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. Computer-assisted 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.