The expression of many chemoreceptor genes depends on the cognate chemoeffector as well as on the growth medium and phase.

Chemoreceptor-based signaling is a major bacterial signal transduction mechanism. Escherichia coli, the traditional model, has five chemoreceptors. Recent genome analyses have shown that many bacteria have a much higher number of chemoreceptors. Pseudomonas putida KT2440 is an alternative model that has 27 chemoreceptors and the cognate chemoeffector is known for many of them. Here, we address the question whether and which factors modulate chemoreceptor gene expression. We report reverse transcriptase quantitative PCR measurements of all KT2440 chemoreceptor genes. Transcript levels of individual chemoreceptors differed largely, namely up to 174-fold between the most and least abundant. The cognate chemoeffectors had three different effects on the expression of their chemoreceptor genes. In some cases, the respective chemoeffectors, shown previously to be C- and/or N-sources, induced the expression. In contrast, for the two inorganic phosphate sensing chemoreceptors, the chemoeffector caused dramatic reduction in expression. For four other receptors, including the three TCA cycle intermediate sensing receptors, the chemoeffector did not cause any significant alterations. In addition, a significant number of receptors were induced in minimal growth medium and in the stationary phase. We show here that environmental cues determine largely chemoreceptor expression. This work will serve as reference for analogous studies in other bacteria.

Two different mechanisms mediate chemotaxis to inorganic phosphate in Pseudomonas aeruginosa.

Inorganic phosphate (Pi) is a central signaling molecule that modulates virulence in various pathogens. In Pseudomonas aeruginosa, low Pi concentrations induce transcriptional alterations that increase virulence. Also, under low Pi levels, P. aeruginosa exhibits Pi chemotaxis-a process mediated by the two non-paralogous receptors CtpH and CtpL. Here we show that the two receptors operate via different mechanisms. We demonstrate that the ligand binding domain (LBD) of CtpH but not CtpL binds Pi directly. We identify the periplasmic ligand binding protein PstS as the protein that binds in its Pi loaded state to CtpL, resulting in receptor stimulation. PstS forms part of the Pi transporter and has thus a double function in Pi transport and chemotaxis. The affinity of Pi for CtpH was modest whereas that for PstS very high, which may explain why CtpH and CtpL mediate chemotaxis to high and low Pi concentrations, respectively. The pstS/ctpH double mutant was almost devoid of Pi taxis, indicating that PstS is the only CtpL Pi-shuttle. Chemotaxis mechanisms based on indirect ligand recognition were unambiguously identified in enterobacteria. The discovery of a similar mechanism in a different bacterial order, involving a different chemoreceptor type and chemoeffector suggests that such systems are widespread.

Identification of ligands for bacterial sensor proteins.

Bacteria have evolved a variety of different signal transduction mechanisms. However, the cognate signal molecule for the very large amount of corresponding sensor proteins is unknown and their functional annotation represents a major bottleneck in the field of signal transduction. The knowledge of the signal molecule is an essential prerequisite to understand the signalling mechanisms. Recently, the identification of signal molecules by the high-throughput protein screening of commercially available ligand collections using differential scanning fluorimetry has shown promise to resolve this bottleneck. Based on the analysis of a significant number of different ligand binding domains (LBDs) in our laboratory, we identified two issues that need to be taken into account in the experimental design. Since a number of LBDs require the dimeric state for ligand recognition, it has to be assured that the protein analysed is indeed in the dimeric form. A number of other examples demonstrate that purified LBDs can contain bound ligand which prevents further binding. In such cases, the apo-form can be generated by denaturation and subsequent refolding. We are convinced that this approach will accelerate the functional annotation of sensor proteins which will help to understand regulatory circuits in bacteria.

McpQ is a specific citrate chemoreceptor that responds preferentially to citrate/metal ion complexes.

Chemoreceptors are at the beginning of chemosensory pathways that mediate chemotaxis. Pseudomonas putida KT2440 is predicted to have 27 chemoreceptors, most of which uncharacterized. We have previously identified McpS as chemoreceptor for Krebs cycle intermediates. Citrate is primarily present in the environment as metal complex, which, however, is not recognized by McpS. We show here that the McpS paralogue McpQ recognizes specifically citrate and citrate/metal2+ complexes. The McpQ ligand binding domain (McpQ-LBD) binds citrate/metal2+ complexes with higher affinity than citrate. McpQ-LBD is present in a monomer-dimer equilibrium and citrate and particularly citrate/Mg2+ binding stabilize the dimer. The bacterium showed much stronger responses to citrate/Mg2+ than to citrate and mcpQ inactivation caused a dramatic reduction in chemotaxis. Responses to Krebs cycle intermediates are thus mediated by the broad range McpS and McpQ that responds specifically to an intermediate not recognized by McpS. Interesting parallels exist to the paralogous amino acid chemoreceptors of Pseudomonas aeruginosa and Bacillus subtilis. Whereas one paralogue recognizes most amino acids, the remaining paralogue binds specifically one of the few acids not recognized by the broad range receptors. Therefore, chemotaxis to compound families by the concerted action of broad and narrow range receptors may represent a general mechanism.

