RESUMEN
Expression of the gene for the extracellular alkaline protease (aprE) of Bacillus subtilis is subject to regulation by many positive and negative regulators. We have found that aprE expression was increased by disruption of the glutamine synthetase gene glnA. The increase in aprE expression was attributed to a decreased in expression of scoC, which encodes a negative regulator of aprE expression. The glnA effect on scoC expression was abolished by further disruption of tnrA, indicating that aprE expression is under global regulation through TnrA. In the scoC background, however, aprE expression was decreased by glnA deletion, and it was shown that the decrease was due to a defect in positive regulation by DegU. Among the genes that affect aprE expression through DegU, the expression of degR, encoding a protein that stabilizes phosphorylated DegU, was inhibited by glnA deletion. It was further shown that the decrease in degR expression by glnA deletion was caused by inhibition of the expression of sigD, encoding the sigma(D) factor, which is required for degR expression. In accordance with these findings, the expression levels of aprE-lacZ in glnA scoC degR and scoC degR strains were identical. These results led us to conclude that glnA deletion brings about two effects on aprE expression, i.e., a positive effect through inhibition of scoC expression and a negative effect through inhibition of degR expression, with the former predominating over the latter.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/antagonistas & inhibidores , Proteínas Bacterianas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Glutamato-Amoníaco Ligasa/fisiología , Proteínas de Transporte de Membrana/biosíntesis , Factores de Transcripción/antagonistas & inhibidores , Animales , Fusión Artificial Génica , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Secuencia de Bases , Eliminación de Gen , Genes Reporteros , Glutamato-Amoníaco Ligasa/genética , Datos de Secuencia Molecular , Octodon , Regiones Promotoras Genéticas , Proteínas Represoras/genética , Proteínas Represoras/fisiología , Factor sigma/fisiología , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
Bacillus subtilis DegS-DegU belongs to a bacterial two-component system that controls many processes, including the production of exocellular proteases and competence development. It was found that when the glutamine synthetase gene glnA, which is involved in nitrogen regulation, was disrupted, the expression of the response regulator degU gene was increased. Deletion analysis and 5'-end mapping of the degU transcripts showed that the increase was caused by induction of a promoter (P2) located before the degU gene. Disruption of tnrA, a global regulator of nitrogen regulation, eliminated the P2 promoter induction by the glnA mutation. The fact that the P2 promoter is under nitrogen regulation was demonstrated by an increase in P2 expression with nitrogen-limited growth. It was also found by primer extension analysis that degU was transcribed by another promoter, P3, that is located downstream of P2. Efficient expression of P3 was dependent on phosphorylated DegU, as inactivation of the sensor kinase gene, degS, resulted in the loss of degU expression, although less efficient stimulation of degU expression was also observed with an enhanced level of DegU in a degS-deficient mutant. The promoter located upstream of the degSU operon, designated the P1 promoter here, was insensitive to glnA and degU mutations. These results suggest that degU expression is controlled by the three promoters under different growth conditions.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/biosíntesis , Regulación Bacteriana de la Expresión Génica , Nitrógeno/metabolismo , Regiones Promotoras Genéticas , Fusión Artificial Génica , Bacillus subtilis/genética , Bacillus subtilis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Secuencia de Bases , Northern Blotting , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Reporteros , Glutamato-Amoníaco Ligasa/genética , Datos de Secuencia Molecular , Mutagénesis Insercional , Mutagénesis Sitio-Dirigida , ARN Bacteriano/biosíntesis , ARN Bacteriano/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Represoras/genética , Eliminación de Secuencia , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/genéticaRESUMEN
The Bacillus subtilis aprE gene, which encodes the extracellular alkaline protease, is regulated by many positive and negative transcriptional regulators. SenS is one such positive regulator consisting of 65 amino acids. We found that the senS gene on a multicopy plasmid, pSEN24, caused an increase in aprE expression in strains carrying the upstream region of aprE up to -340 with respect to the transcription initiation site but not in a strain carrying the region up to -299, which is within the binding site of the negative regulator ScoC (Hpr). Epistatic analysis showed that the pSEN24 effect was lost in a scoC-deleted mutant. In accordance with these results, the scoC transcription level as assayed by a scoC-lacZ fusion and Northern analysis was greatly reduced in the cells carrying pSEN24. From these results we conclude that multicopy senS enhances aprE expression by suppressing the transcription of scoC.