Regulation of Porin Gene Expression by the Two-Component Regulatory System EnvZ/OmpR

ABSTRACT FlhF proteins are putative GTPases that are often necessary for one or more steps in flagellar organelle development in polarly flagellated bacteria. In Campylobacter jejuni, FlhF is required for σ54-dependent flagellar gene expression and flagellar biosynthesis, but how FlhF influences these processes is unknown. Furthermore, the GTPase activity of any FlhF protein and the requirement of this speculated activity for steps in flagellar biosynthesis remain uncharacterized. We show here that C. jejuni FlhF hydrolyzes GTP, indicating that these proteins are GTPases. C. jejuni mutants producing FlhF proteins with reduced GTPase activity were not severely defective for σ54-dependent flagellar gene expression, unlike a mutant lacking FlhF. Instead, these mutants had a propensity to lack flagella or produce flagella in improper numbers or at nonpolar locations, indicating that GTP hydrolysis by FlhF is required for proper flagellar biosynthesis. Additional studies focused on elucidating a possible role for FlhF in σ54-dependent flagellar gene expression were conducted. These studies revealed that FlhF does not influence production of or signaling between the flagellar export apparatus and the FlgSR two-component regulatory system to activate σ54. Instead, our data suggest that FlhF functions in an independent pathway that converges with or works downstream of the flagellar export apparatus-FlgSR pathway to influence σ54-dependent gene expression. This study provides corroborative biochemical and genetic analyses suggesting that different activities of the C. jejuni FlhF GTPase are required for distinct steps in flagellar gene expression and biosynthesis. Our findings are likely applicable to many polarly flagellated bacteria that utilize FlhF in flagellar biosynthesis processes.

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Genetic Evidence for an Alternative Citrate-Dependent Biofilm Formation Pathway in Staphylococcus aureus That Is Dependent on Fibronectin Binding Proteins and the GraRS Two-Component Regulatory System

Infection and Immunity ◽

10.1128/iai.01370-07 ◽

2008 ◽

Vol 76 (6) ◽

pp. 2469-2477 ◽

Cited By ~ 44

Author(s):

Robert M. Q. Shanks ◽

Michael A. Meehl ◽

Kimberly M. Brothers ◽

Raquel M. Martinez ◽

Niles P. Donegan ◽

...

Keyword(s):

Gene Expression ◽

Staphylococcus Aureus ◽

Biofilm Formation ◽

Binding Proteins ◽

Regulatory System ◽

Tca Cycle ◽

Formation Pathway ◽

Two Component ◽

Fibronectin Binding ◽

Tca Cycle Intermediates

ABSTRACT We reported previously that low concentrations of sodium citrate strongly promote biofilm formation by Staphylococcus aureus laboratory strains and clinical isolates. Here, we show that citrate promotes biofilm formation via stimulating both cell-to-surface and cell-to-cell interactions. Citrate-stimulated biofilm formation is independent of the ica locus, and in fact, citrate represses polysaccharide adhesin production. We show that fibronectin binding proteins FnbA and FnbB and the global regulator SarA, which positively regulates fnbA and fnbB gene expression, are required for citrate's positive effects on biofilm formation, and citrate also stimulates fnbA and fnbB gene expression. Biofilm formation is also stimulated by several other tricarboxylic acid (TCA) cycle intermediates in an FnbA-dependent fashion. While aconitase contributes to biofilm formation in the absence of TCA cycle intermediates, it is not required for biofilm stimulation by these compounds. Furthermore, the GraRS two-component regulator and the GraRS-regulated efflux pump VraFG, identified for their roles in intermediate vancomycin resistance, are required for citrate-stimulated cell-to-cell interactions, but the GraRS regulatory system does not impact the expression of the fnbA and fnbB genes. Our data suggest that distinct genetic factors are required for the early steps in citrate-stimulated biofilm formation. Given the role of FnbA/FnbB and SarA in virulence in vivo and the lack of a role for ica-mediated biofilm formation in S. aureus catheter models of infection, we propose that the citrate-stimulated biofilm formation pathway may represent a clinically relevant pathway for the formation of these bacterial communities on medical implants.

