scholarly journals Phosphorylation of the Group A Streptococcal CovR Response Regulator Causes Dimerization and Promoter-Specific Recruitment by RNA Polymerase

2006 ◽  
Vol 188 (13) ◽  
pp. 4620-4626 ◽  
Author(s):  
Asiya A. Gusa ◽  
Jinxin Gao ◽  
Virginia Stringer ◽  
Gordon Churchward ◽  
June R. Scott
Keyword(s):  
Rna Polymerase ◽  
Virulence Factors ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Regulatory System ◽  
In Vitro Transcription ◽  
Wild Type ◽  
Group A ◽  
The Difference

ABSTRACT The group A streptococcus (GAS), Streptococcus pyogenes, is an important human pathogen that causes infections ranging in severity from self-limiting pharyngitis to severe invasive diseases that are associated with significant morbidity and mortality. The pathogenic effects of GAS are mediated by the expression of virulence factors, one of which is the hyaluronic acid capsule (encoded by genes in the has operon). The expression of these virulence factors is controlled by the CovR/S (CsrR/S) two-component regulatory system of GAS which regulates, directly or indirectly, the expression of about 15% of the genome. CovR is a member of the OmpR/PhoB family of transcriptional regulators. Here we show that phosphorylation by acetyl phosphate results in dimerization of CovR. Dimerization was not observed using a D53A mutant of CovR, indicating that D53 is the site of phosphorylation in CovR. Phosphorylation stimulated binding of CovR to a DNA fragment containing the promoter of the has operon (Phas) approximately twofold. Binding of CovR D53A mutant protein to Phas was indistinguishable from the binding of wild-type unphosphorylated CovR. In vitro transcription, using purified GAS RNA polymerase, showed that wild-type CovR repressed transcription, and repression was stimulated more than sixfold by phosphorylation. In the presence of RNA polymerase, binding at Phas of phosphorylated, but not unphosphorylated, CovR was stimulated about fourfold, which accounts for the difference in the effect of phosphorylation on repression versus DNA binding. Thus, regulation of Phas by CovR is direct, and the degree of repression of Phas is controlled by the phosphorylation of CovR.

scholarly journals Transcriptional Activation of the Bacillus subtilis spoIIG Promoter by the Response Regulator Spo0A Is Independent of the C-Terminal Domain of the RNA Polymerase Alpha Subunit

1998 ◽  
Vol 180 (17) ◽  
pp. 4760-4763 ◽  
Author(s):  
Dean A. Rowe-Magnus ◽  
Mario Mencía ◽  
Fernando Rojo ◽  
Margarita Salas ◽  
George B. Spiegelman
Keyword(s):  
Bacillus Subtilis ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Response Regulator ◽  
In Vitro Transcription ◽  
Alpha Subunit ◽  
Deletion Mutants ◽  
Wild Type ◽  
Initiation Mechanism

ABSTRACT In vitro transcription from the spoIIG promoter byBacillus subtilis RNA polymerase reconstituted with wild-type alpha subunits and with C-terminal deletion mutants of the alpha subunit was equally stimulated by the response regulator Spo0A. Some differences in the structure of open complexes formed by RNA polymerase containing alpha subunit mutants were noted, although the wild-type and mutant polymerases appeared to use the same initiation mechanism.

scholarly journals TrxR, a New CovR-Repressed Response Regulator That Activates the Mga Virulence Regulon in Group A Streptococcus

2008 ◽  
Vol 76 (10) ◽  
pp. 4659-4668 ◽  
Author(s):  
Temekka V. Leday ◽  
Kathryn M. Gold ◽  
Traci L. Kinkel ◽  
Samantha A. Roberts ◽  
June R. Scott ◽  
...  
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Luciferase Reporter ◽  
Infection Model ◽  
Insertion Mutation ◽  
Wild Type ◽  
Virulence Attenuation ◽  
Group A

