Genome analyses indicate that many bacteria possess an elevated number of chemoreceptors, suggesting that these species are able to perform chemotaxis to a wide variety of compounds. The scientific community is now only beginning to explore this diversity and to elucidate the corresponding physiological relevance. The discovery of histamine chemotaxis in the human pathogen Pseudomonas aeruginosa provides insight into tactic movements that occur within the host. Since histamine is released in response to bacterial pathogens, histamine chemotaxis may permit bacterial migration and accumulation at infection sites, potentially modulating, in turn, quorum-sensing-mediated processes and the expression of virulence genes. As a consequence, the modulation of histamine chemotaxis by signal analogues may result in alterations of the bacterial virulence. As the first report of bacterial histamine chemotaxis, this study lays the foundation for the exploration of the physiological relevance of histamine chemotaxis and its role in pathogenicity.

Quorum sensing is a bacterial communication mechanism that controls genes, enabling bacteria to live as communities, such as biofilms. Homoserine lactone (HSL) molecules function as quorum-sensing signals for Gram-negative bacteria. Plants also produce previously unidentified compounds that affect quorum sensing. We identified rosmarinic acid as a plant-derived compound that functioned as an HSL mimic. In vitro assays showed that rosmarinic acid bound to the quorum-sensing regulator RhlR of Pseudomonas aeruginosa PAO1 and competed with the bacterial ligand N-butanoyl-homoserine lactone (C4-HSL). Furthermore, rosmarinic acid stimulated a greater increase in RhlR-mediated transcription in vitro than that of C4-HSL. In P. aeruginosa, rosmarinic acid induced quorum sensing-dependent gene expression and increased biofilm formation and the production of the virulence factors pyocyanin and elastase. Because P. aeruginosa PAO1 infection induces rosmarinic acid secretion from plant roots, our results indicate that rosmarinic acid secretion is a plant defense mechanism to stimulate a premature quorum-sensing response. P. aeruginosa is a ubiquitous pathogen that infects plants and animals; therefore, identification of rosmarinic acid as an inducer of premature quorum-sensing responses may be useful in agriculture and inform human therapeutic strategies.

SummaryThe PctC chemoreceptor of Pseudomonas aeruginosa mediates chemotaxis with high specificity to gammaaminobutyric 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 nonpathogenic 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.

Although it is well established that one- and two-component regulatory systems participate in regulating biofilm formation, there also exists evidence suggesting that chemosensory pathways are also involved. However, little information exists about which chemoreceptors and signals modulate this process. Here we report the generation of the complete set of chemoreceptor mutants of Pseudomonas putida KT2440 and the identification of four mutants with significantly altered biofilm phenotypes. These receptors are a WspA homologue of Pseudomonas aeruginosa, previously identified to control biofilm formation by regulating c-di-GMP levels, and three uncharacterized chemoreceptors. One of these receptors, named McpU, was found to mediate chemotaxis towards different polyamines. The functional annotation of McpU was initiated by high-throughput thermal shift assays of the receptor ligand binding domain (LBD). Isothermal titration calorimetry showed that McpU-LBD specifically binds putrescine, cadaverine and spermidine, indicating that McpU represents a novel chemoreceptor type. Another uncharacterized receptor, named McpA, specifically binds 12 different proteinogenic amino acids and mediates chemotaxis towards these compounds. We also show that mutants in McpU and WspA-Pp have a significantly reduced ability to colonize plant roots. Data agree with other reports showing that polyamines are signal molecules involved in the regulation of bacteria-plant communication and biofilm formation.

Methyltransferases of the CheR family and methylesterases of the CheB family control chemoreceptor methylation, and this dynamic posttranslational modification is necessary for proper chemotaxis of bacteria. Studies with enterobacteria that contain a single CheR or CheB show that, in addition to binding at the methylation site, some chemoreceptors bind CheR or CheB through additional high-affinity sites at distinct pentapeptide sequences in the chemoreceptors. We investigated the recognition of chemoreceptors by CheR proteins in the human pathogen Pseudomonas aeruginosa PAO1. Of the four methyltransferases in PAO1, we detected an interaction only between CheR2 and the chemoreceptor methyl-accepting chemotaxis protein B (McpB), which contains the pentapeptide GWEEF at its carboxyl terminus. Furthermore, CheR2 was also the only paralog that methylated McpB in vitro, and deletion of the pentapeptide sequence abolished both the CheR2-McpB interaction and the methylation of McpB. When clustered according to protein sequence, bacterial CheR proteins form two distinct families-those that bind pentapeptide-containing chemoreceptors and those that do not. These two families are distinguished by an insertion of three amino acids in the β-subdomain of CheR. Deletion of this insertion in CheR2 prevented its interaction with and methylation of McpB. Pentapeptide-containing chemoreceptors are common to many bacteria species; thus, these short, distinct motifs may enable the specific assembly of signaling complexes that mediate different responses.

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/metal complexes. The McpQ ligand binding domain (McpQ-LBD) binds citrate/metal complexes with higher affinity than citrate. McpQ-LBD is present in a monomer-dimer equilibrium and citrate and particularly citrate/Mg binding stabilize the dimer. The bacterium showed much stronger responses to citrate/Mg 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.

SummaryChemotaxis is an essential mechanism that enables bacteria to move toward favorable ecological niches. Escherichia coli, the historical model organism for studying chemotaxis, has five well-studied chemoreceptors. However, many bacteria with different lifestyle have more chemoreceptors, most of unknown function. Using a high throughput screening approach, we identified a chemoreceptor from Pseudomonas putida KT2440, named McpH, which specifically recognizes purine and its derivatives, adenine, guanine, xanthine, hypoxanthine and uric acid. The latter five compounds form part of the purine degradation pathway, permitting their use as sole nitrogen sources. Isothermal titration calorimetry studies show that these six compounds bind McpH-Ligand Binding Domain (LBD) with very similar affinity. In contrast, non-metabolizable purine derivatives (caffeine, theophylline, theobromine), nucleotides, nucleosides or pyrimidines are unable to bind McpH-LBD. Mutation of mcpH abolished chemotaxis toward the McpH ligands identified -a phenotype that is restored by complementation. This is the first report on bacterial chemotaxis to purine derivatives and McpH the first chemoreceptor described that responds exclusively to intermediates of a catabolic pathway, illustrating a clear link between metabolism and chemotaxis. The evolution of McpH may reflect a saprophytic lifestyle, which would have exposed the studied bacterium to high concentrations of purines produced by nucleic acid degradation.

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