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The senX3–regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence

Microbiology ◽

10.1099/mic.0.26245-0 ◽

2003 ◽

Vol 149 (6) ◽

pp. 1423-1435 ◽

Cited By ~ 122

Author(s):

Tanya Parish ◽

Debbie A. Smith ◽

Gretta Roberts ◽

Joanna Betts ◽

Neil G. Stoker

Keyword(s):

Gene Expression ◽

Mycobacterium Tuberculosis ◽

Mutant Strain ◽

Regulatory System ◽

Normal Growth ◽

Growth Defect ◽

Stress Genes ◽

Two Component ◽

Whole Genome Microarray ◽

Immunocompetent Mice

Two-component regulatory systems have been widely implicated in bacterial virulence. To investigate the role of one such system in Mycobacterium tuberculosis, a strain was constructed in which the senX3–regX3 system was deleted by homologous recombination. The mutant strain (Tame15) showed a growth defect after infection of macrophages and was attenuated in both immunodeficient and immunocompetent mice. Competitive hybridization of total RNA from the wild-type and mutant strains to a whole-genome microarray was used to identify changes in gene expression resulting from the deletion. One operon was highly up-regulated in the mutant, indicating that regX3 probably has a role as a repressor of this operon. Other genes which were up- or down-regulated were also identified. Many of the genes showing down-regulation are involved in normal growth of the bacterium, indicating that the mutant strain is subject to some type of growth slow-down or stress. Genes showing differential expression were further grouped according to their pattern of gene expression under other stress conditions. From this analysis 50 genes were identified which are the most likely to be controlled by RegX3. Most of these genes are of unknown function and no obvious motifs were found upstream of the genes identified. Thus, it has been demonstrated that the senX3–regX3 two-component system is involved in the virulence of M. tuberculosis and a number of genes controlled by this system have been identified.

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The roles of SsrA–SsrB and OmpR–EnvZ in the regulation of genes encoding the Salmonella typhimurium SPI-2 type III secretion system

Microbiology ◽

10.1099/mic.0.26397-0 ◽

2003 ◽

Vol 149 (9) ◽

pp. 2385-2396 ◽

Cited By ~ 102

Author(s):

Junkal Garmendia ◽

Carmen R. Beuzón ◽

Javier Ruiz-Albert ◽

David W. Holden

Keyword(s):

Gene Expression ◽

Salmonella Typhimurium ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Regulatory System ◽

Secretion System ◽

Host Cells ◽

Environmental Signals ◽

Type Iii ◽

Two Component

The type III secretion system (TTSS) encoded by Salmonella typhimurium pathogenicity island 2 (SPI-2) is expressed after bacterial entry into host cells. The SPI-2 TTSS secretes the translocon components SseBCD, which translocate across the vacuolar membrane a number of effector proteins whose action is required for intracellular bacterial replication. Several of these effectors, including SifA and SifB, are encoded outside SPI-2. The two-component regulatory system SsrA–SsrB, encoded within SPI-2, controls the expression of components of the SPI-2 TTSS apparatus as well as its translocated effectors. The expression of SsrA–B is in turn regulated by the OmpR–EnvZ two-component system, by direct binding of OmpR to the ssrAB promoter. Several environmental signals have been shown to induce in vitro expression of genes regulated by the SsrA–B or OmpR–EnvZ systems. In this work, immunoblotting and flow cytometry were used to analyse the roles of SsrA–B and OmpR–EnvZ in coupling different environmental signals to changes in expression of a SPI-2 TTSS translocon component (SseB) and two effector genes (sifA and sifB). Using single and double mutant strains the relative contribution of each regulatory system to the response generated by low osmolarity, acidic pH or the absence of Ca2+ was determined. SsrA–B was found to be essential for the induction of SPI-2 gene expression in response to each of these individual signals. OmpR–EnvZ was found to play a minor role in sensing these signals and to require a functional SsrA–B system to mediate their effect on SPI-2 TTSS gene expression.

