POSTERS - BLAST XI

Poster # Lab Presenter Title Page #1 Gladys

Alexandre Amber Bible Modulation of clumping and flocculation behavior by a

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

52

2 Karlheinz Altendorf

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

53

3 Judy Armitage

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

54

4 Judy Armitage

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

5 Tatsuo Atsumi

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

56

6 CancelledPoster

57

7 Shannon Au

Shannon AuKwok Ho Lam

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

58

8 Carl Bauer

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

59

9 Robert Belas

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

60

10 Robert Belas

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

61

11 Howard Berg

Richard Branch Binding cooperativity in the bacterial flagellar motor 62

12 Howard Berg

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

63

13 David Blair

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

64Blair of the flagellar stator

14 David Blair

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

65

15 David Blair

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

66

16 David Blair

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

67

17 Nyles Charon

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

68

18 Brian Crane

Xiaoxiao Li Building soluble models of chemoreceptors 69

19 Brian Crane

Ria Sircar Probing the structure of the flagellar switch complex 70

20 Sean Crosson

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

71

21 Rick Dahlquist

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

22 John Dow

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

73

23 Roger Draheim

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

74

POSTERS - BLAST XI

Poster # Lab Presenter Title Page #24 Thierry

EmonetMichael Sneddon Overcoming complexity in systems biology modeling

and simulation with NFsim75

25 Joseph Falke

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

76

26 Joseph Falke

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

77

27 Joseph Falke

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

78

28 Rasika Harshey

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

79

29 Rasika Harshey

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

80

30 Caroline Harwood

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

81

31 Caroline Harwood

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

82

32 Gerald Hazelbauer

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

83

33 Michio Homma

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

84

34 Michio Homma

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

85

driven flagella in Vibrio alginolyticus35 Christine

JosenhansWiebke Behrens Role of the proposed Helicobacter pylori energy sensor

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

86

36 Kirsten Jung

Kirsten Jung Autoinducer-mediated signaling in Vibrio harveyi 87

37 Barbara Kazmierczak

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

88

38 Duncan Krause

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

89

39 Chunhao Li

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

90

40 Jan Liphardt

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

91

41 Jun Liu

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

92

42 Jun Liu

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

93

43 LuhuaLai

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

94

44 Michael Manson

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

95

POSTERS - BLAST XI

Poster # Lab Presenter Title Page #45 Michael

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

46 Richard Marconi

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

97

47 Richard Marconi

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

98

48 Jonathan McMurry

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

99

49 Vincent Méjean

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

100

50 Steven Norris

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

101

51 Christopher O'Connor

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

102

52 Qi Ouyang

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

103

53 Chankyu Park

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

104

54 Chankyu Park

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

105

55 Steven Porter

Steven Porter The GacS phosphorelay of Pseudomonas aeruginosa 106Porter

56 Simon Rainville

Guillaume Paradis Taking control of the bacterial flagellar motor 107

57 Birgit Scharf

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

108

58 Birgit Scharf

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

109

59 Tom Shimizu

Milena Lazova Response rescaling in bacterial chemotaxis 110

60 LotteSøgaard-Andersen

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

111

61 Claudia Studdert

Claudia Studdert Tsr constructions with symmetric heptad deletions display full function

112

62 Lynmarie Thompson

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

113

63 Kai Thormann

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

114

64 Yuhai Tu

Ganhui Lan Mechanical and kinetic principles of bacterial flagellar motor operation

115

65 Mandy Ward

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

116

66 Zhaomin Yang

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

117

POSTERS - BLAST XI

Poster # Lab Presenter Title Page #67 Igor

ZhulinDavi R. Ortega Investigating structural properties of CheW with

molecular dynamics and NMR118

68 David Zusman

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

119

51

POSTER ABSTRACTS

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

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

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

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53

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

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

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

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

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

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

Lab: Karlheinz Altendorf

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Lab: Judy Armitage

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

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

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

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

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

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

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

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

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

57

BLAST XI Poster #6

Poster Cancelled

58

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

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

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

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

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

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

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

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

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

Lab: Robert Belas ___________

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

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

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

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

Lab: Robert Belas ___________

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

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

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

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

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

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

Lab: Howard Berg

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

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

Lab: Howard Berg

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

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

Lab: David F. Blair

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

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

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

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

Lab: David F. Blair

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

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

Lab: David F. Blair

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

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

Lab: Nyles Charon

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

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

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

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

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

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

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

Lab: Brian Crane ____

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

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

Lab: Brian Crane ____

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

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

Lab: Sean Crosson ____

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

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

Lab: Frederick Dahlquist ____

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

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

Lab: John Dow

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

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

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

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

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

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

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

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

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

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

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

Lab: Thierry Emonet ____

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

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

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

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

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

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

Lab: Joseph Falke

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

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

Support Provided by: NIH R01 GM-040731

Lab: Joseph Falke

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

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

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

Lab: Joseph Falke

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

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

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

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

Lab: Rasika M. Harshey

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

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

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

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

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

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

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

Lab: Caroline Harwood ____

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

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

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

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

Lab: Michio Homma____

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

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

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

Lab: Michio Homma____

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

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

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

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

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

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

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

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

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

Lab: Kirsten Jung ____

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

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

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

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

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

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

Lab: Barbara Kazmierczak ____

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

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

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

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

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

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

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

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

Lab: Duncan Krause ____

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Lab: Richard Marconi____

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

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

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

Lab: Jonathan McMurry____

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

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

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

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

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

Lab: Steven Norris ____

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

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

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

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

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

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

Lab: Qi Ouyang ____

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

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

Lab: Chankyu Park ___

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

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

Lab: Chankyu Park ___

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

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

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

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

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

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

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

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

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

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

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Lab: Simon Rainville ___

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

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

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

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

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

Lab: Birgit Sharf ____

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

While single asymmetric deletions abolished serine chemotaxis and caused serious alterations

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

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

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

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

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

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Lab: Lynmarie Thompson

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

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

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

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

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

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

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

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

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

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

CA 93106-9510

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

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

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