ABSTRACT Coordinate regulation of virulence factors by the group A streptococcus (GAS) Streptococcus pyogenes is important in this pathogen's ability to cause disease. To further elucidate the regulatory network in this human pathogen, the CovR-repressed two-component system (TCS) trxSR was chosen for further analysis based on its homology to a virulence-related TCS in Streptococcus pneumoniae. In a murine skin infection model, an insertion mutation in the response regulator gene, trxR, led to a significant reduction in lesion size, lesion severity, and lethality. Curing the trxR mutation restored virulence comparable to the wild-type strain. The trxSR operon was defined in vivo, and CovR was found to directly repress its promoter in vitro. DNA microarray analysis established that TrxR activates transcription of Mga-regulated virulence genes, which may explain the virulence attenuation of the trxR mutant. This regulation appears to occur by activation of the mga promoter, Pmga, as demonstrated by analysis of a luciferase reporter fusion. Complementation of the trxR mutant with trxR on a plasmid restored expression of Mga regulon genes and restored virulence in the mouse model to wild-type levels. TrxR is the first TCS shown to regulate Mga expression. Because it is CovR repressed, TrxR defines a new pathway by which CovR can influence Mga to affect pathogenesis in the GAS.

scholarly journals The Secreted Esterase of Group A Streptococcus Is Important for Invasive Skin Infection and Dissemination in Mice

2009 ◽  
Vol 77 (12) ◽  
pp. 5225-5232 ◽  
Author(s):  
Hui Zhu ◽  
Mengyao Liu ◽  
Paul Sumby ◽  
Benfang Lei
Keyword(s):  
Virulence Factors ◽  
Type Strain ◽  
Wild Type Strain ◽  
Group A Streptococcus ◽  
Regulatory System ◽  
Wild Type ◽  
Subcutaneous Infection ◽  
Systemic Dissemination ◽  
Gene Regulatory System ◽  
Group A

ABSTRACT Virulence factors regulated by the CovRS/CsrRS two-component gene regulatory system contribute to the invasive diseases caused by group A Streptococcus (GAS). To determine whether the streptococcal secreted esterase (Sse), an antigen that protects against subcutaneous GAS infection, is one of these virulence factors, we investigated the phenotype of a nonpolar sse deletion mutant strain (Δsse). In addition, we examined the effects of covS mutation on sse expression. As assessed using a mouse model of subcutaneous infection, the virulence of the Δsse strain is attenuated and the overall pathology is reduced. Furthermore, GAS was detected in the blood and spleens from mice subcutaneously infected with the parental strain, whereas mice subcutaneously infected with the Δsse strain had no GAS present in their blood and spleens. The ability of the mutant to survive in the subcutis of mice appeared to be compromised. The growth of the Δsse strain in rich and chemically defined media and nonimmune human blood and sera was slower than that of the wild-type strain. Complementation restored the phenotype of the Δsse strain to that of the wild-type strain. The wild-type, Δsse, and complement strains had no detectable SpeB activity. Expression of Sse is negatively controlled by CovRS. These findings suggest that Sse is a CovRS-regulated virulence factor that is important for the virulence of GAS in subcutaneous infection and plays an important role in severe soft tissue infections and systemic dissemination of GAS from the skin.

scholarly journals CovR Activation of the Dipeptide Permease Promoter (PdppA) in Group A Streptococcus

2006 ◽  
Vol 189 (4) ◽  
pp. 1407-1416 ◽  
Author(s):  
Asiya A. Gusa ◽  
Barbara J. Froehlich ◽  
Devak Desai ◽  
Virginia Stringer ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Activation Of Transcription ◽  
Genes Encoding ◽  
Group A ◽  
Transcription In Vitro ◽  
Dna Template ◽  
Regulated Promoters

