Cloning and characterisation of the lpxA gene in Campylobacter jejuni.
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Lipopolysaccharides (LPS) are important structural components of the outer membrane of Gram-negative bacteria. Lipid A anchors LPS molecules in the outer membrane and is also the sole LPS component necessary for endotoxic shock induction. UDP-N-acetylglucosamine acyltransferase is encoded by the lpxA gene. In Escherichia coli K12 and Salmonella typhimurium, this enzyme completes the first step in lipid A synthesis. The lpxA gene product is essential for the production ofLPS in these organisms as null mutations in lpxA are lethal. The aim of the study was to characterise lipid A biosynthesis in Campylobacter jejuni. C. jejuni appears to contain three lipid A types, one of which is similar to the lipid A species in E. coli. In this study it was proposed that C. jejuni contains an enzyme that is functionally analogous to LpxA from E. coli. The initial aim was to clone the gene necessary for the production of LpxA in C. jejuni. An E. coli temperature-sensitive mutant defective in LpxA activity provided a model with which to study the effect of acyltransferase deficiency on cell viability. A C. jejuni plasmid expression library was electroporated into E. coli lpxA mutant SM101 cells and transformants were screened for their ability to survive at the previous non-permissive temperature (42°C) on minimal media containing ampicillin. Plasmid DNA from one transformant able to restore the full length LPS in lpxA defect in SM101 was analysed. Nucleotide sequence has shown the gene order to be JabZ-lpxA as in E. coli and the predicted amino acid sequence from the 'lpxA' gene from C. jejuni displayed strong amino acid similarity to the 3' end of the UDP-N-acetylglucosamine acyltransferase protein from E. coli, S. typhimurium, Rickettsia rickettsii, Yersinia enterocolitica and Haemophilus influenzae. While this gene from C. jejuni F38011 was capable of rescuing the temperature-sensitive defect of KLC 4177 (SM101), growth curves and viable plate counts reveal that rescue is not complete. A second allele of lpxA, cloned from C. jejuni strain NZRM 1958 has shown a similar arrangement of genes (fabZ-lpxA) although differences in primary amino acid sequence between the alleles were revealed.