Regulation of the ribA gene encoding GTP cyclohydrolase II by the soxRS locus in Escherichia coli

We isolated a promoter that is inducible by paraquat, a superoxide-generating agent, from Escherichia coli using the promoter-probe plasmid pRS415. Sequence analysis revealed that the promoter derives from the ribA gene encoding GTP cyclohydrolase II, which is the first enzyme in the biosynthetic pathway of riboflavin. We fused the lacZ gene with the ribA promoter to monitor the expression of the gene in the single-copy state. LacZ expression from the ribA promoter was induced about eight-fold by 200 microM paraquat. Other known superoxide generators, menadione and plumbagin, also induced the expression of beta-galactosidase in the fusion strain. On the other hand, no significant induction was observed following treatment with hydrogen peroxide, ethanol or heat shock. Induction of beta-galactosidase was significantly reduced by the introduction of a delta sox-8::cat or soxS3::Tn10 mutation into the fusion strain, indicating that the ribA gene is a member of the soxRS 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 of E. coli increased more than three-fold on treatment with paraquat. This increase was dependent on the soxRS locus, and reflects the increase in transcript levels. However, flavin pools did not change significantly. A possible role for ribA induction during superoxide stress is discussed.