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. 2001 Jul;183(14):4134-41.
doi: 10.1128/JB.183.14.4134-4141.2001.

OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis

M Fuangthong  1 , S Atichartpongkul, S Mongkolsuk, J D Helmann
Affiliations

OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis

M Fuangthong et al. J Bacteriol. 2001 Jul.

Abstract

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 sigma(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 (sigma(B), PerR, and OhrR) that contribute to peroxide stress responses.

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Figures

FIG. 1
FIG. 1
Roles of OhrA, OhrB, and AhpCF in protection against ROS. The sensitivity of each indicated strain was measured as a zone of growth inhibition in a disk diffusion assay. Filters contained either 0.4 M CHP (A), 1.6 M H2O2 (B), or 0.5 M paraquat (C). The data shown are representative of three experiments. The error bars indicate the standard deviations from duplicate samples. PQ, paraquat.
FIG. 2
FIG. 2
Northern analysis ohr region genes. Expression of ohrA (A), ohrB (B), and ohrR (C) was measured using 10 μg of total RNA from each sample separated on a 1% formaldehyde gel. RNA was transferred to a nylon membrane and hybridized with a radiolabeled DNA fragment containing the coding region of each gene. Arrows indicate the major transcript of each gene. Cells were either uninduced (none) or were treated with 100 μM CHP, 100 μM tert-butyl hydroperoxide (t-BuOOH), 100 μM H2O2, 4% ethanol, or 4% NaCl for 15 min as indicated.
FIG. 3
FIG. 3
Primer extension analysis of the ohrA promoter. Cells were grown and treated as described for Fig. 2 prior to RNA isolation. The major alkyl peroxide responsive transcriptional start point for the ohrA gene corresponds to position −27 relative to the start codon, in agreement with previously published start site mapping data (38). The origin of the larger band is not clear, but may be due to readthrough transcription from the upstream proAB operon.
FIG. 4
FIG. 4
Effect of an ahpC mutation on induction of ohrA by organic hydroperoxides. β-Galactosidase activities were assayed in various mutants (bars: gray, ohrA, HB574; black, ohrA ohrB, HB2003; white, ohrA ahpC, HB2008; cross-hatched, ohrA ohrB ahpC, HB2010). Cells were grown to mid-log phase, and various concentrations of CHP or tert-butyl hydroperoxide (tBOOH) were added to the cultures for 15 min at 37°C with shaking. The data shown are representative of triplicate determinations.
FIG. 5
FIG. 5
ohrR encodes a MarR-like repressor of ohrA. (A) Schematic of the ohrA ohrR ohrB region. PA indicates a ςA-dependent promoter element; PB indicates a ςB-dependent promoter. (B) alignment of OhrR with other closely related MarR family members. The abbreviations used are as follows (strain; GenBank accession number): OhrR Bs (B. subtilis; E69857), OhrRa Pa (P. aeruginosa PAO1; D83290), OhrRb Pa (P. aeruginosa PAO1; G83292), OhrR Ac (Acinetobacter sp. strain ADP1; CAA70318), OhrR Sc (Streptomyces coelicolor; CAB87337); OhrR Vc (Vibrio cholerae group O1 strain N16961; B82389), and OhrR Stc (Staphylococcus sciuri strain ATCC 29062). The amino acid sequences were aligned (using CLUSTALW) and conserved residues highlighted using the BoxShade utility.
FIG. 6
FIG. 6
Genetic identification of sequences required for OhrR-mediated repression. The perfect inverted repeat is indicated in capital letters with matching bases identified by a vertical line. (A) In the ohrA promoter, there are two adjacent inverted repeats. The first is imperfect; the second is a perfect inverted repeat (thick arrows). This region also contains two 11-bp direct repeats (thin arrows). The −10 and −35 regions are shown in boldface. (B) The sequence of the mutant promoter region (ohrA∗) is shown with a dashed line to indicate the 15-bp deletion. A new −10 element is created by the deletion. (C) A related, imperfect inverted repeat is found overlapping the ohrR promoter region.
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