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Interacting regulatory networks in the facultative photosynthetic bacterium, Rhodobacter sphaeroides 2.4.1

Biochemical Society Transactions ◽

10.1042/bst0330051 ◽

2005 ◽

Vol 33 (1) ◽

pp. 51-55 ◽

Cited By ~ 18

Author(s):

S. Kaplan ◽

J. Eraso ◽

J.H. Roh

Keyword(s):

Gene Expression ◽

Rhodobacter Sphaeroides ◽

Regulatory Networks ◽

Regulatory System ◽

Photosynthetic Bacterium ◽

Regulatory Elements ◽

Terminal Oxidase ◽

Anaerobic Conditions ◽

Photosynthetic Membrane ◽

Two Component

Regulation of photosynthetic membrane synthesis in Rhodobacter sphaeroides 2.4.1 is dependent on the interactions of numerous regulatory elements, with two of the most important being the cbb3 terminal oxidase and the PrrBAC two-component regulatory system. Here, we reveal that the cbb3 terminal oxidase possesses extensive, additional regulatory activities under anaerobic conditions, and that the PrrBAC system is further involved in the regulation of the expression of more than 20% of the R. sphaeroides genome under anaerobic conditions, extending well beyond functions related to redox gene expression.

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The Two-Component Regulatory System senX3-regX3 Regulates Phosphate-Dependent Gene Expression in Mycobacterium smegmatis

Journal of Bacteriology ◽

10.1128/jb.00190-07 ◽

2007 ◽

Vol 189 (15) ◽

pp. 5495-5503 ◽

Cited By ~ 56

Author(s):

Robert T. Glover ◽

Jordan Kriakov ◽

Scott J. Garforth ◽

Anthony D. Baughn ◽

William R. Jacobs

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Mycobacterium Smegmatis ◽

Transcriptional Control ◽

Response Regulator ◽

Transcriptional Start Site ◽

Regulatory System ◽

Expression Of Genes ◽

Two Component ◽

Regulated Gene Expression

ABSTRACT Phosphate import is required for the growth of mycobacteria and is regulated by environmental inorganic phosphate (Pi) concentrations, although the mechanism of this regulation has not been characterized. The expression of genes involved in Pi acquisition is frequently regulated by two-component regulatory systems (2CRs) consisting of a sensor histidine kinase and a DNA-binding response regulator. In this work, we have identified the senX3-regX3 2CR as a Pi-dependent regulator of genes involved in phosphate acquisition in Mycobacterium smegmatis. Characterization of senX3 mutants with different PhoA phenotypes suggests a dual role for SenX3 as a phosphatase or a phosphodonor for the response regulator RegX3, depending upon Pi availability. Expression of PhoA activity required phosphorylation of RegX3, consistent with a role for phosphorylated RegX3 (RegX3∼P) as a transcriptional activator of phoA. Furthermore, purified RegX3∼P bound to promoter sequences from phoA, senX3, and the high-affinity phosphate transporter component pstS, demonstrating direct transcriptional control of all three genes. DNase I footprinting and primer extension analyses have further defined the DNA-binding region and transcriptional start site within the phoA promoter. A DNA motif consisting of an inverted repeat was identified in each of the promoters bound by RegX3∼P. Based upon our findings, we propose a model for Pi-regulated gene expression mediated by SenX3-RegX3 in mycobacteria.

