The Helicobacter pylori anti-sigma factor FlgM is predominantly cytoplasmic and cooperates with the flagellar basal body protein FlhA.

Helicobacter pylori requires flagellar motility and orientation to persist actively in its habitat. A particular feature of flagella in most Helicobacter species including H. pylori is a membraneous flagellar sheath. The anti-sigma factor FlgM of H. pylori is unusual, since it lacks an N-terminal domain present in other FlgM homologs, e.g., FlgM of Salmonella spp., whose regulatory function is intimately coupled to its secretion through the flagellar type III secretion system. The aim of the present study was to characterize the localization and secretion of the short H. pylori FlgM in the presence of a flagellar sheath and to elucidate its interaction with other flagellar proteins, such as the basal body protein FlhA, which was previously shown to cooperate with FlgM for regulation. H. pylori FlgM was only released into the medium in minor amounts in wild-type bacteria, where the bulk amount of the protein was retained in the cytoplasm. Some FlgM was detected in the flagellar fraction. FlgM was expressed in flhA mutants and was less soluble and differentially localized in bacterial fractions of the flhA mutant in comparison to wild-type bacteria. FlgM-green fluorescent protein and FlgM-V5 translational fusions were generated and expressed in H. pylori. FlgM displayed a predominantly polar distribution and interacted with the C-terminal domain of FlhA (FlhA(C)). We suggest that, in H. pylori, FlgM secretion may not be paramount for its regulatory function and that protein interactions at the flagellar basal body may determine the turnover and localization of functional FlgM.

DNA supercoiling-dependent gene regulation in Chlamydia.

The intracellular pathogen Chlamydia has an unusual developmental cycle marked by temporal expression patterns whose mechanisms of regulation are largely unknown. To examine if DNA topology can regulate chlamydial gene expression, we tested the in vitro activity of five chlamydial promoters at different superhelical densities. We demonstrated for the first time that individual chlamydial promoters show a differential response to changes in DNA supercoiling that correlates with the temporal expression pattern. The promoters for two midcycle genes, ompA and pgk, were responsive to alterations in supercoiling, and promoter activity could be regulated more than eightfold. In contrast, the promoters for three late transcripts, omcAB, hctA, and ltuB, were relatively insensitive to supercoiling, with promoter activity varying by no more than 2.2-fold over a range of superhelicities. To obtain a measure of how DNA supercoiling levels vary during the chlamydial developmental cycle, we recovered the cryptic chlamydial plasmid at different times after infection and assayed its superhelical density. The chlamydial plasmid was most negatively supercoiled at midcycle, with an approximate superhelical density of -0.07. At early and late times, the plasmid was more relaxed, with an approximate superhelicity of -0.03. Thus, we found a correlation between the responsiveness to supercoiling shown by the two midcycle promoters and the increased level of negative supercoiling during mid time points in the developmental cycle. Our results support a model in which the response of individual promoters to alterations in DNA supercoiling can provide a mechanism for global patterns of temporal gene expression in Chlamydia.

Flagellar and global gene regulation in Helicobacter pylori modulated by changes in DNA supercoiling.

In Helicobacter pylori, a host-adapted bacterium with a small genome and few dedicated transcriptional regulators, promoter structure, and gene organization suggested a role for DNA topology in the transcriptional regulation of flagellar genes. H. pylori DNA supercoiling, monitored by a reporter plasmid, was relaxed by novobiocin, an inhibitor of DNA gyrase. A decrease in negative supercoiling coincided with lowered transcription of the late flagellin gene flaA. Targeted mutagenesis that either increased or decreased promoter spacer length in the flaA sigma(28) promoter lowered flaA transcript levels, expression of FlaA protein, and flagella formation. It also changed the promoter response to decreased superhelicity. Supercoiling of reporter plasmid DNA in H. pylori varied with growth phase in liquid culture. H. pylori sigma(28) promoters of various spacer length, as well as other supercoiling-sensitive genes, were differentially transcribed during the growth phases, consistent with supercoiling being associated with growth phase regulation. Genome-wide transcript analysis of wild-type H. pylori under conditions of reduced supercoiling identified flagellar, housekeeping, and virulence genes, the expression of which correlated with supercoiling change and/or growth phase. These data indicate that global supercoiling changes may help coordinate temporal (growth phase-related) regulation of flagellar biosynthesis and other cellular functions in Helicobacter.

Genomic changes during chronic Helicobacter pylori infection.

The gastric pathogen Helicobacter pylori shows tremendous genetic variability within human populations, both in gene content and at the sequence level. We investigated how this variability arises by comparing the genome content of 21 closely related pairs of isolates taken from the same patient at different time points. The comparisons were performed by hybridization with whole-genome DNA microarrays. All loci where microarrays indicated a genomic change were sequenced to confirm the events. The number of genomic changes was compared to the number of homologous replacement events without loss or gain of genes that we had previously determined by multilocus sequence analysis and mathematical modeling based on the sequence data. Our analysis showed that the great majority of genetic changes were due to homologous recombination, with 1/650 events leading to a net gain or loss of genes. These results suggest that adaptation of H. pylori to the host individual may principally occur through sequence changes rather than loss or gain of genes.

