Analysis of a Helicobacter pylori operon incorporating flagellar export genes : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the Institute of Molecular Biosciences at Massey University, New Zealand

Abstract

Motility of Helicobacter species has been shown to be essential for successful colonization of the host. Previous studies indicated that the regulation of flagellar biosynthesis in the human gastric pathogen Helicobacter pylori differs from the suggested model for Gram-negative Enterobacteriaeceae. In this study, the organization of two H. pylori genes involved in export of flagellar structural proteins was investigated. A 7 kb fragment of the H. pylori 17874 genome was cloned. Sequence determination and analysis revealed a putative operon comprising an ORF of unknown function (ORF03), and genes for the isoleucyl-tRNA synthetase (ileS), an Agrobacterium tumefaciens VirB11 homolog (virB11), an ATPase involved in flagellum-specific protein export (flil), a presumptive flagellar export channel component (fliQ), and a homolog of an enzyme necessary for cell wall biosynthesis (murB). The genetic organization of this region was found to be conserved in a panel of clinical H. pylori isolates, and in H. pylori 915 and SS1. The locus was also identified in the genome sequences of the H. pylori strains J99 and 26695. Cotranscription of ORF03, ileS, virB11, fliI, fliQ and murB was demonstrated by RT-PCR. Primer extension experiments identified the major transcription start site, which coincided with the A residue of the initiation codon of ORF03. A promoter element was inferred that resembled the E. coli ơ70 consensus sequence. In addition, a minor transcription start site was detected upstream from ileS. Non-polar mutation of virB11, fliI and fliQ was generated by an allele replacement strategy. Engineered H. pylori fliI and fliQ mutant strains were completely aflagellate and nonmotile, whereas a virB11 mutant still produced flagella and displayed slightly greater motility. The fliI and fliQ mutant strains produced severely reduced levels of flagellin and the hook protein FlgE, although reduction was less stable in the fliI mutant. Production of OMP4, a member of the outer membrane protein family identified in H. pylori 26695, was diminished in both the virB11 and the fliI mutant. This suggested related functions of the putative virulence factor transport protein (VirB11) and the flagellar export component (FliI).