Characterization of the Helicobacter pylori CheZ, CheV1, CheV2, and CheV3 chemotaxis signal transduction proteins

The human gastric pathogen Helicobacter pylori relies on its chemotaxis ability to colonize the stomach. The core chemotaxis proteins: CheA kinase, the CheW coupling protein, and the CheY response regulator have similar functions as in the Escherichia coli model. H. pylori has several accessory chemotaxis proteins, CheZHP and three CheV proteins that have not been extensively characterized and are the subjects of this thesis.;CheZ is a phosphatase that promotes dephosphorylation of phosphorylated CheY (CheY-P). The H. pylori CheZ, called CheZHP, has very poorly conserved amino acid sequence compared to E. coli CheZ. Biochemical analyses of CheZHP, finds that it has the expected phosphatase activity on CheY-P and uses the same active site and mechanism to perform this function. Additionally, CheZHP also dephosphorylates CheA-P and CheV2-P, suggesting that CheZHP is novel to chemotaxis phosphatases by having multiple substrates. In vivo analyses of H. pylori bearing a null allele of cheZHP, showed that it was unable to migrate in soft agar and its swimming was biased toward frequent direction changes as expected for a CheY-P phosphatase mutant. We examined strains with point mutants in the CheZHP active site, and truncated versions of the protein. These were generally better able to migrate in the soft agar suggesting they were not as defective as the cheZHP null mutation. This indicates that the remaining regions in CheZHP mutants are involved in the overall function of CheZHP.;The cellular localization of CheZHP was analyzed using immunofluorescence. CheZHP was consistently found to be localized to the pole in wild type and all chemotaxis and flagellar mutants. This finding is novel because the model CheZ localizes to the pole with known chemotaxis proteins. We did find one protein that affected CheZHP localization, ChePep. This finding suggests that CheZHP and ChePep may form a complex, but the lack of known function for ChePep does not offer any explanation for the importance of CheZHP polar localization.;Also reported here are the localization patterns of the three CheV proteins, CheY, and chemoreceptors. They all localize to the cellular pole and displayed different cytoplasmic dispersion pattern. CheV proteins were dependent on chemoreceptors for polar localization suggesting their association to the chemoreceptor complex. CheY was found to be unevenly distributed in the cytoplasm possibly indicating its movement within the cell. Chemoreceptors were found in cluster or evenly distributed in two H. pylori strains suggesting a more complicated chemoreceptor complex location.