Insertion mutations in Helicobacter pylori flhA reveal strain differences in RpoN-dependent gene expression

Abstract

Flagellar biogenesis in the gastric pathogen Helicobacter pylori involves a transcriptional hierarchy that utilizes all three sigma factors found in this bacterium (RpoD, RpoN and FliA). Transcription of the RpoN-dependent genes requires the sensor kinase FlgS and response regulator FlgR. It is thought that FlgS senses some cellular cue to regulate transcription of the RpoN-dependent flagellar genes, but this signal has yet to be identified. Previous studies showed that transcription of the RpoN-dependent genes is inhibited by mutations in flhA, which encodes a membrane-bound component of the flagellar protein export apparatus. We found that depending on the H. pylori strain used, insertion mutations in flhA had different effects on expression of RpoN-dependent genes. Mutations in flhA in H. pylori strains B128 and ATCC 43504 (the type strain) were generated by inserting a chloramphenicol resistance cassette so as to effectively eliminate expression of the gene (DflhA), or within the gene following codon 77 (designated flhA77) or codon 454 (designated flhA454), which could allow expression of truncated FlhA proteins. All three flhA mutations severely inhibited transcription of the RpoN-dependent genes flaB and flgE in H. pylori B128. In contrast, levels of flaB and flgE transcripts in H. pylori ATCC 43504 bearing either flhA77 or flhA454, but not DflhA, were~60 % of wild-type levels. The FlhA 454 variant was detected in membrane fractions prepared from H. pylori ATCC 43504 but not H. pylori B128, which may account for the phenotypic differences in the flhA mutations of the two strains. Taken together, these findings suggest that only the N-terminal region of FlhA is needed for transcription of the RpoN regulon. Interestingly, expression of an flaB9-9xylE reporter gene in H. pylori ATCC 43504 bearing the flhA77 allele was about eightfold higher than that of a strain with the wild-type allele, suggesting that expression of flaB is not only regulated at the level of transcription but also regulated post-transcriptionally.

Helicobacter pylori FlhA Binds the Sensor Kinase and Flagellar Gene Regulatory Protein FlgS with High Affinity

“…RNA was extracted as previously described (13). Briefly, H. pylori cells were grown on TSA supplemented with 10% horse serum for 18 h. Cells were harvested and resuspended in 100 l of distilled water.…”

“…⌬flhA mutants were constructed in H. pylori strains B128 and ATCC 43504 as previously described (13). Briefly, the region corresponding to 90 nucleotides upstream of the start codon through codon 77 of flhA was replaced with a chloramphenicol transacetylase (cat) cassette.…”

“…Based on characterization of other RpoN-dependent genes (10), phosphorylation of FlgR likely pro-motes multimerization of the protein, which allows it to interact productively with RpoN-RNA polymerase holoenzyme to stimulate transcription. Components of the export apparatus are required for transcription of the RpoN-dependent genes, as deletions of flhB, fliO, and flhA result in decreased expression of these genes in H. pylori (11)(12)(13). The transcription of genes whose products are required late in flagellar biogenesis, which include the major flagellin, is regulated by FliA.…”

“…In a previous study, we showed that a truncated form of the export apparatus component FlhA, consisting of only the first 77 residues of the protein, is sufficient to support transcription of RpoN-dependent genes (13). The 25 amino acid residues at the N terminus of FlhA (FlhA NT ) are predicted to be exposed on the cytoplasmic side of the membrane.…”

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].…”