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

PRESENTER TITLE ABSTRACT PAGE NO.

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

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

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

BREAK Boll, Joseph Michael

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

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

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

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

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

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

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

BREAK

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

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

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

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

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

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

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

BREAK Lertsethtakarn, Paphavee

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

Sukomon, Nattakan

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

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

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

PRESENTER TITLE ABSTRACT PAGE NO.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

PRESENTER TITLE ABSTRACT PAGE NO.

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

Reverse rotation in bacterial flagellar motors at high hydrostatic pressures 38

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

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

BREAK Tipping, Murray Light-powering the flagellar motor 41

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

Schniederberend, Maren

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

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

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

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

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

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

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

Rhodes, Ryan

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

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

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SPEAKER ABSTRACTS

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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Lab: Jay Tang ___________

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

, Tailin Wu2, Zhuoran He2, Lili jiang2

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

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

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

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

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

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

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

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

Lab: Eugene Koonin ____

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

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

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

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

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

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

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

Lab: Joseph Falke

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

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

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

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Lab: Mark Sansom ____

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

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

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

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

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

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

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

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

Lab: Barry Taylor ____

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Lab: Kristen Jung ____

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Phosphorylation-dependent signal transduction via two-component signal transduction

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

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

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

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

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

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

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

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

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

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

Lab: Masayoshi Nishiyama ____

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

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

Lab: Rasika Harshey ____

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

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

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

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

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

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

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

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

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

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

Lab: Michael Eisenbach ____

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

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

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

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

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

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

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

Lab: Oleg Ioshin ____

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

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

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

Lab: David Zusman ____

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

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

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

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

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

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

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

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

Lab: Mark McBride ____

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

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

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

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