Characterisation of the lex2B gene and its role in LPS biosynthesis in Campylobacter jejuni.

Lipopolysaccharides (LPS) are major structural• and virulence components of Gram-negative bacteria. Antigenic and phase variation of LPS, the result of changes in carbohydrate composition and assembly of sugars in LPS, leads to increased cell surface diversity. A gene was isolated from Campylobacter jejuni and Campylobacter coli that had sequence identity to a phase variable gene, lex2B, from Haemophilus influenzae. The lex2B shows sequence similarity to glycosyl and galactosyl transferases, however its function is still unknown. The aim of study was to characterise lex2B in C. jejuni and its potential role in pathogenesis of this enteric pathogen. Previous sequence analysis of C. coli strain M275 revealed the presence of an ORF whose nucleotide sequence showed a high degree of similarity with lex2B from H influenzae and H pylori. The lex2B gene from M275 was shown to be transcribed within aLPS biosynthetic locus, downstream of gmhA and in an opposite orientation to the waaF gene. Nucleotide sequence analysis of an 1.7 kb fragment from C. jejuni strain 928 contained in plasmid pDJ4216 revealed a common arrangement of genes, including lex2B, between C. coli and C. jejuni. The C. jejuni lex2B ORF was shown to consist of 765 nucleotides encoding a polypeptide of 255 amino acids. Comparison of the C. coli and C. jejuni lex2B translated nucleotide sequence revealed strong variation in the second half of the protein. PCR and Southern analysis indicated that the lex2B gene in C. jejuni appears transcriptionally coupled to adjacent genes and physically linked on the genome to genes involved in biosynthesis of the LPS core. Expression studies based on production of lex2B rnRNA (RT-PCR) and expression of the putative Lex2B protein under transcriptional control of the T7 RNA polymerase in E. coli has shown that lex2B is capable of synthesing a polypeptide of the predicted size from sequence analysis. In order to create a null mutation in C. jejuni 928 by homologous recombination, lex2B was cloned into pBluescript II containing a gene encoding resistance to kanamycin in Campylobacter. Based on evidence presented in this study, it is speculated that lex2B contributes to LPS diversity within C. jejuni and C. coli