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Patrice Courvalin - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn1546 by the VanR-VanS Two-Component Regulatory System
Two-Component Signal Transduction, 2014Co-Authors: Michel Arthur, Gerard D. Wright, Patrice Courvalin, Florence Depardieu, Theodore R. Holman, Christopher T. WalshAbstract:Glycopeptide antibiotics vancomycin and teicoplanin are used to treat severe infections caused by gram-positive cocci. Strains displaying the so-called VanA resistance phenotype are inducibly resistant to high levels of vancomycin and teicoplanin. Production of the depsipeptide D-Ala-D-Lac is controlled by the VanR-VanS Two-Component Regulatory System that activates transcription of vancomycin resistance genes in response to the presence of glycopeptides in the culture medium. Response regulators (RRs) of this subclass regulate transcription at specific promoters thought to be recognized by the main form of RNA polymerase holoenzyme, corresponding to Eσ70 in Escherichia coli. The first 122 amino acids at the N terminus of VanS are not related in sequence to other HPKs. This region of VanS contains two clusters of hydrophobic amino acids that could correspond to membrane-spanning regions. Validation of the predicted roles of VanS and VanR in sequential phosphoryl group transfer was obtained by overproduction, purification, and assay of the two proteins. The vanR and vanS genes were introduced into the chromosome of a susceptible strain of Enterococcus faecalis using an integrative vector. trans-Activation of transcriptional fusions carried by plasmids were analyzed based on determination of chloramphenicol acetyltransferase (CAT) activity. Mapping of the 5’ end of mRNA by S1 nuclease protection and by primer extension assays identified one transcriptional start site in the vanS-vanH intergenic region. Cloning of vanR, vanS, vanH, vanA, and vanX upstream from the cat gene in a multicopy vector resulted in high-level transcription of the reporter gene.
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regulation of vanb type vancomycin resistance gene expression by the vans b vanr b two component Regulatory System in enterococcus faecalis v583
Journal of Bacteriology, 1996Co-Authors: Stefan Evers, Patrice CourvalinAbstract:Acquired VanA- and VanB-type glycopeptide resistance in enterococci is due to synthesis of modified peptidoglycan precursors terminating in D-lactate. As opposed to VanA-type strains which are resistant to both vancomycin and teicoplanin, VanB-type strains remain teicoplanin susceptible. We have determined the sequence of a 7,160-bp DNA fragment associated with VanB-type resistance in Enterococcus faecalis V583 that contains seven open reading frames. The distal part encoded the VanH (B), VanB, and VanX (B) proteins that are highly similar to the putative VanH, VanA, and VanX proteins responsible for VanA-type resistance. Upstream from the structural genes for these proteins were the vanY(B) gene encoding a D,D-carboxypeptidase and an open reading frame vanW with an unknown function. The proximal part of the gene cluster coded for the apparent VanS(B)-VanR (B) Two-Component Regulatory System. VanR (B) was related to response regulators of the OmpR subclass, and VanS (B) was related to membrane-associated histidine protein kinases. Analysis of transcriptional fusions with a reporter gene and promoter mapping indicated that the VanR B-VanS B Two-Component Regulatory System activates a promoter located immediately downstream from the vanS B gene. Vancomycin, but not teicoplanin, was an inducer, which explains teicoplanin susceptibility of VanB-type enterococci.
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The VanS-VanR Two-Component Regulatory System controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147
Journal of bacteriology, 1992Co-Authors: Michel Arthur, C Molinas, Patrice CourvalinAbstract:Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR Two-Component Regulatory System encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS Two-Component Regulatory System activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.
Heejeon Hong - One of the best experts on this subject based on the ideXlab platform.
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in vivo characterization of the activation and interaction of the vanr vans two component Regulatory System controlling glycopeptide antibiotic resistance in two related streptomyces species
Antimicrobial Agents and Chemotherapy, 2016Co-Authors: Gabriela Novotna, Min Jung Kwun, Heejeon HongAbstract:The VanR-VanS Two-Component System is responsible for inducing resistance to glycopeptide antibiotics in various bacteria. We have performed a comparative study of the VanR-VanS Systems from two streptomyces strains, Streptomyces coelicolor and Streptomyces toyocaensis, to characterize how the two proteins cooperate to signal the presence of antibiotics and to define the functional nature of each protein in each strain background. The results indicate that the glycopeptide antibiotic inducer specificity is determined solely by the differences between the amino acid sequences of the VanR-VanS Two-Component Systems present in each strain rather than by any inherent differences in general cell properties, including cell wall structure and biosynthesis. VanR of S. coelicolor (VanRsc) functioned with either sensor kinase partner, while VanR of S. toyocaensis (VanRst) functioned only with its cognate partner, S. toyocaensis VanS (VanSst). In contrast to VanRsc, which is known to be capable of phosphorylation by acetylphosphate, VanRst could not be activated in vivo independently of a VanS sensor kinase. A series of amino acid sequence modifications changing residues in the N-terminal receiver (REC) domain of VanRst to the corresponding residues present in VanRsc failed to create a protein capable of being activated by VanS of S. coelicolor (VanSsc), which suggests that interaction of the response regulator with its cognate sensor kinase may require a region more extended than the REC domain. A T69S amino acid substitution in the REC domain of VanRst produced a strain exhibiting weak constitutive resistance, indicating that this particular amino acid may play a key role for VanS-independent phosphorylation in the response regulator protein.
