Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Rust M

PROVIDER: S-EPMC2715733 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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

Rust Melanie M Borchert Sophie S Niehus Eike E Kuehne Sarah A SA Gripp Eugenia E Bajceta Afrodita A McMurry Jonathan L JL Suerbaum Sebastian S Hughes Kelly T KT Josenhans Christine C

Journal of bacteriology 20090522 15

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 pre ...[more]

PMID: 19465658

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Project description:UnlabelledFlagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD (σ(80)), RpoN (σ(54)), and FliA (σ(28)), that temporally coordinates gene expression with the assembly of the flagellum. Previous studies showed that loss of flagellar protein export apparatus components inhibits transcription of flagellar genes. The FlgS/FlgR two-component system activates transcription of RpoN-dependent genes though an unknown mechanism. To understand better the extent to which flagellar gene regulation is coupled to flagellar assembly, we disrupted flagellar biogenesis at various points and determined how these mutations affected transcription of RpoN-dependent (flaB and flgE) and FliA-dependent (flaA) genes. The MS ring (encoded by fliF) is one of the earliest flagellar structures assembled. Deletion of fliF resulted in the elimination of RpoN-dependent transcripts and an ∼4-fold decrease in flaA transcript levels. FliH is a cytoplasmic protein that functions with the C ring protein FliN to shuttle substrates to the export apparatus. Deletions of fliH and genes encoding C ring components (fliM and fliY) decreased transcript levels of flaB and flgE but had little or no effect on transcript levels of flaA. Transcript levels of flaB and flgE were elevated in mutants where genes encoding rod proteins (fliE and flgBC) were deleted, while transcript levels of flaA was reduced ∼2-fold in both mutants. We propose that FlgS responds to an assembly checkpoint associated with the export apparatus and that FliH and one or more C ring component assist FlgS in engaging this flagellar structure.ImportanceThe mechanisms used by bacteria to couple transcription of flagellar genes with assembly of the flagellum are poorly understood. The results from this study identified components of the H. pylori flagellar basal body that either positively or negatively affect expression of RpoN-dependent flagellar genes. Some of these basal body proteins may interact directly with regulatory proteins that control transcription of the H. pylori RpoN regulon, a hypothesis that can be tested by examining protein-protein interactions in vitro.

Project description:UnlabelledHelicobacter pylori contains four genes that are predicted to encode proteins secreted by the autotransporter (type V) pathway. One of these, the pore-forming toxin VacA, has been studied in great detail, but thus far there has been very little investigation of three VacA-like proteins. We show here that all three VacA-like proteins are >250 kDa in mass and localized on the surface of H. pylori. The expression of the three vacA-like genes is upregulated during H. pylori colonization of the mouse stomach compared to H. pylori growth in vitro, and a wild-type H. pylori strain outcompeted each of the three corresponding isogenic mutant strains in its ability to colonize the mouse stomach. One of the VacA-like proteins localizes to a sheath that overlies the flagellar filament and bulb, and therefore, we designate it FaaA (flagella-associated autotransporter A). In comparison to a wild-type H. pylori strain, an isogenic faaA mutant strain exhibits decreased motility, decreased flagellar stability, and an increased proportion of flagella in a nonpolar site. The flagellar localization of FaaA differs markedly from the localization of other known autotransporters, and the current results reveal an important role of FaaA in flagellar localization and motility.ImportanceThe pathogenesis of most bacterial infections is dependent on the actions of secreted proteins, and proteins secreted by the autotransporter pathway constitute the largest family of secreted proteins in pathogenic Gram-negative bacteria. In this study, we analyzed three autotransporter proteins (VacA-like proteins) produced by Helicobacter pylori, a Gram-negative bacterium that colonizes the human stomach and contributes to the pathogenesis of gastric cancer and peptic ulcer disease. We demonstrate that these three proteins each enhance the capacity of H. pylori to colonize the stomach. Unexpectedly, one of these proteins (FaaA) is localized to a sheath that overlies H. pylori flagella. The absence of FaaA results in decreased H. pylori motility as well as a reduction in flagellar stability and a change in flagellar localization. The atypical localization of FaaA reflects a specialized function of this autotransporter designed to optimize H. pylori colonization of the gastric niche.

Project description:FlgM, an antagonist of FliA (also known as σ28), inhibits transcription of bacterial class 3 flagellar genes. It does so primarily through binding to free σ28 to prevent it from forming a complex with core RNA polymerase. We recently identified an FliA homolog (FliATd) in the oral spirochete Treponema denticola; however, its antagonist FlgM remained uncharacterized. Herein, we provide several lines of evidence that TDE0201 functions as an antagonist of FliATd. TDE0201 is structurally similar to FlgM proteins, although its sequence is not conserved. Heterologous expression of TDE0201 in Escherichia coli inhibits its flagellin gene expression and motility. Biochemical and mutational analyses demonstrate that TDE0201 binds to FliATd and prevents it from binding to the σ28-dependent promoter. Deletions of flgM genes typically enhance bacterial class 3 flagellar gene expression; however, deletion of TDE0201 has an opposite effect (e.g., the mutant has a reduced level of flagellins). Follow-up studies revealed that deletion of TDE0201 leads to FliATd turnover, which in turn impairs the expression of flagellin genes. Swimming plate, cell tracking, and cryo-electron tomography analyses further disclosed that deletion of TDE0201 impairs spirochete motility and alters flagellar number and polarity: i.e., instead of having bipolar flagella, the mutant has flagella only at one end of cells. Collectively, these results indicate that TDE0201 is a FlgM homolog but acts differently from its counterparts in other bacteria. IMPORTANCE Spirochetes are a group of bacteria that cause several human diseases. A unique aspect of spirochetes is that they have bipolar periplasmic flagella (PFs), which bestow on the spirochetes a unique spiral shape and distinct swimming behaviors. While the structure and function of PFs have been extensively studied in spirochetes, the molecular mechanism that regulates the PFs' morphogenesis and assembly is poorly understood. In this report, FlgM, an anti-σ28 factor, is identified and functionally characterized in the oral spirochete Treponema denticola. Our results show that FlgM regulates the number and polarity of PFs via a unique mechanism. Identification of FliA and FlgM in T. denticola sets a benchmark to investigate their roles in other spirochetes.

Dataset Information

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

Publications

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

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