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

Abstract

Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors s 54 (RpoN), employed for expression of genes required midway through flagellar assembly, and s 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 FliK 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 DflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the DflhB mutation is not due to the inability to export FliK. 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 FlhB C (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 DfliK 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. INTRODUCTIONBacterial flagellar biosynthesis involves transcriptional hierarchies that coordinate flagellar gene expression with assembly. Flagellar gene hierarchies are well characterized in a few bacteria, including Salmonella enterica serovar Typhimurium (S. typhimurium) and Caulobacter crescentus (Chilcott & Hughes, 2000;Wu & Newton, 1997). Flagellar gene regulation in e-Proteobacteria, such as Helicobacter pylori and Campylobacter jejuni, shares some similarities with these established paradigms but also differs significantly. As in S. typhimurium, H. pylori flagellar genes required late in assembly, such as the major flagellin (FlaA) and filament cap protein, are transcribed by s 28 (FliA)-RNA polymerase holoenzyme (Kim et al., 1999;Leying et al., 1992), and, as in C....

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

“…Furthermore, the lack of YscU/FlhB cleavage does not significantly compromise the secretion of T3S4 proteins (509,626) (Table 7). In contrast, secretion of intermediate and late substrates is often suppressed in YscU/FlhB cleavage mutants, suggesting that the cleavage of YscU/FlhB family members, and thus the reorientation of the PTH loop, is required for the substrate specificity switch (186,330,507,509,548,626) (Table 7). In EPEC and X. campestris pv.…”

Basal Body Structures Differentially Affect Transcription of RpoN- and FliA-Dependent Flagellar Genes in Helicobacter pylori

“…Cells were lysed with three passages through a French press at 10,000 kPa. Unlysed cells were removed by centrifugation for 15 min at 6,000 ϫ g. Membranes were separated from cytoplasmic proteins by two centrifugations for 60 min at 100,000 ϫ g. The supernatant containing the cytoplasmic proteins was concentrated by trichloroacetic acid precipitation as described previously (17). Protein concentrations were determined using the bicinchoninic acid protein assay.…”

“…Transcription of the RpoN regulon is regulated by a twocomponent system composed of a cytoplasmic sensor kinase, FlgS, and the response regulator FlgR (9,12,14,15). In addition to FlgS and FlgR, several components of the flagellar export apparatus are required for transcription of the RpoN regulons in H. pylori and C. jejuni (16), although the export apparatus does not need to be competent for secretion to stimulate transcription of the RpoN-dependent genes (17,18). FlgS is thought to respond to a signal within or near the flagellar T3SS to initiate signal transduction resulting in transcription of the RpoN regulon, though the exact signal that stimulates FlgS kinase activity is currently unknown.…”

Extracellular secretion of protease HtrA fromCampylobacter jejuniis highly efficient and independent of its protease activity and flagellum

“…In case of the C. jejuni deletion mutant, agar plates were amended with 30 μg/ml or chloramphenicol. The H. pylori strain ATCC53405 and its fl agellar mutants were generated and cultivated as described [47,48].…”