The mechanisms by which Bacillus subtilis OhrR, a member of the MarR family of transcription regulators, binds the ohrA operator and is induced by oxidation of its lone cysteine residue by organic hydroperoxides to sulphenic acid are unknown. Here, we describe the crystal structures of reduced OhrR and an OhrR-ohrA operator complex. To bind DNA, OhrR employs a chimeric winged helix-turn-helix DNA binding motif, which is composed of extended eukaryotic-like wings, prokaryotic helix-turn-helix motifs, and helix-helix elements. The reactivity of the peroxide-sensing cysteine is not modulated by proximal basic residues but largely by the positive dipole of helix alpha1. Induction originates from the alleviation of intersubunit steric clash between the sulphenic acid moieties of the oxidized sensor cysteines and nearby tyrosines and methionines. The structure of the OhrR-ohrA operator complex reveals the DNA binding mechanism of the entire MarR family and suggests a common inducer binding pocket.

Bacillus subtilis displays a complex adaptive response to the presence of reactive oxygen species. To date, most proteins that protect against reactive oxygen species are members of the peroxide-inducible PerR and B regulons. We investigated the function of two B. subtilis homologs of the Xanthomonas campestris organic hydroperoxide resistance (ohr) gene. Mutational analyses indicate that both ohrA and ohrB contribute to organic peroxide resistance in B. subtilis, with the OhrA protein playing the more important role in growing cells. Expression of ohrA, but not ohrB, is strongly and specifically induced by organic peroxides. Regulation of ohrA requires the convergently transcribed gene, ohrR, which encodes a member of the MarR family of transcriptional repressors. In an ohrR mutant, ohrA expression is constitutive, whereas expression of the neighboring ohrB gene is unaffected. Selection for mutant strains that are derepressed for ohrA transcription identifies a perfect inverted repeat sequence that is required for OhrR-mediated regulation and likely defines an OhrR binding site. Thus, B. subtilis contains at least three regulons ( B , PerR, and OhrR) that contribute to peroxide stress responses.

Reactive oxygen species induce the expression of detoxification and repair genes critical for life in an aerobic environment. Bacterial factors that sense reactive oxygen species use either thioldisulfide exchange reactions (OxyR, RsrA) or redox labile 2Fe-2S clusters (SoxR). We demonstrate that the reduced form of Bacillus subtilis OhrR binds cooperatively to two adjacent inverted repeat sequences in the ohrA control region and thereby represses transcription. In the presence of organic hydroperoxides, OhrR is inactivated by the reversible oxidation of a single conserved cysteine residue to the corresponding cysteine-sulfenic acid, and perhaps to higher oxidation states.peroxide stress ͉ oxidative stress ͉ peroxiredoxin ͉ alkyl hydroperoxide reductase ͉ Bacillus subtilis

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

The Xanthomonas campestris transcription regulator OhrR contains a reactive cysteine residue (C22) that upon oxidation by organic hydroperoxides (OHPs) forms an intersubunit disulphide bond with residue C127'. Such modification induces the expression of a peroxidase that reduces OHPs to their less toxic alcohols. Here, we describe the structures of reduced and OHP-oxidized OhrR, visualizing the structural mechanism of OHP induction. Reduced OhrR takes a canonical MarR family fold with C22 and C127' separated by 15.5 A. OHP oxidation results in the disruption of the Y36'-C22-Y47' interaction network and dissection of helix alpha5, which then allows the 135 degrees rotation and 8.2 A translation of C127', formation of the C22-C127' disulphide bond, and alpha6-alpha6' helix-swapped reconfiguration of the dimer interface. These changes result in the 28 degrees rigid body rotations of each winged helix-turn-helix motif and DNA dissociation. Similar effector-induced rigid body rotations are expected for most MarR family members.