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A vancomycin photoprobe identifies the histidine kinase VanSsc as a vancomycin receptor
Nature Chemical Biology, 2010Co-Authors: Kalinka Koteva, Heejeon Hong, Ishac Nazi, Donald Hughes, Michael J. Naldrett, Mark J Buttner, Xiao Dong Wang, Gerard D. WrightAbstract:Expression of vancomycin resistance genes is known to be controlled by the Two-Component Regulatory System VanRS, but the identity of the VanS receptor ligand has been controversial. Synthesis of a vancomycin photoaffinity probe has now revealed that vancomycin directly binds VanS to induce the expression of resistance genes. Inducible resistance to the glycopeptide antibiotic vancomycin requires expression of vanH , vanA and vanX , controlled by a Two-Component Regulatory System consisting of a receptor histidine kinase, VanS, and a response regulator, VanR. The identity of the VanS receptor ligand has been debated. Using a synthesized vancomycin photoaffinity probe, we show that vancomycin directly binds Streptomyces coelicolor VanS (VanSsc) and this binding is correlated with resistance and required for vanH , vanA and vanX gene expression.
Reinhold Brückner - One of the best experts on this subject based on the ideXlab platform.
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Activity of the Two-Component Regulatory System CiaRH in Streptococcus pneumoniae R6
Journal of molecular microbiology and biotechnology, 2011Co-Authors: Alexander Halfmann, Anke Schnorpfeil, Miriam Müller, Patrick Marx, Ulrike Günzler, Regine Hakenbeck, Reinhold BrücknerAbstract:The Two-Component Regulatory System CiaRH of Streptococcus pneumoniae affects a variety of processes such as competence development, autolysis, bacteriocin production, host colonization, and virulence. While the targets of the regulator CiaR are known, the role of phosphorylation in CiaR regulation has not been defined. To address this issue, the presumed phosphorylation site of CiaR, aspartic acid at position 51, was replaced by alanine. The mutant CiaRD51A protein was no longer able to activate CiaR-dependent promoters, strongly suggesting that the phosphorylated form of CiaR is active in regulation. However, depending on the growth medium, inactivation of the kinase gene ciaH resulted in a subtle increase of CiaR-dependent promoter activities or in a strong reduction. Therefore, CiaH may act as a kinase or phosphatase and CiaR is apparently able to obtain its phosphate independently of CiaH. On the other hand, promoter measurements in cells with an intact CiaRH System demonstrated a high, nearly constitutive, expression level of the CiaR regulon independent from the growth medium. Thus, in contrast to many other Two-Component Regulatory Systems, CiaRH has apparently evolved to maintain high levels of gene expression under a variety of conditions rather than responding strongly to a signal.
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Identification of genes for small non-coding RNAs that belong to the regulon of the Two-Component Regulatory System CiaRH in Streptococcus
BMC genomics, 2010Co-Authors: Patrick Marx, Regine Hakenbeck, Michael Nuhn, Martá Kovács, Reinhold BrücknerAbstract:Post-transcriptional regulation by small RNAs (sRNAs) in bacteria is now recognized as a wide-spread Regulatory mechanism modulating a variety of physiological responses including virulence. In Streptococcus pneumoniae, an important human pathogen, the first sRNAs to be described were found in the regulon of the CiaRH Two-Component Regulatory System. Five of these sRNAs were detected and designated csRNAs for cia-dependent small RNAs. CiaRH pleiotropically affects β-lactam resistance, autolysis, virulence, and competence development by yet to be defined molecular mechanisms. Since CiaRH is highly conserved among streptococci, it is of interest to determine if csRNAs are also included in the CiaRH regulon in this group of organisms consisting of commensal as well as pathogenic species. Knowledge on the participation of csRNAs in CiaRH-dependent Regulatory events will be the key to define the physiological role of this important control System. Genes for csRNAs were predicted in streptococcal genomes and data base entries other than S. pneumoniae by searching for CiaR-activated promoters located in intergenic regions that are followed by a transcriptional terminator. 61 different candidate genes were obtained specifying csRNAs ranging in size from 51 to 202 nt. Comparing these genes among each other revealed 40 different csRNA types. All streptococcal genomes harbored csRNA genes, their numbers varying between two and six. To validate these predictions, S. mitis, S. oralis, and S. sanguinis were subjected to csRNA-specific northern blot analysis. In addition, a csRNA gene from S. thermophilus plasmid pST0 introduced into S. pneumoniae was also tested. Each of the csRNAs was detected on these blots and showed the anticipated sizes. Thus, the method applied here is able to predict csRNAs with high precision. The results of this study strongly suggest that genes for small non-coding RNAs, csRNAs, are part of the regulon of the Two-Component Regulatory System CiaRH in all streptococci.
