The inducible response to H2O2 stress in Bacillus subtilis is under the control of PerR, one of three Fur homologues in this organism. PerR was purified in both an inactive, metal‐dependent form and an active, metal‐containing form as determined using DNA‐binding assays. Active PerR contains both zinc 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 activity 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 peroxide sensing. Experiments using reporter fusions demonstrate that elevated levels of manganese repress PerR regulon genes and prevent their full induction by H2O2. In contrast, in cells grown with iron supplementation, a PerR‐repressed gene is completely derepressed by H2O2. These results are consistent with the idea that the intracellular form of the PerR metalloprotein, and therefore its hydrogen peroxide sensitivity, can be altered by growth conditions.
PerR is a ferric uptake repressor (Fur) homolog that functions as the central regulator of the inducible peroxide stress response in Bacillus subtilis. PerR has been previously demonstrated to regulate the mrgA, katA, ahpCF, hemAXCDBL, and zosA genes. We now demonstrate that PerR also mediates both the repression of its own gene and that of fur. Whereas PerR-mediated repression of most target genes can be elicited by either manganese or iron, repression of perR and fur is selective for manganese. Genetic studies indicate that repression of PerR regulon genes by either manganese or iron requires PerR and is generally independent of Fur. Indeed, in a fur mutant, iron-mediated repression is enhanced. Unexpectedly, repression of the fur gene by manganese appears to require both PerR and Fur, but only PerR binds to the fur regulatory region in vitro. The fur mutation appears to act indirectly by affecting cellular metal ion pools and thereby affecting PerR-mediated repression. While many components of the perR regulon are strongly induced by hydrogen peroxide, little, if any, induction of fur and perR could be demonstrated. Thus, not all components of the PerR regulon are components of the peroxide stimulon. We suggest that PerR exists in distinct metallated forms that differ in DNA target selectivity and in sensitivity to oxidation. This model is supported by the observation that the metal ion composition of the growth medium can greatly influence the transcriptional response of the various PerR regulon genes to hydrogen peroxide
SummaryThe Zap1 transcription factor is a central player in zinc homeostasis in yeast. This protein regulates the expression of genes involved in zinc accumulation and storage. For most of its target genes, Zap1 activates expression in zinc-limited cells and this function is inhibited in replete cells. Zap1 has two activation domains, AD1 and AD2, which are independently regulated by zinc status. In this study, we characterized AD1 and its regulation by zinc. AD1 was mapped using deletions to residues 332-402 of Zap1. The region required for the zinc responsiveness of this activation domain, designated 'ZRD
AD1, was mapped to residues 182-502. Thus, AD1 is embedded within its larger zinc-responsive domain. Using a combination of in silico analysis, random mutagenesis and site-directed mutagenesis, we identified key residues within ZRD AD1 required for its regulation by zinc. Most of these residues are cysteines and histidines that could potentially serve as Zn(II) ligands. These results suggest that ZRD AD1 senses zinc by direct Zn(II) binding. Consistent with this hypothesis, purified ZRD AD1 bound multiple Zn(II) ions. Finally, our results indicate that, in the context of the full-length Zap1 protein, AD1 and AD2 are both critical to the full control of gene expression in response to zinc.
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