ABSTRACT CovR, the two-component response regulator of Streptococcus pyogenes (group A streptococcus [GAS]) directly or indirectly represses about 15% of the genome, including genes encoding many virulence factors and itself. Transcriptome analyses also showed that some genes are activated by CovR. We asked whether the regulation by CovR of one of these genes, dppA, the first gene in an operon encoding a dipeptide permease, is direct or indirect. Direct regulation by CovR was suggested by the presence of five CovR consensus binding sequences (CBs) near the putative promoter. In this study, we identified the 5′ end of the dppA transcript synthesized in vivo and showed that the start of dppA transcription in vitro is the same. We found that CovR binds specifically to the dppA promoter region (PdppA) in vitro with an affinity similar to that at which it binds to other CovR-regulated promoters. Disruption of any of the five CBs by a substitution of GG for TT inhibited CovR binding to that site in vitro, and binding at two of the CBs appeared cooperative. In vivo, CovR activation of transcription was not affected by individual mutations of any of the four CBs that we could study. This suggests that the binding sites are redundant in vivo. In vitro, CovR did not activate transcription from PdppA in experiments using purified GAS RNA polymerase and either linear or supercoiled DNA template. Therefore, we propose that in vivo, CovR may interfere with the binding of a repressor of PdppA.

scholarly journals A Response Regulator That Represses Transcription of Several Virulence Operons in the Group A Streptococcus

1999 ◽  
Vol 181 (12) ◽  
pp. 3649-3657 ◽  
Author(s):  
Michael J. Federle ◽  
Kevin S. McIver ◽  
June R. Scott
Keyword(s):  
Group A Streptococcus ◽  
Response Regulator ◽  
Regulatory System ◽  
Insertion Mutant ◽  
Multiple Gene ◽  
Streptolysin S ◽  
Streptolysin O ◽  
Group A ◽  
Complement C5a ◽  
Global Regulatory System

ABSTRACT A search for homologs of the Bacillus subtilis PhoP response regulator in the group A streptococcus (GAS) genome revealed three good candidates. Inactivation of one of these, recently identified as csrR (J. C. Levin and M. R. Wessels, Mol. Microbiol. 30:209–219, 1998), caused the strain to produce mucoid colonies and to increase transcription ofhasA, the first gene in the operon for capsule synthesis. We report here that a nonpolar insertion in this gene also increased transcription of ska (encoding streptokinase),sagA (streptolysin S), and speMF (mitogenic factor) but did not affect transcription of slo(streptolysin O), mga (multiple gene regulator of GAS),emm (M protein), scpA (complement C5a peptidase), or speB or speC (pyrogenic exotoxins B and C). The amounts of streptokinase, streptolysin S, and capsule paralleled the levels of transcription of their genes in all cases. Because CsrR represses genes unrelated to those for capsule synthesis, and because CsrA-CsrB is a global regulatory system inEscherichia coli whose mechanism is unrelated to that of these genes in GAS, the locus has been renamed covR, for “control of virulence genes” in GAS. Transcription of thecovR operon was also increased in the nonpolar insertion mutant, indicating that CovR represses its own synthesis as well. All phenotypes of the covR nonpolar insertion mutant were complemented by the covR gene on a plasmid. CovR acts on operons expressed both in exponential and in stationary phase, demonstrating that the CovR-CovS pathway is separate from growth phase-dependent regulation in GAS. Therefore, CovR is the first multiple-gene repressor of virulence factors described for this important human pathogen.

scholarly journals The CiaR Response Regulator in Group B Streptococcus Promotes Intracellular Survival and Resistance to Innate Immune Defenses

2008 ◽  
Vol 191 (7) ◽  
pp. 2023-2032 ◽  
Author(s):  
Darin Quach ◽  
Nina M. van Sorge ◽  
Sascha A. Kristian ◽  
Joshua D. Bryan ◽  
Daniel W. Shelver ◽  
...  
Keyword(s):  
Response Regulator ◽  
Newborn Infants ◽  
Regulatory System ◽  
Intracellular Survival ◽  
Invasive Disease ◽  
Neonatal Meningitis ◽  
Host Defenses ◽  
Wild Type ◽  
Group B

