Requirement of the Flagellar Protein Export Apparatus Component FliO for Optimal Expression of Flagellar Genes in Helicobacter pylori

Tsang, Jennifer; Hoover, Timothy R.

doi:10.1128/jb.01332-13

Cited by 19 publications

(20 citation statements)

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“…Introduction of pflhA in the wild-type background (resulting in the WT/pflhA strain) reduced motility, possibly due to elevated levels of FlhA from the plasmid-based expression method. We have obtained similar results in previous experiments where overexpression of an export apparatus protein in otherwise wild-type cells resulted in decreased motility in soft agar medium (12,19). In contrast to pflhA, the introduction of pflhA ⌬NT in the ⌬flhA mutant (the ⌬flhA/pflhA ⌬NT strain) did not enhance the motility of the strain (Fig.…”

Section: Resultssupporting

confidence: 73%

“…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.…”

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confidence: 99%

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

et al. 2015

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Flagellar biogenesis is a complex process that involves multiple checkpoints to coordinate transcription of flagellar genes with the assembly of the flagellum. In Helicobacter pylori, transcription of the genes needed in the middle stage of flagellar biogenesis is governed by RpoN and the two-component system consisting of the histidine kinase FlgS and response regulator FlgR. In response to an unknown signal, FlgS autophosphorylates and transfers the phosphate to FlgR, initiating transcription from RpoNdependent promoters. In the present study, export apparatus protein FlhA was examined as a potential signal protein. Helicobacter pylori is an epsilonproteobacterium that can cause significant pathologies in the stomach (1-3). Approximately 50% of the world population is infected with H. pylori, although only a small fraction of infected individuals have symptoms. H. pylori possesses 2 to 6 polar flagella that are used to burrow through the mucus layer lining the stomach epithelium. Colonization of the gastric mucosa is dependent on motility, as nonflagellated mutants are unable to colonize (4).The flagellum itself is a complex structure comprised of the basal body, hook, and filament (5). The basal body is located within the cell envelope and contains the flagellar protein export apparatus, the various flagellar rings, the rod, and motor components. The export apparatus is responsible for the secretion of axial components of the flagellum and consists of six proteins located within the inner membrane (FliO, FliP, FliQ, FliR, FlhA, and FlhB) plus three cytoplasmic proteins that bring flagellar substrates to the integral membrane component of the export apparatus (FliH, FliI, and FliJ). The C ring (or switch complex) is located at the cytoplasmic side of the inner membrane. In H. pylori it consists of four proteins, FliG, FliM, FliN, and FliY. In addition to controlling the rotational direction of the flagellum, the C ring works with the soluble components of the export apparatus to bring flagellar substrates to the export apparatus for secretion (6, 7). The rod proteins are the first proteins exported and are followed by hook proteins. The hook serves as a universal joint between the rod and the filament, transmitting torque from the motor to the filament. The filament is assembled after the completion of the hook and involves the minor flagellin FlaB and the major flagellin FlaA. Flagellar biogenesis is a complex process that involves the coordinated expression of over 50 structural and regulatory genes with assembly of the nascent flagellum. In H. pylori, temporal expression of flagellar genes is controlled by the three sigma factors found in the bacterium: RpoD ( 80 ), RpoN ( 54 ), and FliA ( 28 ). Transcription of the early flagellar genes, which encode components of the basal body, is regulated by RpoD. Genes whose products are needed for flagellar biogenesis following assembly of the basal body include components of the hook and a minor flagellin, and transcription of these genes is regulated by R...

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Section: Resultssupporting

confidence: 73%

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confidence: 99%

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

et al. 2015

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“…Transcript levels of flaA, flaB, flgE, flgS, flgR, and fliA were monitored by quantitative reverse-transcription PCR (qRT-PCR) as described previously (27). Primers used are listed in Table S1 in the supplemental material.…”

Section: Methodsmentioning

confidence: 99%

“…Detection of FlhA proteins. H. pylori membrane fractions were collected as previously described (27). Cells were grown for 3 days on TSA plates supplemented with 10% horse serum.…”

Section: Methodsmentioning

confidence: 99%

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

Tsang

Hoover

2015

J Bacteriol

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Flagellar biogenesis in Helicobacter pylori is regulated by a transcriptional hierarchy governed by three sigma factors, RpoD ( 80 ), RpoN ( 54 ), and FliA ( 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 ϳ4-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 ϳ2-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. T he bacterial flagellum is a complex nanomachine powered by an ion-driven rotary motor consisting of about 30 different types of proteins whose copy numbers range from a few to thousands (Fig. 1). The flagellum consists of three basic structures referred to as the basal body, hook, and filament (1). The basal body is an intricate complex that consists of the flagellar rod, rings, a motor, a switch complex, and a specialized type III secretion system (T3SS) that transports flagellar proteins across the cell membrane (1-3). The T3SS, also referred to as the flagellar protein export apparatus, consists of integral membrane proteins (FlhA, FlhB, FliO, FliP, FliQ, and FliR) which form an export pore located within the inner membrane, as well as cytoplasmic components (FliI, FliH, and FliJ) that deliver protein substrates to the export pore (4, 5). During flagellar assembly, the export apparatus initially transports rod-and hook-type substrates across the cell membrane into the lumen of the nascent flagellum (6, 7). Upon completion of the mature hook-basal body (HBB) structure, the export apparatus undergoes a conformational change that is accompanied by a switch in substrate specificity to filament-type substrates.Biogenesis of the bacterial flagellum involves a transcriptional hierarchy that is responsive to checkpoints in assembly so that expression of specific flagellar genes occurs as t...

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“…In H. pylori, its role is played by HP0583. fliO mutants show a no-flagellate nature (Tsang and Hoover, 2014). Since fliO knockout induces a decrease in FlhA and RpoN-regulon (protein involved in biogenesis of flagellar genes) expression in H. pylori cells, causing a reduced level of FlaA and off the hook protein FlgE, it has been postulated that FliO is essential for the transcription of RpoNregulated flagellar genes.…”

Section: H Pylori Flagellar Export Apparatusmentioning

confidence: 99%

Structural Characterization at the Atomic Level of a Molecular Nano-Machine: The State of the Art of <i>Helicobacter Pylori</i> Flagellum Organization

Pulić¹,

Loconte²,

Zanotti³

2014

American Journal of Biochemistry and Biotechnology

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Motility is fundamental for success in colonization and survival of the human pathogen H. pylori, the bacterium that colonizes about half of the human population. Motility is achieved through flagella. In this review the present structural knowledge about the organization of H. pylori flagella is summarized, considering not only the structure of its proteins, but also what is known about flagella of other Gram-negative bacteria. Despite the limited amount of structural information about the H. pylori nano-machinery, sequence alignments and homology modeling allow to investigate the unique structural properties of several H. pylori flagellar proteins.

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Requirement of the Flagellar Protein Export Apparatus Component FliO for Optimal Expression of Flagellar Genes in Helicobacter pylori

Cited by 19 publications

References 42 publications

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

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

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

Structural Characterization at the Atomic Level of a Molecular Nano-Machine: The State of the Art of <i>Helicobacter Pylori</i> Flagellum Organization

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