Methyl-accepting Chemotaxis Proteins Research Articles

Serine Hydroxymethyltransferase ShrA (PA2444) Controls Rugose Small-Colony Variant Formation in Pseudomonas aeruginosa.

Pseudomonas aeruginosa causes many biofilm infections, and the rugose small-colony variants (RSCVs) of this bacterium are important for infection. We found here that inactivation of PA2444, which we determined to be a serine hydroxymethyltransferase (SHMT), leads to the RSCV phenotype of P. aeruginosa PA14. In addition, loss of PA2444 increases biofilm formation by two orders of magnitude, increases exopolysaccharide by 45-fold, and abolishes swarming. The RSCV phenotype is related to higher cyclic diguanylate concentrations due to increased activity of the Wsp chemosensory system, including diguanylate cyclase WspR. By characterizing the PA2444 enzyme in vitro, we determined the physiological function of PA2444 protein by relating it to S-adenosylmethionine (SAM) concentrations and methylation of a membrane bound methyl-accepting chemotaxis protein WspA. A whole transcriptome analysis also revealed PA2444 is related to the redox state of the cells, and the altered redox state was demonstrated by an increase in the intracellular NADH/NAD+ ratio. Hence, we provide a mechanism for how an enzyme of central metabolism controls the community behavior of the bacterium, and suggest the PA2444 protein should be named ShrA for serine hydroxymethyltransferase related to rugose colony formation.

Cellular Stoichiometry of Methyl-Accepting Chemotaxis Proteins in Sinorhizobium meliloti.

The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli, the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilisIMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression in S. meliloti is controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower in S. meliloti than in Escherichia coli and Bacillus subtilis Moreover, the chemoreceptor-to-kinase CheA ratio is different from that of E. coli but similar to that of B. subtilis.

Crystal structure of a substrate-binding protein from Rhodothermus marinus reveals a single α/β-domain

Substrate-binding proteins (SBPs) bind to specific ligands and are associated with membrane protein complexes for transport or signal transduction. Most SBPs recognize substrates by the hinge motion between two distinct α/β domains. However, short SBP motifs are often observed in protein databases, which are located around methyl-accepting chemotaxis protein genes, but structural and functional studies have yet to be performed. Here, we report the crystal structure of an unusually small SBP from Rhodothermus marinus (named as RmSBP) at 1.9 Å. This protein is composed of a single α/β-domain, unlike general SBPs that have two distinct domains. RmSBP exhibits a high structural similarity to the C-terminal domain of the previously reported amino acid bound SBPs, while it does not contain an N-terminal domain for substrate recognition. As a result of the structural comparison analysis, RmSBP has a putative SBP that is different from the previously reported SBP. Our results provide insight into a new class of substrate recognition mechanism by the mini SBP protein.

Methyl-accepting chemotaxis like Rv3499c (Mce4A) protein in Mycobacterium tuberculosis H37Rv mediates cholesterol-dependent survival

Cholesterol, an essential cellular component in macrophages, is exploited for entry and long-term survival of Mycobacterium inside the host. Cholesterol-deficient macrophages can restrict the cholesterol-dependent entry of Mycobacterium. Rv3499c protein in Mycobacterium has high binding affinity for cholesterol. Rv3499c gene is a part of mce4 operon which is reported to act as cholesterol transport system in mycobacteria. Earlier we reported Rv3499c protein to localise on cell wall and facilitate entry of Mycobacterium inside macrophages. Here we performed fold recognition and multiple sequence alignment to find similarity with methyl-accepting chemotaxis protein (MCP). MCP allows detection of level of nutrient in the medium, which in this case is cholesterol. We showed Rv3499c protein expression is important for host cholesterol utilization by Mycobacterium for its survival. Infected female balb/c mice presented increased CFU of Rv3499c overexpressing M. tuberculosis H37Rv marked with early disease conditions and increased lung pathology. Thus, findings suggest specific domain of MCP of Rv3499c help in regulation of downstream PDIM synthesis pathways for ligand utilization by M. tuberculosis H37Rv.

