scholarly journals FlhF and Its GTPase Activity Are Required for Distinct Processes in Flagellar Gene Regulation and Biosynthesis in Campylobacter jejuni

2009 ◽  
Vol 191 (21) ◽  
pp. 6602-6611 ◽  
Author(s):  
Murat Balaban ◽  
Stephanie N. Joslin ◽  
David R. Hendrixson
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Campylobacter Jejuni ◽  
Regulatory System ◽  
Flagellar Gene ◽  
Gtpase Activity ◽  
Gtp Hydrolysis ◽  
Genetic Analyses ◽  
Flagellar Biosynthesis ◽  
Two Component

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.

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

2009 ◽  
Vol 191 (8) ◽  
pp. 2656-2667 ◽  
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.

scholarly journals RocA Binds CsrS To Modulate CsrRS-Mediated Gene Regulation in Group A Streptococcus

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Nicola N. Lynskey ◽  
Jorge J. Velarde ◽  
Meredith B. Finn ◽  
Simon L. Dove ◽  
Michael R. Wessels
Keyword(s):  
Gene Regulation ◽  
Target Genes ◽  
Response Regulator ◽  
Critical Role ◽  
Regulatory Protein ◽  
Regulatory System ◽  
Fine Tuning ◽  
Human Pathogen ◽  
Two Component ◽  
Group A

ABSTRACT The orphan regulator RocA plays a critical role in the colonization and pathogenesis of the obligate human pathogen group A Streptococcus. Despite multiple lines of evidence supporting a role for RocA as an auxiliary regulator of the control of virulence two-component regulatory system CsrRS (or CovRS), the mechanism of action of RocA remains unknown. Using a combination of in vitro and in vivo techniques, we now find that RocA interacts with CsrS in the streptococcal membrane via its N-terminal region, which contains seven transmembrane domains. This interaction is essential for RocA-mediated regulation of CsrRS function. Furthermore, we demonstrate that RocA forms homodimers via its cytoplasmic domain. The serotype-specific RocA truncation in M3 isolates alters this homotypic interaction, resulting in protein aggregation and impairment of RocA-mediated regulation. Taken together, our findings provide insight into the molecular requirements for functional interaction of RocA with CsrS to modulate CsrRS-mediated gene regulation. IMPORTANCE Bacterial two-component regulatory systems, comprising a membrane-bound sensor kinase and cytosolic response regulator, are critical in coordinating the bacterial response to changing environmental conditions. More recently, auxiliary regulators which act to modulate the activity of two-component systems, allowing integration of multiple signals and fine-tuning of bacterial responses, have been identified. RocA is a regulatory protein encoded by all serotypes of the important human pathogen group A Streptococcus. Although RocA is known to exert its regulatory activity via the streptococcal two-component regulatory system CsrRS, the mechanism by which it functions was unknown. Based on new experimental evidence, we propose a model whereby RocA interacts with CsrS in the streptococcal cell membrane to enhance CsrS autokinase activity and subsequent phosphotransfer to the response regulator CsrR, which mediates transcriptional repression of target genes.

scholarly journals 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

2008 ◽  
Vol 76 (6) ◽  
pp. 2469-2477 ◽  
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.

scholarly journals Variation in the Group B Streptococcus CsrRS Regulon and Effects on Pathogenicity

2008 ◽  
Vol 190 (6) ◽  
pp. 1956-1965 ◽  
Author(s):  
Sheng-Mei Jiang ◽  
Nadeeza Ishmael ◽  
Julie Dunning Hotopp ◽  
Manuela Puliti ◽  
Luciana Tissi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Virulence Factors ◽  
Group B Streptococcus ◽  
Regulatory System ◽  
Systemic Infection ◽  
Global Gene Expression ◽  
Pathogenic Potential ◽  
Group B

ABSTRACT CsrRS (or CovRS) is a two-component regulatory system that controls expression of multiple virulence factors in the important human pathogen group B Streptococcus (GBS). We now report global gene expression studies in GBS strains 2603V/R and 515 and their isogenic csrR and csrS mutants. Together with data reported previously for strain NEM316, the results reveal a conserved 39-gene CsrRS regulon. In vitro phosphorylation-dependent binding of recombinant CsrR to promoter regions of both positively and negatively regulated genes suggests that direct binding of CsrR can mediate activation as well as repression of target gene expression. Distinct patterns of gene regulation in csrR versus csrS mutants in strain 2603V/R compared to 515 were associated with different hierarchies of relative virulence of wild-type, csrR, and csrS mutants in murine models of systemic infection and septic arthritis. We conclude that CsrRS regulates a core group of genes including important virulence factors in diverse strains of GBS but also displays marked variability in the repertoire of regulated genes and in the relative effects of CsrS signaling on CsrR-mediated gene regulation. Such variation is likely to play an important role in strain-specific adaptation of GBS to particular host environments and pathogenic potential in susceptible hosts.

