E. coli. These results provide, for the first time, a molecular mechanism by which NO might control bacterial motility.

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BLAST X Tue. Evening Session SYNTHETIC LETHALITY UNCOVERS A NOVEL LINK BETWEEN THE MalT AND OmpR REGULONS Sylvia A. Reimann and Alan J. Wolfe Loyola University Chicago, Maywood, Il

Synthetic lethality (SL) is a genetic term for the inviability of a double mutant combination of two fully viable single mutants. SL is commonly interpreted as redundancy at an essential metabolic step. However, a second, less well-known, class of SL exists: the so-called “defect-damage-repair” (DDR) cycles that link apparently unrelated metabolic pathways (Ting et al., 2008).

We have isolated an SL mutant of the form ompR SL(ompR), where SL(ompR) refers to

a mutation that causes death when present in a cell that lacks the two-component response regulator OmpR. This global regulator is required for proper assembly of the cell envelope and we reasoned that the nature of the SL(ompR) mutation would provide further insight into the role of OmpR and its regulon.

Using a combined genetic/genomic approach, we mapped the SL(ompR) mutation to

malT, which encodes the transcription factor MalT. Because overexpression of the MalT inhibitor MalK did not rescue growth, we proposed that the malT mutation leads to a constitutively active form (MalTc). To test this hypothesis, we deleted the MalT-dependent lamB, which encodes an outer membrane porin, and found that this deletion suppressed the SL of the ompR malTc mutant. Since LamB and OmpR do not perform redundant functions, we propose that the observed SL is of the DDR variety, as follows: the defect (MalTc activity) leads to damage (constitutive expression of LamB) that is repaired by one or more members of the OmpR regulon. To further understand the role of OmpR, we are currently seeking OmpR regulon members that can suppress the SL of the ompR malTc mutant.

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BLAST X Tue. Evening Session A CHEMOTAXIS-LIKE SIGNALING PATHWAY REGULATES THE EXPRESSION OF EXTRACELLULAR MATERIALS IN GEOBACTER SULFURREDUCENS Hoa T. Tran1, Derek R. Lovley2, and Robert M. Weis1 Departments of Chemistry1 and Microbiology2, University of Massachusetts, Amherst, MA 01003, USA.

The chemotaxis pathway that regulates cell motility toward chemical attractants is well-studied in Escherichia coli. By contrast, multiple chemotaxis clusters have been found in many other bacteria, and evidence is accumulating that these cells use chemotaxis-like pathways to regulate diverse cellular functions. The genome of Geobacter sulfurreducens, a δ-Proteobacterium found predominantly in the Fe(III) reducing zone of sedimentary environment contains ~70 chemotaxis gene homologs arranged in 6 major clusters. Cluster 5 (Che5) has a complete set of chemotaxis homologs, including the kinase cheA (1 copy), cheW (2 copies), cheR (1), cheB (1), cheY (3) and four other non-che genes. There are 34 chemoreceptor homologs in the genome, but none is found in the Che5 cluster. Che5-type clusters have been identified in the genomes of several δ-Proteobacteria, yet their functions are not known. Here, we report that G. sulfurreducens Che5 cluster regulates gene expression, and in particular the synthesis of extracellular material that is abundant in OmcS and OmcZ, two c-type cytochromes bound to the outer membrane. OmcS and OmcZ are essential for cell growth in insoluble electron acceptors and for effective electricity production on electrodes. Deletion mutants of the homologs of cheW (gsu2218, gsu2220), cheA (gsu2222) and cheR (gsu2215) increased OmcS production and decreased OmcZ. In contrast, deletion mutants of cheB (gsu2214), one of the three cheYs (gsu2223) and a non-che gene (gsu2216) decreased OmcS and increased OmcZ production. The chemoreceptors that signal through these Che5 proteins are hypothesized to belong to a single MA class. Evidence that supports this idea is based on the phenotypes of deletion mutants in two mcp genes (gsu1704, and gsu2372), which are similar the cheA mutant. Wherever the functional parallels can be drawn, the function of the homologues in Geobacter Che5 signaling pathway is similar to their counterpart in the E. coli chemotaxis pathway. In addition to changes in OmcS and OmcZ expression, the cheA, cheR and cheW (gsu2220) mutants promote cell aggregation and overproduce non-PilA filamentous material. Moreover, microarray data of the cheR mutant, and quantitative RT-PCR data from the other che mutants indicate that the Che5 cluster alter the expression of ~175 genes. A substantial fraction of these are predicted to contain export signals that will result in an to extracellular location. Taken together, these data demonstrate that G. sulfurreducens Che5 cluster, with one class of chemoreceptors, regulates the extracellular matrix material biosynthesis.

This research was supported by the U.S. Department of Energy Office of Science (BER)

under the Cooperative Agreement No. DE-FC02-02ER63446.

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BLAST X Tue. Evening Session THE TWO-COMPONENT REGULATORY SYSTEM BarA/SirA IS AT THE TOP OF A MULTI-FACTORIAL REGULATORY CASCADE CONTROLLING THE EXPRESSION OF THE SPI-1 AND SPI-2 VIRULENCE REGULONS IN SALMONELLA Luary C. Martínez, José L. Puente and Víctor H. Bustamante Departamento de Microbiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México. Cuernavaca, Mor., México. Horizontal gene transfer of pathogenicity islands has been a major event in the evolution of pathogenic bacteria. Integration of regulatory networks to control the expression of the gained genes has represented another essential step in this process. Salmonella Pathogenicity Islands 1 and 2 (SPI-1 and SPI-2) are required at different phases during Salmonella infection in humans and animals. A positive regulatory cascade comprising the SPI-1-encoded regulators HilD, HilA and InvF induces expression of the SPI-1 regulon in response to conditions resembling the intestinal environment, such as growth in Luria-Bertani (LB) rich medium. Two global two-component regulatory systems, OmpR/EnvZ and PhoP/PhoQ, control the expression of the SsrA/B two-component system encoded within SPI-2, which specifically induces the expression of the SPI-2 regulon genes in response to conditions resembling the intracellular environment, mimicked in vitro by growth at low concentrations of phosphate and magnesium. Interestingly, we have recently shown that HilD also induces expression of the SsrA/B system, and thus of the SPI-2 regulon, at late stationary phase in LB cultures, indicating that SPI-2 expression is also controlled by a SPI-1/SPI-2 cross-talk mechanism. The results presented here, together with previous reports, better define the complex and multi-factorial regulatory cascade that controls SPI-1 and SPI-2 expression through HilD. We show that the global two-component system BarA/SirA activates the transcription of two small RNA molecules, csrB and csrC. These molecules counteract the negative effect exerted by the CsrA RNA binding protein on hilD mRNA stability, ensuring the synthesis of the appropriate concentration of HilD required for the expression of HilA and SsrA/B, the central positive regulators of the SPI-1 and SPI-2 regulons, respectively. Furthermore, we demonstrated that growth conditions affecting SPI-1 expression in LB (e.g. low salt, acidic pH or temperatures below 37°C), similarly repress the expression of the SPI-2 regulon. However, while acidic pH seems to negatively regulate the regulatory cascade by affecting the activity of the BarA sensor kinase, growth at low salt concentration or at low temperature seems to directly repress hilD expression through an as yet unidentified mechanism.

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BLAST X Tue. Evening Session INTERACTION OF THE TRANSCRIPTIONAL REGULATORY COMPLEX, FlhDC, WITH ITS TARGET DNA Yi-Ying Lee, and Philip Matsumura Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., M/ C 790, Chicago, Illinois 60612-7344 The bacterial flagellum is the structure that allows bacteria to move and respond to nutritional and chemical signals in their environment. It is a complex suborganelle and the transcriptional regulation of the 40 plus structural genes is organized in a highly regulated cascade. At the top of the hierarchy is the master operon which codes for FlhD and FlhC. These two positive transcriptional regulators form a unique heteroheximeric complex which binds upstream of the -35 region and requires sigma 70 for transcription. This complex has an unusually large ‘footprint’ of 48 base pair and bends the DNA 110 degrees. We have proposed that the DNA bind on the circumference of this toroid shaped FlhDC complex. Although we have determined the sequence 3 footprints on FlhDC regulated promoters, it is not possible to determine a consensus binding site in these 3 sequences. In this study, we have determined which bases are important for DNA binding and activity for FlhDC regulated promoter activity. First, we have divided the FlhDC footprint in the fliA promoter into five segments and found that two of the segments or 40% of the footprint were not required for binding. The remaining 30 base pairs were divided into 3-5 base segments and randomly mutagenized and screened for the ability to bind and activate the fliA promoter. Analysis of these data suggests a consensus of 12A, 15A, 34T, 36A, 37T, 44A, 45T in FlhD4C2 footprint fragment were important for activity. Five of these bases demonstrated high specificity. Finally, this consensus was tested and found to be important in other FlhDC regulated promoter regions.

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BLAST X Wed. Morning Session A NOVEL AMINO ACID BINDING STRUCTURE IN BACTERIAL CHEMOTAXIS George D. Glekas and George W. Ordal Department of Biochemistry, University of Illinois at Urbana-Champaign 409 MSB, Urbana, IL 61801

Simple flagellated bacteria, such as Bacillus subtilis, possess the ability to sense their environment and move to more favorable conditions, where there are, for instance, more nutrients like amino acids, by the process of chemotaxis. The initial stage is binding of an amino acid by receptors on the outside of the cell. This binding causes conformational changes that affects the activity of enzymes on the inside of the cell and alters the movement of the bacteria. The main paradigm for understanding these events is the bacterium Escherichia coli. However, we have discovered that, in fact, the mechanism used by E. coli is not used by most bacteria and that the mechanism used by the distantly related bacterium B. subtilis is more likely to be the general mechanism. Unlike in E. coli, where binding of attractant causes a shift of the receptor polypeptide that goes from the outside of the cell to the cell interior toward the cell interior, attractant causes rotational movement of the receptors without any comparable interior shifting

We seek to understand how this rotational movement occurs in the asparagine receptor

McpB. To do this, we have discovered that the most likely conformation of the exterior part of the receptor is far different from that in the E. coli receptor. Molecular and homology modeling of the McpB sensing domain has led to a structural model that reveals a dual PAS domain structure similar to the crystal structure of the LuxQ sensor. PAS domains are known to be conserved structures capable of binding a great many “small” molecules. Further mutagenetic analyses of putative asparagine-binding residues have not only confirmed the validity of the structural model, they have revealed certain residues that greatly effect chemo-attractant binding. Using both in vivo chemotactic assays and in vitro isothermal titration calorimetry performed on purified mutant receptor exterior regions, three residues, all in the upper PAS domain, have been shown to lower the affinity of the McpB receptor for asparagine. Mutations in the lower PAS domain show no such effect. Further structural and mutagenetic studies have shown a similar dual PAS architecture in the B. subtilis proline receptor McpC, with similar residues responsible for amino acid binding. Extensive homology modeling shows that eight of the ten B. subtilis chemoreceptors have PAS domains in their sensing domains. We are now in the process of modeling the consequences of binding attractant at these residues on the expected structure of the receptor.

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BLAST X Wed. Morning Session STRUCTURE AND FUNCTION OF THE HELICOBACTER PYLORI CHEMORECEPTOR TlpB John Goers, Nathan Henderson, S. James Remington, Karen Ottemann* and Karen Guillemin Institute of Molecular Biology, University of Oregon, Eugene OR, 97403 *Environmental Toxicology, UC Santa Cruz, Santa Cruz, CA 95064

We have shown that the Helicobacter pylori chemoreceptor TlpB is required for chemotaxis away from acid and from the quorum-sensing molecule autoinducer-2. Here we report the determination of an atomic resolution crystal structure for the periplasmic domain of TlpB. The structure reveals a PAS domain inserted into a pair of antiparallel helices and shows unexpected structural homology to the LuxQ receptor from Vibrio harveyi. Furthermore, the PAS domain tightly binds urea, suggesting that the TlpB receptor may be associated with detection of urea. We present mutational and physical evidence for interaction of TlpB with urea and its role in chemotaxis.

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BLAST X Wed. Morning Session THE TM2-HAMP CONNECTION Gus A. Wright, Rachel L. Crowder and Michael D. Manson Department of Biology, Texas A&M University, College Station, TX 77843

The HAMP domain—a conserved protein motif in most Histidine kinases, Adenylate cyclases, Methyl-accepting chemotaxis proteins, and Phosphatases—typically conforms to a helix–connector-helix domain architecture. Recently, an NMR solution structure was determined for the Af1503 HAMP from the archaebacteria Archeoglobus fulgidis (Hulko, et al, Cell 126: 929-940, 2006). This structure revealed that the HAMP domain is a parallel four-helix bundle with a knob-on-knob packing. Similar structures of HAMP domains are proposed to exist in all 5 chemoreceptors of Escherichia coli, including the aspartate chemoreceptor Tar. The HAMP domain of Tar receives information from TM2 and regulates the transmission of the signal to the kinase signaling domain. Mutations were introduced into the TM2-HAMP connector region of Tar to determine if manipulating the input signal into the HAMP affects the output signal to the signaling domain. MLLT residues between R214 and P219 were deleted (-4 through -1), and additional LLT tandem repeats were added up to 8 residues after P219 (+1 through +8). Aspartate sensitivity, rotational bias, mean reversal frequency, and in vivo methylation were measured for these mutants. The results suggest that adding helical turns in this region destabilizes, or “relaxes”, the HAMP/signaling domains; while, removing helical turns from this region stabilizes, or “tightens”, the HAMP/signaling domains. In order to determine if increasing flexibility of the TM2/HAMP connector affects the output signal, a second set of mutants were constructed to replace the MLLT region with four tandem glycine residues (4G). Glycine residues were then subtracted (-4G through -1G) and added (+1G through +5G). Data collected from this experiment suggests that increasing the flexibility of this region dampens the input signal from TM2, in addition to destabilizing the HAMP/signaling domains.

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BLAST X Wed. Morning Session STRUCTURE, ASSEMBLY AND CONFORMATIONAL CHANGES IN CHEMORECEPTORS STUDIED IN INTACT BACTERIAL CELLS USING CRYO-ELECTRON TOMOGRAPHY Cezar M. Khursigara, Xiongwu Wu*, Peijun Zhang, Jon Lefman, Mario J. Borgnia, Yuhai Tu‡, Jacqueline Milne and Sriram Subramaniam National Cancer Institute and *National Heart, Lung and Blood Institute, NIH, Bethesda, MD 20892. ‡T. J. Watson Research Center, IBM, Yorktown Heights, NY 10598

Bacteria respond to changes in their chemical environment by activating an assembly of proteins that collectively represent the bacterial chemotaxis apparatus. In Gram-negative bacteria the core-signaling unit of the chemotaxis machinery is a ternary complex composed of chemoreceptors, CheA and CheW that localize primarily to the poles of the cell and form extended arrays. Using cryo-electron tomography, we describe and compare the architecture, localization and spatial relationship between macromolecular complexes involved in chemotaxis signaling and cellular motility in three different Gram-negative bacteria. In addition, by combining the tomographic analysis with 3D averaging methods we demonstrate that trimeric chemoreceptors in E. coli display two distinct conformations that differ principally in arrangement of the HAMP domains within each trimer.

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BLAST X Wed. Morning Session DISCRETE SIGNAL-ON AND -OFF CONFORMATIONS IN THE AER HAMP DOMAIN Kylie J. Watts, Mark S. Johnson and Barry L. Taylor Dept. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda, CA, USA The PAS-FAD sensor of the aerotaxis receptor, Aer, signals through HAMP and signaling domains that are similar to these domains in other chemoreceptors. Our previous crosslinking studies showed that the AS-1 and AS-2 helices of the Aer-HAMP domain might form a four-helix bundle similar to the Af1503 and Tar HAMP domains. In this study, AS-1 residues were crosslinked to AS-2′ residues in di-cys Aer mutants (using 13 proximal and 4 distal di-cys pairs). The results confirmed a parallel four-helix HAMP bundle for Aer, but one in which AS-2 is rotated compared to the orientation of AS-2 in Af1503 or Tar. We extended our HAMP crosslinking studies to probe for structural differences between the signal-on (CW) and signal-off (CCW) states. In our previous crosslinking studies, we used the oxidant copper phenanthroline, which maintains Aer in the signal-off state. In order to generate snapshots of the signal-on state, CW lesions such as PAS-N85S were engineered into the Aer di-cys and single-cys mutants. When the AS-1 to AS-2′ di-cys crosslinking experiments were repeated in mutants containing N85S, several di-cys pairs showed significant increases in dimer formation rates. These di-cys pairs were located at the distal end of the HAMP four-helix bundle. In contrast, no significant crosslinking changes were observed at the proximal end of the four-helix bundle. The data supports a model in which the distal ends of the HAMP helices move closer together during signal transduction. This could be due to an inward lateral movement of the helices, and may include some element of rotation. However, the entire Aer-HAMP domain does not appear to rotate as has been proposed for Af1503. In Aer, HAMP lesions that lock the receptor in the signal-on (CW) state cluster at the distal end of a HAMP four-helix bundle, indicating a possible site for PAS-HAMP interactions during signal transduction. We used PEG-maleimide to determine in vivo the solvent-accessible surface of the HAMP and proximal signaling domains (residues 206-275). Solvent accessibility was restricted for most AS-2, but not AS-1 or connector, residues in Aer. This indicates that AS-2 residues that are exposed to solvent in Af1503 and Tar, and are predicted to be exposed in an Aer-HAMP model, are buried in vivo in Aer. We are currently investigating whether these residues are buried in a PAS-HAMP contact domain. We are also probing the surface of the HAMP domain in the signal-on state (with N85S) to determine whether there are differences in accessibility between the two signaling states.

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BLAST X Wed. Morning Session INVESTIGATING THE STRUCTURE OF TERNARY COMPLEX OF HISTIDINE KINASE CheA, COUPLING PROTEIN CheW, AND CHEMORECEPTOR BY PULSED DIPOLAR ESR SPECTROSCOPY Jaya Bhatnagar, Peter P. Borbat, Jack H. Freed, Brian R. Crane Cornell University, B 150 Caldwell Hall, Ithaca, NY 14853 A central question in understanding the mechanism of chemotaxis involves the nature of interactions between histidine kinase CheA, adaptor protein CheW and receptors. Pulsed dipolar ESR spectroscopy (PDS) has developed as a valuable technique for structural characterization of protein complexes. PDS provides long-range distance information between spin-labeled residues in the proteins. A set of distance measurements can be subsequently used to model the assembly structure of the whole complex. In our previous work, we demonstrated the success of this approach in predicting the structure of complex of CheW with CheA. We have now applied PDS to the ternary complex formed by CheA, CheW and soluble chemoreceptor fragments. Our results indicate changes in distance distributions from spin-labeled sites on P4, P5 domains and CheW in the presence of unlabeled receptor. Dipolar signals between spin-labeled receptor and CheA∆289 (domains P3, P4 and P5 together) or CheW provide important insights about the relative position and orientation of the three components with respect to each other. Based on this data we have developed a structural model of the ternary complex and the conformational changes CheA undergoes upon binding to receptor.

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BLAST X Wed. Morning Session THE CHEMOTACTIC CORE SIGNALLING COMPLEX IS ULTRASTABLE Annette H. Erbse and Joseph J. Falke Department of Chemistry and Biochemistry, University of Colorado, Boulder, Campus Box 215, Boulder, CO 80309

The chemotatic core complex, composed of the transmembrane receptor, the histidine-kinase CheA and the coupling protein CheW, is the central building block of the extensive, highly cooperative polar signaling clusters in bacteria involved in sensing chemical gradients. Recent studies have shown that the receptors are organized hexagonal arrays of trimers-of-dimers. But it is still unclear how these arrays are formed and stabilized, how CheA and CheW are incorporated into single core complexes and how the complexes are interconnected to build the cooperative signaling network. Here we focus on the stability of the core complex. We show that the isolated, membrane-bound core complex is stable for at least 24 hours, both when it is assembled in vivo and in vitro. All three components are needed to achieve this ultra-stability, which is dependent on electrostatic interactions. By contrast, the stability is independent of ligand binding, receptor methylation or kinase activity. We propose that the assembly of the signaling clusters is cooperative, such that interactions between CheA , CheW and the receptor trimers-of-dimers are needed not only for receptor regulated kinase activity of individual core complexes, but also to position neighbouring complexes during the formation of a multi-linked network. The resulting ultra-stable network is the foundation for the ordered organization and the hypersensitivity of the signaling patches.

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BLAST X Wed. Morning Session ELECTRON CRYOTOMOGRAPHY OF BACTERIAL CHEMOTAXIS ARRAYS Ariane Briegel1, H. Jane Ding1, Zhuo Li1, John Werner2, Zemer Gitai2, D. Prabha Dias1, Rasmus B. Jensen3, Elitza Tocheva1 and Grant Jensen1

1 California Institute of Technology, CA 2 Princeton University, NJ 3 University of Roskilde, Denmark

Motile prokaryotes are able to sense and to respond to ambient conditions through a process known as chemotaxis. Attractants and repellents bind to the sensing domain of methyl-accepting chemotaxis proteins (MCPs), thereby regulating the activity of the histidine kinase CheA. Together with the linking protein CheW, CheA is located at the distal tip of the cytoplasmic signaling domain of the MCPs. If activated, CheA phophorylates CheY (CheY-P), which in turn controls the direction of flagellar rotation. Together with CheA and a linking protein CheW, the MCPs form extended chemotaxis arrays at the cell poles.

Electron cryotomograhy (ECT) makes it possible to visualize chemoreceptor clusters in

prokaryotes in vivo at macromolecular resolution (4-8 nm). While high-resolution 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' cooperative signal amplification and regulation. In order to unambiguously identify the chemotaxis arrays inside cells, we have correlated ECT with fluorescent light microscopy (FLM), using slightly fixed and immobilized Caulobacter crescentus cells with a fusion of the red-fluorescent protein, mCherry, to the C-terminus of the chemoreceptor (McpA). After plunge freezing, we imaged the same cells by ECT. In combination with ECT of near-native wild-type and mutant cells, we used the correlated FLM and ECT approach to identify the chemotactic array, its location and its in-vivo structure. We demonstrate that in wild-type Caulobacter crescentus cells preserved in a near-native state, the chemoreceptors are hexagonally packed with a lattice spacing of 12 nm, just a few tens of nanometers away from the flagellar motor that they control. The arrays were always found on the concave side of the cell, further demonstrating that Caulobacter cells maintain dorsal/ventral as well as anterior/posterior asymmetry. Placing the known crystal structure of a trimer of receptor dimers at each vertex of the lattice accounts well for the density, supporting an array composition unlike the published models for Escherichia coli [1] or Thermotoga maritima [2]. We are now in the process of comparing the chemotaxis arrays of a wide range of bacteria to determine the similarities and differences of these macromolecular assemblies at the ‘mesoscale’ level. 1. Shimizu T. S. et al, Nat Cell Biol 11 (2000) 792. 2. Park, S.-Y. et al, Nat Struct Mol Biol 13 (2006) 400.

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BLAST X Thurs. Morning Session TWO REGULATORY PROTEINS CONTROL THE SWIM-OR-STICK SWITCH IN ROSEOBACTERS Robert Belas University of Maryland Biotechnology Institute, Center of Marine Biotechnology, 701 East Pratt St., Baltimore, MD 21202 Members of the Roseobacter clade of α-Proteobacteria are among the most abundant and ecologically relevant marine bacteria. One of the most salient features of the roseobacters from aspects of marine ecology is their ability to enter into close physical and physiological relationships with “red tide” phytoplankton such as dinoflagellates. For example, Silicibacter sp. TM1040, our model roseobacter, forms a symbiosis with the dinoflagellate Pfiesteria piscicida, such that the dinoflagellate cannot live without TM1040. Aiding TM1040 in development of the symbiosis is a biphasic swim-or-stick lifestyle wherein a genetic regulatory circuit controls whether the bacteria are motile and chemotactic or sessile and develop a biofilm. Bacterial swimming and chemotaxis behavior are initial, essential steps in establishment of the symbiosis. Once near the host surface, motility and flagellar synthesis are downregulated, while biofilm formation and synthesis of an antibiotic are upregulated. The abilities to swim using flagella and to form a biofilm via adhesins have been demonstrated to be important traits for both pathogenic and symbiotic bacteria. While it is generally agreed that motility and biofilm development are mutually exclusive, the molecular mechanisms that underlie the lifestyle switch remain virtually unknown for most bacterial species. We have used genetic screens to search for mutants defective in either the motile or the sessile phenotype, and have discovered many new genes including two previously unknown and novel regulatory proteins, FlaC and FlaD that are envisaged to act together with cyclic dimeric GMP to play important roles in the swim-or-stick switch. FlaC is predicted to function as a response regulator protein, with homology to a protein of Caulobacter crescentus known to be important for cell envelope function. FlaC- cells are skewed towards the motile phase, e.g., their populations have a greater percentage of motile cells and fewer rosettes, and have defects in antibiotic synthesis and biofilm formation. Thus, FlaC determines whether the switch is in the swim or stick position. FlaD is predicted to be a MarR-type DNA-binding protein. Mutations in flaD result in nonmotile cells that synthesize but cannot rotate their flagella, i.e., they produce paralyzed flagella. We hypothesize that FlaD is involved in the function of the flagellar motor, either by (1) acting directly to control transcription of the class IV fliL operon or (2) acting indirectly to control transcription or activity of a protein that acts as a ‘clutch’ to engage or disengage the flagellar motor. The implications of FlaC and FlaD activities in the swim-or-stick strategy and their impact on the symbiosis will be discussed.

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BLAST X Thurs. Morning Session DELETION ANALYSIS OF RcsC REVEALS A NOVEL SIGNALING-PATHWAY CONTROLLING BIOFILM FORMATION IN ESCHERICHIA COLI Ricardo Oropeza, Rosalva Salgado, Ismael Hernandez-Lucas and Edmundo Calva Departmento de Microbiologia Molecular, Instituto de Biotecnologia, Universidad Nacional Autonoma de Mexico. Av. Universidad 2001. Col. Chamilpa. Cuernavaca, Morelos. Mexico, CP 62210

RcsC is a hybrid histidine kinase that forms part of a phosphorelay signal transduction pathway with RcsD and RcsB. Besides the typical domains of a sensor kinase, i.e. the periplasmic (P), linker (L), dimerization and H-containing (A), and ATP- binding (B), RcsC possesses a receiver domain (D) at the carboxy-terminal domain.

In order to study the role played by each of the RcsC domains, four plasmids containing

several of these domains were constructed (i.e. PLAB, LAB, AB and ABD) and transformed in Escherichia coli wild type. Different amounts of biofilm were produced, assessed by crystal violet staining, depending on the RcsC domains expressed by the plasmid.

E. coli transformed with the plasmid expressing the ABD subdomains produced the

highest amount of biofilm, while the lowest amount of biofilm was produced under the control of the PLAB expressing plasmid. This phenotype was observed in the same ratio when the plasmids were transformed in a ΔrcsCDB strain. Several mutants on genes involved in biofilm formation were transformed with this set of plasmids. Biofilm formation was abolished in the pgaABCD and nhaR backgrounds but not in the csrB and uvrY backgrounds. Our results suggest the existence of a signaling pathway depending of RcsC but independent of RcsD and RcsB, activating biofilm formation by the pgaABCD operon.

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BLAST X Thurs. Morning Session REGULATION OF CELL FATE IN BACILLUS SUBTILIS BIOFILMS H. C. Vlamakis1*, C. Aguilar1*, R. Losick2, and R. Kolter1 1Harvard Medical School, Boston, MA, 2Harvard University, Cambridge, MA. *These authors contributed equally to this work.

Many microbial populations differentiate from free-living planktonic cells into surface-associated multicellular communities known as biofilms. Within a biofilm, motile Bacillus subtilis cells differentiate into non-motile chains of cells that form parallel bundles held together by an extracellular matrix. These bundles eventually produce aerial structures that serve as preferential sites for sporulation. By analyzing strains harboring multiple cell-type specific promoter fusions we can visualize the spatial anatomy of at least three physiologically distinct cell populations within mature biofilms. Motile, matrix-producing, and sporulating cells localize to distinct regions within the biofilm and the localization and percentage of each cell type is dynamic. Mutants unable to produce extracellular matrix form unstructured biofilms that are deficient in sporulation. This suggests that in architecturally complex biofilms, spore formation is coupled to the production of extracellular matrix. The coupling of matrix production and sporulation could be explained by the phosphorylation state of the master transcriptional regulator Spo0A. Spo0A is phosphorylated both directly and through a phosphorelay by at least five different histidine kinase proteins. When cells have low levels of Spo0A-P, matrix genes are expressed; however, at higher levels of Spo0A-P, sporulation commences. We have found that a deletion of kinD, a gene encoding one of the kinases that feed into the Spo0A phosphorelay, is sufficient to restore sporulation to matrix-deficient mutants. We hypothesize that KinD is not acting as a kinase under these conditions, but rather functions as a phosphatase to delay sporulation until matrix (or a matrix-encased signal) is sensed.

