RESUMO
The ctc gene of Bacillus subtilis is transcribed in vitro by the minor RNA polymerase holoenzyme forms, E sigma 37 and E sigma 32. To study the expression and regulation of ctc in vivo, we constructed operon and translational fusions of the ctc promoter region to the lacZ gene of Escherichia coli. Our results indicate that ctc is regulated at the transcriptional level, and that this RNA synthesis is maximally induced at the end of the exponential phase of growth under nutritional conditions which inhibit the activity of the tricarboxylic acid cycle. Analysis of in vitro-constructed deletion mutations extending into the ctc promoter region demonstrated that the region required for this regulation is no greater than 53 base-pairs in length. We also compared the expression of ctc to that of another B. subtilis gene, which is transcribed by E sigma 37 and E sigma 32 in vitro, the sporulation gene spoVG. Although the ctc and spoVG promoter regions are recognized by the same forms of RNA polymerase in vitro, our results show that they differ strikingly in the nutritional and genetic requirements for their expression in vivo.
Assuntos
Bacillus subtilis/genética , RNA Polimerases Dirigidas por DNA/genética , Regiões Promotoras Genéticas , Bacillus subtilis/enzimologia , Regulação da Expressão Gênica , Mutação , Plasmídeos , beta-Galactosidase/genéticaRESUMO
Expression of the outer membrane protein OmpF of Escherichia coli K-12 is influenced by a variety of environmental signals. Most of the signals are thought to regulate OmpF expression at the level of transcription initiation. A key element of this regulation is the interaction between the transcriptional factor OmpR and the cis-acting regulatory region of ompF. In this study, we used a combination of DNase I, dimethyl sulfate and hydroxyl radical footprinting analysis and DNA migration retardation assays to identify the bases within the ompF regulatory region that are in contact with OmpR. Our results indicate that the -107 to -39 region of ompF contains three individual binding sites and that a single OmpR-binding site is capable of interacting with two OmpR molecules. We also establish that a single OmpR-binding site is composed of two half-sites and that both half-sites are required for the formation of stable OmpR/DNA complexes. Comparisons of the sequences protected by OmpR indicate that an OmpR-binding site spans approximately 18 bp and has two highly conserved G/C base-pairs that are separated by three nucleotides. Although the three OmpR-binding sites we identified exhibit limited sequence similarity, this may reflect the fact that two of the sites are incapable of binding OmpR independently and can bind OmpR only if adjacent to another OmpR-binding site. Finally, our DNA migration retardation assays suggest that phosphorylation stimulates the cooperative interactions between OmpR molecules bound at neighboring sites. Therefore, this study provides a detailed understanding of how OmpR interacts with its binding sites immediately upstream of ompF and serves as a foundation for studying how phosphorylation of OmpR results in the regulation of ompF expression in response to environmental signals.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação Bacteriana da Expressão Gênica , Sequência de Bases , Sítios de Ligação , Sequência Consenso , Pegada de DNA , Substâncias Macromoleculares , Dados de Sequência Molecular , Alinhamento de Sequência , Relação Estrutura-AtividadeRESUMO
By fusing the transcriptional and translational start signals of lacZ to envZ, we have obtained high-level synthesis of a truncated EnvZ protein (EnvZ115) in which the first 38 amino acids of EnvZ are replaced with the first 8 amino acids of LacZ. Using this construct, we have partially purified the EnvZ115 protein and demonstrated that this protein can be phosphorylated in vitro. We suggest that phosphorylation may be an important feature of EnvZ function.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Bactérias/genética , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Complexos Multienzimáticos , Trifosfato de Adenosina/metabolismo , Escherichia coli/genética , Teste de Complementação Genética , Mutação , Fosforilação , Processamento de Proteína Pós-TraducionalRESUMO
