Coordinated Regulation of gnd , Which Encodes 6-Phosphogluconate Dehydrogenase, by the Two Transcriptional Regulators GntR1 and RamA in Corynebacterium glutamicum

The transcriptional regulation ofCorynebacterium glutamicum gnd , encoding 6-phosphogluconate dehydrogenase, was investigated. Two transcriptional regulators, GntR1 and RamA, were isolated by affinity purification usinggnd promoter DNA. GntR1 was previously identified as a repressor of gluconate utilization genes, includinggnd . Involvement of RamA ingnd expression had not been investigated to date. The level ofgnd mRNA was barely affected by the single deletion oframA . However,gnd expression was downregulated in theramA gntR1 double mutant compared to that of thegntR1 single mutant, suggesting that RamA activatesgnd expression. Two RamA binding sites are found in the 5′ upstream region ofgnd . Mutation proximal to the transcriptional start site diminished the gluconate-dependent induction ofgnd -lacZ . DNase I footprinting assay revealed two GntR1 binding sites, with one corresponding to a previously proposed site that overlaps with the −10 region. The other site overlaps the RamA binding site. GntR1 binding to this newly identified site inhibits DNA binding of RamA. Therefore, it is likely that GntR1 repressesgnd expression by preventing both RNA polymerase and RamA binding to the promoter. In addition, DNA binding activity of RamA was reduced by high concentrations of NAD(P)H but not by NAD(P), implying that RamA senses the redox perturbation of the cell.