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BLAST X Thurs. Morning Session PROTEIN MISFOLDING DONE RIGHT: THE BIOGENESIS OF BACTERIAL AMYLOID FIBERS Xuan Wang, Neal Hammer and Matt Chapman University of Michigan, Department of Molecular, Cellular and Developmental Biology, Ann Arbor, MI, 48109

Many Enterobacteriaceae spp., including E. coli, produce surface-localized amyloid fibers called curli. Curli fibers are associated with biofilm formation, host cell adhesion and invasion, and immune system activation. Unlike disease-associated amyloid formation, curli biogenesis is a directed and highly regulated process. The major curli subunit protein, CsgA, polymerizes into amyloid after interacting the CsgB nucleator protein. CsgB presents an amyloid-like template to CsgA on the cell surface that initiates fiber formation. CsgA has five imperfect repeating units (R1-R5) that are each predicted to form strand-loop-strand structures. Asn and Gln residues in R1 and R5 were found to be required for efficient amyloid formation and for interaction with the CsgB nucleator protein. Furthermore, the polymerization of CsgA was tempered by the presence of conserved aspartic residues in R2, R3 and R4. When these aspartic acid residues were changed to alanine (CsgA*), polymerization was significantly faster in vitro. Even more remarkable was the observation that CsgA* assembled into an amyloid fiber in vivo in the absence of CsgB. The ability of CsgA* to polymerize into amyloid more efficiently, and in the absence of CsgB, was not without consequences. Cells expressing CsgA* grew more slowly when compared to cells expressing wild type CsgA. This analysis suggests that aspartic acid residues can potently inhibit functional amyloid formation. CsgA has apparently evolved to efficiently assemble into an amyloid in vivo only in the presence of CsgB. This suggests an elegant mechanism to control amyloid formation by regulating the temporal and spatial interactions between CsgA and CsgB.

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BLAST X Thurs. Morning Session RHODOBACTER SPHAEROIDES, A BACTERIUM WITH TWO FLAGELLAR SYSTEMS AND MULTIPLE CHEMOTAXIS GENE HOMOLOGS Ana Martínez del Campo1, Sebastian Poggio2, Teresa Ballado1, Aurora Osorio2, Javier de la Mora1, Laura Camarena2 and Georges Dreyfus1 Instituto de Fisiología Celular1, Instituto de Investigaciones Biomédicas2, Universidad Nacional Autónoma de México, 04510 México DF, México.

Rhodobacter sphaeroides has two flagellar systems (fla1 and fla2). One of these systems has been shown to be functional and is required for the synthesis of the well-characterized single subpolar flagellum (fla1), while the other was found only after the genome sequence of this bacterium was completed (fla2). In this work we found that the second flagellar system of R. sphaeroides can be expressed and encodes a functional flagellum. This second flagellar system produces polar flagella that are required for swimming. Phylogenic analysis suggests that the flagellar system that was initially characterized, was in fact, acquired by horizontal transfer from a γ-proteobacterium, while the second flagellar system contains the native genes.

In addition to having two flagellar systems, this photosynthetic bacterium posses several

reiterated chemotactic genes (2 cheB, 3 cheR, 4 cheA and cheW and 6 cheY), which are encoded in three operons (cheOp1, cheOp2 and cheOp3). In spite of this, only some of the gene copies are required when the cell is swimming with the fla1 flagellum. The presence of a second functional flagellum (fla2) suggests that some of these genes could be involved in its tactic control. To test this hypothesis we proceeded to individually mutate each cheY gene. We show evidence that CheY1, CheY2 and CheY5 control de chemotactic behavior mediated by fla2 flagella. Additionally, we identified that open reading frame RSP6099 encodes the fla2 FliM protein. Furthermore CheY1, CheY2 and CheY5 are located within cheOp1, which is not essential for chemotaxis mediated by the fla1 system. This raises the question: What is the role of cheOp1?

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BLAST X Thurs. Morning Session MOTILITY, CHEMOTAXIS AND VIRULENCE OF BORRELIA BURGDORFERI, THE LYME DISEASE SPIROCHETE M. A. Motaleb1, P. Stewart2. A. Bestor2, P. Rosa2 and N. Charon3 1Dept of Microbiology & Immunology, East Carolina University, Greenville, NC 2Human Bacterial Pathogenesis, NIH, RML, Hamilton, MT 3Dept of Microbiology, Immunology & cell Biology, West Virginia University, Morgantown, WV

Borrelia burgdorferi is the causative agent of Lyme disease. It is the most prevalent arthropod borne infection in the United States with 27,444 reported cases on 2007. The disease is a multiple-systemic disorder with various clinical manifestations including erythema migrans rash, arthritis, cardiac, musculoskeletal and neurological manifestations.

B. burgdorferi exists in nature in an enzootic cycle. Ixodes scapularis ticks (commonly

known as deer ticks) acquire the infection when they feed on an infected host, mainly rodents. During subsequent tick feeding, which lasts for several days, B. burgdorferi migrate from the tick midgut, pass through the salivary glands, and are then transmitted to the mammal through the saliva. B. burgdorferi is highly invasive. After being deposited in the skin following a tick bite, the spirochetes can invade many tissues including the joints, heart, and nervous system.

Motility and chemotaxis are critical for bacterial survival and adaptation in diverse

environmental conditions. In several species of bacteria, motility and chemotaxis have been shown to be associated with the disease process. Results obtained using B. burgdorferi with mutations in key motility and chemotaxis genes also indicate that these activities are required for the pathogenesis of Lyme disease. These studies could lead to the development of a novel pharmacological agent to treat/prevent Lyme disease.

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BLAST X Thurs. Morning Session PLEIOTROPIC PHENOTYPES OF A YERSINIA ENTEROCOLICIA FLHD MUTANT INCLUDE REDUCED LETHALITY IN A CHICKEN EMBRYO MODEL Birgit M. Prüß1, Megan K.T. Ramsett1, Nathan J. Carr1, Jyoti G. Iyer1, Penelope S. Gibbs1, Philip Matsumura2, and Shelley M. Horne1 1Veterinary & Microbiological Sciences Department, North Dakota State University, Fargo ND 58108 2Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago IL 60612 The goal of this study was to correlate phenotypes with gene regulation by several flagellar regulators in Yersinia enterocolitica. FlhD/FlhC was initially described as a flagella transcriptional activator and later recognized as a global regulator in several enteric bacteria (1, 2). In Y. enterocolitica, FlhD/FlhC positively affected the expression levels of genes of histidine degradation and pyrimidine biosynthesis, while repressing the urease genes (1). A second protein that is involved in the regulation of flagellar genes, the sigma factor FliA, exhibited a negative effect upon the expression levels of seven plasmid-encoded virulence genes (3). In addition, eight flagellar operons were regulated by FliA. Among the differences to Escherichia coli were a 10 fold regulation of fliZ expression by FliA and a lack of FliA regulation of the flgM operon. Phenotypes relating to FlhD/FlhC and FliA gene regulation were investigated. These phenotypes included growth on carbon and nitrogen sources, and virulence (4). Growth was determined with Phenotype MicroArrays (Biolog). Compared to the wild-type strain, flhD and fliA mutants exhibited increased growth on purines as carbon sources and decreased growth on pyrimidines and histidine as nitrogen sources. Several dipeptides provided differential growth conditions between the wild-type strain and both mutants. Gene regulation was determined for the dpp (dipeptide transport) and opp (oligopeptide transport) genes and was found to correlate with the observed phenotypes. Phenotypes relating to virulence were determined with the chicken embryo lethality assay that was previously established and used for E. coli strains (5). Relative to the wild-type strain, the flhD mutant caused a reduced lethality in this assay, while the fliA mutant caused lethality similar to the wild-type. Mutants were able to colonize infected embryo organs at levels that were comparable to the wild-type. In addition, a mutant in flhB, encoding one component of the flagellar type III secretion system also caused a reduced embryo lethality. Since genes of the type III secretion system are regulated by FlhD/FlhC and not by FliA, we believe that the lethality phenotype of the flhD mutant is due to regulation of the type III secretion genes. 1. V. Kapatral et al., Microbiol. 150, 2289 (2004). 2. B. M. Prüß et al., J. Bacteriol. 185, 534 (2003). 3. S. M. Horne, B. M. Prüß, Arch. Microbiol. 185, 115 (2006). 4. M. K. Townsend et al., BMC Microbiol. 8, 12 (2008). 5. P. S. Gibbs, J. J. Maurer, L. K. Nolan, R. E. Wooley, Avian Dis. 47, 370 (2003).

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BLAST X Thurs. Morning Session REGULATION OF MOTILITY BY QUORUM SENSING IN SINORHIZOBIUM MELILOTI AND ITS ROLE IN SYMBIOSIS ESTABLISHMENT Juan E. González*, Nataliya Gurich, Jennifer L. Morris, Konrad Mueller, and Arati V. Patankar Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas, USA

Quorum sensing is a mechanism widely used by bacteria to coordinate their behavior in response to a particular cell population density. Signal molecules, termed autoinducers, are produced by bacteria, and at a high population density, accumulate in the environment. Once a threshold level of autoinducer is reached, they bind to their cognate transcriptional regulators and activate or repress expression of target genes, thereby preparing the bacteria for behaviors associated with high cell density, such as interacting with eukaryotic hosts.

In Sinorhizobium meliloti, this mechanism is utilized to appropriately modulate gene

expression and permit the establishment of a nitrogen-fixing symbiosis with its host plant Medicago sativa. S. meliloti possesses a quorum-sensing system composed of two transcriptional regulators, SinR and ExpR, and the SinR-controlled autoinducer synthase SinI, which is responsible for the biosynthesis of the signal molecule in the form of an N-acyl homoserine lactone (AHL). These AHLs, in conjunction with the ExpR regulator, control a variety of downstream genes. The concentration of AHLs varies with changes in population density. As a result, expression of quorum-sensing-dependent genes may exhibit different patterns during various stages of bacterial growth. Work in our laboratory has shown that the S. meliloti ExpR/Sin quorum-sensing system regulates over 200 genes, including those involved in exopolysaccharide synthesis, motility and chemotaxis, metal transport, and other metabolic functions, thereby playing an important role during plant-bacteria interactions.

Inoculation of plants with a sinI-deficient strain results in a delay in invasion as well as a

significant reduction in the total number of nodules per plant when compared to the wild type, resulting in plant development deficiencies. Concurrently, expression of most of the motility and chemotaxis genes in the sinI mutant fail to be down-regulated by quorum sensing at high cell population density. Microarray and real-time PCR analyses revealed that the ExpR/Sin system adjusts the expression of the transcriptional regulators VisN/VisR and Rem, which in turn modulate downstream motility genes in a population-density-dependent manner to decrease motility. Recently we have shown that mutating flagellar production in a sinI mutant restores bacterial competency for symbiosis establishment to wild type levels, suggesting that the elimination of flagella during the invasion process is crucial. Therefore, down-regulation of motility and chemotaxis by the ExpR/Sin quorum-sensing system plays an essential role in successful plant invasion by S. meliloti.

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BLAST X Thurs. Evening Session ENGINEERED SINGLE- AND MULTI-CELL CHEMOTAXIS IN E. COLI Shalom D. Goldberg1, Paige Derr2, William F. DeGrado1, and Mark Goulian2,3 1 Department of Biochemistry and Biophysics, University of Pennsylvania School of Medicine, Philadelphia, PA, 19104 2 Department of Physics, University of Pennsylvania, 209 South 33rd Street, Philadelphia, PA 19104 3 Department of Biology, University of Pennsylvania, 433 S University Avenue, Philadelphia, PA 19104

We have engineered the chemotaxis system of E. coli to enable responses to molecules that are not attractants for wild-type cells. The system depends on an artificially introduced enzymatic activity that converts the target molecule into a ligand for an E. coli chemoreceptor, thereby allowing the cells to respond to the new attractant. Two systems, designed to respond to asparagine and to phenylacetyl glycine respectively, showed robust chemotactic responses. In addition, their behavior in a mixed population was suggestive of a “hitchhiker” effect in which cells producing the ligand can induce chemotaxis of neighboring cells lacking the enzymatic activity. This behavior was exploited to design a complex system of two strains that are mutually interdependent for their activity, which functions as a simple microbial consortium.

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BLAST X Thurs. Evening Session PHOTO-ENERGY CONVERSION AND SENSORY TRANSDUCTION OF MICROBIAL RHODOPSINS IN PHOTOSYNTHETIC MICROBES So Young Kim, Keon Ah Lee, Ah Reum Choi, Song-I Han, and Kwang-Hwan Jung Department of Life Science and Interdisciplinary Program of Integrated Biotechnology, Sogang Univeristy, Seoul 121-742, Korea (kjung@sogang.ac.kr)

Microbial rhodopsins, seven transmembrane proteins which contain all-trans/13 cis retinal as a chromophore, have been known for three decades and extensively studies in extreme halophiles. Photosynthetic microbes possess lots of photoactive proteins including chlorophyll-based pigments, phytochromes, phototropin-related blue light receptors, and cryptochromes. Surprisingly, recent genome sequencing projects discovered additional photoactive receptors, retinal-based rhodopsins, in cyanobacterial and algal genera. Analysis of the Anabaena and Chlamyrhodopsin revealed that they have sensory functions, which based on our work with haloarchaeal rhodopsins, may use a variety of signaling mechanisms. Anabaena rhodopsin is interacted with a tetramer of 14kDa soluble transducer (ASRT) and one of their putative functions is a global regulation of phycobilin protein. The Anabaena rhodopsin shows a visible light-absorbing pigment (540-550nm) and it has mixed photochemical reaction of all trans and 13 cis form of retinal in ground state. Two Chlamydomonas rhodopsins are involved in phototaxis and photophobic responses based on electrical measurements by RNAi experiment. The rhodopsins from Gloeobacter violaceus and Acetabularia acetabulum is light-driven proton pump coexisted with photosynthetic machinery. The genes were functionally expressed in Escherichia coli and bound all-trans retinal to form a pigment in the presence of N- and C-terminal MISTIC sequences. Gloeobacter and Acetabularia rhodopsin I showed a light-driven proton pumping activity similar to proteorhodopsin.

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BLAST X Thurs. Evening Session FUNCTION OF MULTIPLE CHEMOTAXIS-LIKE PATHWAYS IN MEDIATING CHANGES IN MOTILITY PATTERNS AND CELLULAR MORPHOLOGY IN AZOSPIRILLUM BRASILENSE Amber N. Bible1, Zhihong Xie1, Matthew Russell1 and Gladys Alexandre1,2

1Department of Biochemistry, Cellular and Molecular Biology and 2Department of Microbiology, The University of Tennessee, Knoxville, TN 37996

Molecular details on bacterial chemotaxis have been derived from studies of model organisms such as Escherichia coli and Bacillus subtilis which genome encode for a single chemotaxis pathway that functions to modulate changes in motility patterns. Comparative genomics analysis indicates that the genome of many bacteria possess multiple chemotaxis-like (Che) pathways. A. brasilense is a plant-associated bacterium that can differentiate in at least four different cell types (swimmer, swarmer, aggregated and cyst cells). Transition from one cell type to the other depends on the environmental (especially nutritional) conditions. One of the 4 Che-like pathways (Che1) encoded within the genome of the alphaproteobacterium A. brasilense was recently shown to regulate changes in motility patterns, cell-to-cell aggregation concomitant with changes in cell length (Bible et al., 2008). We will present evidence for the role of two Che pathways and several chemoreceptors in controlling the ability of cells to modulate multiple cellular responses, including cell length, that suggest that cross-regulation between parallel chemotaxis pathways may function to coordinate and integrate a set of cellular functions. Experimental evidence suggests that proteins that function in the methylation/demethylation of chemoreceptors may have a critical role in this cross-regulation. The implications in the lifestyle of this bacterium will also be discussed in lights of recent experimental evidence obtained. Bible, A. N., Stephens, B. B., Ortega, D. R., Xie, Z. and G. Alexandre (2008) Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense. J Bacteriol 190: 6365-6375.

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BLAST X Thurs. Evening Session PROBING ADAPTATION KINETICS IN VIVO BY FLUORESCENCE RESONANCE ENERGY TRANSFER Thomas S. Shimizu1, Yuhai Tu2 and Howard C. Berg1 1Department of Molecular & Cellular Biology, Harvard University, Cambridge, MA 02138. 2T. J. Watson Research Center, IBM, Yorktown Heights, NY 10598.

Bacteria sense spatial gradients by taking time derivatives of ligand concentrations measured during runs of a random walk1. The remarkable sensitivity to shallow gradients in Escherichia coli has been explained mainly by cooperativity between receptors and ultrasensitivity of the flagellar motor. We have revisited the experimental findings of Block, Segall and Berg2, where the chemotactic response of tethered cells to time-varying stimuli were characterized quantitatively. A simple theoretical model3 that combines robust adaptation4 with an allosteric model of receptor cooperativity5-7 can explain the general features of responses to temporal ramps and oscillatory stimuli.

A notable feature of this model is that the steady-state amplitude of responses to

exponential ramps do not depend on the degree of receptor cooperativity (the parameter N of an MWC-type allosteric model5). The time required to reach this steady state, however, depends inversely on N, so cooperativity speeds up computation of the derivative signal, but does not determine its amplitude. The latter is instead determined by the adaptation kinetics, and this relation allows us to infer quantitative characteristics of adaptation in vivo from measured ramp-response data.

Here we present novel experiments in which the chemotactic responses of E. coli

populations during time-varying stimuli are monitored by fluorescence resonance energy transfer8 (FRET). This approach is far more efficient than the earlier experiments of Block et al.2, in which the chemotactic responses of individual cells were characterized through the stochastic output of the motor. We find that the sensitivity of E. coli to gradients depends strongly on temperature, and using our model framework, we analyze how ultrasensitvity in the adaptation system9 contributes to gradient sensitivity in vivo. REFERENCES

1. Berg, H. C. & Brown, D. A. Chemotaxis in Escherichia coli analysed by three-dimensional tracking. Nature 239, 500-4 (1972).

2. Block, S. M., Segall, J. E. & Berg, H. C. Adaptation kinetics in bacterial chemotaxis. J Bacteriol 154, 312-23 (1983).

3. Tu, Y., Shimizu, T. S. & Berg, H. C. Modeling the chemotactic response of Escherichia coli to time-varying stimuli. Proc Natl Acad Sci U S A 105, 14855-60 (2008).

4. Barkai, N. & Leibler, S. Robustness in simple biochemical networks. Nature 387, 913-7 (1997). 5. Monod, J., Wyman, J. & Changeux, J. P. On the Nature of Allosteric Transitions: A Plausible

Model. J Mol Biol 12, 88-118 (1965). 6. Mello, B. A. & Tu, Y. An allosteric model for heterogeneous receptor complexes: Understanding

bacterial chemotaxis responses to multiple stimuli. Proc Natl Acad Sci U S A 102, 17354-9 (2005).

7. Keymer, J. E., Endres, R. G., Skoge, M., Meir, Y. & Wingreen, N. S. Chemosensing in Escherichia coli: two regimes of two-state receptors. Proc Natl Acad Sci U S A 103, 1786-91 (2006).

8. Sourjik, V., Vaknin, A., Shimizu, T. S. & Berg, H. C. In Vivo Measurement by FRET of Pathway Activity in Bacterial Chemotaxis. Methods Enzymol 423, 363-91 (2007).

9. Emonet, T. & Cluzel, P. Relationship between cellular response and behavioral variability in bacterial chemotaxis. Proc Natl Acad Sci U S A 105, 3304-9 (2008).

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BLAST X Thurs. Evening Session MINOR RECEPTOR SIGNALLING IN E. COLI Silke Neumann, Ned Wingreen* and Victor Sourjik Ruprecht-Karls-Universität Heidelberg, Zentrum für molekulare Biologie Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany * Department of Molecular Biology - Princeton University

Ligand recognition in the chemotaxis pathway of E. coli proceeds through binding of ligands to transmembrane receptors, either directly or indirectly through periplasmic binding proteins. E. coli has five types of receptors, with two high-abundance (or major) receptors – Tsr for serine and Tar for aspartate and maltose – and three low-abundance (or minor) receptors – Tap for dipeptides, Trg for ribose, galactose and glucose, and Aer for redox potential. Together with the histidine kinase CheA, receptors form chemosensory complexes which in turn are organized in tight clusters where receptors of different ligand specificities are intermixed. Signal processing is thought to occur within these receptor clusters through allosteric interactions between receptor dimers. To compare signal processing by minor and major receptors, we systematically investigated responses mediated by Trg and Tap, and by Tar and Tsr in respect to response sensitivity, relation between receptor occupancy and kinase inactivation, dynamic range of the response, adaptation time to a range of stimuli, as well as integration of signals that are sensed by different receptors using an in vivo FRET-based kinase assay. Our experimental analysis shows that signals are amplified and integrated differently by the two receptor populations, but in both cases signal processing can be quantitatively explained by the same allosteric model.

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BLAST X Thurs. Evening Session A SYSTEMS BIOLOGY APPROACH TO UNDERSTANDING HOW BACILLUS MAKES UP ITS MIND Arnaud Chastanet1, Guocheng Yuan2, Thomas M. Norman1, Jun Liu3 and Richard Losick1

1Molecular and Cellular Biology Department, Harvard University, 2Department of Biostatistics and Computational Biology, Harvard School of Public Health, 3Department of Statistics, Harvard University.

Understanding how cells make decisions and differentiate are key biological questions. Mechanisms underlying such behaviors integrate multiple environmental signals in intricate networks in order to appropriately respond to the situations. The sporulation process that takes place in Bacillus subtilis under adverse conditions perfectly exemplifies this kind of question. For this, numerous signals and control systems are integrated at the level of a ”decider” protein called Spo0A, a transcriptional regulator belonging to the two-component systems family. The decision to sporulate is taken during the first two hours after optimal sporulation conditions have been reached. During this time, Spo0A accumulates slowly reaching a high level at hour two. It has been previously shown that while some of its targets are activated at low concentration, thus early on, others are switched on later, when the maximal quantity of the regulator has been achieved. Interestingly, even in optimal conditions, only a fraction of the population will finally decide to sporulate, a phenomenon described as bistability.

We are attempting to understand how this two-stage activation of Spo0A is achieved

through an interdisciplinary approach combining the methods of genetics and mathematics. The time resolved picture of the regulatory process we have obtained has revealed a multiple step process involving successive switches. First, Spo0A activity is rising during log phase, activating some switches. Then under conditions of nutrient limitation, Spo0A is further activated to a mid and variable extent throughout the population. During this period, “low-threshold” genes are turned ON. In an ultimate step, a bistable switch allows Spo0A to be activated to a high level but only in a portion of the population. These cells express high threshold genes and proceed to sporulate.

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POSTER ABSTRACTS

BLAST X ______ Poster #1 THE CHARACTERISATION OF THE DYNAMICS OF THE FliT:FliD:FlhD4C2 INTERACTION AND ITS ROLE IN REGULATING FLAGELLAR ASSEMBLY C. Aldridge 1,2, K. Poonchareon 1,2, S. Saini3, A. Soloyva2, M. Banfield4, T. Minamino5,6, C. V. Rao3, and P. D. Aldridge 1,2 1: Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH. 2: Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH. 3: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois, United States, 61801. 4: Dept. of Biological Chemistry, John Innes Centre, Norwich, NR4 7UH UK. 5: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. 6: Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Each bacterial cell of Salmonella enterica serovar Typhimurium produces a discrete number of complete flagella. Flagellar gene expression in Salmonella is negatively regulated by the Type 3 secretion chaperone FliT. FliT is known to interact with the flagellar structural subunit FliD and the master transcriptional regulator FlhD4C2. In this regulatory circuit FliD is proposed to act as an anti-regulator - a regulatory role similar to that observed for FlgM inhibition of s28 activity. We were interested in determining the kinetics of the FliT:FliD:FlhD4C2 regulatory circuit and how they influence flagellar assembly. We have shown that the FliT:FliD interaction is a 1:1 ratio while in solution FliT is a dimer. Surface plasma resonance (SPR) and analytical ultracentrifugation (AUC) analysis of the dynamics of the FliT:FlhD4C2 interaction showed that it was very different from FliT:FliD. A FliT:FlhD4C2 complex is only observed when excess FliT is added to FlhD4C2. AUC analysis identified that a stable intermediate of a combination of FlhD, FlhC and/or FliT exists. Our data suggests that FliT acts to dissociate the FlhD4C2 complex and that this is a transient interaction allowing for the FlhD4C2 complex to reform. This suggests that a limiting factor in the regulation of FlhD4C2 activity is the concentration of free FliT. To test this model, we have investigated the affect of overexpressing FliT on flagellar gene expression and basal body assembly.

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BLAST X ______ Poster #2 SUBUNIT FEEDBACK CONTROL OF FLAGELLAR FILAMENT ASSEMBLY IN CAULOBACTER CRESCENTUS Phillip D. Aldridge 1,2§ Alexandra Faulds-Pain 1,2, Christine Aldridge 1,2, Giulia Grimaldi 1,2, Christopher Birchall 1,2, Shuichi Nakamura 3, Tomoko Miyata 3, Joe Gray 4, Guanglai Li 5, Jay Tang 5, Keiichi Namba 3,6, Tohru Minamino 3,6 1: Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH 2: Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH 3: Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. 4: Pinnacle, Newcastle University, Framlington Place, Newcastle upon Tyne, United Kingdom, NE2 4HH 5: Physics Department, Brown University, 184 Hope Street, Providence, RI 02912, USA 6: Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. Bacterial flagellar filaments play key roles in surface attachment and host-bacterial interactions as well as motility. Approximately 40% of annotated flagellar systems (n = 278) utilise multiple flagellin variants to assemble their flagellar filaments. Here we have investigated the ability of the model flagellar system of Caulobacter crescentus to assemble its flagellar filament from six flagellins: FljJ, FljK, FljL, FljM, FljN and FljO. A flagellin gene mutant collection of multiple gene deletion combinations, exhibited a range of Mot phenotypes from impaired motility (Mot-/+) to motile (Mot+). Further characterisation of the mutant collection showed: 1) there is no strict requirement for all six flagellins to assemble a filament exhibiting wild type characteristics; 2) a correlation between slower swimming speeds and shorter filament lengths in all ∆fljK ∆fljM Mot+ mutants; and 3) the flagellins FljM – FljO are less stable than FljJ – FljL. Our data suggests that the flagellins FljJ, FljK and FljL play both a regulatory and structural role during filament assembly. In contrast, the flagellins FljM to FljO possess only a structural role. We propose the model that the observed order of multiple flagellin incorporation in C. crescentus, and plausibly other flagellar systems, is a result of the system coupling flagellin synthesis to filament assembly.

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BLAST X ______ Poster #3 FUNCTION OF UNIQUE DOMAINS OF CheA1 FROM A. BRASILENSE IN REGULATING MULTIPLE CELLULAR BEHAVIORS Amber N. Bible and Gladys Alexandre Department of Biochemistry, Cellular, and Molecular Biology at the University of Tennessee, Knoxville The alpha-proteobacterium Azospirillum brasilense contains four different chemotaxis operons. One of the 4 Che-like pathways (Che1) encoded within the genome of the alphaproteobacterium A. brasilense was recently shown to regulate changes in motility patterns, cell-to-cell aggregation (clumping) concomitant with changes in cell length (Bible et al., 2008). Mutations affecting cheA1 decrease chemotaxis, cell length, but lead to an increase in clumping relative to the wild type A. brasilense. Interestingly, CheA1 is a hybrid histidine kinase with two P5-like domains and a REC-like receiver domain at its C-terminus. In addition, the N-terminus of CheA1 comprises a highly conserved polytopic domain of unknown function, suggesting that CheA1 may be a membrane-bound protein. Experimental data indicate that CheA1 is produced as a membrane-bound protein that localizes to the cell pole, similar to other Che1 proteins. Noticeably, the polytopic N-terminal domain of CheA1 is not required for the localization of CheA1 neither to the cell pole, nor for changes in cell length or cell-to-cell aggregation but it is essential for wild-type chemotaxis and aerotaxis behaviors. Data obtained from a combination of in-frame deletions of the C-terminal domains and site-specific mutagenesis approaches with behavioral assays suggest that the second P5-like domain of CheA1 has a role in modulating changes in cell length. A working model for the functions of the N- and C-terminal domains of CheA1 in modulating chemotaxis, aerotaxis, clumping and cell length changes will be presented in lights of recent experimental data. Bible, A. N., Stephens, B. B., Ortega, D. R., Xie, Z. and G. Alexandre (2008) Function of a chemotaxis-like signal transduction pathway in modulating motility, cell clumping, and cell length in the alphaproteobacterium Azospirillum brasilense. J Bacteriol 190: 6365-6375.