Growth phase-dependent and differential transcriptional control of flagellar genes in Helicobacter pylori.

Helicobacter pylori possesses two different flagellin genes, flaA and flaB, which are unlinked on the chromosome and transcribed from sigma(28) and sigma(54) promoters, respectively. Both flagellins are hypothesized to be present in varying amounts in the flagellum, to adapt the physical properties of the flagellar filament to different environmental conditions. The influence of growth phase and environmental conditions on the transcriptional regulation of both flagellin genes has not been investigated so far. Using three different reporter genes as well as Northern blot analyses and RT-PCR, it was determined that both flagellin genes are transcribed in a growth phase-dependent fashion. Growth phase dependency was also found for the flagellar basal body export apparatus gene flhA which is involved in the transcriptional regulation of both flagellin genes. Peak transcription of flaB and flhA occurred earlier during the growth phase than that of flaA, possibly consistent with a hook-proximal localization of the minor flagellin FlaB. Of the reporter gene systems, luciferase fusions reflected best the dynamic regulation patterns of H. pylori flagellin genes. Growth phase in vitro had the strongest influence on transcriptional control of H. pylori flaA and flaB, while differences in supplements to a rich culture medium had only a modest modulatory effect on flagellin gene transcription.

Functional characterization of the antagonistic flagellar late regulators FliA and FlgM of Helicobacter pylori and their effects on the H. pylori transcriptome.

Helicobacter pylori is thought to regulate gene expression with a very small set of regulatory genes. We identified a previously unannotated open reading frame (ORF) in the H. pylori 26695 genome (HP1122) as a putative H. pylori flgM gene (sigma28 factor antagonist) by a motif-based bioinformatic approach. Deletion of HP1122 resulted in a fourfold increase in transcription of the sigma28-dependent major flagellin gene flaA, supporting the function of HP1122 as H. pylori FlgM. Helicobacter pylori FlgM lacks a conserved 20-amino-acid N-terminal domain of enterobacterial FlgM proteins, but was able to interact with the Salmonella typhimurium sigma28 (FliA) and inhibit the expression of FliA-dependent genes in Salmonella. Helicobacter pylori FlgM inhibited FliA to the same extent in a Salmonella strain with an intact flagellar export system and in an export-deficient strain. Helicobacter pylori FliA was able to drive transcription of FliA-dependent genes in Salmonella. The effects of mutations in the H. pylori flgM and fliA genes on the H. pylori transcriptome were analysed using whole genome DNA microarrays. The antagonistic roles of FlgM and FliA in controlling the transcription of the major flagellin gene flaA were confirmed, and two additional FliA/FlgM dependent operons (HP472 and HP1051/HP1052) were identified. None of the three genes contained in these operons has a known function in flagellar biogenesis in other bacteria. Like other motile bacteria, H. pylori has a FliA/FlgM pair of sigma and anti-sigma factors, but the genes controlled by these differ markedly from the Salmonella/Escherichia coli paradigm.

Genome-wide analysis of transcriptional hierarchy and feedback regulation in the flagellar system of Helicobacter pylori.

The flagellar system of Helicobacter pylori, which comprises more than 40 mostly unclustered genes, is essential for colonization of the human stomach mucosa. In order to elucidate the complex transcriptional circuitry of flagellar biosynthesis in H. pylori and its link to other cell functions, mutants in regulatory genes governing flagellar biosynthesis (rpoN, flgR, flhA, flhF, HP0244) and whole-genome microarray technology were used in this study. The regulon controlled by RpoN, its activator FlgR (FleR) and the cognate histidine kinase HP0244 (FleS) was characterized on a genome-wide scale for the first time. Seven novel genes (HP1076, HP1233, HP1154/1155, HP0366/367, HP0869) were identified as belonging to RpoN-associated flagellar regulons. The hydrogenase accessory gene HP0869 was the only annotated non-flagellar gene in the RpoN regulon. Flagellar basal body components FlhA and FlhF were characterized as functional equivalents to master regulators in H. pylori, as their absence led to a general reduction of transcripts in the RpoN (class 2) and FliA (class 3) regulons, and of 24 genes newly attributed to intermediate regulons, under the control of two or more promoters. FlhA- and FlhF-dependent regulons comprised flagellar and non-flagellar genes. Transcriptome analysis revealed that negative feedback regulation of the FliA regulon was dependent on the antisigma factor FlgM. FlgM was also involved in FlhA- but not FlhF-dependent feedback control of the RpoN regulon. In contrast to other bacteria, chemotaxis and flagellar motor genes were not controlled by FliA or RpoN. A true master regulator of flagellar biosynthesis is absent in H. pylori, consistent with the essential role of flagellar motility and chemotaxis for this organism.