Involvement of many chemotaxis sensors in negative chemotaxis to ethanol in Ralstonia pseudosolanacearum Ps29.

Ralstonia pseudosolanacearum Ps29 showed repellent responses to alcohols including methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 1,3-propanediol and prenol. R. pseudosolanacearum Ps29 possesses 22 putative chemoreceptors known as methyl-accepting chemotaxis proteins (MCPs). To identify a MCP involved in negative chemotaxis to ethanol, we measured ethanol chemotaxis of a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29. However, all the mutants showed repellent responses to ethanol comparable to that of the wild-type strain. We constructed a stepwise- and multiple-mcp gene deletion mutant collection of R. pseudosolanacearum Ps29. Analysis of the collection found that an 18-mcp-knockout mutant (strain POC18) failed to respond to ethanol. Complementation analysis using POC18 as the host strain found that introduction of mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 restored the ability of POC18 to respond to ethanol. However, unexpectedly, strain POC10II, harbouring unmarked deletions in 10 mcp genes including mcpA, mcpT, mcp09, mcpM, mcp15 and mcp19 showed repellent responses to ethanol comparable to that of wild-type Ps29. We hypothesised that multiple mcp mutations in POC18 led to a shortage of MCPs required for formation of functional chemoreceptor arrays. When pPS16 (encoding McpP involved in phosphate chemotaxis) was introduced into POC18, POC18(pPS16) did not respond to phosphate. This result supports the hypothesis. But, genetic analysis revealed that MCPs (Mcp07, Mcp13, Mcp20 and Mcp21) are not essential for ethanol chemotaxis. Thus, we conclude that many and unspecified MCPs are involved in negative chemotaxis to ethanol in R. pseudosolanacearum Ps29.

Methyl-accepting chemotaxis proteins: a core sensing element in prokaryotes and archaea.

Chemotaxis is the directed motility by means of which microbes sense chemical cues and relocate towards more favorable environments. Methyl-accepting chemotaxis proteins (MCPs) are the most common receptors in bacteria and archaea. They are arranged as trimers of dimers that, in turn, form hexagonal arrays in the cytoplasmic membrane or in the cytoplasm. Several different classes of MCPs have been identified according to their ligand binding region and membrane topology. MCPs have been further classified based on the length and sequence conservation of their cytoplasmic domains. Clusters of membrane-embedded MCPs often localize to the poles of the cell, whereas cytoplasmic MCPs can be targeted to the poles or distributed throughout the cell body. MCPs play an important role in cell survival, pathogenesis, and biodegradation. Bacterial adaptation to diverse environmental conditions promotes diversity among the MCPs. This review summarizes structure, classification, and structure-activity relationship of the known MCP receptors, with a brief overview of the signal transduction mechanisms in bacteria and archaea.

Scarless deletion of up to seven methyl-accepting chemotaxis genes with an optimized method highlights key function of CheM in Salmonella Typhimurium.

Site-directed scarless mutagenesis is an essential tool of modern pathogenesis research. We describe an optimized two-step protocol for genome editing in Salmonella enterica serovar Typhimurium to enable multiple sequential mutagenesis steps in a single strain. The system is based on the λ Red recombinase-catalyzed integration of a selectable antibiotics resistance marker followed by replacement of this cassette. Markerless mutants are selected by expressing the meganuclease I-SceI which induces double-strand breaks in bacteria still harboring the resistance locus. Our new dual-functional plasmid pWRG730 allows for heat-inducible expression of the λ Red recombinase and tet-inducible production of I-SceI. Methyl-accepting chemotaxis proteins (MCP) are transmembrane chemoreceptors for a vast set of environmental signals including amino acids, sugars, ions and oxygen. Based on the sensory input of MCPs, chemotaxis is a key component for Salmonella virulence. To determine the contribution of individual MCPs we sequentially deleted seven MCP genes. The individual mutations were validated by PCR and genetic integrity of the final seven MCP mutant WRG279 was confirmed by whole genome sequencing. The successive MCP mutants were functionally tested in a HeLa cell infection model which revealed increased invasion rates for non-chemotactic mutants and strains lacking the MCP CheM (Tar). The phenotype of WRG279 was reversed with plasmid-based expression of CheM. The complemented WRG279 mutant showed also partially restored chemotaxis in swarming assays on semi-solid agar. Our optimized scarless deletion protocol enables efficient and precise manipulation of the Salmonella genome. As demonstrated with whole genome sequencing, multiple subsequent mutagenesis steps can be realized without the introduction of unwanted mutations. The sequential deletion of seven MCP genes revealed a significant role of CheM for the interaction of S. Typhimurium with host cells which might give new insights into mechanisms of Salmonella host cell sensing.

