Molecular cloning and characterization of a chemotactic transducer gene in Pseudomonas aeruginosa

Abstract

A Pseudomonas aeruginosa mutant, defective in taxis toward L-serine but responsive to peptone, was selected by the swarm plate method after N-methyl-N-nitrosoguanidine mutagenesis. The mutant, designated PCT1, was fully motile but failed to show chemotactic responses to glycine, L-serine, L-threonine, and L-valine. PCT1 also showed weaker responses to some other commonly occurring L-amino acids than did the wild-type strain PAO1. A chemotactic transducer gene, denoted pctA (Pseudomonas chemotactic transducer A), was cloned by phenotypic complementation of PCT1. Nucleotide sequence analysis showed that the pctA gene encodes a putative polypeptide of 629 amino acids with a calculated mass of 68,042. A hydropathy plot of the predicted polypeptide suggested that PctA may be an integral membrane protein with two potential membrane-spanning regions. The C-terminal domain of PctA showed high homology with the enteric methyl-accepting chemotaxis proteins (MCPs). The most significant amino acid sequence similarity was found in the region of MCPs referred to as the highly conserved domain. Pseudomonas aeruginosa is an obligately aerobic bacterium and is capable of swimming by rotating a single polar flagellum. This organism inhabits a wide range of environmental niches, from soil and water to the human host; it is an opportunistic pathogen that is among the most frequently isolated bacteria in nosocomial infections (8). P. aeruginosa, like most other motile bacteria, exhibits chemotactic responses to a wide range of chemical stimuli, including L-amino acids (5), sugars (22), organic acids (21), thiocyanic and isothiocyanic esters (24), and P i (16). Several lines of evidence suggest the presence of regulatory mechanisms underlying the chemotaxis by P. aeruginosa. The taxis toward L-amino acids is subject to control by nitrogen availability in a manner similar to the control of various enzymes of nitrogen metabolism (5). The strength of the chemotactic responses to glucose and citrate is also dependent on prior growth of the bacterial cells on those carbon sources (22). P i taxis in P. aeruginosa is induced by P i limitation and requires the phoU gene for its negative regulation (17). However, these regulatory mechanisms are still poorly understood at the molecular level.Biochemical and genetic information concerning chemotactic transducers has been accumulated with the enteric bacteria Escherichia coli (3) and Salmonella typhimurium (26), the gram-positive bacterium Bacillus subtilis (12), and the soil bacterium Rhizobium meliloti (11). The methyl-accepting chemotaxis proteins (MCPs) are the best-characterized signal transducers of the chemosensory apparatus (3, 26). These transducers recognize ligand-occupied forms of various periplasmic binding proteins or small molecules directly and are known to be covalently modified by dynamic methylation and demethylation reactions in response to chemical stimuli (6). P. aeruginosa possesses approximately 73-kDa proteins that are labeled with L-[methyl-3 H]methionine in response to...

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

“…Initial control experiments involved the assessment of chemotaxis of these three strains toward alanine. In P. putida, taxis toward alanine is mediated by the PctA chemoreceptor (17). Assays show that the mutation of McpS and its complementation do not alter taxis toward alanine (Fig.…”

Elastin degradation product isodesmosine is a chemoattractant for Pseudomonas aeruginosa

“…All photographs were taken after 5 min. There are three amino acid receptors in P. aeruginosa strain PAO1: PctA, PctB and PctC (Kuroda et al, 1995;Taguchi et al, 1997). Isodesmosine contains lysine residues and chemotaxis towards lysine is directly linked to PctA and PctB, and therefore we hypothesized that the chemotactic response of P. aeruginosa PAO1 is mediated by some of these receptors.…”

“…Quantitative chemotaxis studies with some of these strains confirmed these results (Table S2). Kuroda et al (1995) and Taguchi et al (1997) identified the chemoreceptors PctA, PtcB and PctC associated with PAO1 chemoattraction towards 20 natural amino acids. Recently, Rico-Jiménez et al (2013) demonstrated that the recombinant ligand-binding region of PctC binds the non-proteinogenic amino acid GABA.…”

“…Ten MCPs have been shown to mediate a flagella-based positive chemotaxis response to malate (PA2652), oxygen (Aer and Aer-2), ethylene (TlpQ), chloroethylenes (McpA), amino acids and gamma aminobutyrate (GABA) (PctABC) and inorganic phosphate (CtpH and CtpL) (Alvarez-Ortega & Harwood, 2007;Hong et al, 2004;Kim et al, 2006Kim et al, , 2007Rico-Jiménez et al, 2013;Shitashiro et al, 2005;Taguchi et al, 1997;Wu et al, 2000). The MCPs responsible for detecting the 20 natural L-amino acids in PAO1 are the paralogous PctA, PctB and PctC receptors (Kuroda et al, 1995;Taguchi et al, 1997). PctA recognizes and mediates the response to most of the natural L-amino acids, while PctB is primarily involved in mediating taxis to glutamine and PctC to histidine and proline.…”

Ethylene Chemotaxis in Pseudomonas aeruginosa and Other Pseudomonas Species

“…Recently, we found that Pseudomonas aeruginosa strain PAO1 is repelled by TCE 23) . Genetic analysis revealed that the methyl-accepting chemotaxis proteins (MCPs) PstA, PctB, and PctC, which were identified as MCPs for amino acids 15,25) , serve as the major chemoreceptors for the negative chemotaxis to TCE 24) . Interestingly, the pctA pctB pctC triple mutant of P. aeruginosa strain PAO1 shows an attractive response to TCE 13) .…”