The identification and characterisation of the gmhD gene in Campylobacter jejuni.
Thesis DisciplineCellular and Molecular Biology
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
Lipopolysaccharides (LPS) are an integral component of the Gram-negative bacterial outer membrane and play an important role in maintaining cellular integrity. L-glyceroD- mannoheptose (Hep) is a primary component of the LPS core region and appears to be synthesised from sedoheptulose-7-phosphate via a four-step process. In Escherichia coli, the final step of Hep synthesis is performed by ADP-L-glycero-D-mannoheptose-6- epimerase, encoded by the gmhD gene. The initial aim of this study was to characterise Hep biosynthesis in Campylobacter jejuni. An E. coli K12 isolate defective in gmhD provided a model with which to study Hep biosynthesis in C. jejuni, in particular the last step in Hep biosynthesis catalysed by the enzyme GmhD. To test whether a functional equivalent of this enzyme is present within Campylobacter, a C. jejuni plasmid expression library was introduced into the E. coli K12 gmhD strain. Restored LPS was assessed by measuring changes in novobiocin sensitivity and LPS profiles of the transformants. Partial nucleotide sequencing of the cloned C. jejuni DNA insert revealed an incomplete ORF that, when translated into amino acid sequence, displayed strong similarity to the 3' end of the GmhD protein from Helicobacter pylori, Aquifex aeolicus, and Haemophilus injluenzae. Transformation of the E. coli K12 gmhD mutant with a plasmid (PDV08) containing a 350 bp in-frame deletion of a C. jejuni gmhD allele failed to restore novobiocin resistance or wild-type LPS expression. Based on this evidence, the gene present in C. jejuni has been named gmhD. Nucleotide sequence analysis indicated that one other gene involved in Hep biosynthesis was likely to be present within this insert (gmhC). Analysis of the assembled gmhD DNA sequence from the C. jejuni genome project revealed an ORF of 954 nucleotides, encoding a protein of 318 amino acids with a calculated mass of 36.0 kDA. Amino acid sequence analysis of the C. jejuni GmhD protein revealed an ADP-binding site as found in the GmhD protein of E. coli, Salmonella typhimurium, and Neisseria gonorrhoeae. Growth of the transformants in minimal and nutrient-rich media revealed reduced growth; however, this might be insignificant. Insertion of a kanamycin resistance cassette into pDV08 followed by gene replacement onto the C. jejuni chromosome by homologous recombination would have completed the construction of a null mutation in gmhD of C. jejuni. However, due to time constraints this was not completed.