Stringent factor and proteolysis control of sigma factor RpoS expression in Vibrio cholerae

Vibrio cholerae can colonize the gastrointestinal track of humans and cause the disease cholera. During colonization, the alternative sigma factor, RpoS, controls a process known as “mucosal escape response,” defining a specific spatial and temporal response and effecting chemotaxis and motility. In this report, the expression and proteolytic control of RpoS in V. cholerae was characterized. To date, aspects of proteolysis control, the involved components, and proteolysis regulation have not been addressed for RpoS in V. cholerae. Similar to Escherichia coli, we find that the RpoS protein is subjected to regulated intracellular proteolysis, which is mediated by homologues of the proteolysis-targeting factor RssB and the protease complex ClpXP. As demonstrated, RpoS expression transiently peaks after cells are shifted from rich to minimal growth medium. This peak level is dependent on (p)ppGpp-activated rpoS transcription and controlled RpoS proteolysis. The RpoS peak level also correlates with induction of a chemotaxis gene, encoding a methyl-accepting chemotaxis protein, earlier identified to belong to the mucosal escape response pathway. These results suggest that the RpoS expression peak is linked to (p)ppGpp alarmone increase, leading to enhanced motility and chemotaxis, and possibly contributing to the mucosal escape response.

Mechanism and Role of Globin-Coupled Sensor Signalling.

The discovery of the globin-coupled sensor (GCS) family of haem proteins has provided new insights into signalling proteins and pathways by which organisms sense and respond to changing oxygen levels. GCS proteins consist of a sensor globin domain linked to a variety of output domains, suggesting roles in controlling numerous cellular pathways, and behaviours in response to changing oxygen concentration. Members of this family of proteins have been identified in the genomes of numerous organisms and characterization of GCS with output domains, including methyl accepting chemotaxis proteins, kinases, and diguanylate cyclases, have yielded an understanding of the mechanism by which oxygen controls activity of GCS protein output domains, as well as downstream proteins and pathways regulated by GCS signalling. Future studies will expand our understanding of these proteins both in vitro and in vivo, likely demonstrating broad roles for GCS in controlling oxygen-dependent microbial physiology and phenotypes.

Comparative Genomic and Phenotypic Characterization of Pathogenic and Non-Pathogenic Strains of Xanthomonas arboricola Reveals Insights into the Infection Process of Bacterial Spot Disease of Stone Fruits.

Xanthomonas arboricola pv. pruni is the causal agent of bacterial spot disease of stone fruits, a quarantinable pathogen in several areas worldwide, including the European Union. In order to develop efficient control methods for this disease, it is necessary to improve the understanding of the key determinants associated with host restriction, colonization and the development of pathogenesis. After an initial characterization, by multilocus sequence analysis, of 15 strains of X. arboricola isolated from Prunus, one strain did not group into the pathovar pruni or into other pathovars of this species and therefore it was identified and defined as a X. arboricola pv. pruni look-a-like. This non-pathogenic strain and two typical strains of X. arboricola pv. pruni were selected for a whole genome and phenotype comparative analysis in features associated with the pathogenesis process in Xanthomonas. Comparative analysis among these bacterial strains isolated from Prunus spp. and the inclusion of 15 publicly available genome sequences from other pathogenic and non-pathogenic strains of X. arboricola revealed variations in the phenotype associated with variations in the profiles of TonB-dependent transporters, sensors of the two-component regulatory system, methyl accepting chemotaxis proteins, components of the flagella and the type IV pilus, as well as in the repertoire of cell-wall degrading enzymes and the components of the type III secretion system and related effectors. These variations provide a global overview of those mechanisms that could be associated with the development of bacterial spot disease. Additionally, it pointed out some features that might influence the host specificity and the variable virulence observed in X. arboricola.

