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Title: Mechanistic and functional analysis of Cj0031, a phase variable methyltransferase in Campylobacter jejuni
Author: Anjum, Awais
ISNI:       0000 0004 2738 3750
Awarding Body: University of Leicester
Current Institution: University of Leicester
Date of Award: 2013
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Campylobacter jejuni constitutes the major cause of food-borne diarrheal disease in developed countries. The genome of this species comprises many surface genes having mononucleotide repeat tracts (PolyG/PolyC) which undergo reversible switching between ON and OFF phases, termed phase variation. Phase variation helps bacteria to colonize the host effectively as most phase variable genes are involved in expression of cell surface structures mediating interactions with the host. The mutation rates of phase variable genes are important determinants for genetic diversity and overall fitness of the bacterial population residing in various niches. The major aim of the project was to determine the phase variation rates for the simple sequence repeat tracts of varying lengths in cj0031 and capA of C. jejuni strain 11168. The PV rates were determined by using chromosomally-located reporter construct for cj0031 and by an immunoblotting assay for capA. The mutation rates of a G10 tract were a 1.5 fold higher than a G9 repeat tract in cj0031. Similarly, a 6-fold increase in the PV rate was recorded for G12 in capA over a G10 repeat tract. The mutational spectra for G9 and G10 were predominantly insertions and were shifted to mainly deletions for G11 and G12 repeats. Major shifts in the ON/OFF status of phase variable genes of C. jejuni strain 11168 were detected by performing a multiplex PCR on isolates following passage through chickens. Thirteen novel genotypes found in the output population indicated a high level of genetic diversity was generated by changes in repeat tract lengths of phase variable genes. Bioinformatics analysis of cj0031 revealed a homology to type IIG restriction modification systems. A Southern blot analysis demonstrated that cj0031 possessed methyltransferase activity and led to the conclusion that 5’ CCCGA 3’/5’ CCCGAA 3’ were putative recognition sequences of Cj0031 methyltransferase. An investigation of functional abilities showed that Cj0031 enhanced the capability of adhesion, invasion and biofilm formation without having any affect on the motility of C. jejuni. A 5-fold restriction activity was exerted by Cj0031 on one phage type, showing that this enzyme also possessed restriction activity although this was marginal in comparison to restriction endonucleases in E. coli. It is postulated that Cj0031 mainly controls the phasevarion of other genes in C. jejuni through methylation of target sequences located either in promoter region or intergenic regions near the promoters of such genes, rather than having active involvement in protection of hosts from phages. The high PV rates of cj0031 might be compatible with its role as phasevarion to facilitate the rapid adaptation of C. jejuni to the micro-environment of hosts.
Supervisor: Bayliss, Christopher D. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available