Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa

Abstract

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC1 and PC2, were selected by the swarm plate method after N-methyl-N-nitro-N-nitrosoguanidine mutagenesis. These mutants were fully motile but incapable of swarming, suggesting that they had a defect in the intracellular signalling pathway. Computerassisted capillary assays confirmed that they failed to show behavioral responses to chemical stimuli, including peptone, methyl thiocyanate, and phosphate. Two chemotaxis genes were cloned by phenotypic complementation of PC1 and PC2. From nucleotide sequence analysis, one gene was found to encode a putative polypeptide that was homologous to the enteric CheZ protein, while the other gene was cheY, which had been previously reported (M. N. Starnbach and S. Lory, Mol. Microbiol. 6:459-469, 1992). Deletion and complementation analysis showed that PC1 was a cheY mutant, whereas PC2 had a double mutation in the cheY and cheZ genes. A chromosomal cheZ mutant, constructed by inserting a kanamycin resistance gene cassette into the wild-type gene, changed its swimming direction much more frequently than did wild-type strain PAO1. In contrast, cheY mutants were found to rarely reverse their swimming directions.Chemotaxis is the movement of an organism toward chemical attractants and away from chemical repellents (1). Pseudomonas aeruginosa is attracted to various amino acids (6) and is less strongly responsive to sugars and organic acids (15, 16). The taxis toward several amino acids is subject to control by nitrogen availability in a manner similar to the control of various enzymes of nitrogen metabolism (6) and is mediated by methylation and demethylation of methyl-accepting proteins analogous to those of the enteric bacteria (5). The strengths of the chemotactic responses to glucose and to citrate are also dependent on prior growth of the bacterial cells on those carbon sources (16). The glucose-binding protein has been identified as the glucose chemoreceptor in this organism (28). However, virtually nothing is known about the other chemoreceptors of P. aeruginosa. Our previous work demonstrated that P. aeruginosa is attracted to P i (10). This chemotactic response is induced by P i limitation. The P i -starved cells are also attracted to arsenate (11). Since P i competitively inhibits the response to arsenate, both P i and arsenate are likely to be detected by the same chemoreceptor. Genetic evidence showed that P i taxis in P. aeruginosa is not regulated by the phoB and phoR gene products but requires the phoU gene (12). P. aeruginosa is also repelled by thiocyanic and isothiocyanic esters, including allyl isothiocyanate, ethyl thiocyanate, methyl isothiocyanate, and methyl thiocyanate (19).The molecular mechanisms that underlie bacterial chemotaxis have been studied intensively with the enteric bacteria Escherichia coli (20) and Salmonella typhimurium (30) and to a lesser extent with the gram-positive bacterium Bacillus subtilis (4). However, little is known about the chemotaxis genes in the bacteri...

Initiation of Biofilm Formation by Pseudomonas aeruginosa 57RP Correlates with Emergence of Hyperpiliated and Highly Adherent Phenotypic Variants Deficient in Swimming, Swarming, and Twitching Motilities

“…4A and 5). To control the direction of swimming, P. aeruginosa uses a two-component sensor-regulator system with methyl-accepting chemotaxis proteins similar to those found in enteric bacteria (31,46). Phenotypic differences between the L and the S variants, such as small colony size and defective flagellar and twitching motilities, were observed not only with undefined broth substrates but also with BH mineral salts medium supplemented with succinate or dextrose (data not shown), indicating that the defect is not simply limited to chemotactic transducers.…”

Inorganic polyphosphate is needed for swimming, swarming, and twitching motilities of Pseudomonas aeruginosa

“…In a search for genes affecting twitching motility in P. aeruginosa, two cheY homologues, pilG and pilH, were found which do not affect swimming (39,40). For reversing the flagellar rotation in swimming, a different cheY homologue is involved (47). P. aeruginosa possesses at least two distinct chemotaxis systems: one controls flagellar swimming and presumably swarming; the other supports type IV pili-based twitching.…”

Both chemotaxis and net motility greatly influence the infectivity of Vibrio cholerae

“…Although CheY has an intrinsic phosphatase activity, it is not adequate for the high turnover of CheY-P required for efficient chemotaxis (25). The protein CheZ stimulates the rate of CheY-P dephosphorylation (26,27) and is required for chemotaxis in organisms such as Pseudomonas aeruginosa, E. coli, and S. enterica serovar Typhimurium (28,29). Despite the presence of a single cheZ in the V. cholerae genome, this gene is not required for chemotaxis in V. cholerae (data not shown).…”