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BLAST X ___ Poster #4 HOW DOES THE RHODOBACTER SPHAEROIDES FLAGELLAR MOTOR STOP – USING A CLUTCH OR A BRAKE? M. Brown, T. Pilizota, M. Leake, R. Berry, J. Armitage University of Oxford, Oxford, UK

Unlike most species with bidirectional motors, Rhodobacter sphaeroides employs a unidirectional stop-start flagellar motor, where stops are analogous to tumbles. By controlling when the motor stops, cells can accumulate in areas favourable for their survival.

We asked the question, how do the CheYs stop motor rotation in R. sphaeroides; by

causing the torque-generating units to disengage from the rotor, allowing free rotational movement, or by jamming the rotor, locking it in a particular configuration?

These hypothesises were tested by applying external force (viscous flow or optical

tweezers) to chemotactically stopped motors.

We found that the motor is stopped with a brake mechanism and that approximately 3-4 times more torque acts on the motor when stopped than when it rotates. Furthermore, by monitoring the position of sub-micron beads attached to flagella stubs we discovered that stops can only occur at a number of discrete angles. Analysis is underway to determine the number of steps per revolution.

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BLAST X ______ Poster #5 DYNAMICS OF THE FLAGELLAR MOTOR PROTEIN FliM Nicolas J. Delalez Microbiology Unit, Biochemistry Dept, University of Oxford, South Parks Road, Oxford OX1 3QU, UK Escherichia coli swims by rotating 4-6 long helical filaments to propel the cell through its environment. The flagellar motor that rotates the filament is bidirectional and composed of two parts: the rotor and the stator, the stator being the fixed component against which the rotor spins. The stator is composed of units of two integral membrane proteins, MotA and MotB, the stoichiometry of each unit being (MotA4:MotB2). The rotor is composed of multiple rings, including the C-ring which is localized at the base of the motor and is the switch complex that gives the bidirectionality to the E. coli motor. The C-ring comprises FliG (~ 25 copies), FliM (~34 copies) and FliN (> 100 copies). By using a TIRF microscope, and expressing GFP-MotB from the genome of E. coli in place of the wild-type gene, it has recently been possible to monitor the protein stoichiometry, dynamics and turnover of this stator component with single-molecule precision in functioning bacterial flagellar motors. Repeating these experiments with different flagellar motor proteins will greatly enhance our understanding of the mechanism of this motor. In particular, one of the main questions for our understanding is the mechanism of the switching process. The C-terminus of FliM, a component of the C-ring, has therefore been tagged with Ypet and expressed from the genome. Data will be presented on the construction of this mutant as well as data on its dynamics, turnover and stoichoimetry, and the influence of the response regulator CheY on these features. The consequences of these results and future work will be discussed.

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BLAST X Poster #6 USING CONTROL THEORY TO ELUCIDATE CONNECTIVITY IN R. SPHAEROIDES CHEMOTAXIS Mark A. J. Roberts1, Elias August2, Judith P. Armitage1 and Antonis Papachristodoulou3 1Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, UK 2Control Group, Department of Engineering Science, Oxford University, Parks Road, Oxford, OX1 3PJ, UK 3Oxford Centre for Integrative Systems Biology, Department of Biochemistry, South Parks Road, Oxford, OX1 3QU, UK With an increasing number of sequenced bacterial genomes it becomes evident that the chemotactic sensory mechanism of bacteria is more complex than E. coli. In this poster we describe how ideas from engineering control theory can be used to develop a novel approach for designing experiments in order to elucidate the biochemical network structure of signalling pathways in general. The goal is to develop a systematic approach for finding the best experiment that will delineate the network structure. We then apply this method to the chemotaxis pathway of R. sphaeroides, which has multiple homologues of the E. coli proteins. To achieve this we are constructing, in silico, various possible models of R. sphaeroides chemotaxis that can explain experimental observations. These models include the different possible interactions for the CheB and CheY proteins. Applying results from optimal control theory, we determined the best input (ligand) profile that gives an output which would allow us to discriminate best between the proposed models, aiming to invalidate some of them. This input ligand profile is then administered to R. sphaeroides in a flow cell and the response is measured using a tethered cell assay. We have also developed methods to determine the best initial conditions to discriminate between the models, based on the limitations of what can be implemented biochemically, and these were then also tested in a tethered cell assay. We used the experimental results from these designed tethered cell experiments to invalidate some of the proposed network structures and hence suggest a probable network connectivity for the multiple CheY and CheB proteins within R. sphaeroides. This is an exciting approach to determine network structures in a fast and efficient manner and can be applied to a wide range of signalling pathways as well as potentially allowing chemotaxis pathways in other species using published genomes to generate the necessary models.

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BLAST X Poster #7 BEHAVIOR OF THE FLAGELLAR ROTARY MOTOR NEAR ZERO LOAD Junhua Yuan and Howard Berg Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 The physiology of the flagellar rotary motor has been studied extensively in the regime of relatively high load and low speed. Here, we describe an assay that allows systematic study of the motor near zero load. Sixty-nanometer-diameter gold spheres were attached to hooks of cells lacking flagellar filaments, and light scattered from a sphere was monitored at the image plane of a microscope through a small pinhole. Resurrection experiments were carried out near zero load. Paralyzed motors of cells carrying a motA point mutation were resurrected at 23°C by expression of wild-type MotA, and speeds jumped from zero to a maximum value of about 300 Hz in one step. The temperature dependence of the speed near zero load also was studied and showed a high activation enthalpy comparable to that observed previously in electrorotation experiments. The assay has been modified so that both the speed and the direction of rotation can be monitored near zero load. Switching properties of the flagellar motor near zero load are under investigation.

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BLAST X ______ Poster #8 NUTRIENT SENSING BY A HUMAN GUT SYMBIONT Hongjun Zheng1, Susan Firbank1, Eric Martens2, Edith Diaz1, Rick Lewis1, Jeff Gordon2 and David Bolam1 1. Institute for Cell and Molecular Biosciences, Newcastle University, The Medical School, Framlington Place, Newcastle upon Tyne NE2 4HH, UK 2. Center for Genome Sciences, Washington University, St Louis, USA. The gut microbiota play an important role in human health and nutrition. Bacteroides thetaiotaomicron (Bt) is a dominant gut symbiont whose main sources of carbon and energy are dietary and host polysaccharides, reflected in the presence on the genome of a large number of genes involved in the sensing, acquisition and processing of complex carbohydrates. One of the most striking features of the Bt genome are 33 genes encoding novel hybrid two component systems (HTCS) that contain all of the domains of a classical TCS in a single polypeptide. Custom Genechip arrays reveal that when Bt is grown in the presence of inulin, a β-1,2 linked fructose storage polysaccharide, a specific locus composed of eight genes predicted to be involved in polysaccharide binding, uptake and degradation is activated. The closest regulatory protein to this locus is an HTCS, BT1754. Genetic, biochemical and structural studies reveal that BT1754 is the sensor that controls fructan utilisation and the identity of the signalling molecule itself, as well as the mechanism of signal perception and insights into signal transduction across the cytoplasmic membrane.

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BLAST X Poster #9 THE LeuO REGULON IN SALMONELLA Edmundo Calva, Víctor H. Bustamante, Miguel Ángel De la Cruz, Marcos Fernández-Mora, Mario Alberto Flores-Valdez, Ana Lucía Gallego-Hernández, Carmen Guadarrama, Ismael Hernández-Lucas, Omar Ortega, Alejandra Vázquez, and Ricardo Oropeza. Instituto de Biotecnología UNAM, Cuernavaca, Morelos, Mexico. LeuO is a LysR-type transcriptional regulator, which antagonizes the repressing effect by the H-NS and StpA nucleoid proteins on the Salmonella enterica serovar Typhi ompS1 quiescent porin gene, allowing the transcriptional activation by OmpR among other regulators. LeuO is also a positive regulator for the OmpS2 quiescent porin, for a quiescent putative periplasmic arylsulfate sulfotransferasa (AssT), and for an open reading frame of unknown function, STY3068. It is a negative regulator for the OmpX porin, a thiol peroxidase (Tpx), and for STY1978. These proteins were identified by a whole-genome proteomic analysis and the regulatory effects were confirmed on transcriptional gene reporter fusions. STY 3068 appears to be part of an operon (5’-STY3070-STY3064-3’), as transcriptional regulation occurs upstream, at STY 3070. The assT gene forms a LeuO-dependent operon with STY3371 and STY3372, which are paralogues of the dsbA and dsbB genes, respectively. This operon (5’-assT-STY3371-STY3372-3’) is repressed by OmpR. H-NS represses the expression of ompS1, assT and STY3070 and, on the other hand, activates the expression of tpx and STY1978. Expression of ompX was independent of H-NS. Interestingly, ompS2 is negatively regulated by H-NS in Escherichia coli and in S. Typhimurium, although in S. Typhi other negative regulators are involved aside from H-NS. LeuO specifically binds to the 5´ intergenic regions of ompS1, ompS2, assT, STY3070, ompX, and tpx. LeuO did not bind to the promoter region of STY1978, suggesting that it is regulating in an indirect manner.

Hence, LeuO regulates a cadre of genes which appear to be mostly involved in the

response to stress and in virulence.

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BLAST X Poster #10 EnvZ-OmpR AND CpxA-CpxR REGULATE ompS1 BY DIFFERENTIAL PROMOTER EXPRESSION De la Cruz, M.A, Flores-Valdez, M.A., and Calva, E. Departamento de Microbiología Molecular, Instituto de Biotecnología UNAM Av. Universidad 2001 Col. Chamilpa C.P. 62210 Cuernavaca, Morelos México

The Salmonella enterica ompS1 gene encodes a quiescent porin that belongs to the

OmpC/OmpF family. We recently reported that LeuO, OmpR, H-NS and StpA are regulators of ompS1 expression. We have now detailed the mechanism of OmpR regulation. In vivo, phosphorylated OmpR (OmpR-P) showed to be determinant for the regulation of both ompS1 promoters: OmpR-P activated the P1 promoter and repressed the P2 promoter, in an EnvZ-dependent manner. In vitro, OmpR-P had a seventy two-fold higher binding-affinity to the ompS1 promoter region than OmpR, being 2 to 20-fold higher than to the major porin-encoding ompC and ompF genes, respectively. In addition to EnvZ-OmpR, we found that CpxA-CpxR positively regulated ompS1 expression. CpxR, together OmpR, was necessary for the activation of the P1 promoter. CpxR also activated the P2 promoter, but only in the absence of OmpR. Our model involves LeuO as an antagonist that relieves H-NS and StpA-mediated silencing, allowing the binding of OmpR and CpxR. High osmolarity and acid pH, or both, stimulated ompS1 expression in an OmpR and CpxR-independent manner, yet dependent on DNA supercoiling.

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BLAST X Poster #11 THE COMPLEX HOOK BASAL BODY STRUCTURE OF THE LYME DISEASE SPIROCHETE BORRELIA BURGDORFERI K. Miller1, R. Duda2, R. Hendrix2, M. Motaleb1,3, and N.W. Charon1 1West Virginia University Health Sciences Center, Microbiology and Immunology Department, Box 9177, Room 2077, Morgantown, WV 26506 2University of Pittsburgh, Department of Biological Sciences, 4249 Fifth Avenue, 357A, Crawford Hall, Pittsburgh, PA 15260 3East Carolina University, Department of Microbiology and Immunology, Greenville, NC 27834

FlgE is the structural protein of the flagellar hook of B. burgdorferi. Previous western blot analysis using polyclonal FlgE antibody indicated that the majority of the hook migrated as a high molecular weight complex in SDS-PAGE gels instead of at the position of monomeric FlgE (46 kDa). Both the high molecular complex and a small amount of the monomer were present in wild-type and complemented cells but absent in flgE mutants. These data suggest that FlgE may be cross-linked into oligomers in a manner similar to that found in bacteriophage capsid proteins and in some bacterial pili. High molecular weight FlgE complexes have been observed in other spirochete species (Treponema phagedenis and Treponema denticola), suggesting that this property is conserved. We are interested in determining whether or not FlgE is cross-linked, and what amino acids are involved in the cross-linking.

The hook-basal body complexes were purified using a refined version of methods

previously reported (Sal, et al, 2008). Following purification of periplasmic flagella that contained attached hook-basal body complexes, the filament portions were depolymerized by acidic conditions or the use of urea. Hook-basal body complexes were then isolated by pelleting using an ultracentrifuge recovered in a neutralizing buffer. Electron microscopy of these preparations showed that acidic conditions resulted in more intact hook-basal body complexes than urea treatment, although the high molecular weight forms of FlgE were found using both methods. In order to determine results from each experiment, the experimental samples were run on 10% SDS-PAGE gels. We are using proteolytic finger printing and peptide mapping methods to compare the high molecular weight complexes of FlgE with the monomeric form (histidine-tagged recombinant). Hook-basal body complexes and recombinant FlgE were treated with proteases or hydroxylamine at various temperatures (37, 45, and 65°C) and run in SDS-PAGE gels. Western blotting was used to assess the stability of the FlgE complex as well as to determine the size of (antigenic) peptide fragments resulting from proteolytic digestion. Digest patterns were compared between recombinant FlgE and the isolated hook-basal body complexes.

Refining the hook-basal body isolation procedure resulted in improved hook-basal body

preparations. The FlgE complex is stable under harsh conditions (both urea and acid treatment). Differences and similarities in hydroxylamine digest patterns between recombinant FlgE and purified hook-basal body complexes supports the hypothesis that FlgE is cross-linked.

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BLAST X Poster #12 HOW DO PAS AND HAMP DOMAINS COMMUNICATE? INSIGHTS FROM Aer2, A HEME BASED SENSOR FOR AEROTAXIS Michael Airola (1), Joanne Widom (1), Kylie Watts (2), Brian Crane (1) (1) Dept. of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14850 (2) Dept. of Biochemistry and Microbiology, Loma Linda University, Loma Linda, CA 92350 Thousands of PAS and HAMP domains have been identified yet how these common signal transduction domains function and communicate is still poorly understood. Structural studies of the HAMP domain have been especially difficult due to it often being contained within integral membrane proteins. Recently Aer2, from P. aeruginosa, has been identified as a soluble aerotaxis receptor that binds oxygen directly through a heme moiety located in the PAS domain. Using a combination of spectroscopic and structural techniques we sought to determine if oxygen binding induces large-scale conformational changes. Initial results point to an interaction between the PAS and HAMP domains of Aer2 giving insight to how these widespread signaling modules may function.

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BLAST X Poster #13 STRUCTURE OF SOLUBLE CHEMORECEPTOR SUGGESTS A MECHANISM FOR PROPAGATING CONFORMATIONAL SIGNALS Abiola Pollard and Brian Crane Cornell University, Chemistry Research Bldg. G63, Ithaca, NY 14853 Transmembrane chemoreceptors, also known as methyl-accepting chemotaxis proteins (MCP’s), translate extracellular signals into intracellular responses in the bacterial chemotaxis system. MCP ligand binding domains control the activity of the CheA kinase, situated ~200 Å away, across the cytoplasmic membrane. The 2.15 Å resolution crystal structure of a T. maritima soluble receptor (Tm14) reveals distortions in its dimeric four-helix bundle that provide insight into the conformational states available to MCP’s for propagating signals. A bulge in one helix generates asymmetry between subunits that displaces the kinase-interacting tip, which resides over 100 Å away. The maximum bundle distortion maps to the adaptational region of transmembrane MCP’s where reversible methylation of acidic residues tunes receptor activity. Minor alterations in coiled-coil packing geometry translates the bulge distortion to a >25 Å movement of the tip. The Tm14 structure discloses how alterations in local helical structure, which could be induced by changes in methylation state and/or by conformational signals from membrane proximal regions, can reposition a remote domain that regulates the CheA kinase.

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BLAST X Poster #14 FUNCTIONAL ANALYSIS OF A LARGE NON-CONSERVED REGION OF FlgK (HAP1) FROM RHODOBACTER SPHAEROIDES Castillo, D.J., Ballado, T., Camarena, L., Dreyfus, G. Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, Apdo. Postal 70-243 Cd. Universitaria, 04510, Mexico, D.F., Mexico. The single subpolar flagellum of Rhodobacter sphaeroides shows an enlarged hook-filamentjunction. One of the two proteins that compose this section of the filament is HAP1Rs (FlgKRs) it contains a central non-conserved region of 860 amino acids that makes this protein about three times larger than its homologue in Salmonella enterica serovar Typhimurium. We investigated the role of this central portion of the unusually large HAP1 protein of R. sphaeroides by monitoring the effects of serial deletions in flgKRs, the gene encoding HAP1Rs, on swimming and swarming. Two deletion mutants did not assemble functional flagella, two were paralyzed and five exhibited reduced free-swimming speeds. Some mutants produced unusual swarming patterns on soft agar without or with Ficoll 400. A segment of approximately 200-aa of the central region of HAP1Rs that aligns with the variable region of the flagellin sequence from other γ- and β-proteobacteria was also found. Therefore, it is possible that the origin of this large central domain of HAP1Rs could be associated with an event of horizontal transfer and subsequent duplications and/or insertions.

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BLAST X Poster #15 THE FLAGELLAR MURAMIDASE FROM THE PHOTOSYNTHETIC BACTERIUM RHODOBACTER SPHAEROIDES Javier de la Mora1, Teresa Ballado1, Aldo E. García-Guerrero1, Laura Camarena2 and Georges Dreyfus1 Instituto de Fisiología Celular1, Instituto de Investigaciones Biomédicas2, Universidad Nacional Autónoma de México, 04510, México City, México.

The biogenesis of flagellum from R. sphaeroides is a tightly regulated process and requires the expression of more than 50 genes in a strict hierarchical manner (1). The flagellar rod is composed by four proteins, FlgB, FlgC, FlgF and FlgG. Besides these structural components several more proteins are required for rod assembly among these FlgJ from Salmonella enterica, has been postulated to be a dual function protein: the N-terminal half could function as a scaffold or cap essential for rod assembly and the C-terminal half may function as a muramidase that degrades the peptidoglycan layer to facilitate rod penetration (2, 3).We have previously reported that the FlgJ protein of R. sphaeroides lacks the C-terminal muramidase domain and that mutations in this protein renders a Fla- phenotype (4).The absence of the muramidase domain in this protein, suggests that other polypeptide must accomplish this function. In this work we describe the characterization of an open reading frame (orf) RSP0072, which is located within the flgG operon in R. sphaeroides. The amino acid sequence analysis of this gene product showed the presence of a soluble lytic transglycosylase domain. The deletion of the N-terminal region (112 aa) of the product of RSP0072 renders a non-motile phenotype as determined by swarm assays in soft agar. Electron micrographs revealed the lack of flagellum in the mutant cells. The purified wild-type protein showed lytic activity on extracts of M. lysodeikticus. Interaction assays suggests that the protein encoded by RSP0072 interacts with the flagellar rod-scaffolding protein FlgJ. We propose that the product of RSP0072 is a flagellar muramidase that is exported to the periplasm via the Sec pathway where it interacts with FlgJ to open a gap in the peptidoglycan layer for the subsequent penetration of the nascent flagellar structure (5).

References: 1) Poggio S., et al., 2005, Mol. Microbiol., 58:969-83. 2) Nambu T., et al., 1999, J. Bacteriol., 181:1555-1561. 3) Hirano T., et al., 2001, J. Mol. Biol., 312:359-369. 4) Gonzalez-Pedrajo B., et al., 2002, Biochim. Biophys. Acta, 1579:55-63. 5) De la Mora J., et al., 2007, J. Bacteriol., 189:7998-04.

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BLAST X Poster #16 MODELING SCAFFOLD PHOSPHORYLATION AS AN ADAPTATION MECHANISM IN BACTERIAL CHEMOTAXIS Roger Alexander, Adam Bildersee, and Thierry Emonet Department of Molecular, Cellular, and Developmental Biology, Kline Biology Tower 1054, Yale University, New Haven, CT 06520

Bacterial chemotaxis is one of the best-understood signaling networks in biology. Chemotaxis in E. coli is a model system for thoroughly understanding a behavioral phenotype at the molecular level. Chemotaxis has been studied experimentally in a variety of other species, and modeled in B. subtilis, but no dynamical models of chemotaxis network architectures from other species have yet been built. A key feature of the chemotaxis signal transduction network is adaptation: after a transient response to a step change in input stimulus, its activity returns to its pre-stimulus steady state level, allowing the system to respond to higher stimulus concentrations without saturation. In E. coli, adaptation is mediated through two enzymes, CheR and CheB, that methylate and demethylate the sensory receptors. In this work we consider scaffold phosphorylation as an adaptation mechanism complementary to receptor methylation. The receptors form a sensory array in the membrane at the cell pole; the scaffold protein couples the receptors to the kinase CheA which they activate. In all current dynamical models of chemotaxis, the receptors, scaffolds, and kinases are treated as a single species, a receptor-kinase complex. This is understandable, because in E. coli, the scaffold CheW is a passive mediator of the receptor-kinase interaction. However, in other organisms, the scaffold CheV has not only a CheW scaffold domain, but also a receiver domain that is the target of phosphorylation by the kinase. Bacillus subtilis has both CheW and CheV scaffolds, and phosphorylation of CheV is known to be necessary for adaptation. In B. subtilis, phospho-CheV decouples receptor from kinase, so it actively mediates their interaction. The evolutionary distance between B. subtilis and E. coli is wide, and there are other differences between their chemotaxis network architectures that confound an understanding of the specific role of scaffold phosphorylation. Therefore we choose to focus on a close relative of E. coli, Salmonella enterica serovar typhimurium. The chemotaxis network architecture in Salmonella is almost identical to that in E. coli, except that Salmonella has both CheW and CheV scaffolds. A mutant Salmonella that lacks methylation-based adaptation is able to adapt partially, presumably through its scaffold phosphorylation mechanism. We have built a dynamical model of the chemotaxis network in E. coli that explicitly represents receptor-kinase coupling by the scaffold. We have extended that model to include scaffold phosphorylation in Salmonella. Our model of a Salmonella mutant that lacks methylation exhibits partial adaptation, consistent with experimental results. This work is the first step in a long-range program to explore how evolutionary changes in chemotaxis network architecture affects the dynamics and function of the network. Evolution of dynamic signal transduction networks is an important emerging research area in systems biology.

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BLAST X Poster #17 TESTING THE YIN-YANG MODEL OF SIGNAL TRANSDUCTION IN A BACTERIAL CHEMORECEPTOR CYTOPLASMIC DOMAIN 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 aspartate receptor (Tar) of E. coli and S. typhimurium chemotaxis is a homodimer that assembles to form larger oligomers, most likely a trimer-of dimmers. 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 protein interaction region that binds the CheA kinase. The kinase control module is a 4-helix bundle essential for transmitting the integrated signal output of the HAMP domain and the adaptation sites to the bound histidine kinase CheA. Considerable evidence indicates that structural rearrangments of the subunit-subunit interface within the homodimeric 4-helix bundle are important in signaling. This study further probes the mechanism of signal transduction in the kinase control module of the S. Typhimurium aspartate receptor. The approach mutates the conserved “knob” residues of “sockets” that stabilize helix-helix contacts to alanines, in order to destabilize the packing of adjacent helices in the 4- helix bundle. Knob mutations that lock the receptor in “on” and “off” signaling states are identified by their opposite effects on CheA kinase and receptor methylation rates. The results suggest a novel “Yin-Yang” mechanism in which the helix packing states of the adaptation region (I) and the protein interaction region (II) have opposing effects on receptor on-off switching: stable helix packing in region I and unstable packing in region II drive the receptor into the kinase-activating on-state, while unstable packing in region I and stable packing in region II favor the kinase-inactivating off-state.

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BLAST X Poster #18 CYTOCHROME d BUT NOT CYTOCHROME o RESCUES THE TOLUIDINE BLUE GROWTH SENSITIVITY OF arc MUTANTS IN E. COLI Adrián F. Alvarez, Roxana Malpica, Martha Contreras, Edgardo Escamilla, Everardo Ramírez and Dimitris Georgellis. Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México, 04510 México D.F., México The Arc (anoxic redox control) two-component signal transduction system, consisting of the ArcB sensor kinase and the ArcA response regulator, allows adaptive responses of Escherichia coli to changes of O2 availability. Under anaerobic conditions, the ArcB sensor kinase autophosphorylates and then transphosphorylates ArcA, which in this form acts as a global transcriptional regulator that controls the expression of many operons involved in respiratory or fermentative metabolism. Under aerobic conditions, the system is inactivated through the inhibition of the kinase activity of ArcB by the quinone electron carriers, and the subsequent ArcA dephosphorylation. The arcA gene was previously known as the dye gene because null mutants were growth sensitive to the photosensitizer redox dyes toluidine blue and methylene blue, a phenotype whose molecular basis still remains elusive. Toluidine blue is a redox dye that, in presence of light, allows the production of reactive oxygen species (ROS). In this study we report that the toluidine blue O effect on the arc mutants is light independent, and only observed during aerobic growth conditions. Moreover, seventeen suppressor mutants with restored growth were generated and analyzed. Thirteen of those possessed insertion elements (IS) upstream the cydAB operon, rendering its expression ArcA independent. Finally, it was found that, in contrast to cythocrome d, cythocrome o was not able to confer toluidine blue resistance to arc mutants, thereby representing an intriguing difference between the two terminal oxidases.

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BLAST X Poster #19 SEARCHING THE PHYSIOLOGICAL SIGNAL(S) THAT REGULATE THE ACTIVITY OF THE SENSOR KINASE BarA González-Chávez, R., Rodríguez-Rangel, C., Georgellis, D. Departamento de Genética Molecular, Instituto de Fisiología Celular, UNAM, México, D.F. 04510.Tel. 56-22-57-38. dimitris@ifc.unam.mx The BarA/UvrY two-component system of Escherichia coli comprises the BarA protein as the histidine sensor kinase, and UvrY as the cognate response regulator. The E. coli BarA/UvrY two-component system (TCS) and its homologues in other gram-negative bacteria, such as BarA/SirA of Salmonella, ExpS/ExpA of Erwinia, VarS/VarA of Vibrio, and GacS/GacA of Pseudomonas species, have been shown to positively control expression of the noncoding CsrB and CsrC RNAs in E. coli. These small regulatory RNAs together with the 6.8 kD CsrA protein constitute the Csr (carbon storage regulation) system, a post-transcriptional regulatory system that has profound effects on central carbon metabolism, motility and multicellular behavior of E. coli. No physiological signals able to regulate the BarA sensor kinase and thereby control the UvrY response regulator have so far been identified for any of the orthologous TCSs. However, in a recent study it was demonstrated that pH lower than 5.5 provides an environment that does not allow activation of the BarA/UvrY signaling pathway, providing an important physiological tool for further experimentation in this direction. Here, we present experiments aiming at identifying the environmental signal(s) to which BarA respond. Our results indicate that short fatty acids, such as formate and acetate, act as direct signals for BarA. The implications of our findings on the overall physiology of the cell are be discussed.

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BLAST X Poster #20 THE ROLE OF QUORUM SENSING IN THE CONTROL OF MOTILITY AND PLANT INVASION BY SINORHIZOBIUM MELILOTI Nataliya Gurich* and Juan E. González Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Texas, USA

Quorum sensing, a population density dependent regulation of gene expression, is used by various bacteria to establish symbiotic or pathogenic bacterium-host associations. This mechanism requires the production of signaling molecules called autoinducers. At high cell population densities, autoinducer concentration in the surrounding area reaches a threshold level, which leads to activation of specific transcriptional regulators and the control of numerous phenotypes.

