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Identification and characterization of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa.

ABSTRACT: Two chemotactic transducers for inorganic phosphate (P(i)), designated CtpH and CtpL, have been identified in Pseudomonas aeruginosa. The corresponding genes (ctpH and ctpL) were inactivated by inserting kanamycin and tetracycline resistance gene cassettes into the wild-type genes in the P. aeruginosa PAO1 genome. Computer-assisted capillary assays showed that the ctpH single mutant failed to exhibit P(i) taxis when the concentration of P(i) in the capillary was higher than 5 mM. Conversely, the ctpL single mutant could not respond to P(i) at the concentration of 0.01 mM. The ctpH ctpL double mutant was defective in P(i) taxis at any concentration ranging from 0.01 to 10 mM. To investigate regulation of P(i) taxis, the ctpH and ctpL genes were also disrupted individually in the P. aeruginosa phoU and phoB single mutants. The ctpH phoU and ctpH phoB double mutants were defective in P(i) taxis, regardless of whether the cells were starved for P(i). The ctpL phoU double mutant was constitutive for P(i) taxis, whereas the ctpL phoB double mutant was induced by P(i) limitation for P(i) taxis. The region upstream of ctpL, but not ctpH, contained a putative pho box sequence. Expression of ctpL::lacZ was induced by P(i) limitation in PAO1, while it was constitutive in the phoU mutant. In contrast, the phoB mutant showed only background levels of ctpL::lacZ expression. These results showed that ctpL is involved in the pho regulon genes in P. aeruginosa. The ctpH phoU mutant, which failed to exhibit P(i) taxis, was constitutive for ctpL::lacZ expression, suggesting that the P(i) detection by CtpL requires PhoU. Like PAO1, the phoB and phoU single mutants were constitutive for expression of ctpH::lacZ. Thus, the evidence that the ctpL phoU mutant, but not the ctpL phoB mutant and PAO1, was constitutive for P(i) taxis raised the possibility that PhoU exerts a negative control on P(i) detection by CtpH at the posttranscriptional level.

SUBMITTER: Wu H

PROVIDER: S-EPMC101905 | biostudies-literature | 2000 Jun

REPOSITORIES: biostudies-literature

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Identification and characterization of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa.

Wu H H Kato J J Kuroda A A Ikeda T T Takiguchi N N Ohtake H H

Journal of bacteriology 20000601 12

Two chemotactic transducers for inorganic phosphate (P(i)), designated CtpH and CtpL, have been identified in Pseudomonas aeruginosa. The corresponding genes (ctpH and ctpL) were inactivated by inserting kanamycin and tetracycline resistance gene cassettes into the wild-type genes in the P. aeruginosa PAO1 genome. Computer-assisted capillary assays showed that the ctpH single mutant failed to exhibit P(i) taxis when the concentration of P(i) in the capillary was higher than 5 mM. Conversely, the ...[more]

PMID: 10852870

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Project description:Binding of Pseudomonas aeruginosa strain PAK to mucin has been shown to be mediated by the flagellar cap protein, product of the fliD gene. Since the flagellar cap is very likely an exposed structure, the FliD polypeptide should be recognized by the host immune system, analogous to the recognition of dominant epitopes located in the exposed parts of the flagellin polypeptide within the assembled flagellum. In P. aeruginosa, a number of distinct flagellin variants are made, and these variable sequences presumably allow the newly infected P. aeruginosa to escape recognition by the antibody induced during a previous infection. Since similar mechanisms may direct the selection of FliD variants, we examined the extent of sequence heterogeneity among various FliD sequences among a selected group of P. aeruginosa. The results of PCR and nucleotide sequencing of the fliD region of eight different P. aeruginosa strains (laboratory strains PAK, PAO1, and PA103; clinical strains 1244, CS2, and CS32; cystic fibrosis strains CS29 and MDR) suggested that there were two distinct types of FliD in P. aeruginosa, which we named A type and B type. The results of Western blotting using the polyclonal antibodies raised against the purified FliD of A type (PAK) or B type (PAO1) further confirmed the existence of two distinct antigenic types of FliD proteins, with no cross-reactivity between the two serotypes. Further Western immunoblot analysis of the same strains using polyclonal FliC antibody showed that the strains with A-type FliD possessed a-type FliC and those with B-type FliD had b-type FliC. Similar Western blot analyses of 50 more P. aeruginosa strains obtained from varied sources revealed that all strains contained either A-type or B-type FliD, suggesting the existence of only two types of FliD in P. aeruginosa and indicating that fliC and fliD were coinherited. This limited diversity of FliC and FliD serotypes seems to be a unique feature of flagellar proteins. A chromosomal mutant having an insertion in the fliD gene of P. aeruginosa PAO1 was constructed. The motility defect of this mutant and a previously constructed PAK fliD mutant was better complemented with the fliD gene of the homologous types.

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Identification and characterization of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa.

Publications

Identification and characterization of two chemotactic transducers for inorganic phosphate in Pseudomonas aeruginosa.

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