| Summary: | 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.
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