RESUMEN
The molecular mechanisms of regulation of the genes involved in the biosynthesis of cysteine are poorly characterized in Bacillus subtilis and other gram-positive bacteria. In this study we describe the expression pattern of the B. subtilis cysH operon in response to sulfur starvation. A 6.1-kb polycistronic transcript which includes the cysH, cysP, ylnB, ylnC, ylnD, ylnE, and ylnF genes was identified. Its synthesis was induced by sulfur limitation and strongly repressed by cysteine. The cysH operon contains a 5' leader portion homologous to that of the S box family of genes involved in sulfur metabolism, which are regulated by a transcription termination control system. Here we show that induction of B. subtilis cysH operon expression is dependent on the promoter and independent of the leader region terminator, indicating that the operon is regulated at the level of transcription initiation rather than controlled at the level of premature termination of transcription. Deletion of a 46-bp region adjacent to the -35 region of the cysH promoter led to high-level expression of the operon, even in the presence of cysteine. We also found that O-acetyl-L-serine (OAS), a direct precursor of cysteine, renders cysH transcription independent of sulfur starvation and insensitive to cysteine repression. We propose that transcription of the cysH operon is negatively regulated by a transcriptional repressor whose activity is controlled by the intracellular levels of OAS. Cysteine is predicted to repress transcription by inhibiting the synthesis of OAS, which would act as an inducer of cysH expression. These novel results provide the first direct evidence that cysteine biosynthesis is controlled at a transcriptional level by both negative and positive effectors in a gram-positive organism.
Asunto(s)
Bacillus subtilis/genética , Cisteína/biosíntesis , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Operón , Azufre/metabolismo , Transcripción Genética , Bacillus subtilis/metabolismo , Secuencia de Bases , Cisteína/genética , Cartilla de ADN , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , ARN Bacteriano/química , ARN Bacteriano/genética , Sulfotransferasas/metabolismo , Azufre/farmacología , Regiones Terminadoras GenéticasRESUMEN
Random Tn917 mutagenesis of Bacillus subtilis followed by selection of lipoic acid auxotrophs led to the isolation of the cysH gene. The gene was sequenced and found to encode a phosphoadenylylsulfate sulfotransferase with a molecular mass of 27 kDa. Expression of lacZ fused to the cysH promoter was repressed by cysteine and sulfide and induced by sulfur limitation, indicating that cysH is controlled at the level of transcription.