Abstract
The transcriptional regulation of Corynebacterium glutamicum gnd, encoding 6-phosphogluconate dehydrogenase, was investigated. Two transcriptional regulators, GntR1 and RamA, were isolated by affinity purification using gnd promoter DNA. GntR1 was previously identified as a repressor of gluconate utilization genes, including gnd. Involvement of RamA in gnd expression had not been investigated to date. The level of gnd mRNA was barely affected by the single deletion of ramA. However, gnd expression was downregulated in the ramA gntR1 double mutant compared to that of the gntR1 single mutant, suggesting that RamA activates gnd expression. Two RamA binding sites are found in the 5' upstream region of gnd. Mutation proximal to the transcriptional start site diminished the gluconate-dependent induction of gnd-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 represses gnd 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.