Sinorhizobium meliloti is a gram-negative soil bacterium that can form a nitrogen-fixing

symbiotic association with its host Medicago sativa. The quorum sensing system in S. meliloti is comprised of a transcriptional regulator, SinR and an autoinducer synthase, SinI, which specifies production of N-acyl homoserine lactone (AHL) signaling molecules. In conjunction with AHL, an additional regulator, ExpR, controls expression of several hundred genes. Recent microarray studies in our laboratory described the control of motility and chemotaxis in S. meliloti by quorum sensing through the regulators VisN/VisR and Rem in a population-density-dependent manner. The wild type strain is motile during the early log phase of growth but shuts down flagella synthesis genes during the mid- and late log phases. In contrast, the sinI mutant remains motile throughout all phases of growth, and at the late log phase, 35 motility and chemotaxis genes are upregulated when compared to wild type. This inability to repress flagella production by the sinI mutant leads to severe invasion deficiency. Detailed analysis of microarray results suggested that the inability of the sinI mutant to shut down flagella synthesis might be detrimental to successful plant invasion. Mutation of flagella synthesis in the sinI strain restored invasion efficiency to wild type levels. Therefore, control of motility and chemotaxis by the ExpR/Sin quorum sensing system plays an important role in plant invasion and may provide a competitive edge for strains possessing it.

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BLAST X Poster #21 CHARACTERIZATION OF EtgA, A MURAMIDASE ASSOCIATED WITH THE TYPE III SECRETION SYSTEM OF ENTEROPATHOGENIC ESCHERICHIA COLI Elizabeth García-Gómez, Norma Espinosa, Bertha González-Pedrajo Departamento de Genética Molecular, Instituto de Fisiología Celular, Universidad Nacional Autónoma de México. Ap. Postal 70-243. Ciudad Universitaria, México D. F., 04510. Tel. (5255) 56-22-59-65 egarcia@ifc.unam.mx, bpedrajo@ifc.unam.mx

Enteropathogenic E. coli (EPEC) is a human pathogen that produces severe diarrhea and death among children from developing countries. EPEC infection is characterized by the formation of an attaching and effacing histopathology (A/E lesion) that consists of gut epithelial microvilli destruction, intimate adherence of the bacterium to the enterocyte and the development of an actin-rich pedestal-like structure beneath the adherent non-invasive bacteria.

EPEC utilizes a type three secretion system (T3SS) to translocate effector proteins

directly from the bacterial cytoplasm into the host cell cytosol, subverting diverse enterocytic cell signaling pathways and producing drastic cytoskeletal reorganization. EPEC genes required for the assembly of the T3SS and A/E lesion development are contained on a 35.6 kb pathogenicity island known as the locus of enterocyte effacement (LEE).

The T3SS or injectisome is a macromolecular protein complex that needs to span the

periplasmic space for its assembly; however, the peptidoglycan cell wall constitutes a barrier that allows the free passage of only small proteins. To overcome this obstacle, lytic transglycosylases (LTs) have been identified as specialized enzymes associated with different transport systems. It has been proposed that during T3SS biogenesis a LT facilitates a temporally and spatially controlled opening of the peptidoglycan layer.

In this study, we have identified and characterized a LEE open reading frame with

unknown function, rorf3 (renamed etgA), which encodes a protein with a lytic transglycosylase domain and a signal sequence. A truncated version of EtgA lacking its signal sequence (EtgAns) was purified by nickel chromatography as an N-terminal His-tagged recombinant protein. Lytic activity was determined by zymograms. An invariantly conserved glutamate residue at position 42 was replaced by alanine through site-directed mutagenesis, and its enzymatic activity was evaluated. The effect of over-producing EtgA, EtgAns and EtgAE42A over EPEC growth and secretion was evaluated. Furthermore, the subcellular localization of the protein was determined. Additionally, an etgA null mutant strain was generated to evaluate the role of EtgA in T3SS assembly. Our results show that EtgA is a T3SS associated muralytic enzyme. The highly conserved glutamate residue (catalytic residue) is essential for EtgA function. EPEC growth rate was affected when EtgA was overproduced, but not with EtgAns or EtgAE42A. Our data demonstrate that the protein is secreted by the Sec pathway and that it disrupts the peptidoglycan layer, causing bacterial lysis. Periplasmic localization of EtgAns was determined. Finally, we show that EtgA is essential for efficient protein secretion.

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BLAST X Poster #22 flhE: A PERIPLASMIC CHAPERONE OF FLAGELLIN? Jae-Min Lee* and Rasika Harshey Section of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712

flhE is the last gene in the flhBAE flagellar operon whose first two members encode

components of the Type III secretion pathway in Salmonella typhimurium. The role of flhE is still a mystery. Absence of flhE does not affect swimming motility, but reduces swarming motility. In this study, we show that FlhE is a periplasmic protein, and have localized it within the flagellar basal body. We have found a new ‘green’ phenotype associated with a flhE deletion mutant, which we are using as a tool to understand flhE function. The ‘green’ phenotype refers to green colony color when bacteria are plated on aniline blue-alizarin yellow pH indicator plates. The green color should be indicative of a lowered pH. However, independent pH measurements failed to confirm pH differences between wild-type and flhE strains. Curiously, mutations that prevented assembly but not secretion of flagellar filament subunits (flgK, fliD), eliminated the green color. Immunoblotting and immunostaining assays showed that there was more ‘free’ flagellin in flhE mutant supernatants than in wild-type bacteria isolated from swarm plates. Based on these and other experiments, we hypothesize that FlhE is a periplasmic chaperone for the flagellar filament subunits. We propose that improperly folded filaments interact with dyes on the pH indicator plates giving rise to the ‘green’ phenotype, and are more easily broken when subjected to higher viscous forces on the flagellar filament during swarming motility.

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BLAST X Poster #23 THE Cyclic-di-GMP RECEPTOR PROTEIN YcgR LOCALIZES TO THE FLAGELLAR BASAL BODY AND CHANGES MOTOR BIAS IN SALMONELLA Vincent Nieto* and Rasika Harshey Section of Molecular Genetics and Microbiology, University of Texas at Austin, Texas 78712 Cyclic-di-GMP (cdG) is a bacterial second messenger that plays a central role in the transition between motile and sessile states. In Salmonella enterica, absence of the cdG phosphodiesterase YhjH is known to elevate cdG levels, inhibit motility, and promote biofilm formation. YcgR, a cdG -binding protein, is required for this phenomenon. We show in this study that the absence of YhjH inhibits chemotaxis, and that this inhibition is more pronounced when YcgR is overexpressed from a plasmid. In liquid media, the bacteria were predominantly smooth swimming. Tethering experiments showed a pronounced shift to CCW rotation with a significant slowing of motor rotation. Inhibition of chemotaxis was therefore at the level of either production, or activity of the chemotaxis response regulator CheY~P, which acts at the switch to change the default CCW bias to CW. Expression of a GFP-tagged cdG-binding protein YcgR in a yhjH mutant background, resulted in punctate fluorescent dots along the cell membrane. Presence of the puncta was dependent on the presence of flagellar basal bodies, but not on presence of chemotaxis signaling components, suggesting that YcgR inhibits chemotaxis by acting at the switch. Inhibition of chemotaxis may represent a novel strategy to prepare for a sedentary existence by disfavoring migration away from a substrate on which a biofilm is to be formed.

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BLAST X Poster #24 ANALYSIS OF THE PEPTIDOGLYCAN-BINDING DOMAIN OF THE FLAGELLAR STATOR PROTEIN MotB USING SYSTEMATIC MUTAGENESIS AND CHIMERIC PROTEIN IN ESCHERICHIA COLI Yohei Hizukuri1, John Frederick Morton1, Toshiharu Yakushi1, 2, Seiji Kojima1 and Michio Homma1

1Division of Biological Science, Graduate School of Science, Nagoya University, Furo-Cho, Chikusa-Ku, Nagoya 464-8602, Japan 2Present address: Applied Molecular Bioscience, Graduate School of Medicine, Yamaguchi University, Yamaguchi 753-8515, Japan The bacterial flagellar stator proteins, MotA and MotB, form a complex and are thought to be anchored to the peptidoglycan (PG) layer by the C-terminal conserved peptidoglycan-binding (PGB) motif of MotB. To clarify the role of the C-terminal PGB region of MotB, we performed systematic cysteine mutagenesis of this region in the Escherichia coli MotB. Furthermore, we constructed three chimeric MotB proteins whose PGB regions were replaced with corresponding regions of other PGB proteins, E. coli Pal (peptidoglycan-associated lipoprotein), PomB (Vibrio MotB homolog) or MotY (Vibrio flagellar T-ring protein). Although these chimeric proteins did not complement the motB mutant, we were able to isolate two independent motile revertants from cells producing the MotB-Pal chimera. One is the F172V mutation in the Pal region of the chimera, and this mutation did not affect the ability to bind to PG when introduced into native Pal. The other is the P159L mutation in the MotB region, and Pro159 mutation in native MotB has been reported to affect a spatial positioning of the MotA/MotB stator complex relative to the motor switch complex. We speculated that the PGB region of MotB-Pal chimera was properly aligned by the mutations and the stator and rotor could interact properly. We tried to interpret phenotype of MotB Cys mutants by using the crystal structure of the E. coli Pal, and found that MotB mutants that affected motility were nearly overlapped with the predicted PG-binding residues of Pal. Our results indicate that the PGB regions from functionally distinct proteins, MotB and Pal, works similarly to anchor the stator complex.

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BLAST X Poster #25 ATTEMPT TO PURIFY THE HOOK-BASAL BODY WITH C-RING FROM THE Na+-DRIVEN FLAGELLAR MOTOR Masafumi Koike, Seiji Kojima, Michio Homma Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan. The flagellum is the biggest architecture in the bacterial organ. At the base of each flagellum, there is a rotary motor embedded in the membrane whose energy source is the electrochemical gradient of the specific ion across the membrane. E. coli and Salmonella have the H+ driven motor, and Vibrio alginolyticus has Na+ driven type. The motor is composed of the rotor and the stator, and torque is generated by the interactions between them. The hook-basal body (HBB) is the rotor part of the motor composed of rod, LP-, MS-ring and hook. C-ring is mounted on the cytoplasmic side of the MS-ring of HBB, and believed to be involved in torque generation in rotor side of the motor. It is composed of three proteins; FliM, FliN, and FliG. FliG is the most closely participated in the torque generation among them. To elucidate the mechanism of torque generation, we undertake the isolation of C-ring from the Na+-driven polar flagellum of V. alginolyticus to investigate rotor-stator interactions. We applied the C-ring isolation method established for Salmonella, but that resulted in the HBB without C-ring. It indicates that solubilization by Triton X-100 was harsh for C-ring of Vibrio. Suitable detergents were screened and we found that CHAPS could solubilize HBB with FliG attached. Currently immunoelectron microscopic observation is ongoing to directly detect FliG attached on the MS-ring. Also, we are searching more favorable conditions to isolate remaining C-ring proteins, FliM and FliN.

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BLAST X Poster #26 MECHANISM FOR SORTASE LOCALIZATION AND ROLE IN EFFICIENT PILUS ASSEMBLY IN ENTEROCOCCUS FAECALIS Kimberly A. Kline*† Andrew L. Kau*, Swaine L. Chen*, Birgitta Henriques-Normark†, Staffan Normark†, Michael G. Caparon* and Scott J. Hultgren* *Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110; †Department of Microbiology,Tumor and Cell Biology, Karolinska Institutet, Solna, Sweden Pathogenic streptococci and enterococci primarily rely on the conserved secretory (Sec) pathway for the translocation and secretion of virulence factors out of the cell. Since many of these secreted Gram positive virulence factors are subsequently attached to the bacterial cell surface via sortase enzymes, we sought to investigate the spatial relationship between secretion and cell wall attachment in Enterococcus faecalis. We discovered that Sortase A (SrtA) and Sortase C (SrtC) are co-localized with SecA at single foci in enterococcus. The SrtA-processed substrate aggregation substance accumulated in single foci implying a single site of secretion for these proteins. Furthermore, we show by electron and immunofluorescent microscopy and immunoblot analysis that in the absence of the pilus polymerizing SrtC, pilin subunits also accumulate in single foci. Proteins that localized to single foci in E. faecalis were found to share a positively charged domain flanking a transmembrane helix. Mutation or deletion of this domain in SrtC abolished both its retention at single foci and its function in efficient pilus assembly. We conclude that a positively charged domain adjacent to a transmembrane helix can act as a localization retention signal for the focal compartmentalization of membrane proteins. Finally, we have examined the localization of the secretion and sorting machinery and substrates throughout the bacterial cell cycle and present a model for spatial and temporal organization of the molecular components leading to efficient pilus biogenesis in E. faecalis.

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BLAST X Poster #27 VISUALIZATION OF EXCHANGE OF ROTOR COMPONENT IN FUNCTIONING BACTERIAL FLAGELLAR MOTOR Hajime Fukuoka, Shun Terasawa, Yuichi Inoue, Akihiko Ishijima IMRAM, Tohoku Univ., 2-1-1 Katahira, Aoba-ku, Sendai, 980-5877, Japan

Bacterial flagellum is a supramolecular complex penetrating the bacterial cell envelope, including the cytoplasmic and the outer membranes. Bacterial flagellum consists of a basal body (rotary motor), a helical filament (propeller), and a hook (universal joint). The flagellar motor is driven by the electrochemical potential of H+

or Na+, and the interaction between stator and a

rotor of flagellar motor is thought to generate the motor rotation. Stator parts are exchanged in a functioning motor and the assembly of stator to the motor requires coupling ion for the motor rotation. In this study, we focused on protein dynamics of rotor in functioning bacterial flagellar motors. We constructed GFP-fusions of rotor components, and investigated whether rotor components are exchanged in a functioning motor by Fluorescent Recovery After Photobleaching (FRAP) experiment for a single motor.

We constructed the expression systems of GFP-FliG, FliM-GFP, and GFP-FliN as GFP-

fusions of rotor components. In tethered cells producing each GFP-fusions, we observed the localization of fluorescent spot at the rotational center. Each GFP fusion was probably incorporated into flagellar motor as a rotor component. To observe the exchange of rotor components, we carried out FRAP experiment using evanescent illumination to the motor located at rotational center of the tethered cell. When fluorescent spot of FliM-GFP or GFP-FliN localized at rotational center was photobleached, the fluorescence at the rotational center recovered with the passage of time. On the other hand, the recovery of fluorescence was not observed in cells producing GFP-FliG. These results indicate that some rotor components, FliN and FliM at least, assemble to the motor even after the functional motor is constructed. Probably, in functioning motor, some components of flagellar structure are exchanged dynamically. We would like to clarify turnover rates of rotor components until the annual meeting.

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BLAST X Poster #28 TORQUE RESPONSE OF THE SODIUM-DRIVEN CHIMERIC FLAGELLAR MOTOR IN E.COLI INDUCED BY REVERSIBLE TEMPERATURE CHANGE 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, Katahira 2-1-1, Aoba-ku, Sendai 980-8577, Japan Mechanical step of motor proteins is a key to understand the molecular mechanism of energy transduction from chemical energy into mechanical work. We have demonstrated the 26 steps in a rotation of Na+-driven chimeric flagellar motor (Sowa et al., 2005) and the torque-speed relationship (Inoue et al., 2008) using back-focal-plane interferometry. However, present time resolution is not enough to understand molecular mechanism of the steps of motor proteins including not only flagellar motors but also linear motors as skeletal myosin. One possible option would be to slow down the temperature-dependent processes in a chemo-mechanical cycle by reducing temperature. We report the cooling experiment with a water-cooling chip in a simple and reversible way. A small chip of ~18mm*18mm*2mm was made with glass cover slides and plastic tubes to lead cooling water. This chip was made direct contact on a sample chamber and placed in the light axis of the microscope. By changing temperature of the cooling flow, sample temperature from 23 to -8 degree Celsius was monitored using thermocouple. Temperature change could be applied repeatedly in a time constant < 60 sec. Simultaneous measurement of the motor speed and sample temperature showed the speed change as reported for low temperature (Chen and Berg, 2000). With increasing temperature with hot water, however, sudden drop of speed was measured over ~40 deg C. When the temperature returned back to room temperature, the speed was restored mostly in several minutes. The drop and recovery of the speed were coincided with stepwise change in the generated torque. These results suggest that our cooling system is useful not only for the cooling experiment for improving time resolution but also for the heating experiment to understand heat resistance which might be related to stator dynamics.

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BLAST X Poster #29 THE HELICOBACTER PYLORI FLAGELLAR ANTI-SIGMA FACTOR FlgM REMAINS BACTERIA-ASSOCIATED AND INTERACTS WITH FlhAC Melanie Rust1, Sophie Borchert1, Eugenia Gripp1, Sarah Kühne1, Eike Niehus1, Sebastian Suerbaum1, Kelly T. Hughes2, Christine Josenhans1 1Department for Med. Microbiology and Hospital Epidemiology, Hannover Medical School ,Carl-Neuberg-Strasse 1, D-30625 Hannover, Germany, 2Department of Biology, University of Utah, USA

Helicobacter pylori persistently colonize in the human gastric mucus with the help of their polar flagellar organelles. A particular feature of flagella in most Helicobacter species including H. pylori is the presence of a membraneous flagellar sheath. Previously, flagellar regulators FliA, RpoN and the functional master regulator FlhA were characterized. H. pylori also possesses an anti-sigma factor FlgM, which has an unusually short N-terminus, yet is functional in Salmonella. FlgM in H. pylori fulfills a similar conserved function as an antagonist and binding partner to FliA. However, FlgM of H. pylori is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g. FlgM of Salmonella, whose function is intimately coupled to its secretion through the flagellar type III secretion system.

The aim of the present study was to characterize in more detail the localization and

potential for secretion of the short H. pylori FlgM in the presence of a flagellar sheath. Furthermore we investigated its interaction with other flagellar proteins in the basal body, which may be required for its function in flagellar regulation. FlgM was expressed in flhA mutants and was differentially localized in bacterial fractions of flhA mutant bacteria in comparison to wild type bacteria. H. pylori FlgM was only released into the medium in very minor amounts in wild type bacteria, where the bulk amount of the protein was retained in the cytoplasm, and some FlgM was detected in the flagellar fraction. FlgM-GFP (green fluorescent protein) and FlgM-V5 translational fusions were generated and expressed in H. pylori. FlgM displayed a predominantly polar distribution in microscopy. Evidence was gathered that it is able to interact with the C-terminal domain of the flagellar basal body protein FlhA. We conclude that in H. pylori and possibly in other closely related bacteria, which also possess a truncated FlgM, secretion may not be paramount for the regulatory function of FlgM and that protein interactions at the flagellar basal body, in particular with FlhAC, may determine the turnover and localization of functional FlgM in H. pylori. We gratefully acknowledge the German Research Council, grant Jo344/2-2, for financial support.

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BLAST X Poster #30 LOCALIZATION PATTERNS OF THE HISTIDINE KINASES IN AN ESCHERICHIA COLI CELL Takehiko Inaba1, Satomi Banno2, Hiroyuki Sawaki2, Akiko Yamakawa2, Masayuki Yoshimoto3 Michio Homma3 and Ikuro Kawagishi1,4 1: Res. Cen. Micro-Nano Tech., Hosei Univ.; 2: Natl. Inst. Infect. Dis.; 3: Div. of Biol. Science, Nagoya Univ., 4: Dept. Frontier Biosci., Hosei Univ. E.coli has 30 histidine kinases (HKs). In response to a specific environmental stimulus, each HK controls the activity of the downstream response regulator (RR) by modulating its activities of autophosphorylation on the specific His residue and transfer of the phosphoryl group to the specific Asp residue of RR (Generally referred to as the "two-component" regulatory system or the His-Asp phosphorelay system). The activated form of RR regulates specific cell functions (in most cases, gene expression). Almost all HKs resemble MCPs (methyl-accepting chemotaxis proteins) in that they have two transmembrane regions and form homodimers. CheA and NtrB are cytosolic proteins. CheA forms a chemosensory complex with the trasmembrane chemoreceptors (MCPs) and the adaptor CheW. This complex localizes to the cell pole and forms a huge cluster, which plays a critical role in signal amplification. Do the other HKs also localize to particular regions of the membrane? To answer this question, we constructed HK-GFP fusion proteins and observed the localization via fluorescence microscopy. As mentioned above, cytosolic CheA-GFP localized to the cell pole, whereas the other cytosolic HK NtrB-GFP was diffused throughout the cytoplasm. Although many of the transmembrane HK-GFPs were diffused evenly in the cytoplasmic membrane, some HK-GFPs showed characteristic localization patterns. In particular, the GFP fusions to the anaerobic sensors TorS and ArcB localized to the cell pole. Most of HKs that localized to the pole are of hybrid type, i.e. they have receiver and HPt domains, which are involved in multi-step phophorelay. A series of deletion from TorS-GFP revealed that none of these domains nor the transmitter domain was required for polar localization. Nevertheless, polar localization of such hybrid HKs might provide a basis of signal integration and crosstalk.

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BLAST X Poster #31 THERMOSENSING FUNCTION OF Aer, A REDOX SENSOR OF E. COLI So-ichiro Nishiyama1, Shinji Ohno2, Noriko Ohta3, Akihiko Ishijima4 and Ikuro Kawagishi1, 5 1Department of Frontier Bioscience, Faculty of Bioscience and Applied Chemistry, Hosei University

2Department of Material Chemistry, Faculty of Engineering, Hosei University 3Division of Biological Science, Graduate School of Science, Nagoya University 4Institute of Multidisciplinary, Research for Advanced Materials, Tohoku University 5Department of Frontier Bioscience, Faculty of Engineering, Hosei University

Some motile bacteria can sense temperature and move to temperatures best-suited to growth. This behavior, called thermotaxis, has been extensively studied in Escherichia coli. Our early studies revealed that E. coli thermotaxis is mediated by chemoreceptors that also sense amino acids or sugars: Tsr (serine), Tar (aspartate and maltose) and Trg (ribose and galactose) function as warm sensors, producing counter-clockwise or clockwise flagellar rotation signals upon temperature upshift and downshift, respectively. Tap (dipeptides, pyrimidines) functions as a cold sensor, producing CW and CCW signals upon temperature increases and decreases, respectively. Unique among these temperature sensors, Tar switches from a warm sensor to a cold sensor after adaptation to its ligand, aspartate. Intensive studies of Tar revealed that the receptor’s transmembrane and methylation domains play important roles in thermotactic responses, but what part of the chemoreceptor molecule actually senses temperature, remains unknown.

In this study, we found that the aerotaxis transducer Aer also has temperature-sensing

ability. An otherwise receptor-less strain expressing only aer showed extremely smooth swimming and did not show any thermoresponse. However, after imposing a CW rotational bias by adding the general repellent, glycerol (up to 10% w/v) or by co-expressing a cytoplasmic fragment of Tar that does not mediate a thermoresponse, the cells showed CCW and CW responses to temperature upshifts and downshifts, respectively. These results suggest that Aer functions as a warm sensor, even though, unlike the Tar, Tap, Tsr, and Trg chemoreceptors, Aer does not have a periplasmic ligand-binding domain. Thus, temperature sensing by E. coli chemoreceptors may be a general attribute of their highly-conserved cytoplasmic signaling domain (or their less conserved transmembrane domain).

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BLAST X Poster #32 ATPase ACTIVITY OF T3SS SPECIFIC ATPase InvC Fumio Hayashi, Eri Inobe, Kenji Oosawa Department of Chemistry and Chemical Biology, Graduate school of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu, Gunma, 376-8515, Japan

The type III secretion systems (T3SSs) are widely used by Gram-nagative bacteria, and there are essential systems of many bacterial pathogenic to humans, animals, and plants. The systems are anchored to the bacterial envelope by a multi-ring, and a needle-like extracellular structure facilitates the translocation of the virulence proteins (effectors) to a host cell from the bacterial cytosol. An ATPase that is believed to be in close association with the basal body is involved in the protein translocation.

The specific ATPase of T3SSs in Salmonella enterica serovar Typhimurium is InvC. We

purified InvC, determined the low kcat value of InvC-ATPase activity and the high Km value for ATP, and found 2~3-fold stimulation of InvC-ATPase activity in the presence of phospholipid. To examine the possibility that InvC-ATPase activity is further stimulated by the interaction with an effector or an effecter-specific chaperon, we purified SicP (chaperon) and SptP (effector) and measured InvC-ATPase activity in the presence of SicP or SptP. SicP- or SptP-dependent stimulation of InvC-ATPase activity was not detected yet.

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BLAST X Poster #33 CHARACTERIZATIONS OF THE PSEUDOREVERTANTS FROM SALMONELLA TYPHIMURIUM STRAIN SJW1655 AND SJW1660 WITH THE R- AND THE L-TYPE STRAIGHT FLAGELLAR FILAMENTS Hidetoshi Tomaru, Fumio Hayashi, Eiji Furukawa, Shigeru Yamaguchi & Kenji Oosawa Department of Chemistry and Chemical Biology, Graduate School of Engineering, Gunma University, 1-5-1 Tenjin, Kiryu, Gunma 376-8515, JAPAN A cell of Salmonella typhimurium swims by rotating its flagellar filaments. A wild-type cell carries a left-handed helical filament that is called a normal filament. On the other hand, various mutants carrying different helical shapes were isolated. There are curly, coiled, semi-coiled and two kinds, L- and R-types, of straight filaments known. Transformations of filament helical shape among normal, semi-coiled and curly were observed during tumbling of the cell. To investigate the transformation mechanism of flagellar filaments, we isolated 95 revertants, which recovered their swarming abilities, from Salmonella typhimurium strain SJW1655 with the R-type straight flagellar filament. The numbers of the pseudorevertants carrying another mutation site were 64 and the second mutation sites were 4. Similarly, we isolated 106 revertants (including 101 pseudorevertants) from SJW1660 (the L-type straight flagellar filament). The numbers of the second mutation sites were 19. We also measured the swimming speeds of the pseudorevertants and observed the flagellar shapes isolated from the pseudorevertants. We will discuss, in this meeting, the differences in the distribution of the second mutation sites, the swimming speeds, and the flagellar shapes of the pseudorevertants from between SJW1655 and SJW1660.

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BLAST X Poster #34 RAMAN OPTICAL ACTIVITY AND VIBRATIONAL CIRCULAR DICHROISM OF FLAGELLAR FILAMENTS OF SALMONELLA Tomonori Uchiyama1, Fumio Hayashi1, Masashi Sonoyama2, Yoshiaki Hamada3, Rina K. Dukor4, Laurence A. Nafie4, 5, Kenji Oosawa1(kenji@nms.gunma-u.ac.jp) 1Department of Nano-Material Systems, Gunma University, Kiryu, Japan 2Department of Applied Physics, Nagoya University, Nagoya, Japan 3The Open University of Japan, Chiba, Japan 4BioTools Inc., Jupiter, FL 33458, USA 5Department of Chemistry, Syracuse University, Syracuse, NY 13244-4100, USA

The flagellar filament of Salmonella is an assembly of a single protein, flagellin. There are different helical and straight shape filaments from wild type and mutants. The difference between two types of straight filaments, L- and R-type straight, is the inclination of the flagellin monomer arrangements. The structures of the straight filaments were analyzed by X-ray diffraction and electron microscope, while it is difficult to analyze the structure of helical flagellar filaments by these methods, due to deficiency in the symmetric property of the molecular assembly. On the other hand, Raman optical activity (ROA) and vibrational circular dichroism (VCD) spectroscopies are available new techniques for studying structure and dynamics of chiral molecules and the solution structure of biomacromolecules. In the present study, these methods are employed for investigating the structural characteristics of bacterial flagellar filaments.

In the ROA spectra, intensive peaks were observed only from the L-type filaments, while

no significant signals were observed from flagellin monomer and other shapes of the filaments (R-type straight and normal helical) in our measurement conditions. The intensive signals disappeared or were weakened when the L-type straight filaments were shortened.

In the VCD spectra, intensive peaks in the amide I region were observed from the L-type

filaments. Whereas peaks observed from the R-type filaments were different from those from L-type and only weak peaks were observed in the normal filaments and flagellin monomer. From these results, it is thought that these spectra reflect the differences of structure and the physicochemical properties of flagellin subunits in three types of filaments.