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

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1423-1435 ◽  
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.

scholarly journals FliZ Is a Posttranslational Activator of FlhD4C2-Dependent Flagellar Gene Expression

2008 ◽  
Vol 190 (14) ◽  
pp. 4979-4988 ◽  
Author(s):  
Supreet Saini ◽  
Jonathon D. Brown ◽  
Phillip D. Aldridge ◽  
Christopher V. Rao
Keyword(s):  
Gene Expression ◽  
Sigma Factor ◽  
Flagellar Gene ◽  
Critical Aspect ◽  
Swarming Motility ◽  
Flagellar Biosynthesis ◽  
Serovar Typhimurium ◽  
Flagellar Assembly

ABSTRACT Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the σ28-FlgM checkpoint, which couples the expression of late flagellar (Pclass3) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (Pclass2) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD4C2-dependent activator of Pclass2/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD4C2 posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor σ28 and the filament-cap chaperone/FlhD4C2 inhibitor FliT. Furthermore, we show that the previously described ability of σ28 to activate Pclass2/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping Pclass2 and Pclass3 promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ's role in swimming and swarming motility.

scholarly journals A Regulatory Checkpoint during Flagellar Biogenesis in Campylobacter jejuni Initiates Signal Transduction To Activate Transcription of Flagellar Genes

mBio ◽  
2013 ◽  
Vol 4 (5) ◽  
Author(s):  
Joseph M. Boll ◽  
David R. Hendrixson
Keyword(s):  
Signal Transduction ◽  
Campylobacter Jejuni ◽  
Gene Transcription ◽  
Type Iii Secretion ◽  
Secretion System ◽  
Flagellar Gene ◽  
Content Type ◽  
Regulatory Systems ◽  
Two Component ◽  
Flagellar Biogenesis

ABSTRACTMany polarly flagellated bacteria require similar two-component regulatory systems (TCSs) and σ54to activate transcription of genes essential for flagellar motility. Herein, we discovered that in addition to the flagellar type III secretion system (T3SS), theCampylobacter jejuniflagellar MS ring and rotor are required to activate the FlgSR TCS. Mutants lacking the FliF MS ring and FliG C ring rotor proteins were as defective as T3SS mutants in FlgSR- and σ54-dependent flagellar gene expression. Also, FliF and FliG required each other for stability, which is mediated by atypical extensions to the proteins. A FliF mutant that presumably does not interact with the T3SS protein FlhA did not support flagellar gene transcription, suggesting that FliF-T3SS interactions are essential to generate a signal sensed by the cytoplasmic FlgS histidine kinase. Furthermore, the flagellar T3SS was required for FlgS to immunoprecipitate with FliF and FliG. We propose a model whereby the flagellar T3SS facilitates FliF and FliG multimerization into the MS ring and rotor. As a result, these flagellar structures form a cytoplasmic complex that interacts with and is sensed by FlgS. The synthesis of these structures appears to be a regulatory checkpoint in flagellar biogenesis that the FlgS kinase monitors to initiate signal transduction that activates σ54and expression of genes required for the next stage of flagellation. Given that other polar flagellates have flagellar transcriptional hierarchies that are organized similarly as inC. jejuni, this regulatory checkpoint may exist in a broad range of bacteria to influence similar TCSs and flagellar gene transcription.IMPORTANCEDespite the presence of numerous two-component regulatory systems (TCSs) in bacteria, direct signals sensed by TCSs to activate signal transduction are known for only a minority. Polar flagellates, includingPseudomonas,Vibrio,Helicobacter, andCampylobacterspecies, require a similar TCS and σ54for flagellar gene transcription, but the activating signals for these TCSs are unknown. We explored signals that activate theCampylobacter jejuniFlgSR TCS to initiate σ54-dependent flagellar gene transcription. Our discoveries suggest that the FlgS histidine kinase monitors the formation of the flagellar type III secretion system and the surrounding MS and C rings. The synthesis of these structures creates a regulatory checkpoint in flagellar biogenesis that is sensed by FlgS to ensure proper transcription of the next set of genes for subsequent steps in flagellation. Given the conservation of flagellar-associated TCSs and transcriptional cascades in polar flagellates, this regulatory checkpoint in flagellar biogenesis likely impacts flagellation in a broad range of bacteria.

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

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2385-2396 ◽  
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.

scholarly journals Genome-Wide Analyses of Escherichia coli Gene Expression Responsive to the BaeSR Two-Component Regulatory System

2005 ◽  
Vol 187 (5) ◽  
pp. 1763-1772 ◽  
Author(s):  
Kunihiko Nishino ◽  
Takeshi Honda ◽  
Akihito Yamaguchi
Keyword(s):  
Escherichia Coli ◽  
Global Analysis ◽  
Regulatory System ◽  
Pcr Analysis ◽  
Sequence Motif ◽  
Expression Levels ◽  
Maltose Transport ◽  
Reverse Transcription Pcr ◽  
Flagellar Biosynthesis ◽  
Two Component

ABSTRACT The BaeSR two-component regulatory system controls expression of exporter genes conferring drug resistance in Escherichia coli (S. Nagakubo, K. Nishino, T. Hirata, and A. Yamaguchi, J. Bacteriol. 184:4161-4167, 2002; N. Baranova and H. Nikaido, J. Bacteriol. 184:4168-4176, 2002). To understand the whole picture of BaeSR regulation, a DNA microarray analysis of the effect of BaeR overproduction was performed. BaeR overproduction activated 59 genes related to two-component signal transduction, chemotactic responses, flagellar biosynthesis, maltose transport, and multidrug transport, and BaeR overproduction also repressed the expression of the ibpA and ibpB genes. All of the changes in the expression levels were also observed by quantitative real-time reverse transcription-PCR analysis. The expression levels of 15 of the 59 BaeR-activated genes were decreased by deletion of baeSR. Of 11 genes induced by indole (a putative inducer of the BaeSR system), 10 required the BaeSR system for induction. Combination of the expression data sets revealed a BaeR-binding site sequence motif, 5′-TTTTTCTCCATDATTGGC-3′ (where D is G, A, or T). Several genes up-regulated by BaeR overproduction, including genes for maltose transport, chemotactic responses, and flagellar biosynthesis, required an intact PhoBR or CreBC two-component regulatory system for up-regulation. These data indicate that there is cross-regulation among the BaeSR, PhoBR, and CreBC two-component regulatory systems. Such a global analysis should reveal the regulatory network of the BaeSR system.

Sign in / Sign up

Export Citation Format

Share Document