RESUMEN
Previous studies have shown that the MpeR transcriptional regulator produced by Neisseria gonorrhoeae represses the expression of mtrF, which encodes a putative inner membrane protein (MtrF). MtrF works as an accessory protein with the Mtr efflux pump, helping gonococci to resist high levels of diverse hydrophobic antimicrobials. Regulation of mpeR has been reported to occur by an iron-dependent mechanism involving Fur (ferric uptake regulator). Collectively, these observations suggest the presence of an interconnected regulatory system in gonococci that modulates the expression of efflux pump protein-encoding genes in an iron-responsive manner. Herein, we describe this connection and report that levels of gonococcal resistance to a substrate of the mtrCDE-encoded efflux pump can be modulated by MpeR and the availability of free iron. Using microarray analysis, we found that the mtrR gene, which encodes a direct repressor (MtrR) of mtrCDE, is an MpeR-repressed determinant in the late logarithmic phase of growth when free iron levels would be reduced due to bacterial consumption. This repression was enhanced under conditions of iron limitation and resulted in increased expression of the mtrCDE efflux pump operon. Furthermore, as judged by DNA-binding analysis, MpeR-mediated repression of mtrR was direct. Collectively, our results indicate that both genetic and physiologic parameters (e.g., iron availability) can influence the expression of the mtr efflux system and modulate levels of gonococcal susceptibility to efflux pump substrates.
Asunto(s)
Antibacterianos/farmacología , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica , Proteínas Reguladoras del Hierro/genética , Hierro/metabolismo , Neisseria gonorrhoeae/genética , Proteínas Bacterianas/metabolismo , Farmacorresistencia Bacteriana Múltiple/genética , Sitios Genéticos , Humanos , Proteínas Reguladoras del Hierro/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Neisseria gonorrhoeae/efectos de los fármacos , Operón , Plásmidos , Proteínas Represoras/genética , Proteínas Represoras/metabolismoRESUMEN
DNA-binding proteins that control expression of drug efflux pump genes have been termed "local regulators" as their encoding gene is often located adjacent to the gene(s) that they regulate. However, results from recent studies indicate that they can control genes outside efflux pump-encoding loci, which we term as being "off target." For example, the MtrR repressor was initially recognized for its ability to repress transcription of the mtrCDE-encoded efflux pump operon in the strict human pathogen Neisseria gonorrhoeae, but recent results from genetic and microarray studies have shown that it can control expression of nearly 70 genes scattered throughout the chromosome. One of the off-target MtrR-repressed genes is glnA, which encodes glutamine synthetase. Herein, we confirm the capacity of MtrR to repress glnA expression and provide evidence that such repression is due to its ability to negatively influence the binding of a second DNA-binding protein (FarR), which activates glnA. FarR was previously recognized as a transcriptional repressor of the farAB-encoded efflux pump operon. Thus, two DNA-binding proteins previously characterized as repressors of genes encoding efflux pumps that contribute to gonococcal resistance to antimicrobials can act in an opposing manner to modulate expression of a gene involved in basic metabolism.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/fisiología , Regulación Bacteriana de la Expresión Génica , Glutamato-Amoníaco Ligasa/genética , Neisseria gonorrhoeae/genética , Proteínas Represoras/fisiología , Proteínas Bacterianas/fisiología , Secuencia de Bases , Farmacorresistencia Bacteriana , Datos de Secuencia Molecular , Neisseria gonorrhoeae/efectos de los fármacos , Transcripción GenéticaRESUMEN
The important human pathogen Streptococcus pyogenes (the group A streptococcus or GAS) produces many virulence factors that are regulated by the two-component signal transduction system CovRS (CsrRS). Dissemination of GAS infection originating at the skin has been shown to require production of streptokinase, whose transcription is repressed by CovR. In this work we have studied the interaction of CovR and phosphorylated CovR (CovR-P) with the promoter for streptokinase, Pska. We found that, in contrast to the other CovR-repressed promoters, Pska regulation by CovR occurs through binding at a single ATTARA consensus binding sequence (CB) that overlaps the -10 region of the promoter. Binding of CovR to other nearby consensus sequences occurs upon phosphorylation of the protein, but these other CBs do not contribute to the regulation of Pska by CovR. Thus, binding at a specific site does not necessarily indicate that the site is involved in regulation by CovR. In addition, at Pska, CovR binding to the different sites does not appear to involve cooperative interactions, which simplifies the analysis of CovR binding and gives us insight into the modes of interaction that occur between CovR and its specific DNA-binding sites. Finally, the observation that regulation of transcription from Pska occurs at a very low concentration of phosphorylated CovR may have important implications for the regulation of virulence gene expression during GAS infection.
Asunto(s)
Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Proteínas Represoras/metabolismo , Streptococcus pyogenes/genética , Estreptoquinasa/genética , Proteínas Bacterianas/genética , Secuencia de Bases , Sitios de Unión , Histidina Quinasa , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Datos de Secuencia Molecular , Fosforilación , Unión Proteica , Proteínas Represoras/genética , Streptococcus pyogenes/química , Streptococcus pyogenes/enzimología , Estreptoquinasa/química , Estreptoquinasa/metabolismoRESUMEN
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.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas de Transporte de Membrana/genética , Regiones Promotoras Genéticas/genética , Proteínas Represoras/metabolismo , Streptococcus pyogenes/genética , Streptococcus pyogenes/metabolismo , Secuencia de Bases , Regulación Enzimológica de la Expresión Génica , Datos de Secuencia Molecular , Mutación , Unión ProteicaRESUMEN
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.