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BLAST X Poster #35

Poster Cancelled

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BLAST X Poster #36 CrdC NEGATIVELY REGULATES CheW3 AND CheA3 INTERACTION DURING SIGNAL TRANSDUCTION IN MYXOCOCCUS XANTHUS Jonathan Willett, Susanne Mueller, John Kirby University of Iowa, Department of Microbiology, 3-403 Bowen Science Building 51 Newton Road, Iowa City, IA 52242 Previous work on the Che3 system of Myxococcus xanthus has led to the discovery that a chemosensory signal-transduction system affects developmentally regulated gene expression. The Che3 system contains homologs of the prototypical chemotaxis proteins such as CheA, CheW, CheB, CheR, and MCPs but lacks a CheY homolog. The output of the system consists of a NtrC transcriptional activator termed CrdA. There are several other unique proteins comprising the Che3 pathway besides CrdA, with one of the more interesting being a protein termed CrdC. CrdC is contained in the same transcriptional unit as CheW3. Preliminary data has shown that CrdC interacts strongly CheW3 in yeast-two hybrid experiments. More interestingly, the presence of CrdC in a yeast-three hybrid experiment has been shown to inhibit the interaction between CheA3 and CheW3. We hypothesize that CrdC thereby presents a unique mechanism for regulation of signal transduction through the Che3 pathway by decoupling CheA3 and CheW3.

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BLAST X Poster #37 MOLECULAR ARCHITECTURE OF INTACT FLAGELLAR MOTOR REVEALED BY CRYO-ELECTRON TOMOGRAPHY Jun Liu1, Tao Lin1, Douglas J. Botkin1, Erin McCrum1, Hanspeter Winkler2, Steven J. Norris1 1Department of Pathology and Laboratory Medicine, University of Texas Medical School at Houston, Houston, TX 77225-0708, USA. 2Institute of Molecular Biophysics, Florida State University, Tallahassee, FL, 32306-4380, USA Motility is often important for virulence of bacterial pathogens, and the flagellum is the main organelle for motility in bacteria. Bacterial flagella are helical propellers turned by the flagellar motor, a remarkable nano-machine embedded in the bacterial cell envelope. Powered by the proton gradient across the cytoplasmic membrane, the motor converts electrochemical energy into torque through an interaction between a rotating, cylindrical basal body at the end of the flagellar filament and the stator, a surrounding protein assembly embedded in the cytoplasmic membrane. Of the 50 genes needed to build a functional flagellum, at least 25 produce proteins essential for flagellar assembly. Although structural studies have revealed the stunning complexity of the basal body, flagellar assembly and rotation remain poorly understood at the molecular level, mainly because of the lack of structural information about the membrane-bound stators and the torque-generating mechanism in particular. Here, we present the structures of infectious wild-type and mutant Borrelia burgdoferi organisms and their flagella motors in situ using high throughput Cryo-Electron Tomography (Cryo-ET). By averaging the 3-D images of ~1280 flagellar motors, we obtained a ~3 nm resolution model of the combined stator and rotor structure in its cellular environment. We have also been able to identify distinctive structural changes resulting from the mutation of a flagellar gene. This is direct mapping of a single genetic code change into the 3-D structure of a functioning molecular machine in situ. Our results provide new insights into the motor structure and the molecular basis for bacterial motility.

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BLAST X Poster #38 MINING THE E. COLI GFP FUSION COLLECTION Jason Dobkowski, Aleksandra Sikora, John Brooks, Richard Zielke and Janine Maddock Department of MCDB, University of Michigan, 830 N. University, Ann Arbor, MI 48109 Use of green fluorescent protein (GFP) has greatly increased the ability to visualize protein localization within bacterial cells. A library of GFP fusions, expressed from an inducible promoter, was created with each protein from every open reading frame in the Escherichia coli genome (Kitagawa et al, 2005). The addition of the 26.9 kDa GFP, however, can lead to protein misfolding and aggregation and the formation of polar inclusion bodies. These polar inclusion bodies are often mistaken for bona fide polar localization of the protein fusion. Using a purification and screening approach, we have sorted polarly localized GFP fusions into those that form inclusion bodies and those that are likely real polar proteins. Those that form inclusion bodies are being used to identify interacting partners that co-aggregate with the misfolded protein. Proteins not previously not known to be polar are being verified using immunofluorescence microscopy and the localization of known interacting partners determined. We are particularly interested in the spatial organization of membrane bound histidine kinases involved in sensing environmental stress. Our current line of focus is to determine whether the activation of the signal transduction cascade has any influence of their polar localization.

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BLAST X Poster #39 UNDERSTANDING THE FUNDAMENTAL ELEMENTS OF SIGNALING IN THE Tar CHEMORECEPTOR Christopher Adase, Michael Manson Texas A&M University, 3258 TAMU, BSBE Room 303 The Tar chemoreceptor of Escherichia coli has two different attractants: L-aspartate, which binds directly to the receptor; and maltose, which interacts with the receptor indirectly though maltose-binding protein (MBP). Tar also senses Ni2+ and Co2+ as repellents. The Tsr chemoreceptor interacts directly with L-serine as an attractant and also senses repellents such as indole, acetate, and benzoate in an unknown manner. Tar-Tsr and Tsr-Tar chimeras have been created using the endogenous Nde1 site found at the end of the region encoding their respective HAMP domains. The Tar-Tsr hybrid could sense Ni2+ as a repellent, whereas the Tsr-Tar hybrid could not. Thus, Ni2+ probably interacts, directly or indirectly, with an area within the first 256 amino acids of Tar. Using knockouts of nikA, nikB, and nikC, we tested whether periplasmic NikA is necessary and sufficient for Ni2+ taxis, as has been reported, and whether Ni2+ must be taken up into the cytoplasm. I have also created additional Tar-Tsr chimeras to determine exactly what portion of Tar is involved in Ni2+ sensing. Repellent taxis was assayed using novel microfluidic techniques developed by the Jayaraman lab in the Department of Chemical Engineering at Texas A&M.

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BLAST X Poster #40 LINKING THE TM2 TO HAMP—A TOUGH NUT TO CRACK? Rachel L. Crowder, Gus A. Wright, and Michael D. Manson Department of Biology, Texas A&M University, College Station, TX 77843 The HAMP domain is a widely conserved motif found in transmembrane-signaling proteins in prokaryotes and lower eukaryotes. It consists of a pair of amphipathic helices joined by a flexible linker. Recently, the solution structure of the Archeoglobus fulgidis Af1503 HAMP domain was determined using NMR (Hulko et al, Cell 126: 929-940, 2006). The domain forms a parallel four-helix bundle that packs in a non-canonical knob-on-knob conformation. Several models have been proposed to explain how the four helix bundle transmits the downward piston movement of transmembrane 2 (TM2) of E. coli chemoreceptors into the signaling domain to inhibit CheA kinase activity. The connection between TM2 and the HAMP domain is likely to be important for transducing the input signal from TM2 to HAMP. We hypothesized that increasing the flexibility of the connector should attenuate the output signal. To test this idea, residues between Met-215 Thr-218 of the E. coli aspartate receptor Tar were replaced with four Gly residues. Gly residues were then deleted (-4G through -1G) and added (+1G through +5G). Aspartate chemotaxis, rotational bias of tethered cells, mean reversal frequencies of tethered cells, and in vivo methylation levels were measured. These experiments suggest that increasing flexibility between TM2 and HAMP strongly biases the receptor to the “off” (CCW-signaling) state and decreases aspartate mediated signal transmission between TM2 and HAMP.

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BLAST X Poster #41 ELECTROSTATIC EFFECTS ON SIGNALING MUTATIONS IN THE C-TERMINAL REGION OF THE ESCHERICHIA COLI ASPARTATE CHEMORECEPTOR Andrew L. Seely, Run-Zhi Lai, and Michael D. Manson, Department of Biology, Texas A&M University, College Station, TX 77843

The Escherichia coli aspartate chemoreceptor (Tar) responds to attractant by modulating the rotational bias of the flagellar motor. Previous studies measured the effects on Tar signaling when positive residues in its extreme C-terminal region were either neutralized or changed to negative residues. While this region is important as a linker to the NWETF pentapeptide where methylation and demethylation enzymes bind, it also affects transmembrane signaling. An R505A substitution decreased the receptor’s ability to respond to aspartate by 40%, and an R505E charge reversal completely abolished stimulation by aspartate. It was also noted that R505E may interact with D273 to destabilize the “on” signaling state (Lai, et al, Advanced Publication in Biochemistry, 2008). We hypothesize that secondary mutations within the background of the R505 mutations, and an additional R514A/E mutant, can potentially rescue or exacerbate the ability of mutated Tar to respond to aspartate. Specific mutations will include D273N/R, D263R, R228A/E, R505D and R509E. We will test our hypothesis in vivo through chemotaxis swarm assays, receptor methylation assays, and tethered cell assays. Our hypothesis will also be tested in vitro through CheA-Kinase assays.

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BLAST X Poster #42 INTERACTION OF THE TRANSCRIPTIONAL REGULATORY COMPLEX, FlhDC, WITH ITS TARGET DNA Yi-Ying Lee, and Philip Matsumura Department of Microbiology and Immunology, College of Medicine, University of Illinois at Chicago, 835 S. Wolcott Ave., M/ C 790, Chicago, Illinois 60612-7344 The bacterial flagellum is the structure that allows bacteria to move and respond to nutritional and chemical signals in their environment. It is a complex suborganelle and the transcriptional regulation of the 40 plus structural genes is organized in a highly regulated cascade. At the top of the hierarchy is the master operon which codes for FlhD and FlhC. These two positive transcriptional regulators form a unique heteroheximeric complex which binds upstream of the -35 region and requires sigma 70 for transcription. This complex has an unusually large ‘footprint’ of 48 base pair and bends the DNA 110 degrees. We have proposed that the DNA bind on the circumference of this toroid shaped FlhDC complex. Although we have determined the sequence 3 footprints on FlhDC regulated promoters, it is not possible to determine a consensus binding site in these 3 sequences. In this study, we have determined which bases are important for DNA binding and activity for FlhDC regulated promoter activity. First, we have divided the FlhDC footprint in the fliA promoter into five segments and found that two of the segments or 40% of the footprint were not required for binding. The remaining 30 base pairs were divided into 3-5 base segments and randomly mutagenized and screened for the ability to bind and activate the fliA promoter. Analysis of these data suggests a consensus of 12A, 15A, 34T, 36A, 37T, 44A, 45T in FlhD4C2 footprint fragment were important for activity. Five of these bases demonstrated high specificity. Finally, this consensus was tested and found to be important in other FlhDC regulated promoter regions.

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BLAST X Poster #43 APPLICATION OF BIOLAYER INTERFEROMETRY TO UNDERSTANDING INTERACTIONS AMONG SALMONELLA ENTERICA FLAGELLAR EXPORT APPARATUS PROTEINS Jonathan L. McMurry Department of Chemistry & Biochemistry, Kennesaw State University, Kennesaw, GA Biolayer interferometry (BLI) is an emerging optical biosensing technology for the analysis of dynamic biomolecular interactions. Measurements are made of changes in the interference pattern of white light reflected off of two surfaces, one of which possesses a layer of immobilized protein (or other molecule) and the other an internal reference. Binding of second protein to the immobilized one results in a change in distance between the two surfaces and thus a shift in wavelength of the interference pattern. Kinetic and affinity constants can thus be determined in real-time, without attainment of equilibrium, utilizing small, recoverable quantities of label-free biomolecules. Using a commercial BLI biosensor, interactions among proteins involved in flagellar export in Salmonella enterica were investigated. Among interactions measured were those between two proteins involved in specificity switching, FliK and FlhB. FliK binding to wild-type FlhB and two variants (N269A and P270A) surprisingly evidenced similar kinetic profiles with submicromolar KDs resulting from fast-on and fast-off rate constants. Other interactions between export proteins, e.g., FliH-FliI, FliH-FliJ, demonstrated stable, low kd binding, as expected. Additional efforts include screening for interactions too weak to allow for copurification or other traditional binding assays, attempts to demonstrate interactions between export proteins and substrates and analysis of more-than-pairwise interactions. Further application of BLI to the investigation of flagellar export protein dynamics will allow for better understanding of the mechanism of export.

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BLAST X Poster #44 A CROSS-SPECIES COMPARISION OF CHEMOTACTIC BEHAVIOR Julie Simons and Paul Milewski University of Wisconsin—Madison, Department of Mathematics, 480 Lincoln Dr., Madison, WI 53706 Understanding the population-level behavior of bacteria is of importance not only in exploring how simple organisms can perform complex behavior, but also to be able to optimize their potential for bioremediation and other uses. We are interested in modeling the chemotactic behavior of Rhodobacter sphaeroides and the better-understood Escherichia coli using a partial differential equation model known as the Keller-Segel model and experimental data, with the aim of being able to make a cross-species comparison. Swarm-plate experiments with uniform concentrations of the chemoattractant L-aspartate were performed for both bacteria over several concentrations of aspartate. Separately, growth experiments in liquid cultures were undertaken to quantify differences in aspartate concentration dependent growth between the two species. From the data we are able to determine parameter values for a Keller-Segel model and thus quantify differences between non-chemotactic diffusive behavior, growth effects, and chemotactic behavior. This quantitative modeling work comparing population-level behavior of these bacteria allows one to deduce metabolic function parameters in agar, which are not possible to find experimentally and not incorporated in many previous models. We find that a significant proportion of the E. coli wild-type population appears to be non-chemotactic whereas the R. sphaeroides wild-type population appears to be primarily chemotactic, something not explored in other studies. Our parameters also indicate a joint saturation of growth and chemotaxis, which we postulate is a common evolutionary result. These findings provide a platform from which to explore incorporating cell-level knowledge into macro-scale behavioral models and the effects of heterogeneity of populations.

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BLAST X Poster #45 TETHERED MYCOPLASMA Daisuke Nakane and Makoto Miyata Department of Biology, Graduate School of Science, Osaka City University

Mycoplasma mobile is a pathogenic flask-shaped bacterium 0.8 micron long. They bind to solid surfaces by “legs” sticking out from the base of membrane protrusion, “neck”, and glide by a unique mechanism. Recently, we proposed a working model, power stroke model, where the cells are propelled by many legs repeatedly catching and releasing the solid surface, driven by the energy of ATP hydrolysis. Here, to detect the movement of legs, we reduced the number of working legs and amplified the leg movement.

When the cells were treated by 0.1% Tween 60, they were elongated from 0.8 micron to

2.0-4.0 micron in 15 min with the extension of cytoskeletal structures. A previous study showed that the direct binding target of mycoplasma is a sialic acid, and the addition of free sialic acids dissociated gliding cells from the solid surface. Here, in the presence of 0.25-1 mM of sialic acid, the elongated cells pivoted widely, plausibly resulting from the reduction the leg number. The pivoting ceased when strong light was applied in the presence of a fluorescence dye, suggesting that the pivoting is caused by the gliding legs. Azide and the antibody against the gliding machinery affected the distribution of pivoting angle differently, although both reduce the gliding speed. On the basis of these results, the movements of legs will be discussed.

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BLAST X Poster #46 MOLECULAR SHAPES OF Gli123 AND Gli521 INVOLVED IN GLIDING MOTILITY OF MYCOPLASMA MOBILE Takahiro Nonaka, Jun Adan-Kubo, Makoto Miyata Department of Biology, Graduate School of Science, Osaka City University 3-3-138 Sugimoto Sumiyoshi-ku, Osaka-shi, JAPAN Mycoplasma mobile has no flagella or pili, and its genome contains no genes related to known bacterial motility. However, M. mobile binds to solid surfaces and glides smoothly and continuously, with a unique mechanism. M. mobile cells form a membrane protrusion at the leading pole. Three huge proteins, Gli123 (123 kDa), Gli349 (349 kDa), and Gli521 (521 kDa), localize at neck, the base of protrusion, and form the gliding machinery. These proteins are suggested to have the roles of scaffold for other gliding proteins, glass binding, and force transmission, respectively. In this study, we purified Gli123 and Gli521 proteins from M. mobile cells by biochemical procedures, rotary shadowed, and observed their molecular shapes by transmission electron microscopy. The Gli123 molecule shaped asymmetrical oval, 31 nm long and 16 nm wide. The Gli521 molecule consisted of three flexible arms about 130 nm long, each of them had spherical part at the distal end. This shape is reminiscent of clathrin-triskelion which is widely distributed in eukaryotic cells. Clathrin molecules self-assemble, control the membrane shape and form a vesicle. The characteristic molecular shape of Gli521 may suggest that this protein forms a two dimensional sheet like clathrin, and plays a critical role to form the membrane protrusion in a M. mobile cell.

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BLAST X Poster #47 THE ESSENTIAL NATURE OF THE WALK/WALR SIGNAL TRANSDUCTION PATHWAY IS LINKED TO CELL WALL HYDROLASE ACTIVITY IN STAPHYLOCOCCUS AUREUS Aurélia Delauné 1, Olivier Poupel 1, Adeline Mallet 2, Sarah Dubrac1, and Tarek Msadek 1 Biology of Gram-positive Pathogens 1, Department of Microbiology, Plateforme de Microscopie Ultrastructurale 2, Imagopole, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France

Staphylococcus aureus, a major Gram-positive human pathogen, is a leading cause of both nosocomial and community infections due to its considerable capacity for adaptation. One of the principal mechanisms involved in this process are the so-called two-component systems, bacterial signal transduction pathways with a sensor histidine kinase that is autophosphorylated in response to specific environmental stimuli and then transfers the phosphoryl group to its cognate response regulator, which consequently regulates target gene expression. The WalKR two-component system is well conserved and specific to low G+C Gram-positive bacteria, including Bacillus subtilis, Staphylococcus aureus and Streptococcus pneumoniae. This system has been shown to be essential for cell viability.

We have recently demonstrated that the WalKR system positively controls autolytic

activity, and identified ten genes belonging to the WalKR regulon that appear to be involved in S. aureus cell wall degradation. Reasoning that the essential nature of this signaling pathway may be related to its role as a master regulatory system for cell wall metabolism, we tested whether uncoupling autolysin gene expression from WalKR-dependent regulation could compensate for the essential nature of the system. Several genes from the WalKR regulon were expressed from a cadmium chloride-inducible promoter in a WalKR-independent manner. Candidate genes were identified whose expression allowed cells to grow in the absence of WalKR.

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BLAST X Poster #48 THE GraS/GraR TWO-COMPONENT SYSTEM AND DERMASEPTIN RESISTANCE IN STAPHYLOCOCCUS AUREUS Mélanie Falord 1, Pierre Joanne 2, Chahrazade El Amri 2, and Tarek Msadek 1 Biology of Gram-positive Pathogens 1, Department of Microbiology, Institut Pasteur, 25 rue du Dr. Roux, 75015 Paris, France Peptidome de la peau des amphibiens 2, FRE 2852, CNRS, Université Pierre et Marie Curie, 2 place Jussieu, 75251 Paris 75005, France Staphylococcus aureus, a major Gram-positive human pathogen, is a leading cause of both nosocomial and community infections due to its considerable capacity for adaptation. S. aureus is able to resist Cationic Anti-Microbial Peptides (CAMPs) by increasing its positive cell surface charges through D-alanylation of wall teichoic acids and lysylination of phospholipids, leading to electrostatic repulsion of CAMPs. Synthesis of the major enzymes involved in these mechanisms (DltA, MprF) is positively controlled by the GraS/GraR two-component system. Two-component systems are bacterial signal transduction pathways with a sensor histidine kinase that is autophosphorylated in response to specific environmental stimuli and then transfers the phosphoryl group to its cognate response regulator which consequently regulates target gene expression. In S. aureus, GraS is involved in CAMP sensing, promoting bacterial resistance through GraR. Here, we have shown that a ∆graRS mutant in S. aureus is sensitive to Colistin and Dermaseptins. Moreover, we demonstrated that the graRS genes are part of a three-gene operon also containing graX, a gene with unknown function but essential to the system, and defined the operon transcriptional start site. To define the mechanism by which GraS, GraR and GraX confer CAMP resistance to S. aureus, the three proteins were overexpressed and purified to test their in vitro interactions. A potential GraR binding site upstream from the vraFG operon, known to be controlled by GraS/GraR, was identified both by in silico analysis and lacZ transcriptional fusion experiments.

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BLAST X Poster #49 CHARACTERIZATION OF SUPPRESSORS OF THE MotB(D33E) MUTATION, A PUTATIVE PROTON-BINDING RESIDUE OF THE BACTERIAL FLAGELLAR MOTOR Yong-Suk Che, Shuichi Nakamura, Yusuke Morimoto, Nobunori Kami-ike, Keiichi Namba and Tohru Minamino Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan MotA and MotB form the stator of the proton-driven bacterial flagellar motor, which conducts protons and couples proton flow to motor rotation. Asp-33 of Salmonella Typhimurium MotB, which is a putative proton-binding site, is critical for torque generation. However, how does the protonation of Asp could drive the conformational changes requiring for torque generation is largely unknown. Here, we carried out genetic and motility analysis of a slow motile motB(D33E) mutant and its pseudorevertants. First, we confirmed that MotB(D33E) forms the complex with MotA in the cytoplasmic membrane. Then, we isolated suppressor mutants from the motB(D33E) mutant and identified the suppressor mutation sites. Next, we characterized the torque-speed relationship of the flagellar motors of wild-type, motB(D33E) mutant and its suppressors by the bead assays. As a result, we found that while the wild-type motor torque was almost constant over a wide range of rotation rate, the MotB(D33E) mutation caused ≈40% reduction in near-stall torque and a sharp decline in the torque-speed curve with an apparent maximal rotation rate of ≈20 Hz. Furthermore, we also found that the second-site mutations could recover the near-stall torque but not the sharp decline of torque-speed curve and the maximum rotation rate. These results together suggested that MotB(D33E) mutation reduced both proton-conducting activity and torque generation step involving the stator-rotor interactions coupled with protonation/deprotonation of Glu-33 and the second-site mutations could recover the torque generation step but not the proton-conducting activity. Recently, to measure the proton-conducting activity of the motB(D33E) mutant and its pseudorevertants, we developed a novel system to monitor intracellular pH of cells overexpressing MotA/MotB mutant proteins utilizing pH-sensitive GFP (pHluorin). Experimental results will also be discussed.

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BLAST X Poster #50 STRUCTURE OF FliJ, A CYTOPLASMIC COMPONENT OF THE FLAGELLAR TYPE III PROTEIN EXPORT APPARATUS OF SALMONELLA Tatsuya Ibuki1, 2, Masafumi Shimada1, 2, Tohru Minamino1, 2, Katsumi Imada1, 2 and Keiichi Namba1, 2 Grad. Sch. of Frontier Biosci., Osaka Univ. 1, Dynamic NanoMachine Project, ICORP, JST2

The flagellum is a motile organelle composed of the basal body rings and the tubular axial structure. The axial component proteins synthesized in the cytoplasm are transferred into the central channel of the flagellum by the flagellar type III protein-export apparatus for self-assembly at the growing end. The apparatus is composed of six transmembrane proteins (FlhA, FlhB, FliO, FliP, FliQ, FliR) and three soluble components (FliH, FliI, FliJ). FliJ is an essential component for protein export. Although FliJ is thought to be a general chaperone, its function is still unclear. Here we report a crystal structure of FliJ. We obtained hexagonal bi-pyramid crystals of FliJ with N-terminal extra three residues, and determined the structure at 2.1-Å resolution using anomalous diffraction data from a mercury derivative collected at SPring-8 BL41XU. FliJ consists of two α-helices, one is a 13-turn helix and the other a 21-turn helix, which form a coiled-coil structure. FliJ has a remarkable structural similarity to the γ subunit of F0F1-ATPsynthase. The other soluble components FliH and FliI are also known to have similarity to other components of F0F1-ATPsynthase. The structure of FliI closely resembles those of the α/β subunits, and the amino-acid sequence of FliH has two regions that are similar to those of the b and δ subunits, respectively. We will discuss details of the structure and possible functional mechanism of FliJ, and similarity between the flagellar export apparatus and F0F1-ATPsynthase.

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BLAST X Poster #51 CRYOEM STRUCTURE OF THE HOOK-FILAMENT JUNCTION OF SALMONELLA Fumiaki Makino (1), Takayuki Kato (1), Keiichi Namba (1) 1: Grad. Sch. of Frontier Biosci., Osaka Univ. The bacterial flagellum is a biological nanomachine for the locomotion of bacteria. The flagellum consists of three functional parts: the basal body as a rotary motor, the filaments as a helical propeller, and the hook as a universal joint that connects the above-mentioned two parts. The filament is a tubular structure made of a single protein, FliC. It transforms into various helical forms in response to mechanical perturbation by reversal of motor rotation. The hook is also a tubular structure made of a single protein, FlgE, but is more flexible than the filament, allowing it to transmit motor torque to the filament regardless of its orientation. The hook-filament junction made of two proteins, FlgK and FlgL, connects these two mechanically distinct structures. It works as a mechanical adapter, allowing the two parts to go through dynamic conformational changes independently. To understand the adapter mechanism, we have analyzed the structure of the hook-filament junction by electron cryomicroscopy (cryoEM). We needed a Salmonella strain that produces short filaments for efficient data collection. We used a strain, MMC1660, which produces short filament due to a significantly low efficiency of FliC expression controlled by the addition of tetracycline. We established a procedure to purify the hook-basal body with short filament, collected cryoEM images and carried out image analysis. We will show the cryoEM structure of the hook-filament junction complex.

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BLAST X Poster #52 EFFECT OF INTRACELLULAR pH ON THE TORQUE-SPEED RELATIONSHIP OF BACTERIAL PROTON-DRIVEN FLAGELLAR MOTOR Shuichi Nakamura1,2, Nobunori Kami-ike2, Jun-ichi Yokota1,2, Seishi Kudo3 ,Tohru Minamino1,2, and Keiichi Namba1,2 1Graduate School of Frontier Bioscience, Osaka University 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan, 2Dynamic NanoMachine Project, ICORP, JST, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan, 3Faculty of Engineering, Toin University of Yokohama, 1614 Kurogane-cho, Aoba, Yokohama 225-8502, Japan The flagellar motor of Salmonella is a rotary nanomachine driven by proton motive force. It has been shown that an increase in the intracellular proton concentration abolishes the motor function, suggesting that the stator complex has intracellular proton-binding sites by which intracellular protons kinetically interfere with the rate of proton translocation for the motor rotation. In this study, to understand the coupling mechanism of proton flux with torque generation, we have investigated the effect of intracellular pH on the rotation rate of the flagellar motor. Intracellular pH was manipulated by adding potassium benzoate, which crosses the cytoplasmic membrane in the neutral form and equilibrates the intra- and extracellular pH without changing proton motive force. We showed that the rotation rates at low loads sharply decreased as the external pH decreased in the presence of benzoate, suggesting that a high intracellular proton concentration lowered the proton translocation rate. Also, computer simulation by a simple kinetic model suggested that the decrease in intracellular pH interferes with proton dissociation from an intracellular proton-binding site of the stator. We conclude that the intracellular pH is a critical factor that determines the flagellar rotation rate and that proton dissociation from the motor is a rate-limiting step in the mechanochemical reaction cycle.

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BLAST X Poster #53 FLUORESCENCE IMAGING OF ASSEMBLY AND DISASSEMBLY OF THE BACTERIAL FLAGELLAR PROTEIN EXPORT ATPase FliI TO THE FLAGELLAR BASAL BODY Shinsuke Yoshimura, Tohru Minamino, Keiichi Namba Grad. Schl. of Frontier Biosci., Osaka Univ.

For construction of the bacterial flagellum, most of the flagellar proteins are translocated into the central channel of the growing structure by the flagellar protein export apparatus. FliI ATPase forms a complex with its regulator FliH and facilitates the initial entry of export substrates into the export gate made of six membrane proteins. The FliH/FliI complex also binds to a C ring protein, FliN, through the FliH-FliN interaction for efficient export. However, it remains unclear how these reactions proceed within the cell. In this study, we constructed FliI-CFP and FliI-YFP fusion proteins and analyzed their localization by fluorescence microscopy. A few bright spots within each cell suggested that many of them are bound to the C ring, and breaching experiments showed their rapid assembly and disassembly. We confirmed that both FliH and the C ring are required for the localization, but faint spots observed even in the absence of the C ring suggested binding of the FliI hexamer to the gate. FliI-YFP formed a complex with FliH∆1 missing residues 2-10 but the complex did not show the localization. FliH∆1 interacted with neither FliN nor the gate-forming proteins. Alanine-scanning mutagenesis of FliH revealed that only two residues, Trp-7 and Trp-10, are responsible for these interactions. Taken all together, hydrophobic interactions of two Trp residues of FliH with other export components seem to drive the cycling reaction of assembly/disassembly of FliI for efficient export.

