Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors sigma(54) (RpoN), employed for expression of genes required midway through flagellar assembly, and sigma(28) (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhB(C)). The hook length control protein FIX interacts with FlhB(C) to modulate the substrate specificity of the export apparatus. FlhB(C) undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the Delta flhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the Delta flhB mutation is not due to the inability to export FIX. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN- and FliA-dependent reporter genes. In the strain producing FlhB N265A, expression of RpoN- and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhB P266G. H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhBC (FlhB(CC)) and can substitute for FlhB(CC) in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the Delta fliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhB(CC) in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus.