Chemotaxis in the human gastric pathogen Helicobacter pylori: different roles for CheW and the three CheV paralogues, and evidence for CheV2 phosphorylation.
The complete genome sequence of Helicobacter pylori has revealed the presence of a novel set of chemotaxis genes including three cheV paralogues. CheV is a bi-functional protein, the N-terminal domain being homologous to the signalling-complex linker protein CheW, while the C-terminal domain is homologous to the response-regulator CheY, but its precise function in chemotaxis is unknown. In this study, each of the three cheV paralogues were insertionally inactivated in strain 26695 to determine their importance in the chemotactic signal-transduction pathway of H. pylori. Mutation of HP0019 (cheV1) had a severe inhibitory effect on chemotaxis, as determined by a swarm-plate assay. In contrast, strains carrying single mutations in either cheV2 (HP0616) or cheV3 (HP0393) displayed wild-type swarming behaviour, as did a cheV2/cheV3 double mutant. However, expression of the cheV2 or cheV3 genes in Escherichia coli resulted in an inhibition of chemotaxis in a wild-type strain, indicating their role in chemotaxis, although these genes were unable to complement isogenic E. coli cheW or cheY mutants. The product of cheV2/HP0616 was overexpressed in E. coli and purified to homogeneity. Protein fluorescence quenching experiments showed that CheV2 was capable of binding acetyl phosphate, a small-molecule phosphodonor. The measured K(m) for acetyl phosphate was 21 mM. It is concluded that in the absence of a cheZ gene, the CheV proteins could act as phosphate sinks to control the cellular level of phospho-CheY in H. pylori. However, only CheV1 was critical for chemotaxis, indicating a specific role distinct from the other paralogues in the signal-transduction pathway. Significantly, none of the CheV proteins could substitute for the loss of CheW, as an H. pylori cheW null mutant was non-chemotactic.
Pittman
Marc S
MS
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1.
Goodwin
Matthew
M
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1.
Kelly
David J
DJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield S10 2TN, UK1.
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology*; D001483:Base Sequence; D002630:Chemotactic Factors / Q000235:genetics / Q000502:physiology*; D002633:Chemotaxis / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005756:Gastritis / Q000382:microbiology; D015870:Gene Expression; D005798:Genes, Bacterial; D016481:Helicobacter Infections / Q000382:microbiology; D016480:Helicobacter pylori / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology*; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010766:Phosphorylation; D010802:Phylogeny; D017386:Sequence Homology, Amino Acid; D014774:Virulence / Q000235:genetics / Q000502:physiology
D001426:Bacterial Proteins; C489103:CheW protein, E coli; D002630:Chemotactic Factors; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C087011:cheV protein, bacteria; C051236:CheW protein, Bacteria