Three of Four GlnR Binding Sites Are Essential for GlnR-Mediated Activation of Transcription of the Amycolatopsis mediterranei nas Operon

InAmycolatopsis mediterranei U32, genes responsible for nitrate assimilation formed one operon,nasACKBDEF , whose transcription is induced by the addition of nitrate. Here, we characterized GlnR as a direct transcriptional activator for thenas operon. The GlnR-protected DNA sequences in the promoter region of thenas operon were characterized by DNase I footprinting assay, the previously deducedStreptomyces coelicolor double 22-bp GlnR binding consensus sequences comprisinga1 ,b1 ,a2 , andb2 sites were identified, and the sites were then mutated individually to test their roles in both the binding of GlnRin vitro and the GlnR-mediated transcriptional activationin vivo . The results clearly showed that only three GlnR binding sites (a1 ,b1 , andb2 sites) were required by GlnR for its specific binding to thenas promoter region and efficient activation of the transcription of thenas operon in U32, while thea2 site seemed unnecessary.