Cloning and characterization of the Helicobacter pylori flbA gene, which codes for a membrane protein involved in coordinated expression of flagellar genes

Flagellar motility has been shown to be an essential requirement for the ability of Helicobacter pylori to colonize the gastric mucosa, While some flagellar structural components have been studied in molecular detail, nothing was known about factors that play a role in the regulation of flagellar biogenesis. We have cloned and characterized an H. pylori homolog (named flbA) of the lcrD/flbF family of genes. Many proteins encoded by these genes are known to be involved in flagellar biogenesis or secretion of virulence associated proteins via type III secretion 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 sequence similarity with other known members of the LcrD/FlbF protein family, An isogenic strain with a mutation in the flbA gene was constructed by disruption of the gene with a kanamycin resistance cassette and electroporation-mediated allelic exchange mutagenesis. The mutant strain expressed neither the FlaA nor the FlaB flagellin protein, The expression of the FlgE hook protein was reduced in comparison with the wild-type strain, and the extent of this reduction was growth phase dependent, The flbA gene disruption was shown to downregulate the expression of these flagellar genes on the transcriptional level, The flbA mutants were aflagellate and completely nonmotile, Occasionally, assembled hook structures could be observed, indicating that export of axial flagellar filament components was still possible in the absence of the flbA gene product, The hydrophilic part of the FlbA protein was expressed in Escherichia coli, purified, and used to raise a polyclonal rabbit antiserum against the FlbA protein. Western blot experiments with this antiserum indicated that the FlbA protein is predominantly associated with the cytoplasmic membrane in H. pylori. The antiserum cross-reacted with two other proteins (97 and 43 kDa) whose expression,vas not affected by the flbA gene disruption and which might represent further H. pylori homologs of the LcrD/FlbF protein family.