Energy Taxis toward Host-Derived Nitrate Supports a Salmonella Pathogenicity Island 1-Independent Mechanism of Invasion

ABSTRACTSalmonella enterica serovar Typhimurium can cross the epithelial barrier using either the invasion-associated type III secretion system (T3SS-1) or a T3SS-1-independent mechanism that remains poorly characterized. Here we show that flagellum-mediated motility supported a T3SS-1-independent pathway for entering ileal Peyer’s patches in the mouse model. Flagellum-dependent invasion of Peyer’s patches required energy taxis toward nitrate, which was mediated by the methyl-accepting chemotaxis protein (MCP) Tsr. Generation of nitrate in the intestinal lumen required inducible nitric oxide synthase (iNOS), which was synthesized constitutively in the mucosa of the terminal ileum but not in the jejunum, duodenum, or cecum. Tsr-mediated invasion of ileal Peyer’s patches was abrogated in mice deficient for Nos2, the gene encoding iNOS. We conclude that Tsr-mediated energy taxis enables S. Typhimurium to migrate toward the intestinal epithelium by sensing host-derived nitrate, thereby contributing to invasion of Peyer’s patches.

Direct sensing and signal transduction during bacterial chemotaxis toward aromatic compounds in Comamonas testosteroni.

Micro-organisms sense and chemotactically respond to aromatic compounds. Although the existence of chemoreceptors that bind to aromatic attractants and subsequently trigger chemotaxis have long been speculated, such a chemoreceptor has not been demonstrated. In this report, we demonstrated that the chemoreceptor MCP2901 from Comamonas testosteroni CNB-1 binds to aromatic compounds and initiates downstream chemotactic signaling in addition to its ability to trigger chemotaxis via citrate binding. The function of gene MCP2901 was investigated by genetic deletion from CNB-1 and genetic complementation of the methyl-accepting chemotaxis protein (MCP)-null mutant CNB-1Δ20. Results showed that the expression of MCP2901 in the MCP-null mutant restored chemotaxis toward nine tested aromatic compounds and nine carboxylic acids. Isothermal titration calorimetry (ITC) analyses demonstrated that the ligand-binding domain of MCP2901 (MCP2901LBD) bound to citrate, and weakly to gentisate and 4-hydroxybenzoate. Additionally, ITC assays indicated that MCP2901LBD bound strongly to 2,6-dihydroxybenzoate and 2-hydroxybenzoate, which are isomers of gentisate and 4-hydroxybenzoate respectively that are not metabolized by CNB-1. Agarose-in-plug and capillary assays showed that these two molecules serve as chemoattractants for CNB-1. Through constructing membrane-like MCP2901-inserted Nanodiscs and phosphorelay activity assays, we demonstrated that 2,6-dihydroxybenzoate and 2-hydroxybenzoate altered kinase activity of CheA. This is the first evidence of an MCP binding to an aromatic molecule and triggering signal transduction for bacterial chemotaxis.

Twitching motility and cAMP levels: signal transduction through a single methyl-accepting chemotaxis protein.

The Pseudomonas aeruginosa Chp chemosensory system regulates twitching motility, intracellular adenosine 3('') 5(')-cyclic monophosphate (cAMP) levels and is postulated to be involved in directional twitching towards phosphatidylethanolamine (PE). Because PilJ is the only methyl-accepting chemotaxis protein (MCP) identified in the Chp system, we determined the role of PilJ in mediating signal transduction for the distinct outputs of this system. Mutants that lack the periplasmic domain of PilJ (pilJΔ74-273) showed lower levels of cAMP but retained directional twitching towards PE. While initial studies revealed reduced twitching motility by PilJΔ74-273, this was due to decreased cAMP levels. Our data illustrate the importance of the periplasmic domain of PilJ in regulating cAMP. This is the first time a defined domain within PilJ has been identified as having a distinct role in signal transduction.