ABSTRACT Group B Streptococcus (GBS) is major cause of invasive disease in newborn infants and the leading cause of neonatal meningitis. To gain access to the central nervous system (CNS), GBS must not only subvert host defenses in the bloodstream but also invade and survive within brain microvascular endothelial cells (BMEC), the principal cell layer composing the blood-brain barrier (BBB). While several GBS determinants that contribute to the invasion of BMEC have been identified, little is known about the GBS factors that are required for intracellular survival and ultimate disease progression. In this study we sought to identify these factors by screening a random GBS mutant library in an in vitro survival assay. One mutant was identified which contained a disruption in a two-component regulatory system homologous to CiaR/CiaH, which is present in other streptococcal pathogens. Deletion of the putative response regulator, ciaR, in GBS resulted in a significant decrease in intracellular survival within neutrophils, murine macrophages, and human BMEC, which was linked to increased susceptibility to killing by antimicrobial peptides, lysozyme, and reactive oxygen species. Furthermore, competition experiments with mice showed that wild-type GBS had a significant survival advantage over the GBS ΔciaR mutant in the bloodstream and brain. Microarray analysis comparing gene expression between wild-type and ΔciaR mutant GBS bacteria revealed several CiaR-regulated genes that may contribute to stress tolerance and the subversion of host defenses by GBS. Our results identify the GBS CiaR response regulator as a crucial factor in GBS intracellular survival and invasive disease pathogenesis.

scholarly journals A Chemokine-Degrading Extracellular Protease Made by Group A Streptococcus Alters Pathogenesis by Enhancing Evasion of the Innate Immune Response

2008 ◽  
Vol 76 (3) ◽  
pp. 978-985 ◽  
Author(s):  
Paul Sumby ◽  
Shizhen Zhang ◽  
Adeline R. Whitney ◽  
Fabiana Falugi ◽  
Guido Grandi ◽  
...  
Keyword(s):  
Immune Response ◽  
Innate Immune Response ◽  
Group A Streptococcus ◽  
Cell Envelope ◽  
Innate Immune ◽  
Infection Model ◽  
Dependent Manner ◽  
Wild Type ◽  
Group A

ABSTRACT Circumvention of the host innate immune response is critical for bacterial pathogens to infect and cause disease. Here we demonstrate that the group A Streptococcus (GAS; Streptococcus pyogenes) protease SpyCEP (S. pyogenes cell envelope protease) cleaves granulocyte chemotactic protein 2 (GCP-2) and growth-related oncogene alpha (GROα), two potent chemokines made abundantly in human tonsils. Cleavage of GCP-2 and GROα by SpyCEP abrogated their abilities to prime neutrophils for activation, detrimentally altering the innate immune response. SpyCEP expression is negatively regulated by the signal transduction system CovR/S. Purified recombinant CovR bound the spyCEP gene promoter region in vitro, indicating direct regulation. Immunoreactive SpyCEP protein was present in the culture supernatants of covR/S mutant GAS strains but not in supernatants from wild-type strains. However, wild-type GAS strains do express SpyCEP, where it is localized to the cell wall. Strain MGAS2221, an organism representative of the highly virulent and globally disseminated M1T1 GAS clone, differed significantly from its isogenic spyCEP mutant derivative strain in a mouse soft tissue infection model. Interestingly, and in contrast to previous studies, the isogenic mutant strain generated lesions of larger size than those formed following infection with the parent strain. The data indicate that SpyCEP contributes to GAS virulence in a strain- and disease-dependent manner.

scholarly journals Unraveling the Regulatory Network in Streptococcus pyogenes: the Global Response Regulator CovR Represses rivR Directly

2006 ◽  
Vol 189 (4) ◽  
pp. 1459-1463 ◽  
Author(s):  
Samantha A. Roberts ◽  
Gordon G. Churchward ◽  
June R. Scott
Keyword(s):  
Gene Expression ◽  
Rna Polymerase ◽  
Streptococcus Pyogenes ◽  
Regulatory Network ◽  
Group A Streptococcus ◽  
Response Regulator ◽  
Master Regulator ◽  
Global Response ◽  
Conserved Sequences ◽  
Group A

ABSTRACT The response regulator CovR acts as a master regulator of virulence in Streptococcus pyogenes by repressing transcription of approximately 15% of the group A streptococcus genome directly or indirectly. We demonstrate that phosphorylated CovR represses transcription of rivR directly by binding to conserved sequences located downstream from the promoter to block procession of RNA polymerase. This establishes the first link in a regulatory network where CovR interacts directly with other proteins that modulate gene expression.

scholarly journals Properties of Escherichia coli RNA polymerase from a strain devoid of the stringent response alarmone ppGpp.

