Development of new approaches to identify and characterise Campylobacter jejuni genes.

Campylobacter jejuni is now recognised as a major cause of food-borne enteritis. Characteristics of Campylobacter spp. make them less amenable to genetic analysis using standard molecular biology techniques. These problems can prevent the isolation and characterisation of campylobacter loci. In the absence of an efficient method for the generation of random mutations, the only mutagenesis strategy available is that which targets specific genes. The two approaches described were developed specifically for the analysis of specific genes. The polymerase chain reaction (PCR) with degenerate oligonucleotide primers (PCRDOP/inverse PCR mutagenesis (IPCRM) and inverse PCR strategies are designed to isolate and utilise small regions of DNA for the defined mutagenesis of targeted campylobacter genes. Moreover with the development of the IPCR mutagenesis, a small amount of sequence is the only requirement for the application of this approach. To evaluate the potential of both of these approaches, they were used to isolate and characterise potential virulence genes from C.jejuni. This study focused on the role of two classes of putative virulence determinants, stress response proteins of the HtrA family and response regulator proteins which are involved in the adaptation of bacteria to differing environmental stresses. A defined htrA mutant was constructed and phenotypic analysis established that this gene is not involved in the response to either temperature or oxidative stress, unlike many other homologues of HtrA. Three response regulator genes were isolated during this study, one of which is thought to encode CheY. Attempts to construct regX2 and regX3 mutants were unsuccessful. The flanking regions of htrA, regX2 and regX3 were isolated to facilitate the further characterisation of these loci. The work described in this study has verified that the PCRDOP/IPCRM and IPCR methodologies represent powerful approaches for the genetic analysis of Campylobacter spp.