Molecular and Biochemical Characterization of Laboratory and Field Mutants of Botrytis cinerea Resistant to Fludioxonil.

Botrytis cinerea is a filamentous phytopathogen with a high risk of developing resistance to fungicides. The phenylpyrrole fungicide fludioxonil has been reported to have excellent activity against B. cinerea and increasingly has been applied to control gray mold in China. In this study, molecular and biochemical characteristics of laboratory and field mutants of B. cinerea resistant to fludioxonil has been investigated. During 2012 to 2014, B. cinerea isolates collected from Jiangsu and Shandong Provinces in China were tested in vitro for sensitivity to fungicides commonly used to suppress gray mold of cucumber and tomato. Among the 75 isolates collected from cucumber in 2013, two were highly resistant (HR) to fludioxonil. Of the 308 isolates collected from tomato in 2014, four were fludioxonil-HR. This was the first time that B. cinerea isolates HR to fludioxonil had been detected in the field. Six fludioxonil-resistant mutants were obtained in the laboratory by selection on fungicide-amended media. These mutants exhibited stable resistance to fludioxonil, as indicated by resistance factor values that ranged from 34.38 to >10,000. In comparison with fludioxonil-sensitive isolates of B. cinerea, all field and laboratory mutants showed reduced fitness, as defined by mycelial growth, sporulation, virulence, and sensitivity to osmotic stress. When treated with fludioxonil at 1 μg/ml, sensitive isolates showed increased glycerol contents in mycelium and expression levels of Bchog1, while levels in field and laboratory HR mutants increased only slightly. Sequences of the Bos1 gene of field and laboratory fludioxonil-HR mutants showed that mutations in field mutants were located in the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) domains of the N-terminal region, whereas mutations in the laboratory mutants were distributed in HAMP domains or in the HATPase_c domain of the C-terminal region. These results will enhance our understanding of the resistance mechanism of B. cinerea to fludioxonil.

Borrelia burgdorferi CheD Promotes Various Functions in Chemotaxis and the Pathogenic Life Cycle of the Spirochete.

Borrelia burgdorferi possesses a sophisticated chemotaxis signaling system; however, the roles of the majority of the chemotaxis proteins in the infectious life cycle have not yet been demonstrated. Specifically, the role of CheD during host colonization has not been demonstrated in any bacterium. Here, we systematically characterized the B. burgdorferi CheD homolog using genetics and biochemical and mouse-tick-mouse infection cycle studies. Bacillus subtilis CheD plays an important role in chemotaxis by deamidation of methyl-accepting chemotaxis protein receptors (MCPs) and by increasing the receptor kinase activity or enhancing CheC phosphatase activity, thereby regulating the levels of the CheY response regulator. Our biochemical analysis indicates that B. burgdorferi CheD significantly enhances CheX phosphatase activity by specifically interacting with the phosphatase. Moreover, CheD specifically binds two of the six MCPs, indicating that CheD may also modulate the receptor proteins. Although the motility of the cheD mutant cells was indistinguishable from that of the wild-type cells, the mutant did exhibit reduced chemotaxis. Importantly, the mutant showed significantly reduced infectivity in C3H/HeN mice via needle inoculation. Mouse-tick-mouse infection assays indicated that CheD is dispensable for acquisition or transmission of spirochetes; however, the viability of cheD mutants in ticks is marginally reduced compared to that of the wild-type or complemented cheD spirochetes. These data suggest that CheD plays an important role in the chemotaxis and pathogenesis of B. burgdorferi We propose potential connections between CheD, CheX, and MCPs and discuss how these interactions play critical roles during the infectious life cycle of the spirochete.

Chemotactic Signaling by Single-Chain Chemoreceptors.