Identification of a Chemoreceptor for C2 and C3 Carboxylic Acids.

Chemoreceptors are at the beginnings of chemosensory signaling cascades that mediate chemotaxis. Most bacterial chemoreceptors are functionally unannotated and are characterized by a diversity in the structure of their ligand binding domains (LBDs). The data available indicate that there are two major chemoreceptor families at the functional level, namely, those that respond to amino acids or to Krebs cycle intermediates. Since pseudomonads show chemotaxis to many different compounds and possess different types of chemoreceptors, they are model organisms to establish relationships between chemoreceptor structure and function. Here, we identify PP2861 (termed McpP) of Pseudomonas putida KT2440 as a chemoreceptor with a novel ligand profile. We show that the recombinant McpP LBD recognizes acetate, pyruvate, propionate, and l-lactate, with KD (equilibrium dissociation constant) values ranging from 34 to 107 µM. Deletion of the mcpP gene resulted in a dramatic reduction in chemotaxis toward these ligands, and complementation restored a native-like phenotype, indicating that McpP is the major chemoreceptor for these compounds. McpP has a CACHE-type LBD, and we present data indicating that CACHE-containing chemoreceptors of other species also mediate taxis to C2 and C3 carboxylic acids. In addition, the LBD of NbaY of Pseudomonas fluorescens, an McpP homologue mediating chemotaxis to 2-nitrobenzoate, bound neither nitrobenzoates nor the McpP ligands. This work provides further insight into receptor structure-function relationships and will be helpful to annotate chemoreceptors of other bacteria.

Specific gamma-aminobutyrate chemotaxis in pseudomonads with different lifestyle.

The PctC chemoreceptor of Pseudomonas aeruginosa mediates chemotaxis with high specificity to gamma-aminobutyric acid (GABA). This compound is present everywhere in nature and has multiple functions, including being a human neurotransmitter or plant signaling compound. Because P. aeruginosa is ubiquitously distributed in nature and able to infect and colonize different hosts, the physiological relevance of GABA taxis is unclear, but it has been suggested that bacterial attraction to neurotransmitters may enhance virulence. We report the identification of McpG as a specific GABA chemoreceptor in non-pathogenic Pseudomonas putida KT2440. As with PctC, GABA was found to bind McpG tightly. The analysis of chimeras comprising the PctC and McpG ligand-binding domains fused to the Tar signaling domain showed very high GABA sensitivities. We also show that PctC inactivation does not alter virulence in Caenorhabditis elegans. Significant amounts of GABA were detected in tomato root exudates, and deletion of mcpG reduced root colonization that requires chemotaxis through agar. The C. elegans data and the detection of a GABA receptor in non-pathogenic species indicate that GABA taxis may not be related to virulence in animal systems but may be of importance in the context of colonization and infection of plant roots by soil-dwelling pseudomonads.

High specificity in CheR methyltransferase function: CheR2 of Pseudomonas putida is essential for chemotaxis, whereas CheR1 is involved in biofilm formation.

Chemosensory pathways are a major signal transduction mechanism in bacteria. CheR methyltransferases catalyze the methylation of the cytosolic signaling domain of chemoreceptors and are among the core proteins of chemosensory cascades. These enzymes have primarily been studied Escherichia coli and Salmonella typhimurium, which possess a single CheR involved in chemotaxis. Many other bacteria possess multiple cheR genes. Because the sequences of chemoreceptor signaling domains are highly conserved, it remains to be established with what degree of specificity CheR paralogues exert their activity. We report here a comparative analysis of the three CheR paralogues of Pseudomonas putida. Isothermal titration calorimetry studies show that these paralogues bind the product of the methylation reaction, S-adenosylhomocysteine, with much higher affinity (KD of 0.14-2.2 µM) than the substrate S-adenosylmethionine (KD of 22-43 µM), which indicates product feedback inhibition. Product binding was particularly tight for CheR2. Analytical ultracentrifugation experiments demonstrate that CheR2 is monomeric in the absence and presence of S-adenosylmethionine or S-adenosylhomocysteine. Methylation assays show that CheR2, but not the other paralogues, methylates the McpS and McpT chemotaxis receptors. The mutant in CheR2 was deficient in chemotaxis, whereas mutation of CheR1 and CheR3 had either no or little effect on chemotaxis. In contrast, biofilm formation of the CheR1 mutant was largely impaired but not affected in the other mutants. We conclude that CheR2 forms part of a chemotaxis pathway, and CheR1 forms part of a chemosensory route that controls biofilm formation. Data suggest that CheR methyltransferases act with high specificity on their cognate chemoreceptors.