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BLAST X Poster #54 ANALYSIS OF HELICOBACTER PYLORI LACKING ALL FOUR CHEMORECEPTORS Karen M. Ottemann UC Santa Cruz Helicobacter pylori is an epsilon proteobacter that uses chemotaxis and motility to infect human stomachs and cause ulcer disease. Based on genome sequencing and annotation, H. pylori is predicted to have four chemoreceptors. Here we describe the construction and characterization of mutants lacking one, two, three, or all four chemoreceptors in strain mG27, and a comparison of chemoreceptor expression across many H. pylori strains. We find that one chemoreceptor is sufficient to confer soft-agar migration in our standard Brucella Broth/Fetal Bovine Serum soft agar. This finding suggests that ligands for each chemoreceptor are found in this milieu. We also find that mutants lacking some chemoreceptor have an altered stopping frequency, as noted by Schweinitzer et al (J Bac 2008 190:3244). Mutants lacking all four chemoreceptors are non-chemotactic and completely smooth swimming, supporting that the four MA-domain proteins are the only chemoreceptors in this system. Our work thus shows experimentally that H. pylori possesses four chemoreceptors, and sets the stage for dissecting what each chemoreceptor senses.

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BLAST X Poster #55 CHEMOTAXIS TO PYRIMIDINES AND IDENTIFICATION OF A CYTOSINE CHEMORECEPTOR IN PSEUDOMONAS PUTIDA X. Liu, P.L. Wood, J.V. Parales, and R.E. Parales Department of Microbiology, University of California, Davis, CA, 95616 Pseudomonads are motile bacteria that are widespread in nature and are known for their catabolic versatility. These organisms have a conserved chemotaxis system that is homologous to that present in Escherichia coli. Unlike E. coli, however, which has only one set of chemotaxis (che) genes in a single gene cluster, Pseudomonas species have multiple che gene homologues organized in several unlinked gene clusters. In addition, genome sequence analyses have revealed that Pseudomonas genomes encode numerous MCP-like proteins, suggesting that these organisms can also sense and respond to a wide range of chemicals and environmental conditions. For example, Pseudomonas aeruginosa PAO1 has 26 MCP-like genes and Pseudomonas putida F1 has 27. We have been studying the chemotactic responses of P. putida strains to a variety of carbon and nitrogen sources and initiated a study in which we have individually deleted each of the 27 MCP-like genes in P. putida F1 and tested for mutant taxis phenotypes. We use both qualitative and quantitative chemotaxis assays to measure the responses, including a new quantitative capillary assay carried out in 96-well plates. In this study we demonstrated that P. putida strains F1 and PRS2000 are attracted to cytosine, but not thymine or uracil. The chemotactic response to cytosine was constitutively expressed under all tested growth conditions. In contrast, P. aeruginosa PAO1 was not chemotactic to any of these pyrimidines. Chemotaxis assays with a mutant strain of P. putida F1 in which the putative methyl-accepting chemotaxis protein-encoding gene Pput_0623 was deleted revealed that this gene (designated mcpC) encodes a chemoreceptor for positive chemotaxis to cytosine. Complementation of the F1ΔmcpC mutant with the wild-type gene restored chemotaxis to cytosine. In addition, introduction of this gene into P. aeruginosa PAO1 conferred the ability to respond to cytosine. To our knowledge, this is the first report of a chemoreceptor for cytosine.

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BLAST X Poster #56 REACTIVE ALDEHYDES AND MOTILITY IN ESCHERICHIA COLI K12 Changhan Lee, Junghoon Lee, Jongchul Shin, Insook Kim, Kwanghee Baek, and Chankyu Park Department of Biological Sciences, KAIST, Daejon, Korea Graduate School of Biotechnology, Kyung Hee University, Yongin, Korea The short chain carbohydrates such as glyoxal and methylglyoxal are generated in vivo from various sugars either by an oxidative stress or by a cellular metabolism, which are believed to be removed by the glutathione-dependent glyoxalase system. We isolated a number of E. coli mutants conferring resistance or sensitivity to these aldehydes and found that some of them affect motility. In the case of glyoxalse I mutant (gloA) that is deficient in the removal of aldehyde using glutathione, significant reductions in the free-swimming as well as in swarming behaviors were observed. When we introduced the flhDC-lacZ fusion contained in phage Lambda into the wild-type and gloA strains, expression of LacZ was considerably reduced in the gloA mutant compared to that of wild type, suggesting that a decrease in flagellar expression is responsible for the impaired motility. This is further confirmed by the reduced expression of flagellin as detected by anti-flagellin antiserum. We observed similar effects of other glyoxal-related genes on motility, suggesting a possibility that an intracellular level of aldehyde is somehow associated with flagellar gene expression. Cellular redox change due to an aldehyde, affecting motility, will be discussed.

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BLAST X Poster #57 TAKING CONTROL OF THE BACTERIAL FLAGELLAR MOTOR Mathieu Gauthier, Dany Truchon, Alexandre Bastien, Simon Rainville Laval University, Physics department and COPL, Pavillon Optique Photonique, 2139, 2375, rue de la Terrasse, Quebec, Quebec, G1V 0A6, Canada The bacterial flagellar motor is a fairly complex machine, requiring 40-50 genes for its expression, assembly and control. Furthermore, it is 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. Here, we report significant progress towards the development of a unique in vitro system to study quantitatively the bacterial flagellar motor. Our system consists of a filamentous Escherichia coli bacterium partly introduced inside a micropipette. 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 submicrometer-sized hole in the wall of the bacterium, thereby granting us access to the inside of the cell and the control over the proton-motive force that powers the motor. Using a patch-clamp amplifier, we applied an external voltage between the inside and the outside of the micropipette. If the hole in the bacterium is open, that voltage should translate into a membrane potential powering the motors outside of the micropipette. As we varied the applied potential, variations in the motor's rotation speed were observed. For these preliminary results, the rotation speed was observed directly using video microscopy of fluorescently labeled filaments. That system opens numerous possibilities to study the flagellar motor and other membrane components.

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BLAST X Poster #58 CHARACTERIZATION OF THE PERIPLASMIC DOMAIN OF THE SENSOR KINASE CpxA: ROLE OF CONSERVED RESIDUES IN CpxA ACTIVITY Malpica R and Raivio TL. Department of Biological Sciences, University of Alberta, CW405 Biological Sciences Building, Edmonton, Alberta, Canada T6G 2E9. The Cpx two-component signal transduction system plays a major role in the conservation of cell envelope integrity, as well in the regulation of surface structure assembly, cellular attachment and pathogenesis in Escherichia coli. This system is comprised of the inner-membrane sensor kinase CpxA and the cytosolic response regulator CpxR. Envelope stress caused by misfolded and mislocalized proteins activates the Cpx pathway, which also responds to alkaline pH and overexpression of the outer membrane lipoprotein NlpE. In the presence of these activating cues, CpxA autophosphorylates at a conserved His residue and then transphosphorylates CpxR (CpxR-P). In turn, CpxR-P regulates the expression of several genes such as periplasmic protein folding and degrading factors involved in envelope protein manteinance under adverse conditions. It is known that in the absence of stress, the periplasmic protein CpxP, whose expression is positively controlled by this pathway, inhibits the activation of the Cpx system. The periplasmic domain of CpxA (CpxA-pd), which contains 133 residues, has been proposed as the signal reception site and therefore as the regulatory element of the kinase and phosphatase activities of CpxA. To explore the role of CpxA-pd components in signal sensing and CpxA activity, we generated mutants that carry substitution mutations in highly conserved residues of this domain. The phenotypes of these mutants were evaluated, using alkaline pH or NlpE as inducing cues and the activatable cpxP´-lacZ fusion as a reporter of Cpx pathway activity. Remarkably, the substitution of Phe108, Gly130 and Pro164 by Ala lead to a constitutively active phenotype. Also, mutants in other residues displayed a significant increase on pathway activity in the absence of the inducing cues. Thus, we have identified relevant CpxA-pd elements for both signalling and the overall enzymatic activity of CpxA.

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BLAST X Poster #59 CHARACTERIZATION OF FliZ AS AN ACTIVATOR OF FLAGELLAR GENES IN SALMONELLA ENTERICA SEROVAR TYPHIMURIUM Supreet Saini1, Phillip Aldridge2,3, and Christopher Rao1 Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801,1 Centre for Bacterial Cell Biology, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom,2 Institute for Cell and Molecular Biosciences, Newcastle University, Framlington Place, Newcastle upon Tyne NE2 4HH, United Kingdom3

Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the σ28-FlgM checkpoint, which couples the expression of late flagellar (Pclass3) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (Pclass2) gene expression to assembly in Salmonella enterica serovar Typhimurium (Salmonella). We also demonstrate that significant cross talk exists between different secretion systems in Salmonella with FliZ as the cross talk element. Specifically, we show that FliZ is also an activator of Salmonella Pathogenicity Island-1 (SPI1) encoded virulence genes and the fim, lpf, std, saf, bcf, stb, stc, stf, and sth loci encoded fimbriae in Salmonella. We demonstrate that FliZ is an FlhD4C2-dependent activator of Pclass2/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD4C2 posttranslationally which leads to faster induction of Pclass2/middle genes. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor σ28 and the filament-cap chaperone/ FlhD4C2 inhibitor FliT. We correlate our gene expression experiments by discussing the role of FliZ in flagellar biosynthesis during swimming and swarming motility. FliZ was found to effect SPI1 activity levels in a HilD dependent posttranslational manner and its effect on type I fimbriae gene expression was found to be FimZ dependent.

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BLAST X Poster #60 LOCALIZATION OF THE CHEMOTAXIS PROTEINS IN BACILLUS SUBTILIS Kang Wu and Christopher Rao University of Illinois at Urbana-Champaign The Bacillus subtilis chemotaxis pathway utilizes three proteins – CheC, CheD, and CheV - not found in Escherichia coli. These proteins are thought to be involved in two methylation-independent adaptation systems not found in E. coli. While the functions of these proteins have been characterized to some degree, the details concerning their interactions within the polar signaling complex are still unknown. To better understand these interactions, we are investigating the factors influencing the localization of the B. subtilis chemotaxis proteins using immunofluorescence and fluorescent protein fusions. Of significance, we have constructed functional fluorescent protein fusions to CheC and CheD and also explored their co-localization using two-color immunofluorescence. Using these approaches, we have quantified the localization of these proteins in a number of different mutants. This poster will discuss some of our preliminary results regarding the factors influencing localization of the chemotaxis proteins in B. subtilis.

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BLAST X Poster #61 RcdA STRUCTURE AND FUNCTION IN REGULATED CtrA PROTEOLYSIS James A. Taylor*, Jeremy D. Wilbur†, Kathleen R. Ryan* * Department of Plant & Microbial Biology, University of California, Berkeley, Berkeley, CA 94720-3102 † Graduate Group in Biophysics, University of California, San Francisco, San Francisco, CA 94158 The Caulobacter crescentus master cell cycle regulator CtrA must undergo cyclic activation and deactivation to drive orderly progression through the division cycle. CtrA is essential for viability and directly activates or represses the transcription of ~100 genes. However, it also blocks the initiation of DNA replication, so CtrA activity must be eliminated from the cell before chromosome replication can occur. To this end, CtrA is rapidly degraded specifically at the G1-S cell cycle transition by the ClpXP protease. Regulated CtrA proteolysis in vivo requires two other factors, the single-domain response regulator CpdR and RcdA, a conserved protein of unknown function. Although each of these proteins is cytoplasmic, they all colocalize at one pole of the cell during CtrA degradation. We are investigating the role of RcdA in CtrA proteolysis. RcdA was proposed to act as an adaptor bridging CtrA and ClpXP to promote CtrA degradation. However, RcdA is not necessary for CtrA proteolysis by ClpXP in vitro and does not change the rate of CtrA degradation. We have therefore taken a structure-function approach to learn how RcdA contributes to CtrA proteolysis in vivo. Using X-ray crystallography, we have found that RcdA is a dimer, and each monomer consists of a three-helix bundle. Peptides at the N- and C-termini and a peptide linking helices two and three are disordered in the crystal structure. We have created point mutations and deletions to alter conserved surface features of RcdA. We expressed these proteins in a Caulobacter ∆rcdA mutant to identify regions of RcdA necessary for regulated CtrA degradation and for the polar localization of CtrA and RcdA itself. We have identified three types of rcdA mutants: 1) those that permit CtrA degradation and protein localization, 2) those that cannot support either CtrA degradation or protein localization, and 3), those that allow CtrA degradation without polar accumulation of either RcdA or CtrA. Surprisingly, RcdA does not need to be stably located at the cell pole to promote CtrA proteolysis. These results suggest that RcdA can act via transient interaction with other degradation proteins at the pole, or that RcdA’s function can be performed anywhere in the cell. For example, RcdA could inhibit an unknown negative regulator of CtrA proteolysis. We are examining the rcdA mutants in further detail and are screening for additional proteins that regulate CtrA degradation in Caulobacter.

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BLAST X Poster #62 MODELLING MCP SIGNALLING MECHANISMS WITH HIGH-THROUGHPUT SIMULATION OF Tar TM2 Benjamin A Hall, Mark SP Sansom Department of Biochemistry, University of Oxford, South Parks Road, OX1 3QU Transmembrane helices play a multiple vital roles in cell function, including intercell signalling processes, channel gating and active transport. As such, there exists a considerable amount of data on the biological function and structural properties of naturally occurring helices and their mutants. Simulation studies can provide insight into the dynamics and behaviour of biomolecular systems in a variety of environments, but however such analyses are computationally expensive and typically difficult to automate. Coarse grain simulations are becoming an increasingly popular tool for understanding the properties of biological systems, overcoming canonical limits of atomistic simulations such as timescale or system size. Both such techniques involve several manual steps, including system build, simulation set up and analysis. Here we present Sidekick, a piece of software which automates these processes to allow for the set up of massive numbers of coarse grain simulations on the basis of a small set of input sequences, or a single sequence and a scanning mutation. We demonstrate the use of this software to approach the mechanism of signalling in the MCPs, based on existing mutational data for TM2 from different MCPs and organisms. By observing the change in positions and orientations of the helix with different mutations, we propose that a piston model dominates the signalling event, though there may be a lesser role for rotation of the helix.

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BLAST X Poster #63 STRUCTURAL EVIDENCE SUGGESTS THAT ANTIACTIVATOR ExsD FROM PSEUDOMONAS AERUGINOSA IS A DNA BINDING PROTEIN Robert C. Bernhards¶, Xing Jing¶, Nancy J. Vogelaar¶, Howard Robinson#, Florian D. Schubot¶* ¶Department of Biological Sciences, Virginia Polytechnic Institute & State University, Washington Street, Blacksburg, VA 24060; #Biology Department, Brookhaven National Laboratory, Upton, NY 11973-5000. The opportunistic pathogen P.aeruginosa utilizes a type III secretion system (T3SS) to support acute infections in predisposed individuals. In this bacterium expression of all T3SS-related genes is dependent on the AraC-type transcriptional activator ExsA. Prior to host contact, the T3SS is inactive and ExsA is repressed by the antiactivator protein ExsD. The repression, thought to occur through direct interactions between the two proteins, is relieved upon opening of the type III secretion (T3S) channel when secretion chaperone ExsC sequesters ExsD. We have solved the crystal structure of Δ20ExsD, a protease-resistant fragment of ExsD that lacks only the twenty amino terminal residues of the wild type protein at 2.6 Å. Surprisingly the structure revealed similarities between ExsD and the DNA binding domain of transcriptional repressor KorB. A model of an ExsD-DNA complex constructed on the basis of this homology produced a realistic complex that is supported by the prevalence of conserved residues in the putative DNA binding site and the results of differential scanning fluorimetry studies. Our findings challenge the currently held model that ExsD solely acts through interactions with ExsA and raise new questions with respect to the underlying mechanism of ExsA regulation.

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BLAST X Poster #64 A NOVEL PAS-GGDEF-EAL PROTEIN INVOLVED IN REGULATION OF MOTILITY IN PSEUDOMONAS PUTIDA Herrera Seitz, K 1 and Shingler, V 2 1 IIB, FCEyN, Univ. Nac. del Mar del Plata, 2 Molecular Biology Department, Umeå University, Sweden. Chemotaxis allows motile bacteria to respond to chemical gradients to relocate themselves near the source of nutrients. Soil Pseudomonads are known to be able to grow in a wide variety of carbon sources, including many that are considered environmentally toxic. Unlike previously characterized chemotactic responses in Pseudomonas strains, taxis of P. putida CF600 and P. putida KT2440 towards methyl-phenols is dependent upon its ability to metabolize the compound, rather than on a classical ligand-binding chemoreceptor. E. coli metabolism-dependent taxis responses are mediated by the Aer receptor that is closely related to chemoreceptors, but which contains a FAD-binding PAS sensory domain. P. putida possesses three aer-like genes. During analysis of the Aer-like receptors of P. putida, Aer-1 was found to be encoded in a dicistonic operon with PP2258, a PAS-GGDEF-EAL domain protein. Our attention was drawn to PP2258 because a null mutant was found to exhibit a general motility defect on solid, but not in liquid, media (Sarand et al., 2008). GGDEF- and EAL-domains are associated with diguanylate cyclase and phosphodiesterase activities that are involved in turnover of the near ubiquitous bacterial second messenger c-di-GMP. The levels of c-di-GMP can modify cells behavior and motility; therefore we reasoned that PP2258 link to motility via c-di-GMP signaling. As a first approach to test this idea, the biochemical properties of wild type and mutant derivatives of PP2258 were studied using over expression of the protein both in E. coli and P. putida. When PP2258 was over expressed in E. coli or P. putida cells, c-di-GMP levels were markedly increased compared to those of control cells, although accumulation of c-di-GMP was much lower in E. coli than in P. putida extracts. Alanine substitutions of the GGDEF domain associated with c-di-GMP synthesis causes a major decrease c-di-GMP accumulation, while an alanine substitution in EAL domain associated with c-di-GMP hydrolysis led to a >7-fold increase in accumulation. Together, these results suggest that PP2258 could be one of the rare examples of a dual GGDEF-EAL domain protein where both domains are catalytically active. References: Sarand, I., Österberg, S., Holmqvist, S., Holmfeldt, P. Skärfstad, E., Parale, R. E., & Shingler, V. (2008) Metabolism-dependent taxis towards (methyl)phenols is coupled through the most abundant of three polar localized Aer-like proteins of Pseudomonas putida. Environ. Microbiology. 10:1320-1334

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BLAST X Poster #65 IN VIVO STUDY OF THE TWO-COMPONENT SIGNALLING NETWORK IN E. COLI Erik Sommer and Victor Sourjik Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Contact: e.sommer@zmbh.uni-heidelberg.de Two-component systems are the most widespread sensing systems in prokaryotes and lower eukaryotes, with multiple members of this class being present in one organism. We are interested in investigating the interconnection among different two-component signalling pathways in Escherichia coli. To map interactions between the pathways in vivo and to study relative cellular distribution of their proteins, we assay real-time dynamics of protein interactions and their dependencies on stimulation using fluorescence imaging and fluorescence resonance energy transfer (FRET)- and fluorescence recovery after photobleaching (FRAP)-microscopy. Additionally, intracellular processing of sensed stimuli with regard to amplification, integration and possible cross-talk between the systems will be investigated. Such analysis will help to establish an integral picture of cell signalling performed by prokaryotic organisms.

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BLAST X Poster #66 GENE REGULATION BY ESCHERICHIA COLI RESPONSE REGULATOR PhoB Hua Han1,2, Timothy R. Mack1,2, Rong Gao1.3, and Ann M. Stock1,3 1Center for Advanced Biotechnology and Medicine, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA, 2Department of Biochemistry, Graduate School of Biomedical Sciences, UMDNJ-Robert Wood Johnson Medical School, and 3Howard Hughes Medical Institute.

Structural analysis of the Escherichia coli response regulator transcription factor PhoB indicates that the protein dimerizes in two different orientations, both of which are mediated by the receiver domain. The two dimers exhibit two-fold rotational symmetry: one involves the α4-β5-α5 surface and the other involves the α1/α5 surface. The α4-β5-α5 dimer is observed when the protein is crystallized in the presence of the phosphoryl analog BeF3

- while the α1/α5 dimer is observed in its absence. From these studies a model of the inactive and active states of PhoB has been proposed that involves the formation of two distinct dimers. In order to gain further insight into the roles of these dimers we have engineered a series of mutations in PhoB intended to selectively perturb each of them. Our results indicate that perturbations to the α4-β5-α5 surface disrupt phosphorylation-dependent dimerization and DNA binding as well as PhoB mediated transcriptional activation of phoA, while perturbations to the α1/α5 surface do not. Furthermore, experiments with a GCN4 leucine zipper/PhoB chimera protein indicate that PhoB is activated through an intermolecular mechanism. Together these results support a model of activation of PhoB in which phosphorylation promotes dimerization via the α4-β5-α5 face which in turn enhances DNA binding to a pair of direct-repeat half-sites – a model that we propose to be common for most all OmpR/PhoB transcription factors. These data contrast with a recent proposal that the α1/α5 dimer corresponds to the active form of PhoB, a conclusion derived from structural analysis of constitutively active mutant PhoB proteins (Arribas-Bosacoma et al., 2007 J. Mol. Biol. 366:626-641). We have also examined the kinetics of gene expression of several PhoB-regulated genes under conditions of phosphate limitation.

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BLAST X Poster #67 NEW REPORTER RESIDUES OF TRIMER FORMATION BY ESCHERICHIA COLI MCPs Diego A. Massazza1, John S. Parkinson2, Claudia A. Studdert1 1 Instituto de Investigaciones Biológicas, Universidad Nacional de Mar del Plata, Argentina 2 Biology Department, University of Utah It is currently accepted that E. coli chemoreceptors are organized in vivo in trimer-of-dimer arrangements. Using receptors bearing a cysteine reporter residue near the trimer axis, we previously demonstrated efficient formation of 2- and 3-subunit crosslinking products upon treatment of intact cells with the trifunctional maleimide reagent TMEA. In this work, we assessed the in vivo crosslinking behavior of receptors bearing cysteine reporters at different positions along their cytoplasmic domains. We found that the formation of 3-subunit crosslinking products declined with increasing distance of the reporter from the cytoplasmic tip. This result suggests that the in vivo arrangement of the trimer of dimers resembles that observed in the crystal structure of the Tsr cytoplasmic domain, in which the dimers contact one another at the tip and splay apart in the regions that are farther from the tip. We also found that stimulation with repellents immediately before the TMEA treatment caused a slight but reproducible increase in crosslinking efficiency. This behavior is consistent with previous observations suggesting that receptor dimers within the trimer move closer together after repellent stimuli. In cells co-expressing different marked receptors, we analyzed the formation of mixed crosslinking products. When the cysteine reporters were located the same distance from the tip, mixed crosslinking products formed with high efficiency. Mixed products did not form between reporters at different distances from the tip. This result suggests that there are no significant vertical displacements within the trimer of dimers, nor sufficient flexibility between the receptors to get crosslinking between reporters at diferent levels in the dimers.

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BLAST X Poster #68 SYSTEMATIC DETECTION OF PROTEINS THAT LOCALIZE AT THE ATTACHMENT ORGANELLE REGIONS OF MYCOPLASMA PNEUMONIAE BY FLUORESCENT-PROTEIN TAGGING Tsuyoshi Kenri1, Atsuko Horino1, Mayumi Kubota1, Yuko Sasaki1, Daisuke Nakane2 and Makoto Miyata2

1. Department of Bacterial Pathogenesis and Infection Control, National Institute of Infectious Disease, 4-7-1, Gakuen, Musashimurayama, Tokyo, 208-0011, Japan 2. Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka, 558-8585, Japan Mycoplasma pneumoniae is a cell wall-less bacterium with minimum range of genome size required for self-replication. It is also known as a general cause of bronchitis and pneumonia in humans. Pathogenicity of this bacterium is largely depends on its ability to attach to respiratory epithelial cells (cytadherence). The M. pneumoniae cells attached to cell surface also exhibit gliding motility. The cytadherence and gliding motility are mediated by a unique cell terminal structure of this bacterium, the attachment organelle, which is a membrane protrusion supported by internal cytoskeletal structures. Since the attachment organelle duplicates before cell division, the organelle formation is thought to be coordinated with cell cycles. A number of protein components of the attachment organelle (cytadherence-related proteins) have been identified so far (HMW1, HMW2, HMW3, P1, P30, P65, P200, P24, P41, P90(B) and P40(C)). However, configuration and fine structure of these proteins in the organelle are not fully understood. In addition, the studies of Triton X-100 insoluble fraction of M. pneumoniae cells, cross-linking analysis of P1 adhesin protein and isolation of various gliding mutants are suggesting a possibility that there are more protein factors that associate with the attachment organelle components. In this study, to explore whether there are more proteins that localize at the organelle, we perform systematic fluorescent-protein tagging analysis for M. pneumoniae ORFs. At present, we have finished the localization analysis of 620 ORFs (about 90% of total ORFs). In this analysis, we observed that about 50 ORF products including previously known 9 cytadherence-related proteins localized at the attachment organelle region. The other ORF products identified are 10 proteins involved in DNA, RNA and protein synthesis, 20 homologs of variety of enzymes, 2 chaperons and 15 proteins of unknown function. Probably, some of these proteins may be the components of the attachment organelle and have function in formation of the organelle, cytadherence, gliding motility and coordination of cell cycle.

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BLAST X Poster #69 MAPPING THE SIGNAL TRANSDUCTION PATHWAY WITHIN THE PAS DOMAIN OF THE Aer RECEPTOR Asharie J. Campbell, Kylie J. Watts, Mark S. Johnson, and Barry L. Taylor Dept. Microbiology and Mol. Genetics, Loma Linda University, Loma Linda CA, USA The E. coli Aer receptor senses redox changes through an FAD-binding PAS domain, and transmits this redox status to the HAMP and signaling domains. Little is known about the conformational changes that take place within the PAS domain. In this study, we used error-prone PCR mutagenesis to find residues critical for PAS FAD-binding, sensing and signal transduction. We screened 10,000 colonies for function, measured expression for 1,300 clones, sequenced 400 mutants, and found 84 Aer aerotaxis-defective mutants that had just one amino acid substitution. Of these, there were 72 substitutions in the PAS domain, 11 in the F1 region and 1 in the TM region. The swimming behavior of the cells expressing these mutant proteins included those that 1) were locked in a smooth swimming (CCW), "signal-off" state (60/84), 2) had increased tumbling (CW) frequency (4/84) or 3) were locked in the CW "signal-on" state (20/84). Approximately half (49/84) of the Aer mutants were functionally rescued by Tar. Mutant proteins (11/84) that expressed at levels less than 30% of wild-type Aer showed enhanced protein degradation rates. These replacements had altered side-chain polarity, mapped to positions on or near loops and, with one exception, yielded a CCW (signal-off) phenotype. In contrast, all but one of the CW (signal-on) mutants were stable, and clustered in three localized regions: 1) in the putative FAD-binding cleft, 2) on the rear surface of the FAD-binding cleft and 3) at or near a loop in the N-terminal Cap region. When expressed at a 1:1 ratio with wild-type Aer, three CW-locked mutants were dominant, abolishing wild-type mediated aerotaxis. Most but not all of the FAD-binding lesions resulted in an unstable protein; those that were most stable were located outside of the putative FAD-binding cleft. The localization of gain-of-function (CW) lesions to three distinct clusters suggests that conformational changes in these specific regions mimic the signal-on state of the PAS sensor. If so, signaling within the Aer-PAS sensor would begin in the FAD-binding cleft and propagate outward to the N-Cap loop. Previously, we found that removing part of the N-Cap mimics the signal-on state. Thus, an attractive model is one where FAD reduction in the PAS domain initiates a conformational change that propagates to the N-Cap loop, which, in turn, acts as a hinge around which the N-cap moves, unmasking the signal-on state.