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Transcription Analysis of a Lantibiotic Gene Cluster from Bifidobacterium longum DJO10A

Applied and Environmental Microbiology ◽

10.1128/aem.00571-11 ◽

2011 ◽

Vol 77 (17) ◽

pp. 5879-5887 ◽

Cited By ~ 28

Author(s):

Ju-Hoon Lee ◽

Xiulan Li ◽

Daniel J. O'Sullivan

Keyword(s):

Gene Expression ◽

Response Regulator ◽

Bifidobacterium Longum ◽

Regulatory System ◽

Broth Culture ◽

Northern Hybridization ◽

Transcription Start ◽

Transcription Analysis ◽

Content Type ◽

Two Component

ABSTRACTBifidobacterium longumDJO10A was previously demonstrated to produce a lantibiotic, but only during growth on agar media. To evaluate the feasibility of production of this lantibiotic in broth media, a transcription analysis of thelanAgene was undertaken. Comparative microarray analysis of broth and agar cultures ofB. longumDJO10A revealed that the lantibiotic production, modification, transport/peptidase, and immunity genes were significantly upregulated in agar cultures, while the two-component regulatory genes were expressed equally under both conditions. This suggested that the signal transduction regulatory system should function in broth cultures. Real-time PCR and Northern hybridization confirmed thatlanAgene expression was significantly repressed in broth cultures. A crude lantibiotic preparation from an agar-grown culture was obtained, and its antimicrobial spectrum analysis revealed a broad inhibition range. Addition of this extract to broth cultures ofB. longumDJO10A inducedlanAgene expression in a dose-dependent fashion. Subinoculation using >10% of an induced broth culture maintainedlanAexpression. The expression oflanAwas log-phase specific, being significantly downregulated in stationary phase. Transcription start analysis oflanArevealed a 284-bp 5′ untranslated region, which was proposed to be involved in repression of transcription, while an inverted repeat structure located at bp −75 relative to the transcription start was strategically located to likely function as a binding site for the two-component response regulator. Understanding the transcription regulation of thislanAgene is the first step toward enabling production of this novel and potentially interesting lantibiotic in broth cultures.

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Activation of the Campylobacter jejuni FlgSR Two-Component System Is Linked to the Flagellar Export Apparatus

Journal of Bacteriology ◽

10.1128/jb.01689-08 ◽

2009 ◽

Vol 191 (8) ◽

pp. 2656-2667 ◽

Cited By ~ 44

Author(s):

Stephanie N. Joslin ◽

David R. Hendrixson

Keyword(s):

Gene Expression ◽

Campylobacter Jejuni ◽

Response Regulator ◽

Regulatory System ◽

Two Component System ◽

Regulatory Cascade ◽

Regulatory Systems ◽

Two Component ◽

Flagellar Protein ◽

Flagellar Genes

ABSTRACT Activation of σ54-dependent gene expression essential for formation of flagella in Campylobacter jejuni requires the components of the inner membrane-localized flagellar export apparatus and the FlgSR two-component regulatory system. In this study, we characterized the FlgS sensor kinase and how activation of the protein is linked to the flagellar export apparatus. We found that FlgS is localized to the C. jejuni cytoplasm and that His141 of FlgS is essential for autophosphorylation, phosphorelay to the cognate FlgR response regulator, motility, and expression of σ54-dependent flagellar genes. Mutants with incomplete flagellar export apparatuses produced wild-type levels of FlgS and FlgR, but they were defective for signaling through the FlgSR system. By using genetic approaches, we found that FlgSR activity is linked to and downstream of the flagellar export apparatus in a regulatory cascade that terminates in expression of σ54-dependent flagellar genes. By analyzing defined flhB and fliI mutants of C. jejuni that form flagellar export apparatuses that are secretion incompetent, we determined that formation of the apparatus is required to contribute to the signal sensed by FlgS to terminate in activation of expression of σ54-dependent flagellar genes. Considering that the flagellar export apparatuses of Escherichia coli and Salmonella species influence σ28-dependent flagellar gene expression, our work expands the signaling activity of the apparatuses to include σ54-dependent pathways of C. jejuni and possibly other motile bacteria. This study indicates that these apparatuses have broader functions beyond flagellar protein secretion, including activation of essential two-component regulatory systems required for expression of σ54-dependent flagellar genes.

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