Shota Oku

Identification of Chemotaxis Sensory Proteins for Amino Acids in Pseudomonas fluorescens Pf0-1 and Their Involvement in Chemotaxis to Tomato Root Exudate and Root Colonization

Pseudomonas fluorescens Pf0-1 showed positive chemotactic responses toward 20 commonly-occurring l-amino acids. Genomic analysis revealed that P. fluorescens Pf0-1 possesses three genes (Pfl01_0124, Pfl01_0354, and Pfl01_4431) homologous to the Pseudomonas aeruginosa PAO1 pctA gene, which has been identified as a chemotaxis sensory protein for amino acids. When Pf01_4431, Pfl01_0124, and Pfl01_0354 were introduced into the pctA pctB pctC triple mutant of P. aeruginosa PAO1, a mutant defective in chemotaxis to amino acids, its transformants showed chemotactic responses to 18, 16, and one amino acid, respectively. This result suggests that Pf01_4431, Pfl01_0124, and Pfl01_0354 are chemotaxis sensory proteins for amino acids and their genes were designated ctaA, ctaB, and ctaC, respectively. The ctaA ctaB ctaC triple mutant of P. fluorescens Pf0-1 showed only weak responses to Cys and Pro but no responses to the other 18 amino acids, indicating that CtaA, CtaB, and CtaC are major chemotaxis sensory proteins in P. fluorescens Pf0-1. Tomato root colonization by P. fluorescens strains was analyzed by gnotobiotic competitive root colonization assay. It was found that ctaA ctaB ctaC mutant was less competitive than the wild-type strain, suggesting that chemotaxis to amino acids, major components of root exudate, has an important role in root colonization by P. fluorescens Pf0-1. The ctaA ctaB ctaC triple mutant was more competitive than the cheA mutant of P. fluorescens Pf0-1, which is non-chemotactic, but motile. This result suggests that chemoattractants other than amino acids are also involved in root colonization by P. fluorescens Pf0-1.

Identification of Pseudomonas fluorescens chemotaxis sensory proteins for malate, succinate, and fumarate, and their involvement in root colonization.

Pseudomonas fluorescens Pf0-1 exhibited chemotactic responses to l-malate, succinate, and fumarate. We constructed a plasmid library of 37 methyl-accepting chemotaxis protein (MCP) genes of P. fluorescens Pf0-1. To identify a MCP for l-malate, the plasmid library was screened using the PA2652 mutant of Pseudomonas aeruginosa PAO1, a mutant defective in chemotaxis to l-malate. The introduction of Pfl01_0728 and Pfl01_3768 genes restored the ability of the PA2652 mutant to respond to l-malate. The Pfl01_0728 and Pfl01_3768 double mutant of P. fluorescens Pf0-1 showed no response to l-malate or succinate, while the Pfl01_0728 single mutant did not respond to fumarate. These results indicated that Pfl01_0728 and Pfl01_3768 were the major MCPs for l-malate and succinate, and Pfl01_0728 was also a major MCP for fumarate. The Pfl01_0728 and Pfl01_3768 double mutant unexpectedly exhibited stronger responses toward the tomato root exudate and amino acids such as proline, asparagine, methionine, and phenylalanine than those of the wild-type strain. The ctaA, ctaB, ctaC (genes of the major MCPs for amino acids), Pfl01_0728, and Pfl01_3768 quintuple mutant of P. fluorescens Pf0-1 was less competitive than the ctaA ctaB ctaC triple mutant in competitive root colonization, suggesting that chemotaxis to l-malate, succinate, and/or fumarate was involved in tomato root colonization by P. fluorescens Pf0-1.

Identification of CtpL as a chromosomally-encoded chemoreceptor for 4-chloroaniline and catechol in Pseudomonas aeruginosa PAO1

ABSTRACT Bacterial chemotaxis influences the ability of bacteria to survive and thrive in most environments, including polluted ones. Despite numerous reports of the phenotypic characterization of chemotactic bacteria, only a few molecular details of chemoreceptors for aromatic pollutants have been described. In this study, the molecular basis of chemotaxis toward an environmentally toxic chlorinated aromatic pollutant, 4-chloroaniline (4CA), was evaluated. Among the three Pseudomonas spp. tested, Pseudomonas aeruginosa PAO1 exhibited positive chemotaxis both to the nonmetabolizable 4CA, where 4-chloroacetanilide was formed as a dead-end transformation product, and to the metabolizable catechol. Molecular analysis of all 26 mutants with a disrupted methyl-accepting chemotaxis gene revealed that CtpL, a chromosomally encoded chemoreceptor, was responsible for the positive chemotactic response toward 4CA. Since CtpL has previously been described to be a major chemoreceptor for inorganic phosphate at low concentrations in PAO1, this report describes a fortuitous ability of CtpL to function toward aromatic pollutants. In addition, its regulation not only was dependent on the presence of the chemoattractant inducer but also was regulated by conditions of phosphate starvation. These results expand the range of known chemotactic transducers and their function in the environmental bacterium PAO1.

