Molecular genetics approaches to the investigation of Campylobacter jejuni

This thesis describes the utilisation of two different methods for the isolation of C. jejuni genes. In the first method, a Lambda ZAP II library of C. jejuni strain H132 DNA was screened with convalescent antiserum to isolate novel genes for antigenic proteins which are likely to be synthesized in vivo. The second method used PCR with degenerate oligonucleotide primers (PCRDOP) to isolate a fragment of a known conserved gene.;Several Lambda ZAP II clones of C. jejuni DNA which synthesized products recognised by convalescent antiserum were successfully isolated and rescued into the pBluescript phagemid. In vitro transcription/translation and immunoprecipitation analysis confirmed that the clones could synthesize products which reacted with convalescent antiserum. The transposon, TnphoA, was used to mutagenise the ORFs and identify any which could encode exported C. jejuni peptides. It was revealed that at least three of the positive clones could produce C. jejuni proteins which were exported from E. coli. The ORFs were partially sequenced to determine any similarities with known exported products which may be involved in virulence. One of the ORFs could encode a protein with N-terminal similarity to Campylobacter flagellins and, upstream from this gene, an ORF encoding an ATP-binding transport protein was found.;PCRDOP was used successfully to isolate a fragment of the C. jejuni recA gene. The fragment was sequenced and amino acids substitutions were found which may cause the RecA protein to have constitutive protease activity for the LexA repressor.;It is hoped that the development of molecular genetics techniques for the investigation of C. jejuni physiology and pathogenesis will lead to a greater understanding of the mechanisms by which Campylobacter cause disease.