The two-component regulatory system consisting of OmpR and EnvZ controls the differential expression of major outer membrane porin proteins OmpF and OmpC of Escherichia coli K-12. We have isolated and characterized two mutations in rpoA, the gene encoding the alpha subunit of RNA polymerase, that decrease the expression of OmpF. These mutations have a number of properties that distinguish them from previously isolated rpoA mutations that affect porin expression. The rpoA203 mutation decreases the expression of porin genes ompF and ompC and also decreases the expression of the malE and phoA genes. In contrast, rpoA207 decreases the expression of ompF but does not affect ompC, malE, or phoA transcription. Our results suggest that mutations at various positions in the alpha subunit may affect the OmpR-dependent transcription of ompF and ompC differently and may be useful for analyzing the mechanism underlying their differential expression in response to medium osmolarity.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , RNA Polimerases Dirigidas por DNA/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Alelos , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , DNA Bacteriano , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/crescimento & desenvolvimento , Cinética , Dados de Sequência Molecular , Mutagênese , Óperon , Reação em Cadeia da Polimerase , Porinas , Transcrição GênicaRESUMO
We have generated a mutant form of the OmpR regulatory protein, OmpRD55E, that is active independent of the EnvZ kinase. Notably, the pattern of OmpF and OmpC expression can be altered simply by changing the level of this mutant protein in the cell. This result supports a key prediction of the current model of porin regulation, which states that the differential regulation of OmpF and OmpC is a direct consequence of the cellular level of the active form of OmpR.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Proteínas de Escherichia coli , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Complexos Multienzimáticos , Porinas/genética , Fatores de Transcrição/fisiologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Dosagem de Genes , Mutação , Fenótipo , Fosforilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Transcription of the genes that encode the major outer membrane porin proteins OmpF and OmpC of Escherichia coli is regulated in response to changes in medium osmolarity by EnvZ and OmpR. EnvZ functions to sense environmental conditions and to relay this information to the DNA-binding protein OmpR. We have used a truncated EnvZ protein (EnvZ115), which is defective in sensory function but able to communicate with OmpR, to study the biochemical interactions between these two proteins and their effects on transcription from the ompF promoter. We show that purified EnvZ115 can phosphorylate OmpR in the presence of ATP. In addition, EnvZ115 stimulates the ability of OmpR to activate ompF transcription in vitro. Using antibodies specific for EnvZ, we have purified the wild-type protein and have shown that it is also an OmpR kinase. These results provide a prokaryotic example of a transmembrane sensory protein that functions as a protein kinase and suggest a mechanism by which EnvZ communicates with OmpR in signal transduction.
Assuntos
Proteínas da Membrana Bacteriana Externa/fisiologia , Escherichia coli/genética , Proteínas Quinases/fisiologia , Transcrição Gênica , Fosforilação , SolubilidadeRESUMO
The two-component regulatory system, OmpR-EnvZ, of Escherichia coli K-12 regulates the expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein which acts as both an activator and a repressor to control ompF transcription. In this article, we describe a new OmpR-binding site that is located between 384 to 351 bp upstream from the ompF start point of transcription. Inactivation of this site by insertion of a 22-bp fragment prevents the repression of ompF expression conferred by the dominant negative mutation, envZ473. On the basis of the location of this binding site, the presence of bent DNA in the ompF regulatory region (T. Mizuno, Gene 54:57-64, 1987), and the fact that mutations altering integration host factor result in constitutive ompF expression (P. Tsui, V. Helu, and M. Freundlich, J. Bacteriol. 170:4950-4953, 1988), we propose that the negative regulation of ompF involves a DNA loop structure.
