Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.702654
Title: Regulation and function of a novel gene cluster in Campylobacter jejuni
Author: White, Michael Alexander
ISNI:       0000 0004 6058 6517
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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Abstract:
The genome of Campylobacter jejuni NCTC 11168 contains a small three gene operon encoding a series of seemingly un-related proteins not typically found in bacteria. Immediately upstream of this operon are two genes encoding a small DNA-binding protein and a membrane bound cytoplasmic protein, proposed to act as a sensor-regulator system. The DNA-binding protein tightly represses the expression of the structural operon under normal growth conditions and its removal results in a strong (>100-fold) up-regulation of the operon. The operon contains 3 structural genes which encode for proteins not typically found in microbial genomes, cj0423 encodes a small membrane protein, cj0424 encodes a periplasmic lipid binding MORN domain protein (Membrane Occupation and Recognition Nexus) and cj0425 encodes for a periplasmic cystatin like protein (cysteine protease inhibitor). Bioinformatic analysis revealed that the structural genes within this cluster are highly variable between Campylobacter jejuni and Campylobacter coli strains yet the regulatory mechanisms appear highly conserved. The only structural gene which appears conserved between Campylobacter strains is cj0423, which shows architectural similarity to phage derived holin proteins. Initially the project focused on the hypothesis that the genes were responsible for defending against cationic antimicrobial peptides (CAMPs). This however proved to be incorrect and a series of mutant and complementation constructs were produced in a newly genome sequenced laboratory variant of C. jejuni (designated NCTC 11168-DJK for this study). To assay for the roles of the genes, a series of LacZ reporter constructs were produced to screen for potential inducers of the operon. The Biolog© phenotype microarray system was also adapted for use as a high throughput screening system using the LacZ reporter. During the course of this work the cystatin like protein Cj0425 was shown to not inhibit the activity of the archetypal cysteine protease papain. Both bioinformatic analysis and in-vitro pulldown assays were unable to locate a native binding partner for the protein, implying its role may be targeted to foreign proteins or perhaps unrelated to protease inhibition. A series of novel assays were developed to examine the possibility that the operon may be involved in defending against foreign derived periplasmic-acting toxins such as those secreted by the type VI secretion system from other Gram-negative bacteria.
Supervisor: Kelly, Dave ; Rafferty, John Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.702654  DOI: Not available
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