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BLAST X Poster #70 MODULATING TWO-COMPONENT SIGNAL OUTPUT WITH PROTEIN-MEMBRANE INTERACTIONS Roger R. Draheim*, Morten H. H. Nørholm, Salomé C. Botelho, Karl Enquist, and Gunnar von Heijne Center for Biomembrane Research, Department of Biochemistry and Biophysics, The Arrhenius Laboratories for Natural Sciences, Stockholm, University, 10691, Stockholm, Sweden The most prevalent type of environmental sensor in prokaryotic organisms is the two-component system (TCS). A canonical TCS consists of a membrane-spanning sensor histidine kinase (SHK) and a cytoplasmic response regulator (RR). TCSs have been shown to regulate a diverse array of virulence factors, therefore identifying two-component signaling pathways that lead to enhanced pathogenicity is essential to understanding complex host-pathogen interactions. The specific hypothesis examined is that SHK-membrane interactions can be identified and harnessed to modulate SHK signal output in a predictable manner. If individual SHK signal output could be directly manipulated, then two-component signaling pathways could be rapidly unraveled in any pathogenic organism of interest. Three discrete steps are proposed to establish a broadly-applicable methodology. The first will identify interactions between TM2 of a well-characterized SHK (e.g., EnvZ) and the cell membrane using a glycosylation-mapping technique. The second will identify HAMP-membrane interactions within EnvZ using a translocon-challenge method. Protein-membrane interactions that are identified will be confirmed using circular dichroism and fluorescent techniques. The third will harness protein-membrane interactions identified during the first two to directly and incrementally modulate SHK signal output. This experimentation will determine the feasibility of coupling modulated SHK output with transcriptional profiling to rapidly unravel two-component signaling pathways in any pathogenic organism of interest. A high-throughput approach using fluorescent transcriptional reporter systems has been established to expedite these steps. The long-term goal of this research is to create a method for rapidly identifying the signaling pathways that regulate the virulence of pathogenic microorganisms. This research will lead to a better understanding of complex host-pathogen interactions and will result in the detection of previously unidentified therapeutic targets.

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BLAST X Poster #71

Poster Cancelled

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BLAST X Poster #72 A MECHANICAL AND GENETIC STUDY OF ESCHERICHIA COLI SWARMING MOTILITY Matthew Copeland and Douglas B. Weibel Department of Biochemistry, University of Wisconsin-Madison, WI 53706 USA Bacterial swarming is a phenotype associated with the motility of bacteria across surfaces in search of resources. In this abstract we describe two approaches to understand this phenotype in Escherichia coli; a ‘mechanistic’ approach to elucidate potential flagella/flagella interactions between adjacent swarming cells and a genetic investigation to chart the expression of genes unique to the swarming phenotype.

In contrast to swimming motility, swarming bacteria cells migrate cooperatively across

surfaces. The role of physical interactions in the coordination of swarming motility is unknown. It has been shown that swarming bacteria align along their long axis and move as multicellular rafts from which the characteristic dynamic swirling patterns of swarming emerge. The alignment of cells may facilitate or accompany the intercellular bundling of flagella between adjacent cells and play a role in the characteristic, coordinated movement observed during swarming motility. To test this hypothesis, we have created strains of E. coli with fluorescent flagella and are using space- and time-resolved fluorescence resonance energy transfer (FRET) to measure flagella/flagella interactions.

Iron starvation is known to signal swarmer cell differentiation in Vibrio parahaemolyticus

and the genes for iron uptake and metabolism have been shown to be elevated in swarming populations of Salmonella typhimurium. We have found that iron metabolism and iron acquisition genes are upregulated in swarming cells of E. coli versus planktonic cells. A specific role for iron in E. coli swarming cells is unknown. Like V. parahaemolyticus, iron starvation may be a signal for swarmer cell differentiation in E. coli or swarming development and motility may require intracellular levels of iron in excess of those necessary for growth under vegetative conditions. We are exploring the role of iron in E. coli swarming using a combination of chemical and gene expression techniques and strains containing iron metabolism gene knockouts.

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BLAST X Poster #73 PROBING HYDRODYNAMIC INTERACTRIONS BETWEEN SWIMMING BACTERIA USING MICROFULIDICS Abishek Muralimohan, Douglas B. Weibel Department of Biochemistry, University of Wisconsin-Madison 433 Babcock Drive, Madison, WI 53706, U.S.A. Populations of self propelled motile bacteria have been shown to exhibit collective swimming behavior, leading to large scale fluid motion and fluid transport1. Such collective behavior arises from hydrodynamic interactions (HI) between the individual bacterial cells. While computational studies have been performed on HI between pairs of swimming cells2, there have been no reports on experimental measurements of HI. Here, we describe a PDMS based microfluidic device that promotes pair wise interactions between swimming bacteria. The device generates collisions between pairs of cells and HI between them is quantified by the length scale (persistence) over which the two cells swim together as a single unit. We have used this device to quantify the strength of HI between pairs of filamentous E. coli hcb437 cells based on a number of parameters including cell length, angle of initial contact, trajectory, and velocity. Our observations indicate two subpopulations based on the persistence length of interaction: long persisters are characterized by low angles of contact (<5°) and long cells (>9.0 µm); short persisters are characterized by a wider range of contact angles (10° – 60°) and shorter cells (6 µm -9 µm). We believe that our microfluidics-based approach is well suited to the study of bacterial interactions at a single cell level. A broader understanding of HI between motile bacteria will shed light on other forms of collective motility, including swarming. 1. Underhill P, et. al. (2008) Phys. Rev. Lett. 100: 248101. 2. Ishikawa T, et al. (2007) Biophys. J. 93, 2217.

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BLAST X Poster #74 EXAMINATION OF PHOSPHORYLATION IN THE Dif CHEMOTAXIS-LIKE SYSTEM IN MYXOCOCCUS XANTHUS Wesley P. Black and Zhaomin Yang Department of Biological Sciences, Virginia Polytechnic and State University, Life Sciences I, Washington Street, Blacksburg, VA 24061 Myxococcus xanthus is able to move on surfaces using social (S) gliding motility, which is powered by the retraction of type IV pili (Tfp). Exopolysaccharides (EPS), also essential for S motility, have been proposed to function as the anchor and trigger for Tfp retraction in M. xanthus. EPS production in M. xanthus is regulated by the Dif chemotaxis-like signal transduction pathway. DifA, DifC and DifE, homologous to methyl-accepting chemotaxis proteins (MCPs), CheW and CheA respectively, are positive regulators of EPS production. DifD and DifG, which are respective homologs of CheY and CheC, are negative regulators of EPS production. It was demonstrated previously that DifD (CheY-like) does not function downstream of DifE (CheA-like) in the regulation of EPS production.

The purpose of this study was to examine the phosphorylation of heterologously

expressed and purified Dif proteins in vitro. Protein phosphorylation, phosphate transfer and dephosporylation were monitored using γ-32P-ATP. We demonstrate the autophosphorylation of DifE, which is an atypical CheA-like kinase with an extra domain. Unlike observations in other systems, the presence of both DifA (MCP-like) and DifC (CheW-like) had inhibitory rather than stimulatory effects on DifE autophosphorylation. In addition, we show that the phosphate from DifE-phosphate can be transferred to DifD (CheY-like). Lastly, DifG, which is similar to the CheC phosphatase, accelerates the dephosphorylation of DifD. These results are consistent with a model where the DifE kinase positively regulates EPS production, while DifD and DifG function collectively to divert phosphate away from an unidentified downstream phosphorylation substrate of DifE.

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BLAST X Poster #75 EVOLUTION OF CHEMOTAXIS PROTEINS ON A MICRO SCALE Brian Cantwell and Igor Zhulin University of Tennessee, Department of Microbiology, Knoxville, TN Oak Ridge National Laboratory, Joint Institute for Computational Sciences, Oak Ridge, TN Computational analysis of very closely related genomes offers the advantage of more accurate tracing of evolutionary events. The chemotaxis system of enteric bacteria Escherichia coli and Salmonella enterica are extremely well studied, and the availability of over fifty sequenced genomes of Enterobacteriacea offers an opportunity to determine the microevolutionary trends in chemotaxis of enterics. Similarly, seventeen sequenced genomes are available for the family Shewanellaceae for which Shewanella oneidensis MR-1 has been most studied by genetic and biochemical methods. In this work we examine the evolution of the chemotaxis systems of the Enterobacteriaceae and Shewanellaceae using computational biology methods. Protein sequences of chemotaxis proteins and receptors were extracted from non-redundant database by matching to domain models and organized into orthologous groups based on reciprocal BLAST hits, genome context, and phylogenetic relationships. All Enterobacteriacea contain a single set of chemotaxis proteins and chemoreceptors orthologous to the E. coli Tsr, Tap, and Aer proteins. Most enteric species contain numerous additional chemoreceptors including multiple orthologs of the E. coli Trg protein. Shewanella species have one common set of chemotaxis proteins with some species having an additional set of chemotaxis proteins. Two chemoreceptors are common among all seventeen Shewanella species with an additional ten chemoreceptors found in at least fifteen of the seventeen sequenced genomes. To examine the evolutionary trends within chemotaxis proteins, we compared orthologous proteins by computing pairwise percentage identity and comparing domains across species, genus, and family lines. As expected, the domains with highest conservation include the catalytic domains of the core chemotaxis proteins as well as the signaling subdomain of the chemoreceptors. The most divergent domains include the P2 domains of CheA, sensory domains of chemoreceptors, HAMP domains, and the methylation subdomains of the Aer proteins.

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BLAST X Poster #76 EVOLUTION OF SIGNAL TRANSDUCTION IN A BACTERIAL GENUS Harold Shanafield, Luke E. Ulrich and Igor B. Zhulin National Institute for Computational Sciences, University of Tennessee – Oak Ridge National Laboratory The recent completion of sequencing of multiple genomes in the Shewanella genus provides a unique opportunity to study evolution at a much finer scale than previously possible. Using a bi-directional best BLAST hit approach at the protein domain rather than traditional whole-protein sequence level we analyzed the evolutionary relationships of proteins predicted to be involved in signal transduction. Based on these relationships, we have determined a core set of 99 proteins across the first 11 sequenced Shewanella genomes that were highly conserved in both domain architecture and protein sequence. The core included one of the several chemotaxis systems found in Shewanella and several two-component regulatory systems. A large group of orthologous signal transduction proteins across multiple genomes showed some primary sequence drift and were classified as “significant similarity”, and finally there were several unique signal transduction proteins in each organism. We also quantified a recent disproportionate loss of signal transduction genes in Shewanella denitrificans OS217 above and beyond the overall reduction in that organism’s genome size, and an enrichment of signal transduction genes in Shewanella amazonensis SB2B. Possible relationships of the observed changes with the metabolism and environment are discussed.

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BLAST X Poster #77 THE CHEMOSENSORY RECEPTOR FrzCD INTERACTS WITH TWO A-MOTILITY PROTEINS, AglZ AND AgmU

Beiyan Nan and David R. Zusman Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA. The Frz chemosensory system of Myxococcus xanthus controls directed motility by regulating cellular reversals. FrzCD, the Frz system chemoreceptor, plays a central role in the Frz signal transduction pathway. Recently, evidence was obtained that AglZ, an A-motility protein, and FrzS, an S-motility protein, are localized in separate complexes that change their positions as cells move forward and reverse. We were interested in studying how FrzCD might communicate with these motility complexes. To gain information on these interactions, we have been doing pull-down experiments using Myxococcus cell extracts and GST-tagged FrzCD as bait. The GST-FrzCD interacting proteins were identified by mass spectrometry. Five proteins were found to reproducibly bind to GST-FrzCD besides FrzA, FrzB and FrzE. Two of these were A-motility associated protein, AglZ and AgmU. The interactions were confirmed using formaldehyde cross-linking, which showed that FrzCD interacts directly with the N-terminal pseudo-receiver domain of AglZ and two TPR clusters of AgmU. These results provide preliminary evidence for a direct role in the control of the A-motility system by the FrzCD receptor. The roles of the other FrzCD-interacting proteins remain to be identified.

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BLAST X Poster #78 PHENOTYPIC CHARACTERIZATION OF ALL SINGLE MUTANTS OF TWO COMPONENT SYSTEM PROTEINS IN NEUROSPORA CRASSA Barba C., Chavez-Canales M., Salas G., Hernandez C., Sanchez O and Georgellis D. Department of Molecular Genetics, Instituto de Fisiologia Celular, UNAM

Perception and response to environmental stimuli is essential for the growth and survival of all organisms. The sensing and processing of these stimuli are carried out by molecular circuits within the cell, which detect, amplify and integrate them into a specific response. In prokaryotes these molecular circuits are typically organized by protein pairs, "sensory kinase" proteins (SK) and "response regulator" proteins (RR) that belong to the large family of two component systems (TCS). This organization implies that each SK activates its cognate RR, and thus providing specificity to signal propagation and output. However, an interesting variation of TCS architecture is observed in filamentous fungi where multiple SKs appear to use a single phosphotransfer protein to relay signals to a few RRs. Therefore, the question of whether a specific signal can generate a specific response, or whether the various signals sensed by individual SKs result in the same response, is raised. To explore this intriguing question we used Neurospora crassa, which has eleven SKs, one phosphotransfer protein and three RRs, as our model. Here, by using single mutants of all SKs and RRs, we demonstrate that these signaling cascades are involved in the regulation of various developmental processes, and in responses to environmental conditions, such as osmotic, oxidative and fungicide stress.

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BLAST X ____________ Poster #79 AFM STUDY OF MYCOPLASMA MOBILE’S GLIDING MOTILITY Charles Lesoil(1), Hiroshi Sekiguchi(1), Takahiro Nonaka(2), Makoto Miyata(2), Toshiya Osada(1), Atsushi Ikai(3) (1) Department of life science, Graduate school of Bioscience and Biotechnology, Tokyo

Institute of Technology. (2) Department of Biology , Graduate school of Science, Osaka City University (3) Innovation Research Center, Tokyo Institute of Technology

Mycoplasma mobile is a parasitic bacterium that lacks the peptidoglycan layer but still

presents recognizable flask-shape cell morphology. It glides along cell or glass surfaces at an average speed of 2.0 to 4.5 μm/s towards the tapered end of the cell called the head, with a unique mechanism. Recent studies have identified four proteins Gli23, Gli349 and Gli521 that are involved in this system, and a model for gliding motility has been presented, but more experimental data are needed to obtain the arrangement and detailed role of each protein in this system.

In this study, we propose a novel approach to investigate the gliding motility system of

M. mobile using an AFM (Atomic Force Microscope) both as an imaging and a force measurement device.

AFM Images of biotinylated M. mobile cells were obtained through immobilization on a

Streptavidin modified mica surface. Pictures showing the morphology of individual Gli349 and Gli521 molecules in dried and liquid conditions were also obtained and were consistent with previous electron micrographs of the proteins. Investigation of the interaction between Gli molecules and Sialyllactose, the direct binding target in gliding was also conducted using AFM tips decorated with Gli349 or Gli521 molecules and the results showed a specific interaction between Gli349 and Sialyllactose, whereas Gli521 did not show any interaction. Nano indentation of living cells was also performed and revealed a great variation in the local stiffness of the cell, consistent with the available information about M. mobile’s cytoskeleton.

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BLAST X PARTICIPANT LIST

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

Gladys Alexandre The University of Tennessee M407 Walters Life Sciences 1414 West Cumberland Ave. Knoxville, TN 37996 Phone: (865) 974-0866 Fax: (865) 974-6306 galexan2@utk.edu

Michael Airola Cornell University G63 S.T. Olin Laboratory Chemistry Research Building Ithaca, NY 14853 Phone: (607) 255-4970 mva5@cornell.edu

Adrian Alvarez Instituto de Fisiologia Celular - UNAM Ciudad Universitaria México D.F. 04510 México Phone: +525556225738 aalvarez@ifc.unam.mx

Christine Aldridge Newcastle University The Medical School Framlington Place Newcastle upon Tyne NE2 4HH United Kingdom Phone: +44 1912227704 Fax: +44 1912227424 christine.aldridge@ncl.ac.uk

Angel Andrade UNAM Circuito interior Cd Universitaria México City 05100 México Phone: +5256225965 aandrade@live.com.mx

Phillip Aldridge Newcastle University The Medical School Framlington Place Newcastle upon tyne NE2 4HH United Kingdom Phone: +44 1912227704 Fax: +44 1912227424 p.d.aldridge@ncl.ac.uk

Judy Armitage Oxford South Parks Road Oxford OX1 3QU United Kingdom Phone: +44 1865 613293 armitage@bioch.ox.ac.uk

Roger Alexander Yale University Kline Biology Tower 1054 New Haven, CT 06511 Phone: (404) 217-5664 roger.alexander@yale.edu

Teresa Ballado UNAM Instituto de Fisiologia Celular Circuito Exterior S/N Cd. Universitaria México City 04510 México Phone: (5255) 56225618 Fax: (5255) 56225611 tballado@ifc.unam.mx

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Rina Barak Weizmann Institute of Science Herzel St. Rehovot 76100 Israel Phone: 972-8-9342710 Fax: 972-8-9344112 rina.barak@weizmann.ac.il

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

Carlos Arturo Barba Ostria Instituto de Fisiologia Celular - UNAM Ciudad Universitaria México D.F. 04510 México Phone: +525556225738 cbarba@ifc.unam.mx

Paola Bisicchia Trinity College Lincoln Place Gate Smurfit Institute of Genetics Dublin 2 Ireland Phone: 003538962447 paolabisicchia@yahoo.it

Robert Belas University of Maryland Biotechnology Institute 701 East Pratt Street Baltimore, MD 21202 Phone: (410) 234-8876 belas@umbi.umd.edu

Wesley Black Virginia Tech Life Sciences I Washington St. Blacksburg, VA 24061 Phone: (540) 231-9381 weblack@vt.edu

James Berleman University of Iowa 51 Newton Rd. Iowa City, IA 52242 Phone: (404) 372-4836 berleman@gmail.com

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

Jaya Bhatnagar Cornell University Chemistry Research Bldg. Ithaca, NY 14853 Phone: (607) 255-4970 jb394@cornell.edu

Richard Branch University of Oxford Clarendon Laboratory Parks Road Oxford OX1 3PU United Kingdom Phone: +44 01865272357 r.branch1@physics.ox.ac.uk

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

Karen Camargo Universidad Nacional Autonoma de México C.U. Instituto de Fisiologia Celular México Distrito Federal 04510 México Phone: 52 556225618 karen_100490@hotmail.com

Mostyn Brown University of Oxford South Parks Road Oxford OX1 3QU United Kingdom Phone: +44 01865 275 298 mostyn.brown@bioch.ox.ac.uk

Asharie Campbell Loma Linda University 11021 Campus Street Loma Linda, CA 92350 Phone: (909) 558-1000 ajohnson07b@llu.edu

Iryna Bulyha Max Planck Institute for Terrestrial Microbiology Karl-von-Frisch-Strasse, 8 35043 Marburg Germany Phone: +496421178222 Fax: +496421178209 bulyha@mpi-marburg.mpg.de

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

Victor Bustamante Universidad Nacional Autonoma de México Av. Universidad 2001 Colonia Chamilpa Cuernavaca, Morelos 62210 México Phone: (52) 777 329 16 27 victor@ibt.unam.mx

Brian Cantwell University of Tennessee WLS 437 Knoxville, TN 37996-0845 Phone: (865) 974-7687 Fax: (865) 974-4007 bcantwe1@utk.edu

Edmundo Calva Instituto de Biotecnología UNAM Av. Universidad 2001 Cuernavaca 62210 México Phone: +52-777-329-1645 Fax: +52-777-313-8673 ecalva@ibt.unam.mx

C. Britt Carlson HHMI/UMDNJ Center for Advanced Biotechnology and Medicine 679 Hoes Lane, Room 324 Piscataway, NJ 08854 Phone: (732) 235-4206 carlson@cabm.rutgers.edu

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David Castillo Universidad Nacional Autónoma de México IFC-UNAM México D.F., C.P. 04510 México City 04510 México Phone: (+52) 55 562 Fax: (+52) 55 56225611 castillo@ifc.unam.mx

Brian Crane Cornell University Baker Laboratory Ithaca, NY 14850 Phone: (607) 254-8634 Fax: (607) 255-1248 bc69@cornell.edu

Matt Chapman University of Michigan 830 North University Ann Arbor, MI 48109 Phone: (734) 764-7592 Fax: (734) 647-0884 chapmanm@umich.edu

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

Arnaud Chastanet Harvard University 16 Divinity ave Biolabs Cambridge, MA 02138 Phone: (617) 384-7622 Fax: (617) 496-4642 arnaud@mcb.harvard.edu

Rachel Crowder Texas A&M University 3258 TAMU College Station, TX 77843 Phone: (979) 845-1249 rcrowder@tamu.edu

Yong-Suk Che Osaka University Suita1-3 Yamadaoka Osaka 565-0871 Japan Phone: +81-6-6879-4625 yongsuk@fbs.osaka-u.ac.jp

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

Matthew Copeland University of Wisconsin-Madison 473 Biochemistry Addition 433 Babcock Drive Madison, WI 53706 Phone: (608) 263-2636 mfcopeland@wisc.edu

Miguel De la Cruz Instituto de Biotecnología UNAM Av Universidad 2001 Col Chamilpa Cuernavaca 62210 México Phone: 52 777 3 29 16 27 mike@ibt.unam.mx

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Javier De la Mora Universidad Nacional Autonoma de México C.U., Instituto de Fisiologia Celular México, Distrito Federal 04510 México Phone: 52 556225618 fbravo@ifc.unam.mx

Edith Diaz-Mireles Newcastle University Medical School Framlington Place Newcastle Upon Tyne NE2 4HH United Kingdom Phone: +44 0191-2228947 Fax: +44 0191-2227424 edith.diaz-mireles@ncl.ac.uk

Nicolas Delalez University of Oxford Biochemistry Dept, Microbiology Unit, South Parks Road Oxford OX1 3QU United Kingdom Phone: +44 01865613315 nicolas.delalez@bioch.ox.ac.uk

Jason Dobkowski University of Michigan 830 N. University Ann Arbor, MI 48103 Phone: (734) 647-5677 jdobkow@umich.edu

Aurelia Delaune Institut Pasteur 25 Rue du Dr. Roux 75015 Paris France Phone: 33 1 45 68 88 48 Fax: 33 1 45 68 89 38 aureliad@pasteur.fr

Roger Draheim Stockholm University DBB - von Heijne Group Svante Arrhenius vag 12, plan 4 10691 Stockholm Sweden Phone: +4686747656 rogerdraheim@gmail.com

Kevin Devine Trinity College Dublin Dublin 2 Ireland Phone: (353)-1-896-1872 Fax: (353)-1-6714968 kdevine@tcd.ie

Georges Dreyfus UNAM Instituto de Fisiologia Celular Circuito exterior s/n Cd. Universitaria México City 04510 México Phone: (5255) 56225618 gdreyfus@ifc.unam.mx

Miguel Diaz UNAM Ciudad Universitaria Apartado postal 70-243 México 04510 México Phone: (52) 56225965 madiaz@ifc.unam.mx

Sarah Dubrac Institut Pasteur 25 Rue du Dr. Roux 75015 Paris France Phone: (33) 1-45-68-88-48 Fax: (33) 1 45 68 89 38 sdubrac@pasteur.fr

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Michael Eisenbach Weizmann Institute of Science PO Box 26 Rehovot 76100 Israel Phone: 972-8-9343923 Fax: 972-8-9472722 m.eisenbach@weizmann.ac.il

Ana Gallego Instituto de Biotecnologia, UNAM Av Universidad 2001 Cuernavaca 62210 México Phone: 52 777 3291627 analgh@ibt.unam.mx

Annette Erbse UC Boulder 76 Chemistry Boulder, CO 80309 Phone: (303) 492-3597 erbse@colorado.edu

Elizabeth García-Gómez Universidad Nacional Autonoma de México, Instituto de Fisiología Celular Ciudad Universitaria, México D. F Ap. Postal 70-243. México 04510 México Phone: (55)56225965 egarcia@ifc.unam.mx

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

Aldo García-Guerrero Universidad Nacional Autónoma de México Instituto de Fisiología Celuar Circuito Exterior s/n Cd. Universitaria México 04510 México Phone: (5255) 56225618 aldog.2602@gmail.com

Melanie Falord Institut Pasteur 25 Rue du Dr. Roux 75015 Paris France Phone: 33 1 44 38 94 87 Fax: 33 1 45 68 89 38 mfalord@pasteur.fr

Mathieu Gauthier Universite Laval Pavillon Alexandre-Vachon Quebec QC G1K7P4 Canada Phone: 418-656-2131 mathieu.gauthier.5@ulaval.ca

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

Meztlli Gaytán UNAM Ap. postal 70-243 Ciudad Universitaria México D.F México 04510 México Phone: +52 56225965 ogaytan@ifc.unam.mx

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Dimitris Georgellis UNAM Circuito exterior S/N México D.F. 04510 México Phone: +52 55 5622 5738 dimitris@ifc.unam.mx

Ricardo González UNAM Instituto de Fisiología Celular Ciudad Universitaria México 04510 México Phone: 52 5556225738 rgonzalez@ifc.unam.mx

Zemer Gitai Princeton University LTL-355 Washington Rd Princeton, NJ 08540 Phone: (609) 258-9420 zgitai@princeton.edu

Bertha Gonzalez-Pedrajo National Autonomous University of México Apartado Postal 70-243 México 04510 México Phone: 5255 56225965 Fax: 5255 56225611 bpedrajo@ifc.unam.mx

George Glekas University of Illinois Urbana-Champaign 409 Medical Sciences Building 506 S. Mathews Ave. Ubana, IL 61801 Phone: (217) 333-0268 glekas@illinois.edu

Nataliya Gurich University of Texas at Dallas RL11, 800 W. Campbell, Rd. Richardson, TX 75080 Phone: (972) 883-6291 nxg010400@utdallas.edu

Shalom Goldberg University of Pennsylvania School of Medicine 422 Curie Blvd. Philadelphia, PA 19104 Phone: (215) 898-3495 shalom@mail.med.upenn.edu

Benjamin Hall University of Oxford South Parks Road Oxford OX1 3QU United Kingdom Phone: +441865275380 Fax: +441865275273 benjamin.hall@bioch.ox.ac.uk

Juan Gonzalez University of Texas at Dallas RL11, 800 W. Campbell, Rd. Richardson, TX 75080 Phone: (972) 883-2526 jgonzal@utdallas.edu

Hua Han UMDNJ/CABM Rm 326, CABM 679 Hoes Lane Piscataway, NJ 08854 Phone: (732) 235-4206 han@cabm.rutgers.edu

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

Penelope Higgs Max-Planck-Institute for Terrestrial Microbiology Karl-von-Frisch Strasse D35043 Marburg Germany Phone: +49 6421 178301 Fax: +49 6421 178309 higgs@mpi-marburg.mpg.de

Caroline Harwood University of Washington Dept of Microbiology- Box 357242 1705 NE Pacific Street Seattle, WA 98112 Phone: (206) 221-2848 csh5@u.washington.edu

Yohei Hizukuri Nagoya University NagoyaFuro-Cho, Chikusa-Ku 464-8602 Japan Phone: 81-52-789-3543 Fax: 81-52-789-3001 4hiz1103@bunshi3.bio.nagoya-u.ac.jp

Fumio Hayashi Gunma University Kiryu1-5-1 Tenjin 376-8515 Japan Phone: +81-277-30-1663 Fax: +81-277-30-1663 hayashi@chem-bio.gunma-u.ac.jp

Shelley Horne North Dakota State University NDSU Vet & MIcro Sci PO Box 6050 - Dept 7690 Fargo, ND 58108 Phone: (701) 231-6741 Fax: (701) 231-9692 sm.horne@ndsu.edu

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

Basarab Hosu Harvard University 16 Divinity Avenue Bio Labs Buliding Rom 3068 Cambridge, MA 02138 Phone: (617) 495-6127 Fax: (617) 496-1114 ghosu@mcb.harvard.edu

María Herrera Seitz Universidad Nacional de Mar del Plata Funes 3250 Mar del Plata 7600 Argentina Phone: 54 223 4753030 Fax: 54 223 4753150 khseitz@mdp.edu.ar

Colin Hughes Cambridge University Dept of Pathology Cambridge CB4 3AH United Kingdom Phone: 44-1223-338538 ch@mole.bio.cam.ac.uk

