Regulation of theribA gene encoding GTP cyclohydrolase II by thesoxRS locus inEscherichia coli

Abstract

We isolated a promoter that is inducible by paraquat, a superoxide-generating agent, fromEscherichia coli using the promoter-probe plasmid pRS415. Sequence analysis revealed that the promoter derives from theribA gene encoding GTP cyclohydrolase II, which is the first enzyme in the biosynthetic pathway of riboflavin. We fused thelacZ gene with theribA promoter to monitor the expression of the gene in the single-copy state. LacZ expression from theribA promoter was induced about eight-fold by 200 µM paraquat. Other known superoxide generators, menadione and plumbagin, also induced the expression ofβ-galactosidase in the fusion strain. On the other hand, no significant induction was observed following treatment with hydrogen peroxide, ethanol or heat shock. Induction ofβ-galactosidase was significantly reduced by the introduction of a Δsox-8::cat orsoxS3::Tn10 mutation into the fusion strain, indicating that theribA gene is a member of thesoxRS regulon. The transcriptional start site was determined by primer extension analysis and putative binding sites for SoxS in both orientations were identified. GTP cyclohydrolase II activity in soluble extracts ofE. coli increased more than three-fold on treatment with paraquat. This increase was dependent on thesoxRS locus, and reflects the increase in transcript levels. However, flavin pools did not change significantly. A possible role forribA induction during superoxide stress is discussed.