2008 ◽  
Vol 55 (2) ◽  
pp. 317-323 ◽  
Author(s):  
Agnieszka Szalewska-Pałasz
Keyword(s):  
Escherichia Coli ◽  
Rna Polymerase ◽  
Stringent Response ◽  
In Vitro Transcription ◽  
Complex Stability ◽  
Wild Type ◽  
Guanosine Tetraphosphate ◽  
Sigma 70 ◽  
In Vitro Performance

The stringent response alarmone guanosine tetraphosphate (ppGpp) affects transcription from many promoters. ppGpp binds directly to the transcription enzyme of Escherichia coli, RNA polymerase. Analysis of the crystal structure of RNA polymerase with ppGpp suggested that binding of this nucleotide may result in some conformational or post-translational alterations to the enzyme. These changes might affect in vitro performance of the enzyme. Here, a comparison of the in vitro properties of RNA polymerases isolated from wild type and ppGpp-deficient bacteria shows that both enzymes do not differ in i) transcription activity of various promoters (e.g. sigma(70)-rrnB P1, lambdapL, T7A1), ii) response to ppGpp, iii) promoter-RNA polymerase open complex stability. Thus, it may be concluded that ppGpp present in the bacterial cell prior to purification of the RNA polymerase does not result in the alterations to the enzyme that could be permanent and affect its in vitro transcription capacity.

scholarly journals Bordetella pertussis fim3gene regulation by BvgA: Phosphorylation controls the formation of inactive vs. active transcription complexes

2015 ◽  
Vol 112 (6) ◽  
pp. E526-E535 ◽  
Author(s):  
Alice Boulanger ◽  
Kyung Moon ◽  
Kimberly B. Decker ◽  
Qing Chen ◽  
Leslie Knipling ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Bordetella Pertussis ◽  
Transcription Initiation ◽  
Response Regulator ◽  
Gene Activation ◽  
Virulence Gene ◽  
In Vitro Transcription ◽  
Transcription Complexes

Two-component systems [sensor kinase/response regulator (RR)] are major tools used by microorganisms to adapt to environmental conditions. RR phosphorylation is typically required for gene activation, but few studies have addressed how and if phosphorylation affects specific steps during transcription initiation. We characterized transcription complexes made with RNA polymerase and theBordetella pertussisRR, BvgA, in its nonphosphorylated or phosphorylated (BvgA∼P) state at Pfim3, the promoter for the virulence genefim3(fimbrial subunit), using gel retardation, potassium permanganate and DNase I footprinting, cleavage reactions with protein conjugated with iron bromoacetamidobenzyl-EDTA, and in vitro transcription. Previous work has shown that the level of nonphosphorylated BvgA remains high in vivo under conditions in which BvgA is phosphorylated. Our results here indicate that surprisingly both BvgA and BvgA∼P form open and initiating complexes with RNA polymerase at Pfim3. However, phosphorylation of BvgA is needed to generate the correct conformation that can transition to competent elongation. Footprints obtained with the complexes made with nonphosphorylated BvgA are atypical; while the initiating complex with BvgA synthesizes short RNA, it does not generate full-length transcripts. Extended incubation of the BvgA/RNA polymerase initiated complex in the presence of heparin generates a stable, but defective species that depends on the initial transcribed sequence offim3. We suggest that the presence of nonphosphorylated BvgA down-regulates Pfim3activity when phosphorylated BvgA is present and may allow the bacterium to quickly adapt to the loss of inducing conditions by rapidly eliminating Pfim3activation once the signal for BvgA phosphorylation is removed.

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