Bacterial chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family operate in commingled clusters that enable cells to detect and track environmental chemical gradients with high sensitivity and precision. MCP homodimers of different detection specificities form mixed trimers of dimers that facilitate inter-receptor communication in core signaling complexes, which in turn assemble into a large signaling network. The two subunits of each homodimeric receptor molecule occupy different locations in the core complexes. One subunit participates in trimer-stabilizing interactions at the trimer axis, the other lies on the periphery of the trimer, where it can interact with two cytoplasmic proteins: CheA, a signaling autokinase, and CheW, which couples CheA activity to receptor control. As a possible tool for independently manipulating receptor subunits in these two structural environments, we constructed and characterized fused genes for the E. coli serine chemoreceptor Tsr that encoded single-chain receptor molecules in which the C-terminus of the first Tsr subunit was covalently connected to the N-terminus of the second with a polypeptide linker. We showed with soft agar assays and with a FRET-based in vivo CheA kinase assay that single-chain Tsr~Tsr molecules could promote serine sensing and chemotaxis responses. The length of the connection between the joined subunits was critical. Linkers nine residues or shorter locked the receptor in a kinase-on state, most likely by distorting the native structure of the receptor HAMP domain. Linkers 22 or more residues in length permitted near-normal Tsr function. Few single-chain molecules were found as monomer-sized proteolytic fragments in cells, indicating that covalently joined receptor subunits were responsible for mediating the signaling responses we observed. However, cysteine-directed crosslinking, spoiling by dominant-negative Tsr subunits, and rearrangement of ligand-binding site lesions revealed subunit swapping interactions that will need to be taken into account in experimental applications of single-chain chemoreceptors.

Hypothetical Protein BB0569 Is Essential for Chemotaxis of the Lyme Disease Spirochete Borrelia burgdorferi.

The Lyme disease spirochete Borrelia burgdorferi has five putative methyl-accepting chemotaxis proteins (MCPs). In this report, we provide evidence that a hypothetical protein, BB0569, is essential for the chemotaxis of B. burgdorferi. While BB0569 lacks significant homology to the canonical MCPs, it contains a conserved domain (spanning residues 110 to 170) that is often evident in membrane-bound MCPs such as Tar and Tsr of Escherichia coli. Unlike Tar and Tsr, BB0569 lacks transmembrane regions and recognizable HAMP and methylation domains and is similar to TlpC, a cytoplasmic chemoreceptor of Rhodobacter sphaeroides. An isogenic mutant of BB0569 constantly runs in one direction and fails to respond to attractants, indicating that BB0569 is essential for chemotaxis. Immunofluorescence, green fluorescent protein (GFP) fusion, and cryo-electron tomography analyses demonstrate that BB0569 localizes at the cell poles and is required for chemoreceptor clustering at the cell poles. Protein cross-linking studies reveal that BB0569 forms large protein complexes with MCP3, indicative of its interactions with other MCPs. Interestingly, analysis of B. burgdorferi mcp mutants shows that inactivation of either mcp2 or mcp3 reduces the level of BB0569 substantially and that such a reduction is caused by protein turnover. Collectively, these results demonstrate that the domain composition and function of BB0569 are similar in some respects to those of TlpC but that these proteins are different in their cellular locations, further highlighting that the chemotaxis of B. burgdorferi is unique and different from the Escherichia coli and Salmonella enterica paradigm. Spirochete chemotaxis differs substantially from the Escherichia coli and Salmonella enterica paradigm, and the basis for controlling the rotation of the bundles of periplasmic flagella at each end of the cell is unknown. In recent years, Borrelia burgdorferi, the causative agent of Lyme disease, has been used as a model organism to understand spirochete chemotaxis and its role in infectious processes of the disease. In this report, BB0569, a hypothetical protein of B. burgdorferi, has been investigated by using an approach of genetic, biochemistry, and cryo-electron tomography analyses. The results indicate that BB0569 has a distinct role in chemotaxis that may be unique to spirochetes and represents a novel paradigm.

Characterization of the Vibrio fischeri Fatty Acid Chemoreceptors, VfcB and VfcB2.