Bioavailability of pollutants and chemotaxis.

The exposure of bacteria to pollutants induces frequently chemoattraction or chemorepellent reactions. Recent research suggests that the capacity to degrade a toxic compound has co-evolved in some bacteria with the capacity to chemotactically react to it. There is an increasing amount of data which show that chemoattraction to biodegradable pollutants increases their bioavailability which translates into an enhancement of the biodegradation rate. Pollutant chemoreceptors so far identified are encoded on degradation or resistance plasmids. Genetic engineering of bacteria, such as the transfer of chemoreceptor genes, offers thus the possibility to optimize biodegradation processes.

Evidence for chemoreceptors with bimodular ligand-binding regions harboring two signal-binding sites.

Chemoreceptor-based signaling is a central mechanism in bacterial signal transduction. Receptors are classified according to the size of their ligand-binding region. The well-studied cluster I proteins have a 100- to 150-residue ligand-binding region that contains a single site for chemoattractant recognition. Cluster II receptors, which contain a 220- to 300-residue ligand-binding region and which are almost as abundant as cluster I receptors, remain largely uncharacterized. Here, we report high-resolution structures of the ligand-binding region of the cluster II McpS chemotaxis receptor (McpS-LBR) of Pseudomonas putida KT2440 in complex with different chemoattractants. The structure of McpS-LBR represents a small-molecule binding domain composed of two modules, each able to bind different signal molecules. Malate and succinate were found to bind to the membrane-proximal module, whereas acetate binds to the membrane-distal module. A structural alignment of the two modules revealed that the ligand-binding sites could be superimposed and that amino acids involved in ligand recognition are conserved in both binding sites. Ligand binding to both modules was shown to trigger chemotactic responses. Further analysis showed that McpS-like receptors were found in different classes of proteobacteria, indicating that this mode of response to different carbon sources may be universally distributed. The physiological relevance of the McpS architecture may lie in its capacity to respond with high sensitivity to the preferred carbon sources malate and succinate and, at the same time, mediate lower sensitivity responses to the less preferred but very abundant carbon source acetate.

Bacterial chemotaxis towards aromatic hydrocarbons in Pseudomonas.

Bacterial chemotaxis is an adaptive behaviour, which requires sophisticated information-processing capabilities that cause motile bacteria to either move towards or flee from chemicals. Pseudomonas putida DOT-T1E exhibits the capability to move towards different aromatic hydrocarbons present at a wide range of concentrations. The chemotactic response is mediated by the McpT chemoreceptor encoded by the pGRT1 megaplasmid. Two alleles of mcpT are borne on this plasmid and inactivation of either one led to loss of this chemotactic phenotype. Cloning of mcpT into a plasmid complemented not only the mcpT mutants but also its transfer to other Pseudomonas conferred chemotactic response to high concentrations of toluene and other chemicals. Therefore, the phenomenon of chemotaxis towards toxic compounds at high concentrations is gene-dose dependent. In vitro experiments show that McpT is methylated by CheR and McpT net methylation was diminished in the presence of hydrocarbons, what influences chemotactic movement towards these chemicals.

Diversity at its best: bacterial taxis.

Bacterial taxis is one of the most investigated signal transduction mechanisms. Studies of taxis have primarily used Escherichia coli and Salmonella as model organism. However, more recent studies of other bacterial species revealed a significant diversity in the chemotaxis mechanisms which are reviewed here. Differences include the genomic abundance, size and topology of chemoreceptors, the mode of signal binding, the presence of additional cytoplasmic signal transduction proteins or the motor mechanism. This diversity of chemotactic mechanisms is partly due to the diverse nature of input signals. However, the physiological reasons for the majority of differences in the taxis systems are poorly understood and its elucidation represents a major research need.

Low prevalence of vancomycin-resistant enterococci in clinical samples from hospitalized patients of the Canary Islands, Spain

Over the last decade vancomycin-resistant enterococci (VRE) have emerged as nosocomial pathogens. The aim of this study was to determine the prevalence of VRE in clinical samples from hospitalized patients in the Canary Islands. From April to November 2000, 437 enterococci were isolated from patients hospitalized at the four main health care centers in those islands. Identification to the species level was performed with the GPS-TA (Vitek 1) or the Wider I system. A PCR assay was used to determine the genotype of glycopeptide resistance ( vanA, vanB, vanC1, and vanC2/C3 genes). Only three (0.7%) VRE were detected: one vanA Enterococcus faecalis, and two vanC1 Enterococcus gallinarum. To our knowledge, this is the first VRE study carried out in the Canary Islands hospitals, and the results showed a low prevalence of VRE (AU)

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