Michel Arthur - One of the best experts on this subject based on the ideXlab platform.
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Regulation of Glycopeptide Resistance Genes of Enterococcal Transposon Tn1546 by the VanR-VanS Two-Component Regulatory System
Two-Component Signal Transduction, 2014Co-Authors: Michel Arthur, Gerard D. Wright, Patrice Courvalin, Florence Depardieu, Theodore R. Holman, Christopher T. WalshAbstract:Glycopeptide antibiotics vancomycin and teicoplanin are used to treat severe infections caused by gram-positive cocci. Strains displaying the so-called VanA resistance phenotype are inducibly resistant to high levels of vancomycin and teicoplanin. Production of the depsipeptide D-Ala-D-Lac is controlled by the VanR-VanS Two-Component Regulatory System that activates transcription of vancomycin resistance genes in response to the presence of glycopeptides in the culture medium. Response regulators (RRs) of this subclass regulate transcription at specific promoters thought to be recognized by the main form of RNA polymerase holoenzyme, corresponding to Eσ70 in Escherichia coli. The first 122 amino acids at the N terminus of VanS are not related in sequence to other HPKs. This region of VanS contains two clusters of hydrophobic amino acids that could correspond to membrane-spanning regions. Validation of the predicted roles of VanS and VanR in sequential phosphoryl group transfer was obtained by overproduction, purification, and assay of the two proteins. The vanR and vanS genes were introduced into the chromosome of a susceptible strain of Enterococcus faecalis using an integrative vector. trans-Activation of transcriptional fusions carried by plasmids were analyzed based on determination of chloramphenicol acetyltransferase (CAT) activity. Mapping of the 5’ end of mRNA by S1 nuclease protection and by primer extension assays identified one transcriptional start site in the vanS-vanH intergenic region. Cloning of vanR, vanS, vanH, vanA, and vanX upstream from the cat gene in a multicopy vector resulted in high-level transcription of the reporter gene.
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The CroRS Two-Component Regulatory System Is Required for Intrinsic β-Lactam Resistance in Enterococcus faecalis
Journal of bacteriology, 2003Co-Authors: Yannick Comenge, Richard Quintiliani, Lionnel Dubost, Jean-paul Brouard, Jean-emmanuel Hugonnet, Michel ArthurAbstract:Enterococcus faecalis produces a specific penicillin-binding protein (PBP5) that mediates high-level resistance to the cephalosporin class of beta-lactam antibiotics. Deletion of a locus encoding a previously uncharacterized Two-Component Regulatory System of E. faecalis (croRS) led to a 4,000-fold reduction in the MIC of the expanded-spectrum cephalosporin ceftriaxone. The cytoplasmic domain of the sensor kinase (CroS) was purified and shown to catalyze ATP-dependent autophosphorylation followed by transfer of the phosphate to the mated response regulator (CroR). The croR and croS genes were cotranscribed from a promoter (croRp) located in the rrnC-croR intergenic region. A putative seryl-tRNA synthetase gene (serS) located immediately downstream from croS did not appear to be a target of CroRS regulation or to play a role in ceftriaxone resistance. A plasmid-borne croRp-lacZ fusion was trans-activated by the CroRS System in response to the presence of ceftriaxone in the culture medium. The fusion was also induced by representatives of other classes of beta-lactam antibiotics and by inhibitors of early and late steps of peptidoglycan synthesis. The croRS null mutant produced PBP5, and expression of an additional copy of pbp5 under the control of a heterologous promoter did not restore ceftriaxone resistance. Deletion of croRS was not associated with any defect in the synthesis of the nucleotide precursor UDP-MurNAc-pentapeptide or of the D-Ala(4)-->L-Ala-L-Ala-Lys(3) peptidoglycan cross-bridge. Thus, the croRS mutant was susceptible to ceftriaxone despite the production of PBP5 and the synthesis of wild-type peptidoglycan precursors. These observations constitute the first description of Regulatory genes essential for PBP5-mediated beta-lactam resistance in enterococci.