Bacteria use a wide variety of methyl-accepting chemotaxis proteins (MCPs) to mediate their attraction to or repulsion from different chemical signals in their environment. The bioluminescent marine bacterium Vibrio fischeri is the monospecific symbiont of the Hawaiian bobtail squid, Euprymna scolopes, and encodes a large repertoire of MCPs that are hypothesized to be used during different parts of its complex, multistage lifestyle. Here, we report the initial characterization of two such MCPs from V. fischeri that are responsible for mediating migration toward short- and medium-chain aliphatic (or fatty) acids. These receptors appear to be distributed among only members of the family Vibrionaceae and are likely descended from a receptor that has been lost by the majority of the members of this family. While chemotaxis greatly enhances the efficiency of host colonization by V. fischeri, fatty acids do not appear to be used as a chemical cue during this stage of the symbiosis. This study presents an example of straight-chain fatty acid chemoattraction and contributes to the growing body of characterized MCP-ligand interactions.

Structural basis for amino-acid recognition and transmembrane signalling by tandem Per–Arnt–Sim (tandem PAS) chemoreceptor sensory domains

Chemotaxis, mediated by methyl-accepting chemotaxis protein (MCP) receptors, plays an important role in the ecology of bacterial populations. This paper presents the first crystallographic analysis of the structure and ligand-induced conformational changes of the periplasmic tandem Per-Arnt-Sim (PAS) sensing domain (PTPSD) of a characterized MCP chemoreceptor. Analysis of the complex of the Campylobacter jejuni Tlp3 PTPSD with isoleucine (a chemoattractant) revealed that the PTPSD is a dimer in the crystal. The two ligand-binding sites are located in the membrane-distal PAS domains on the faces opposite to the dimer interface. Mutagenesis experiments show that the five strongly conserved residues that stabilize the main-chain moiety of isoleucine are essential for binding, suggesting that the mechanism by which this family of chemoreceptors recognizes amino acids is highly conserved. Although the fold and mode of ligand binding of the PTPSD are different from the aspartic acid receptor Tar, the structural analysis suggests that the PTPSDs of amino-acid chemoreceptors are also likely to signal by a piston displacement mechanism. The PTPSD fluctuates between piston (C-terminal helix) `up' and piston `down' states. Binding of an attractant to the distal PAS domain locks it in the closed form, weakening its association with the proximal domain and resulting in the transition of the latter into an open form, concomitant with a downward (towards the membrane) 4 Å piston displacement of the C-terminal helix. In vivo, this movement would generate a transmembrane signal by driving a downward displacement of the transmembrane helix 2 towards the cytoplasm.

Identification of the mcpA and mcpM genes, encoding methyl-accepting proteins involved in amino acid and l-malate chemotaxis, and involvement of McpM-mediated chemotaxis in plant infection by Ralstonia pseudosolanacearum (formerly Ralstonia solanacearum phylotypes I and III).

Sequence analysis has revealed the presence of 22 putative methyl-accepting chemotaxis protein (mcp) genes in the Ralstonia pseudosolanacearum GMI1000 genome. PCR analysis and DNA sequencing showed that the highly motile R. pseudosolanacearum strain Ps29 possesses homologs of all 22 R. pseudosolanacearum GMI1000 mcp genes. We constructed a complete collection of single mcp gene deletion mutants of R. pseudosolanacearum Ps29 by unmarked gene deletion. Screening of the mutant collection revealed that R. pseudosolanacearum Ps29 mutants of RSp0507 and RSc0606 homologs were defective in chemotaxis to l-malate and amino acids, respectively. RSp0507 and RSc0606 homologs were designated mcpM and mcpA. While wild-type R. pseudosolanacearum strain Ps29 displayed attraction to 16 amino acids, the mcpA mutant showed no response to 12 of these amino acids and decreased responses to 4 amino acids. We constructed mcpA and mcpM deletion mutants of highly virulent R. pseudosolanacearum strain MAFF106611 to investigate the contribution of chemotaxis to l-malate and amino acids to tomato plant infection. Neither single mutant exhibited altered virulence for tomato plants when tested by root dip inoculation assays. In contrast, the mcpM mutant (but not the mcpA mutant) was significantly less infectious than the wild type when tested by a sand soak inoculation assay, which requires bacteria to locate and invade host roots from sand. Thus, McpM-mediated chemotaxis, possibly reflecting chemotaxis to l-malate, facilitates R. pseudosolanacearum motility to tomato roots in sand.