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)

ABSTRACT 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.

Degradation of chloroanilines by toluene dioxygenase from Pseudomonas putida T57

In this study, we investigated the ability of Pseudomonas putida toluene dioxygenase to oxidize chloroanilines. Toluene-induced P. putida T57 cells degraded 4-chloroaniline (4CA) more rapidly than toluene-non-induced cells, suggesting that toluene dioxygenase pathway was involved in 4CA degradation. Escherichia coli harboring P. putida T57 genes encoding toluene dioxygenase complex (todC1C2BA) showed 4CA degradation activity, demonstrating that toluene dioxygenase oxidizes 4CA. Thin-layer chromatography (TLC) and mass spectrometry (MS) analyses identified 4-chlorocatechol and 2-amino-5-chlorophenol as reaction products, suggesting that toluene dioxygenase catalyzes both 1,2- and 2,3-dioxygenation of 4CA. A plasmid containing the entire tod operon (todC1C2BADE) was introduced to P. putida T57 to enhance its ability to degrade 4CA. Resulting P. putida T57 (pHK-C1C2BADE) showed 250-fold higher 4CA degradation activity than P. putida T57 parental strain. P. putida T57 (pHK-C1C2BADE) degraded 2-chloroaniline (2CA), 3-chloroaniline (3CA), and 3,4-dichloroaniline (34DCA) as well as 4CA, but not 3,5-dichloroaniline (35DCA). The order of the degradation rate was: 4CA > 3CA > 2CA > 34DCA.

Identification and characterization of chemosensors for D-malate, unnatural enantiomer of malate, in Ralstonia pseudosolanacearum

Ralstonia pseudosolanacearum Ps29 is attracted by nonmetabolizable d-malate, an unnatural enantiomer. Screening of a complete collection of single-mcp-gene deletion mutants of Ps29 revealed that the RSc1156 homologue is a chemosensor for d-malate. An RSc1156 homologue deletion mutant of Ps29 showed decreased but significant responses to d-malate, suggesting the existence of another d-malate chemosensor. McpM previously had been identified as a chemosensor for l-malate. We constructed an RSc1156 homologue mcpM double deletion mutant and noted that this mutant failed to respond to d-malate; thus, the RSc1156 homologue and McpM are the major chemosensors for d-malate in this organism. To further characterize the ligand specificities of the RSc1156 homologue and McpM, we constructed a Ps29 derivative (designated K18) harbouring deletions in 18 individual mcp genes, including mcpM and RSc1156. K18 harbouring the RSc1156 homologue responded strongly to l-tartrate and d-malate and moderately to d-tartrate, but not to l-malate or succinate. K18 harbouring mcpM responded strongly to l-malate and d-tartrate and moderately to succinate, fumarate and d-malate. Ps29 utilizes l-malate and l-tartrate, but not d-malate. We therefore concluded that l-tartrate and l-malate are natural ligands of the RSc1156 homologue and McpM, respectively, and that chemotaxis toward d-malate is a fortuitous response by the RSc1156 homologue and McpM in Ps29. We propose re-designation of the RSc1156 homologue as McpT. In tomato plant infection assays, the mcpT deletion mutant of highly virulent R. pseudosolanacearum MAFF106611 was as infectious as wild-type MAFF106611, suggesting that McpT-mediated chemotaxis does not play an important role in tomato plant infection.

Identification of boric acid as a novel chemoattractant and elucidation of its chemoreceptor in Ralstonia pseudosolanacearum Ps29

Chemotaxis enables bacteria to move toward more favorable environmental conditions. We observed chemotaxis toward boric acid by Ralstonia pseudosolanacearum Ps29. At higher concentrations, the chemotactic response of R. pseudosolanacearum toward boric acid was comparable to or higher than that toward L-malate, indicating that boric acid is a strong attractant for R. pseudosolanacearum. Chemotaxis assays under different pH conditions suggested that R. pseudosolanacearum recognizes B(OH)3 (or B(OH3) + B(OH)4−) but not B(OH)4− alone. Our previous study revealed that R. pseudosolanacearum Ps29 harbors homologs of all 22R. pseudosolanacearum GMI1000 mcp genes. Screening of 22 mcp single-deletion mutants identified the RS_RS17100 homolog as the boric acid chemoreceptor, which was designated McpB. The McpB ligand-binding domain (LBD) was purified in order to characterize its binding to boric acid. Using isothermal titration calorimetry, we demonstrated that boric acid binds directly to the McpB LBD with a KD (dissociation constant) of 5.4 µM. Analytical ultracentrifugation studies revealed that the McpB LBD is present as a dimer that recognizes one boric acid molecule.

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.