Roles of metal ions and hydrogen peroxide in modulating the interaction ofthe Bacillus subtilis PerR peroxide regulon repressor with operator DNA

The inducible response to H2O2 stress in Bacillus subtilis is under the con trol of PerR, one of three Fur homologues in this organism. PerR was purifi ed in both an inactive, metal-dependent form and an active, metal-containin g form as determined using DNA-binding assays. Active PerR contains both zi nc and iron and is designated PerR:Zn,Fe. Added manganous ion competes for binding to the iron site and can restore DNA-binding activity to the metal- dependent form of PerR, presumably generating PerR:Zn,Mn. The DNA-binding a ctivity of PerR:Zn,Fe is eliminated by exposure to H2O2 whereas PerR:Zn,Mn is comparatively resistant. DNA-binding activity can be restored by a thiol -reducing agent, suggesting that redox-active cysteines are involved in per oxide sensing. Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full indu ction by H2O2. In contrast, in cells grown with iron supplementation, a Per R-repressed gene is completely derepressed by H2O2. These results are consi stent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.