Induction of the sufA operon encoding Fe-S assembly proteins by superoxide generators and hydrogen peroxide: involvement of OxyR, IHF and an unidentified oxidant-responsive factor

Abstract

A promoter (sufAp), inducible by various oxidants, directs transcription of the sufABCDSE operon encoding an alternative Fe-S cluster assembly system in Escherichia coli. Superoxide generators and H2O2 induced expression of sufA-lacZ even in DeltasoxRS and DeltaoxyR mutants, suggesting participation of an additional regulator(s) in oxidant induction of the sufA operon. Through deletion and linker scanning mutagenesis, we found three cis-acting oxidant-responsive elements (OREs). ORE-I lies between -236 and -197 nucleotides from the transcription start site, overlapping extensively with the OxyR binding site reported previously. ORE-II (-156 to -127) was found to be the site of IHF action. ORE-III (-56 to -35) had no predictable binding sites for known regulators. Gel mobility shift assays with a 50 bp DNA probe containing ORE-III revealed the presence of an ORE-III-specific factor that binds only when cells are treated with oxidants. S1 mapping analysis revealed that phenazine methosulphate (PMS) and H2O2 induced sufA expression by more than 40-fold. In a DeltaoxyR mutant, sufA was still induced more than 10-fold. Fur, a ferric uptake regulator that negatively regulates this operon in response to iron availability, did not mediate the oxidant induction. Deletion of the suf operon caused cells to be more sensitive to superoxide-generating agents without affecting sensitivity to H2O2. From these results, we propose that the oxidant induction of the sufA operon is mediated through OxyR, IHF, plus an unidentified oxidant-responsive factor, and that the suf gene products are needed to defend cells against oxidative stress caused by superoxide generators.