Assuntos
Proteínas da Membrana Bacteriana Externa/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Genes Reguladores , Sequências Reguladoras de Ácido Nucleico , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/metabolismo , Sequência de Bases , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Proteínas de Ligação a DNA/metabolismo , Desoxirribonuclease I , Genótipo , Dados de Sequência Molecular , Transcrição GênicaRESUMO
A new paradigm, termed two-component regulatory systems, is emerging from the study of signal transduction in bacteria. A simple example of such a system is provided by the Omp regulon of Escherichia coli. This regulon, which controls the expression of the major outer membrane porin proteins OmpF and OmpC in response to changes in osmolarity, includes the inner membrane protein EnvZ (a receptor kinase) and the DNA-binding protein OmpR (a transcriptional activator). Although we do not know what "ligand" is sensed in the Omp system, we can trace the signal transduction pathway from the receptor at the cell surface directly to regulatory sequences within the DNA. Perhaps signal transduction in bacteria can serve as a simple archetype for understanding certain functions performed by receptor kinases and phosphorylated DNA-binding proteins in higher organisms.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Escherichia coli , Regulação Bacteriana da Expressão Gênica , Modelos Biológicos , Complexos Multienzimáticos , Proteínas Quinases/fisiologia , Transdução de Sinais , Proteínas da Membrana Bacteriana Externa/biossíntese , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/genética , Escherichia coli/fisiologia , Células Eucarióticas/fisiologia , Genes Bacterianos , Fosforilação , Proteínas Quinases/genética , Processamento de Proteína Pós-Traducional , Receptores de Superfície Celular/fisiologia , Fatores de Transcrição/fisiologiaRESUMO
The two-component regulatory proteins OmpR and EnvZ of Escherichia coli K-12 regulate expression of the major outer membrane porin protein, OmpF. OmpR is a DNA-binding protein that is involved in both the positive and negative control of ompF transcription. EnvZ is a histidine kinase that phosphorylates OmpR in response to environmental signals. We used DNA migration retardation analysis to examine the interactions of OmpR and the phosphorylated form of OmpR (OmpR-P) with the regulatory region immediately upstream of the ompF promoter. Our results indicate that the binding of OmpR to this regulatory region is cooperative and that phosphorylation significantly stimulates these cooperative interactions. Moreover, although phosphorylation increases the intrinsic binding of OmpR to a single OmpR-binding site, the primary role of phosphorylation in ompF regulation is to facilitate cooperative interactions between OmpR molecules bound at adjacent sites. Based on these results, we propose a model to explain how the phosphorylation of OmpR could stimulate the occupancy of specific sites in the ompF regulatory region, thereby resulting in the activation or repression of ompF transcription under the appropriate environmental conditions.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Dados de Sequência Molecular , Fosforilação , Sequências Reguladoras de Ácido NucleicoRESUMO
Signal transduction in the bacterial Omp, Che, and Ntr systems involves the phosphorylation and dephosphorylation of response regulators (OmpR, CheY and CheB, NRI) that share a homologous domain. We show that in the Omp system, the transmembrane sensor EnvZ, catalyzes both the phosphorylation of OmpR and the dephosphorylation of OmpR-P. The phosphorylation reaction proceeds by a mechanism shared with the Ntr and Che kinases, NRII, and CheA. EnvZ can phosphorylate NRI and can stimulate transcription from the glnAp2 promoter, and similarly, CheA can phosphorylate OmpR and can stimulate transcription from the ompF promoter. OmpR-P formed by either CheA or EnvZ is much more stable than CheY-P and NRI-P, but is rapidly hydrolyzed to OmpR and Pi by EnvZ in the presence of ATP, ADP, or nonhydrolyzable analogs of ATP. Because EnvZ is normally a transmembrane receptor with a periplasmic sensory domain, our results suggest that the role of EnvZ may be to control the intracellular concentration of OmpR-P in response to environmental signals.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias , Proteínas de Escherichia coli , Complexos Multienzimáticos , Transdução de Sinais , Transativadores , Fatores de Transcrição/metabolismo , Transcrição Gênica , Proteínas da Membrana Bacteriana Externa/genética , Fatores Quimiotáticos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Histidina Quinase , Proteínas de Membrana/metabolismo , Proteínas Quimiotáticas Aceptoras de Metil , Proteínas PII Reguladoras de Nitrogênio , Fosfoproteínas Fosfatases/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Proteínas Quinases/metabolismoRESUMO
Two-component systems, sensor kinase-response regulator pairs, dominate bacterial signal transduction. Regulation is exerted by phosphorylation of an Asp in receiver domains of response regulators. Lability of the acyl phosphate linkage has limited structure determination for the active, phosphorylated forms of receiver domains. As assessed by both functional and structural criteria, beryllofluoride yields an excellent analogue of aspartyl phosphate in response regulator NtrC, a bacterial enhancer-binding protein. Beryllofluoride also appears to activate the chemotaxis, sporulation, osmosensing, and nitrate/nitrite response regulators CheY, Spo0F, OmpR, and NarL, respectively. NMR spectroscopic studies indicate that beryllofluoride will facilitate both biochemical and structural characterization of the active forms of receiver domains.