RESUMEN
Genes encoding one or more Ser/Thr protein kinases have been identified recently in many bacteria, including one (stk) in the human pathogen Streptococcus pyogenes (group A streptococcus [GAS]). We report that in GAS, stk is required to produce disease in a murine myositis model of infection. Using microarray and quantitative reverse transcription-PCR (qRT-PCR) studies, we found that Stk activates genes for virulence factors, osmoregulation, metabolism of α-glucans, and fatty acid biosynthesis, as well as genes affecting cell wall synthesis. Confirming these transcription studies, we determined that the stk deletion mutant is more sensitive to osmotic stress and to penicillin than the wild type. We discuss several possible Stk phosphorylation targets that might explain Stk regulation of expression of specific operons and the possible role of Stk in resuscitation from quiescence.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Respuesta al Choque Térmico/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Streptococcus pyogenes/enzimología , Streptococcus pyogenes/patogenicidad , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Modelos Animales de Enfermedad , Tolerancia a Medicamentos , Femenino , Humanos , Ratones , Datos de Secuencia Molecular , Miositis/microbiología , Miositis/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Penicilinas/farmacología , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Infecciones Estreptocócicas/microbiología , Infecciones Estreptocócicas/patología , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/fisiología , Virulencia , Factores de Virulencia/genética , Factores de Virulencia/metabolismoRESUMEN
Selection of possible targets for vaccine and drug development requires an understanding of the physiology of bacterial pathogens, for which the ability to manipulate expression of essential genes is critical. For Streptococcus pyogenes (the group A streptococcus [GAS]), an important human pathogen, the lack of genetic tools for such studies has seriously hampered research. To address this problem, we characterized variants of the inducible Ptet cassette, in both sense and antisense contexts, as tools to regulate transcription from GAS genes. We found that although the three-operator Ptet construct [Ptet(O)3] had low uninduced expression, its induction level was low, while the two-operator construct [Ptet(O)2] was inducible to a high level but showed significant constitutive expression. Use of Ptet(O)3 in the chromosome allowed us to demonstrate previously that RNases J1 and J2 are required for growth of GAS. Here we report that the uninduced level from the chromosomally inserted Ptet(O)2 construct was too high for us to observe differential growth. For the highly expressed histone-like protein (Hlp) of GAS, neither chromosomal insertion of Ptet(O)2 or Ptet(O)3 nor their use on a high-copy-number plasmid to produce antisense RNA specific to hlp resulted in adequate differential expression. However, by replacing the ribosome binding site of hlp with an engineered riboswitch to control translation of Hlp, we demonstrated for the first time that this protein is essential for GAS growth.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Genes Esenciales , Genética Microbiana/métodos , Streptococcus pyogenes/crecimiento & desarrollo , Streptococcus pyogenes/metabolismo , Proteínas de Unión al ADN/genética , Expresión Génica , Regiones Promotoras Genéticas , Riboswitch , Streptococcus pyogenes/genéticaRESUMEN
We found that the global regulatory two-component signal transduction system CovRS mediates the ability of group A streptococcus (GAS) to grow under two stresses encountered during infection: iron starvation and the presence of LL-37. We also showed that CovRS regulates transcription of the multimetal transporter operon that is important for GAS growth in a low concentration of iron.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Proteínas Bacterianas/metabolismo , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Hierro/metabolismo , Proteínas Represoras/metabolismo , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/crecimiento & desarrollo , Proteínas Bacterianas/genética , Catelicidinas , Regulación Bacteriana de la Expresión Génica , Histidina Quinasa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Represoras/genética , Streptococcus pyogenes/efectos de los fármacos , Streptococcus pyogenes/metabolismo , Transcripción GenéticaRESUMEN
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
CS1 pili are important virulence factors of enterotoxigenic Escherichia coli strains associated with human diarrheal disease. They are the prototype for a family of pili that share extensive sequence similarity among their structural and assembly proteins. Only four linked genes, cooB, cooA, cooC, and cooD, are required to produce CS1 pili in E. coli K-12. To identify amino acids important for the function of the major pilin CooA, we used alanine substitution mutagenesis targeting conserved residues in the N and C termini of the protein. To test function, we examined cooA mutants for the ability to agglutinate bovine erythrocytes. Each hemagglutination-negative (HA(-)) cooA mutant was examined to identify its assembly pathway defect. CooA has been shown to be degraded in the absence of CooB (K. Voegele, H. Sakellaris, and J. R. Scott, Proc. Natl. Acad. Sci. USA 94:13257-13261, 1997). We found several HA(-) cooA mutants that produced no detectable CooA, suggesting that recognition by CooB is mediated by residues in both the N and C termini of CooA. In addition, we found that alanine substitution for some of the conserved residues in the C-terminal motif "AGxYxG(x(6))T," which is found in all subunits of this pilus family, had no effect on pilus formation. However, alanine substitution for some of the alternating hydrophobic residues within this motif prevented CooA from interacting with CooD, which serves as both the tip adhesin and nucleation protein for pilus formation. Thus, it appears that some, but not all, of the residues in both the N and C termini of CooA play a critical role in the intermolecular interactions of the major pilin with the other structural and assembly proteins. We anticipate that the results obtained here for CS1 pili in enterotoxigenic E. coli will help develop an understanding of the pilus assembly pathway used by CS1 family members in several important human pathogens.