RESUMO
The Bacillus subtilis lmrAB operon is involved in multidrug resistance. LmrA is a repressor of its own operon, while LmrB acts as a multidrug efflux transporter. LmrA was produced in Escherichia coli cells and was shown to bind to the lmr promoter region, in which an LmrA-binding site was identified. Genome-wide screening involving DNA microarray analysis allowed us to conclude that LmrA also repressed yxaGH, which was not likely to contribute to the multidrug resistance. LmrA bound to a putative yxaGH promoter region, in which two tandem LmrA-binding sites were identified. The LmrA regulon was thus determined to comprise lmrAB and yxaGH. All three LmrA-binding sites contained an 18-bp consensus sequence, TAGACCRKTCWMTATAWT, which could play an important role in LmrA binding.
Assuntos
Bacillus subtilis/genética , Regulação Bacteriana da Expressão Gênica , Proteínas Associadas à Resistência a Múltiplos Medicamentos/fisiologia , Proteínas Repressoras/metabolismo , Proteínas Repressoras/fisiologia , Sequência de Aminoácidos , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/fisiologia , Sequência de Bases , Sítios de Ligação , Clonagem Molecular , Sequência Consenso , Escherichia coli/genética , Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Dados de Sequência Molecular , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Análise de Sequência com Séries de Oligonucleotídeos , Óperon , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Regulon , Proteínas Repressoras/genéticaRESUMO
Bacillus subtilis TnrA is a global regulator that responds to the availability of nitrogen sources and both activates and represses many genes during nitrogen-limited growth. In order to obtain a holistic view of the gene regulation depending on TnrA, we performed a genome-wide screening for TnrA-regulated genes associated with a TnrA box. A combination of DNA microarray hybridization and a genome-wide search for TnrA boxes allowed us to find 36 TnrA-regulated transcription units associated with a putative TnrA box. Gel retardation assaying, using probes carrying at least one putative TnrA box and the deletion derivatives of each box, indicated that 17 out of 36 transcription units were likely TnrA targets associated with the TnrA boxes, two of which (nasA and nasBCDEF) possessed a common TnrA box. The sequences of these TnrA boxes contained a consensus one, TGTNANAWWWTMTNACA. The TnrA targets detected in this study were nrgAB, pucJKLM, glnQHMP, nasDEF, oppABCDF, nasA, nasBCDEF and ywrD for positive regulation, and gltAB, pel, ywdIJK, yycCB, yttA, yxkC, ywlFG, yodF and alsT for negative regulation, nrgAB and gltAB being well-studied TnrA targets. It was unexpected that the negatively regulated TnrA targets were as many as the positively regulated targets. The physiological role of the TnrA regulon is discussed.
