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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.

SUBMITTER: Masduki A

PROVIDER: S-EPMC176688 | biostudies-other | 1995 Feb

REPOSITORIES: biostudies-other

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Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

Masduki A A Nakamura J J Ohga T T Umezaki R R Kato J J Ohtake H H

Journal of bacteriology 19950201 4

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 ...[more]

PMID: 7860605

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Project description:Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions.Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14?hepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14.Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing.

Dataset Information

Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

Publications

Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

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