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|Title:||CJ0700 is a remote Chez Orthologue involved in Campylobacter jejuni chemotaxis signal transduction|
|Authors:||Jama, Abdullahi Said|
|Presented at:||University of Leicester|
|Abstract:||The major food borne pathogen Campylobacter jejuni utilizes chemotaxis to colonise the chicken gastrointestinal tract. Similar to the Escherichia coli chemotaxis system, C. jejuni produces a CheA-CheW-CheY backbone that transduces signals from surface chemoreceptors to direct flagellar rotational direction. In contrast to E. coli, C. jejuni also expresses CheV and CheA has a response regulator domain. The phosphorylation level of CheY in E. coli is modulated by the phosphatase CheZ, however until recently C. jejuni was thought to lack a CheZ homologue. The Hp0170 protein of Helicobacter pylori was shown to be a CheZ remote homologue. As C. jejuni Cj0700 has homology with HP0170, it was postulated that Cj0700 acts as a CheZ orthologue in campylobacters. The aim of this project was to characterise the role of Cj0700 in C. jejuni chemotaxis and demonstrate the phosphatase activity of the protein on C. jejuni CheY, CheV and the response regulator domain of CheA (CheA-RR). A mutant (Δcj0700) and the cognate complement (Δcj0700, Cj0046::cj0700) were constructed in C. jejuni NCTC 11168. No growth differences were seen between Δcj0700, ΔcheY and wild-type. On semi-solid agar the Δcj0700 mutant strain showed reduced motility relative to the wild-type and this phenotype was reversed in the complemented strain. The effect of a mutation in cj0700 was also demonstrated in C. jejuni strains 81-176 and 81116. Cj0700 was expressed as a His-tagged protein and was found to dephosphorylate C. jejuni CheY protein. Expressed Cj0700 also dephosphorylated CheA-RR-P and CheV-P, but less efficiently than CheY-P. The ability of Cj0700 to interact with CheY, CheA-RR and CheV was investigated using an in vitro pull down assay and also in a two hybrid system in E. coli. Using these approaches Cj0700 was shown to interact with CheY, CheA-RR and CheV, with the strength of interaction correlating with the observed cognate phosphatase activity. Although attempts to crystallise Cj0700 were not successful, a combination of bioinformatics, CD and NMR approaches indicated that expressed Cj0700 contains significant levels of disorder. These findings indicate that cj0700 has a role in C. jejuni chemotaxis and shows that Cj0700 can promote dephosphorylation of CheY and hence is likely to be a CheZ orthologue. Furthermore, Cj0700 also modulates the phosphorylation level of the response regulator domain on CheA and may also affect the related domain in CheV.|
|Rights:||Copyright © the author. All rights reserved.|
|Appears in Collections:||Theses, Dept. of Genetics|
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