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The VanS-VanR Two-Component Regulatory System controls synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147
Journal of bacteriology, 1992Co-Authors: Michel Arthur, C Molinas, Patrice CourvalinAbstract:Plasmid pIP816 of Enterococcus faecium BM4147 confers inducible resistance to vancomycin and encodes the VanH dehydrogenase and the VanA ligase for synthesis of depsipeptide-containing peptidoglycan precursors which bind the antibiotic with reduced affinity. We have characterized a cluster of five genes of pIP816 sufficient for peptidoglycan synthesis in the presence of vancomycin. The distal part of the van cluster encodes VanH, VanA, and a third enzyme, VanX, all of which are necessary for resistance. Synthesis of these enzymes was regulated at the transcriptional level by the VanS-VanR Two-Component Regulatory System encoded by the proximal part of the cluster. VanR was a transcriptional activator related to response regulators of the OmpR subclass. VanS stimulated VanR-dependent transcription and was related to membrane-associated histidine protein kinases which control the level of phosphorylation of response regulators. Analysis of transcriptional fusions with a reporter gene and RNA mapping indicated that the VanR-VanS Two-Component Regulatory System activates a promoter used for cotranscription of the vanH, vanA, and vanX resistance genes.
Richard T. Marconi - One of the best experts on this subject based on the ideXlab platform.
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The Hpk2-Rrp2 Two-Component Regulatory System of Treponema denticola: a potential regulator of environmental and adaptive responses.
Molecular oral microbiology, 2010Co-Authors: Juni Sarkar, Jesse Frederick, Richard T. MarconiAbstract:Treponema denticola levels in the gingival crevice become elevated as periodontal disease develops. Oral treponemes may account for as much as 40% of the total bacterial population in the periodontal pocket. The stimuli that trigger enhanced growth of T. denticola, and the mechanisms associated with the transmission of these signals, remain to be defined. We hypothesize that the T. denticola open reading frames tde1970 (histidine kinase) and tde1969 (response regulator) constitute a functional Two-Component Regulatory System that regulates, at least in part, responses to the changing environmental conditions associated with the development of periodontal disease. The results presented demonstrate that tde1970 and tde1969 are conserved, universal among T. denticola isolates and transcribed as part of a seven-gene operon in a growth-phase-dependent manner. tde1970 undergoes autophosphorylation and transfers phosphate to tde1969. Henceforth, the proteins encoded by these open reading frames are designated as Hpk2 and Rrp2 respectively. Hpk2 autophosphorylation kinetics were influenced by environmental conditions and by the presence or absence of a PAS domain. It can be concluded that Hpk2 and Rrp2 constitute a functional Two-Component System that contributes to environmental sensing.
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Analysis of a Growth-Phase-Regulated Two-Component Regulatory System in the Periodontal Pathogen Treponema denticola
Journal of bacteriology, 2008Co-Authors: Jesse Frederick, Elizabeth A. Rogers, Richard T. MarconiAbstract:Nothing is currently known regarding the global Regulatory networks of Treponema denticola and other oral spirochetes. In this report, we assess the properties and potential phosphotransfer capability of a putative Two-Component Regulatory System (TCS) of T. denticola that is formed by the products of open reading frames tde0032 (a sensor kinase) and tde0033 (a response regulator), henceforth designated AtcS and AtcR, respectively. Using PCR and DNA sequence analyses, atcS and atcR were demonstrated to be widely distributed and conserved among T. denticola isolates. Reverse transcription-PCR (RT-PCR) analyses revealed that these genes are cotranscribed and may also be expressed as part of a larger operon that includes several flanking genes. Analyses using 5' rapid amplification of cDNA ends identified the transcriptional start sites for these operons and provided evidence that some of these genes may be independently transcribed from internal promoters. Real-time RT-PCR and Western blot analysis revealed significant upregulation of atcRS during late-stage growth, indicating growth-phase-dependent expression. Lastly, the phosphorelay capability of the AtcRS System was assessed and demonstrated using recombinant proteins. AtcS was found to undergo autophosphorylation and to transfer phosphate to AtcR. These analyses represent the first description of a functional TCS in an oral spirochetes and provide insight into the transcriptional Regulatory mechanisms of these important bacteria.
