CLONING AND CHARACTERIZATION OF THE HELICOBACTER-PYLORI FLBA GENE, WHICH CODES FOR A MEMBRANE-PROTEIN INVOLVED IN COORDINATED EXPRESSION OFFLAGELLAR GENES

Flagellar motility has been shown to be an essential requirement for t he ability of Helicobacter pylori to colonize the gastric mucosa, Whil e some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regula tion of flagellar biogenesis. We have cloned and characterized an H. p ylori homolog (named flbA) of the lcrD/flbF family of genes. Many prot eins encoded by these genes are known to be involved in flagellar biog enesis or secretion of virulence associated proteins via type III secr etion systems, The H. pylori flbA gene (2,196 bp) is capable of coding for a predicted 732-amino-acid, 80.9-kDa protein that has marked sequ ence similarity with other known members of the LcrD/FlbF protein fami ly, An isogenic strain with a mutation in the flbA gene was constructe d by disruption of the gene with a kanamycin resistance cassette and e lectroporation-mediated allelic exchange mutagenesis. The mutant strai n expressed neither the FlaA nor the FlaB flagellin protein, The expre ssion of the FlgE hook protein was reduced in comparison with the wild -type strain, and the extent of this reduction was growth phase depend ent, The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level, The flbA mutan ts were aflagellate and completely nonmotile, Occasionally, assembled hook structures could be observed, indicating that export of axial fla gellar filament components was still possible in the absence of the fl bA gene product, The hydrophilic part of the FlbA protein was expresse d in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with thi s antiserum indicated that the FlbA protein is predominantly associate d with the cytoplasmic membrane in H. pylori. The antiserum cross-reac ted with two other proteins (97 and 43 kDa) whose expression,vas not a ffected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.