139

Tatsuya Ibuki Osaka University suitayamadaoka 1-3 565-0871 Japan Phone: 81 06-6879-4625 Fax: 81 06-6879-4652 tatsuya@fbs.osaka-u.ac.jp

Katy Juarez IBT-UNAM Av. Universidad 2001, Chamilpa Cuernavaca 62210 México Phone: (52) 5556227240 katy@ibt.unam.mx

Katsumi Imada Osaka University Suita1-3 Yamadaoka 565-0871 Japan Phone: +81-6-6879-4625 Fax: +81-6-6879-4652 kimada@fbs.osaka-u.ac.jp

Jung Kwang-Hwan Sogang University Mapo-Gu, Shinsu-Dong 1 R1203 121-742 Seoul Republic of Korea Phone: 82-2-705-8795 Fax: 82-2-704-3601 kjung@sogang.ac.kr

Takehiko Inaba Hosei University Koganei Midori-cho 3-11-15 184-0003 Japan Phone: +81-42-387-7173 takehiko.inaba.13@k.hosei.ac.jp

Alla Kaserer University of Oklahoma 620 Parrington Oval Norman, OK 73019 Phone: (405) 325-1532 Fax: (405) 325-6111 alla@ou.edu

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

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

Christine Josenhans Medical Uiniversity Hannover Carl-Neuberg-Strasse 1 30625 Hannover Germany Phone: +495115324354 Fax: +495115324354 josenhans.christine@mh-hannover.de

Tsuyoshi Kenri National Institute of Infectious Disease Musashimurayama4-7-1 Gakuen, 208-0011 Japan Phone: 81-42-561-0771 Fax: 81-42-565-3315 kenri@nih.go.jp

140

Cezar Khursigara National Institutes of Health 50 South Drive Bldg 50/Rm 4306 Bethesda, MD 20892 Phone: (301) 594-2236 khursigarac@mail.nih.gov

Changhan Lee KAIST CA Daejeon 4101, BMRC, 373-1 Gusung-dong, Yusung-gu 90504 Republic of Korea Phone: +82-42-350-2669 changhanlee@hotmail.com

John Kirby University of Iowa 51 Newton Road Iowa City, IA 52242 Phone: (319) 335-7818 Fax: (319) 335-9006 john-kirby@uiowa.edu

Jae-Min Lee University of Texas at Austin 1 University Station A5000 Austin, TX 78712-0162 Phone: (512) 471-6799 jaeminlee@mail.utexas.edu

Kimberly Kline Washington Univ School of Medicine Department of Molecular Microbiology Box 8230 St. Louis, MO 63110 Phone: (314) 266-0639 k-kline@borcim.wustl.edu

Yi-Ying Lee University of Illinoise at Chicago 835 S Wolcott (M/C 790) Chicago, IL 60612 Phone: (312) 413-0288 yyinglee@uic.edu

Masafumi Koike Nagoya University Chikusa-ku Nagoya 464-8602 Japan Phone: 81 0527892992 Fax: 81 0527893001 4koike@bunshi4.bio.nagoya-u.ac.jp

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

Seiji Kojima Nagoya University Furo-cho 1, Chikusa-ku Nagoya 464-8602 Japan Phone: 81-52-789-2992 Fax: 81-52-789-3001 4seiji@bunshi4.bio.nagoya-u.ac.jp

Janine Maddock University of Michigan 830 North University Ann Arbor, MI 48109 Phone: (734)-936-8068 maddock@umich.edu

141

Fumiaki Makino Osaka University Suita1-3 Yamadaoka, Suita Osaka 565-0871 Japan Phone: 81-06-6879-4625 h1839@fbs.osaka-u.ac.jp

Diego Massazza Universidad Nacional de Mar del Plata Funes 3250 Mar del Plata 7600 Argentina Phone: 54 223 4753030 Fax: 54 223 4753150 diegomassazza@hotmail.com

Roxana Malpica University of Alberta CW405 Biological Sciences Building Edmonton AB T6G2E9 Canada Phone: 780-492 4339 malpica@ualberta.ca

Emilia Mauriello University of California, Berkeley 31 Koshland Hall Berkeley, CA 94720 Phone: (510) 643-5457 emilia-mauriello@berkeley.edu

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

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

Luary Martinez UNAM-Instituto de Biotecnologia Av. Universidad 2001, Colonia Chamilpa Cuernavaca 62210 México Phone: 52-777 329 16 27 luary@ibt.unam.mx

Paul Milewski University of Wisconsin 480 Lincoln Dr. Madison, WI 53706 Phone: (608) 262-3220 milewski@math.wisc.edu

Ana Martinez del Campo UNAM Instituto de Fisiologia Celular Circuito exterior s/n Cd. Universitaria México City 04510 México Phone: (5255) 56225618 acampo@ifc.unam.mx

Kelly Miller West Virginia Univ., Health Sciences Center Box 9177, Room 2077 Morgantown, WV 26506 Phone: (304) 293-5959 kmille49@mix.wvu.edu

142

Makoto Miyata Osaka City University 3-3-138 Sugimoto Sumiyoshi-ku Osaka 558-8585 Japan Phone: +81(6)6605 3157 Fax: +81(6)6605 3158 miyata@sci.osaka-cu.ac.jp

Daisuke Nakane Osaka City University 3-3-138 Sugimoto Sumiyoshi-ku Osaka 558-8585 Japan Phone: +81 6 6605 3157 Fax: +81 6 6605 3158 nakane@sci.osaka-cu.ac.jp

Md Motaleb East Carolina University 600 Moye Blvd BT 116 Greenville, NC 27834 Phone: (252) 744-3129 Fax: (252) 744-3535 motalebm@ecu.edu

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

Tarek Msadek Institut Pasteur 25 Rue du Dr. Roux 75015 Paris France Phone: 33 1 45 68 88 09 Fax: 33 1 45 68 89 38 tmsadek@pasteur.fr

Silke Neumann Ruprecht-Karls-Universität Heidelberg Im Neuenheimer Feld 282 69120 Heidelberg Germany Phone: +49 6221546856 s.neumann@zmbh.uni-heidelberg.de

Abishek Muralimohan University of Wisconsin - Madison 433 Babcock Dr. Madison, WI 53706 Phone: (608) 263-2636 muralimohan@wisc.edu

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

Shuichi Nakamura Osaka Univevrsity Suita1-3,Yamadaoka Osaka, 565-0871 Japan Phone: +81-6-6879-4625 naka@fbs.osaka-u.ac.jp

So-ichiro Nishiyama Hosei University 3-7-2 Kajino-cho, Koganei Koganei 184-8584 Japan Phone: +81-42-387-7173 Fax: +81-42-387-7002 soichiro.nishiyama.s3@k.hosei.ac.jp

143

Takahiro Nonaka Osaka City University 3-3-138 Sugimoto Sumiyoshi-ku Osaka 558-8585 Japan Phone: +81-(0)6-6605-2575 nonaka@sci.osaka-cu.ac.jp

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

Luis Alberto Núñez Oreza Instituto de Fisiologia Celular - UNAM Ciudad Universitaria México D.F. 04510 México Phone: +525556225738 lanoreza@hotmail.com

Chankyu Park KAIST 95014 CA Daejoen 4101, BMRC, KAIST 373-1, Gusung-dong, Yusung-gu Republic of Korea Phone: +82-42-350-2669 nasa@kaist.ac.kr

Ricardo Oropeza Instituto de Biotecnologia Universidad Nacional Autonoma de México Av. UNIVERSIDAD # 2001 col. Chamilpa Cuernavaca 62210 México Phone: 52-777-329-16-27 oropeza@ibt.unam.mx

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

Davi Ortega University of Tennessee / ORNL 2856 Brock Av Knoxville, TN 37919 Phone: (865) 384-9507 dortega@utk.edu

Jonathan Partridge University of Texas at Dallas 800 W Campbell Road Richardson, TX 75080 Phone: (972) 883-2519 j.partridge@gmail.com

Karen Ottemann UC Santa Cruz 1156 High Street METX Santa Cruz, CA 95064 Phone: (831) 459-3482 ottemann@ucsc.edu

Gabriela Peña-Sandoval Universidad Nacional Autonoma de México Circuito Exterior s/n Ciudad Universitaria, Copilco México City 04510 México Phone: (52) 55 5622 5738 gsandova@ifc.unam.mx

144

Abiola Pollard Cornell University Chemistry Research Building G63 S.T. Olin Laboratory Ithaca, NY 14853 Phone: (607) 255-4970 amp65@cornell.edu

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

Steven Porter University of Oxford South Parks Road Oxford OX1 3QU United Kingdom Phone: 01865 275298 steven.porter@bioch.ox.ac.uk

Sylvia Reimann Loyola University Chicago 2160 S. First Ave Maguire Bldg 105, Rm 3822 Maywood, IL 60153 Phone: (708) 216-0845 Fax: (708) 216-9574 sreimann@lumc.edu

Birgit Prüß North Dakota State University 1523 Centennial Blvd. Fargo, ND 58108 Phone: (701) 231-7848 birgit.pruess@ndsu.edu

S. James Remington University of Oregon Institute of Molecular Biology Eugene, OR 97403 Phone: (541) 346-5190 jremington@uoxray.uoregon.edu

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

Peter Reuven Weizmann Institute of Science Ullman Bldg 11-a Rehovot 76100 Israel Phone: 00972 8 934 2701 Fax: 00972 8 934 4112 peter.reuven@weizmann.ac.il

Everardo Ramírez Universidad Nacional Autonoma de México Cto. Exterior, Cd. Universitaria. Coyoacán, D. F. México, D. F. 04510 México Phone: 525556225738 evergrinch@yahoo.com.mx

Mark Roberts University of Oxford South Parks Road Oxford OX1 3QU United Kingdom Phone: 44 1865 613367 Fax: 44 1865 613338 mark.roberts@bioch.ox.ac.uk

145

Claudia Rodríguez UNAM, Instituto de Fisiología Celular Ciudad Universitaria, Col Copilco Distrito Federal 04510 México Phone: 52 01 55 56 22 57 38 Fax: 52 01 55 56 22 56 11 crangel@ifc.unam.mx

Oscar Sánchez UNAM Cto. Ext. S/N Ciudad Universitaria México, D.F. 04510 México Phone: 525556225738 Fax: 525556225611 oscars_69_2@hotmail.com

Mariana Romo Instituto de Fisiología Celular, UNAM Circuito Externo S/N Col Copilco México City 04510 México Phone: 52 55 62 25 965 mromoc@ifc.unam.mx

Birgit Scharf Virginia Polytechnic Institute and State University Life Science I Washington Street Blacksburg, VA 24061 Phone: (617) 495-4217 Fax: (617) 496-1114 bscharf@vt.edu

Kathleen Ryan UC Berkeley Plant & Microbial Biology 251 Koshland Hall Berkeley, CA 94720 Phone: (510) 643-9387 Fax: (510) 642-4995 kryan@nature.berkeley.edu

Florian Schubot Virginia Tech Life Science I, Room 125 Washington Street Blacksburg, VA 24061 Phone: (540) 231-2393 fschubot@vt.edu

Supreet Saini University of Illinois, Urbana Champaign 114 Roger Adams Lab, Box C-3 MC-712, 600 South Mathews Av Urbana, IL 61801 Phone: (217) 244-7528 saini3@uiuc.edu

Andrew Seely Texas A&M University MS 3258 TAMU BSBE Room 303 College Station, TX 77843 Phone: (979) 845-1249 aseely@mail.bio.tamu.edu

Griselda Salas Universidad Nacional Autónoma de México Cto. Exterior, Cd. Universitaria Coyoacán, D. F. México, D. F. 04510 México Phone: 525556225738 gsalas@ifc.unam.mx

Harold Shanafield University of Tennessee 1414 West Cumberland F437 Knoxville, TN 37996 Phone: (865) 974-7687 hshanafi@utk.edu

146

Thomas Shimizu Harvard University 16 Divinity Ave (BL3063) Cambridge, MA 02138 Phone: (617) 495-4217 Fax: (617) 496-1114 tshimizu@mcb.harvard.edu

Stephen Spiro University of Texas at Dallas 800 W Campbell Road Richardson, TX 75080 Phone: (972) 883-6896 stephen.spiro@utdallas.edu

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

Diane Stassi NIH 6701 Rockledge Dr. Room 3202, MSC 7808 Bethesda, MD 20892 Phone: (301) 435-2514 Fax: (301) 480-0940 stassid@csr.nih.gov

Julie Simons University of Wisconsin 480 Lincoln Dr. Madison, WI 53706 Phone: (608) 263-3239 simons@math.wisc.edu

Ann Stock UMDNJ Robert Wood Johnson Medical School Center for Advanced Biotech. and Medicine 679 Hoes Lane Piscataway, NJ 07976 Phone: (732) 235-4844 Fax: (732) 235-5289 stock@cabm.rutgers.edu

Lotte Søgaard-Andersen Max Planck Institute for Terrestrial Microbiology Karl-von-Frisch Str. 35043 Marburg Germany Phone: +49 6421 178 201 Fax: +49 6421 178 209 sogaard@mpi-marburg.mpg.de

Claudia Studdert Universidad Nacional de Mar del Plata Funes 3250 Mar del Plata 7600 Argentina Phone: 54 223 4753030 Fax: 54 223 4753150 studdert@mdp.edu.ar

Erik Sommer University of Heidelberg Im Neuenheimer Feld 282 69120 Heidelberg Germany Phone: +49 6221 546856 e.sommer@zmbh.uni-heidelberg.de

Kalin Swain University of Colorado, Boulder Dept of Chemistry and Biochemistry Campus Box 215 Boulder, CO 80309 Phone: (303) 492-3592 Fax: (303) 493-5894 swain@colorado.edu

147

Hendrik Szurmant The Scripps Research Institute 10550 N Torrey Pines Rd MEM-116 La Jolla, CA 92037 Phone: (858) 784-7904 Fax: (858) 784-7966 szurmant@scripps.edu

Alejandra Vazquez Ramos Universidad Nacional Autónoma de México Av. Universidad #2034 Col. Chamilpa Cuernavaca 62210 México Phone: 52 777 329 1627 Fax: 52 777 331 38673 avazquez@ibt.unam.mx

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

Juan-Jesus Vicente Ruiz University of California, Berkeley 31 Koshland Hall Berkeley, CA 94720 Phone: (510) 643-5457 juanjesus.vicente@gmail.com

Kai Thormann Max Planck Institute For Terrestrial Microbiology Karl-von-Frisch-Strasse D-35043 Marburg Germany Phone: +49 6421 178 302 Fax: +49 6421 178 309 thormann@mpi-marburg.mpg.de

Hera Vlamakis Harvard Medical School 200 Longwood Ave. D1-219 Boston, MA 02115 Phone: (617) 432-4359 hera_vlamakis@hms.harvard.edu

Hoa Tran University of Massachusetts Morrill IV N 639 N.Plesant St. Amherst, MA MA 01003 Phone: (413) 545-9647 htt@chem.umass.edu

George Wadhams Oxford University South Parks Road Oxford OX1 3QU United Kingdom Phone: +44 01865 613329 george.wadhams@bioch.ox.ac.uk

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

Kylie Watts Loma Linda University Dept of Microbiology & Mol Genetics AHBS 120 Loma Linda, CA 92350 Phone: (909) 558-1000 Fax: (909) 558-4035 kwatts@llu.edu

148

Ann West University of Oklahoma 620 Parrington Oval Norman, OK 73019 Phone: (405) 325-1529 Fax: (405) 325-6111 awest@ou.edu

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

Jonathan Willett University of Iowa 51 Newton Road Iowa City, IA 52242 Phone: (319) 335-7938 jonathan-willett@uiowa.edu

Shinsuke Yoshimura Osaka University Suita 3-#601, Yamadaoka 1-chome Osaka 565-0871 Japan Phone: 81-06-6879-4625 Fax: 81-06-6879-4652 yoshimura@fbs.osaka-u.ac.jp

Alan Wolfe Loyola University Chicago Maguire Center 2160 South First Avenue Maywood, IL 60153 Phone: (708) 216-5814 Fax: (708) 216-9574 awolfe@lumc.edu

Junhua Yuan Harvard University 16 Divinity Ave, BioLabs 3063 Cambridge, MA 02138 Phone: (617) 495-4217 jyuan@mcb.harvard.edu

Gus Wright Texas A&M University MS 3258 BSBE 303 College Station, TX 77843 Phone: (979) 845-1249 gwright@mail.bio.tamu.edu

David Zusman University of California 16 Barker Hall #3204 Berkeley, CA 94720-3204 Phone: (510) 642-2293 Fax: (510) 643-6334 zusman@berkeley.edu

Kang Wu University of Illinois, Urbana Champaign 114 RAL Box C-3 (M/C 712) 600 S Mathews Ave, Urbana, IL 61801 Phone: (217) 244-7528 kangwu@uiuc.edu

149

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

150

INDEX

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

151

A Adase, Christopher, 90, 130 - Manson, Michael Airola, Michael, 63, 130 - Crane, Brian Aldridge, Christine, 52, 130 - Aldridge, Phillip Aldridge, Phillip, 52, 53, 130 - Aldridge, Phillip Alexander, Roger, 67, 130 - Emonet, Thierry Alexandre, Gladys, 47, 54, 130 - Alexandre, Gladys Alvarez, Adrian, 69, 130 - Georgellis, Dimitris Andrade, Angel, 130 - González-Pedrajo, Bertha Armitage, Judy, 4, 55, 56, 57, 130 - Armitage, Judy

B Ballado, Teresa, 130 - Dreyfus, Georges Barak, Rina, 132 - Eisenbach, Michael Barba Ostria, Carlos Arturo, 129, 132 - Georgellis, Dimitris Belas, Robert, 37, 132 - Belas, Robert Berleman, James, 12, 132 - Kirby, John Bhatnagar, Jaya, 34, 132 - Crane, Brian Bible, Amber, 47, 132 - Alexandre, Gladys Bisicchia, Paola, 7, 132 - Devine, Kevin Black, Wesley, 125, 132 - Yang, Zhaomin Bollinger, Tarra, 149 - Matsumura, Philip Bourret, Bob, 2, 132 - Bourret, Bob Branch, Richard, 20, 132 - Berry, Richard Briegel, Ariane, 36, 133 - Jensen, Grant Brown, Mostyn, 55, 133 - Armitage, Judith Bulyha, Iryna, 9, 133 - Søgaard-Andersen, Lotte Bustamante, Victor, 133 - Puente, Jose Luis

C Calva, Edmundo, 38, 60, 61, 133 - Calva, Edmundo Camargo, Karen, 133 - Dreyfus, Georges Campbell, Asharie Johnson, 120, 133 - Taylor, Barry Campodonico, Eva, 133 - Zusman, David Cantwell, Brian, 126, 133 - Zhulin, Igor Carlson, C. Britt, 133 - Stock, Ann Castillo, David, 65, 134 - Dreyfus, Georges Chapman, Matt, 40, 134 - Chapman, Matt Chastanet, Arnaud, 50, 134 - Losick, Rich Che, Yong-Suk, 100, 134 - Namba, Keiichi Copeland, Matthew, 123, 134 - Weibel, Douglas Crane, Brian, 34, 63, 64, 134 - Crane, Brian Crosson, Sean, 6, 134 - Crosson, Sean Crowder, Rachel, 91, 134 - Manson, Michael

D Dahlquist, Rick, 134 - Dahlquist, Rick

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

152

De la Cruz, Miguel, 61, 134 - Calva, Edmundo De la Mora, Javier, 66, 135 - Dreyfus, Georges Delalez, Nicolas, 56, 135 - Armitage, Judith Delauné, Aurelia, 98, 135 - Msadek, Tarek Devine, Kevin, 7, 135 - Devine, Kevin Diaz, Miguel, 135 - González-Pedrajo, Bertha Diaz-Mireles, Edith, 59, 135 - Bolam, David Dobkowski, Jason, 9, 135 - Maddock, Janine Draheim, Roger, 121, 135 - von Heijne, Gunnar Dreyfus, Georges, 41, 65, 66, 135 - Dreyfus, Georges Dubrac, Sarah, 8, 135 - Msadek, Tarek

E Eisenbach, Michael, 21, 136 - Eisenbach, Michael Erbse, Annette, 35, 136 - Falke, Joseph

F Falke, Joseph, 35, 68, 136 - Falke, Joseph Falord, Mélanie, 99, 136 - Msadek, Tarek Fukuoka, Hajime, 78, 136 - Ishijima, Akihiko

G Gallego, Ana, 136 - Calva, Edmundo García-Gómez, Elizabeth, 72, 136 - González-Pedrajo, Bertha García-Guerrero, Aldo, 136 - Dreyfus, Georges Gauthier, Mathieu, 19, 136 - Rainville, Simon Gaytán, Meztlli, 136 - González-Pedrajo, Bertha Georgellis, Dimitris, 3, 69, 70, 137 - Georgellis, Dimitris Gitai, Zemer, 14, 137 - Gitai, Zemer Glekas, George, 29, 137 - Ordal, George Goldberg, Shalom, 45, 137 - DeGrado, William Gonzalez, Juan, 44, 71, 137 - Gonzalez, Juan González, Ricardo, 70, 137 - Georgellis, Dimitris González-Pedrajo, Bertha, 72, 137 - González-Pedrajo, Bertha Gurich, Nataliya, 71, 137 - Gonzalez, Juan

H Hall, Benjamin, 113, 137 - Sansom, Mark Han, Hua, 117, 137 - Stock, Ann Harshey, Rasika, 73, 74, 138 - Harshey, Rasika Harwood, Caroline, 138 - Harwood, Carrie Hayashi, Fumio, 83, 84, 85, 138 - Oosawa, Kenji Hazelbauer, Gerald, 138 - Hazelbauer, Gerald Herrera Seitz, María, 115, 138 - Shingler, Victoria Higgs, Penelope, 10, 138 - Higgs, Penelope Hizukuri, Yohei, 75, 138 - Homma, Michio Horne, Shelley, 138 - Prüß, Birgit Hosu, Basarab, 138 - Berg, Howard

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

153

Hughes, Colin, 138 - Hughes, Colin

I Ibuki, Tatsuya, 101, 139 - Namba, Keiichi Imada, Katsumi, 16, 139 - Namba, Keiichi Inaba, Takehiko, 81, 139 - Kawagishi, Ikuro Inoue, Yuichi, 79, 139 - Ishijima, Akihiko

J Josenhans, Christine, 80, 139 - Josenhans, Christine Juarez, Katy, 139 - Juarez, Katy Jung, Kwang-Hwan, 46, 139 - Jung, Kwang -Hwan

K Kaserer, Alla, 23, 139 - West, Ann Kenney, Linda, 139 - Kenney, Linda Kenri, Tsuyoshi, 119, 139 - Takahashi, Motohide Khursigara, Cezar, 32, 140 - Subrmaniam, Sriram Kirby, John, 12, 86, 87, 140 - Kirby, John Kline, Kimberly, 77, 140 - Hultgren, Scott Koike, Masafumi, 76, 140 - Homma, Michio Kojima, Seiji, 17, 140 - Homma, Michio

L Lee, Changhan, 107, 140 - Park, Chankyu Lee, Jae-Min, 73, 140 - Harshey, Rasika Lee, Yi-Ying, 28, 93, 140 - Matsumura, Philip Liu, Jun, 88, 140 - Liu, Jun

M Maddock, Janine, 89, 140 - Maddock, Janine Makino, Fumiaki, 102, 141 - Namba, Keiichi Malpica, Roxana, 109, 141 - Raivio, Tracy Manson, Michael, 31, 90, 91, 92, 141 - Manson, Michael Martínez del Campo, Ana, 41, 141 - Dreyfus, Georges Martinez, Luary, 27, 141 - Puente, Jose Luis Massazza, Diego, 118, 141 - Studdert, Claudia Mauriello, Emilia, 13, 141 - Zusman, David McMurry, Jonathan, 94, 141 - McMurry, Jonathan Milewski, Paul, 95, 141 - Milewski, Paul Miller, Kelly, 62, 141 - Charon, Nyles Miyata, Makoto, 96, 97, 142 - Miyata, Makoto Motaleb, Md, 42, 142 - Motaleb, Md Msadek, Tarek, 8, 98, 99, 142 - Msadek, Tarek Muralimohan, Abishek, 124, 142 - Weibel, Douglas

N Nakamura, Shuichi, 103, 142 - Namba, Keiichi

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

154

Nakane, Daisuke, 96, 142 - Miyata, Makoto Nan, Beiyan, 128, 142 - Zusman, David Neumann, Silke, 49, 142 - Sourjik, Victor Nieto, Vincent, 74, 142 - Harshey, Rasika Nishiyama, So-ichiro, 82, 142 - Kawagishi, Ikuro Nonaka, Takahiro, 97, 143 - Miyata, Makoto Núñez Oreza, Luis Alberto, 143 - Georgellis, Dimitri

O O'Neill, Peggy, 149 - Matsumura, Philip Oropeza, Ricardo, 38, 143 - Calva, Edmundo Ortega, Davi, 143 - Zhulin, Igor Ottemann, Karen, 105, 143 - Ottemann, Karen

P Parales, Rebecca, 106, 143 - Parales, Rebecca Park, Chankyu, 107, 143 - Park, Chankyu Parkinson, John, 143 - Parkinson, John Partridge, John, 24, 143 - Spiro, Stephen Peña-Sandoval, Gabriela, 3, 143 - Georgellis, Dimitris Pollard, Abiola, 64, 144 - Crane, Brian Porter, Steven, 4, 144 - Armitage, Judith Prüß, Birgit, 43, 144 - Prüß, Birgit

R Rainville, Simon, 19, 108, 144 - Rainville, Simon Ramírez, Everardo, 144 - Georgellis, Dimitris Rao, Christopher, 15, 110, 111, 144 - Rao, Christopher Reimann, Sylvia, 25, 144 - Wolfe, Alan Remington, James, 30, 144 - Remington, James Reuven, Peter, 21, 144 - Eisenbach, Michael Roberts, Mark, 57, 144 - Armitage, Judith Rodríguez, Claudia, 145 - Georgellis, Dimitris Romo, Mariana, 145 - González-Pedrajo, Bertha Ryan, Kathleen, 112, 145 - Ryan, Kathleen

S Saini, Supreet, 110, 145 - Rao, Christopher Salas, Griselda, 145 - Georgellis, Dimitris Sánchez, Oscar, 145 - Georgellis, Dimitris Scharf, Birgit, 145 - Scharf, Birgit Schubot, Florian, 114, 145 - Schubot, Florian Seely, Andrew, 92, 145 - Manson, Michael Shanafield, Harold, 127, 145 - Zhulin, Igor Shimizu, Thomas, 48, 146 - Berg, Howard Silversmith, Ruth, 146 - Bourret, Robert Simons, Julie, 95, 146 - Milewski, Paul Søgaard-Andersen, Lotte, 9, 146 - Søgaard-Andersen, Lotte

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

155

Sommer, Erik, 116, 146 - Sourjik, Victor Spiro, Stephen, 24, 146 - Spiro, Stephen Stassi, Diane, 146 - Stassi, Diane Stock, Ann, 117, 146 - Stock, Ann Studdert, Claudia, 118, 146 - Studdert, Claudia Swain, Kalin, 68, 146 - Falke, Joseph Szurmant, Hendrik, 5, 147 - Szurmant, Hendrik

T Taylor, Barry, 33, 120, 147 - Taylor, Barry Thormann, Kai, 18, 147 - Thormann, Kai Tran, Hoa, 26, 147 - Weis, Robert Tu, Yuhai, 22, 147 - Tu, Yuhai

V Vazquez Ramos, Alejandra, 147 - Puente, Jose Luis Vicente Ruiz, Juan-Jesus, 147 - Zusman, David Vlamakis, Hera, 39, 147 - Kolter, Roberto

W Wadhams, George, 147 - Wadhams, George Watts, Kylie, 33, 147 - Taylor, Barry West, Ann, 23, 148 - West, Ann Willett, Jonathan, 87, 148 - Kirby, John Wolfe, Alan, 25, 148 - Wolfe, Alan Wright, Gus, 31, 148 - Manson, Michael Wu, Kang, 111, 148 - Rao, Christopher

Y Yang, Zhaomin, 11, 125, 148 - Yang, Zhaomin Yoshimura, Shinsuke, 104, 148 - Namba, Keiichi Yuan, Junhua, 58, 148 - Berg, Howard

Z Zusman, David, 13, 128, 148 - Zusman, David