Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible

Abstract

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.

FIG. 1.

Interaction of PerR with the perR regulatory region. (A) The −35 and −10 regions of the perR promoter are underlined, and the two overlapping Per box elements are indicated. The A residue start site for transcription is in bold. Regions of both DNA strands protected against DNase I digestion by bound PerR protein are indicated by broken double lines. (B) Primer extension mapping of the perR transcriptional start site (arrow) indicates transcript initiation with the A residue indicated in the sequence to the left. (C) DNase I footprint of purified PerR binding to the perR promoter. PerR was added at the concentrations (nanomolar) indicated in the presence of 10 μM Mn(II). Shown are results from footprinting on the bottom strand; however, top-strand analysis was also performed (data not shown). The regions protected against digestion, as determined by alignment with G+A sequencing ladders (data not shown), are summarized in panel A.

FIG. 2.

Interaction of PerR with the fur regulatory region. (A) The −35 and −10 elements of the fur promoter are underlined, and the transcriptional start sites are in bold. The Per box upstream of the −35 element is indicated. Regions of both DNA strands protected against DNase I digestion by bound PerR protein are indicated by broken double lines. (B) Primer extension mapping of the transcription start sites of the fur gene. Transcription initiates at the indicated A and G residues. (C) DNase I footprint of PerR binding to the fur operator region. The results shown are for the bottom strand. Purified PerR was added at the concentrations indicated in the presence of 10 μM Mn(II). The bold line adjacent to the G+A ladder indicates the position of the Per box, and the protected region is bracketed.

FIG. 3.

Metal selectivity of gene regulation in resuspension experiments. Cells were resuspended in minimal medium either containing no added Mn(II) or Fe(III) (open circles) or containing 5 μM Mn(II) (filled triangles), 10 μM Fe(III) (filled squares), or both (filled circles). Strains contained mrgA-cat-lacZ (A), katA-cat-lacZ (B), zosA-cat-lacZ (C), ahpC-cat-lacZ (D), hemA-cat-lac (E), perR-cat-lacZ (F), or fur-cat-lacZ (G). Samples were taken at the times indicated and assayed for β-Gal activity. The results shown are representative of at least three independent experiments; error bars represent the standard error of the mean of duplicate samples. Panel H is a Northern blot analysis of the fur transcript from cultures collected 3 h after resuspension in minimal medium containing the indicated metal ion supplementation.

FIG. 4.

Roles of PerR and Fur in metalloregulation of PerR regulon genes. Resuspension experiments were performed as described in the legend to Fig. 3 with all seven promoter fusions (indicated at the bottom of panel C) in either the wild-type (A) background or the fur (B) or perR (C) mutant strain background. All samples were measured 3 h. after resuspension and normalized to the level in medium lacking manganese and iron supplementation (white bars; absolute values are shown above the bars). The cells were resuspended in minimal medium either containing (from left to right) no added Mn(II) or Fe(III) (white bars) or containing 10 μM Fe(III) (hatched), 5 μM Mn(II) (stippled), or both (black). The data in panel A are the same as those shown for the 3-h time point in Fig. 3.

FIG. 5.

Effects of mntR and fur mutations on metalloregulation of the fur gene. Overnight cultures of strains carrying the fur-cat-lacZ reporter fusion were diluted 1:100 into minimal medium with either 1 μM Fe(III) (A) or 10 μM Fe(III) (B) and Mn(II) at 0, 0.1, 1, 10, 100, or 1,000 μM (left to right). Cells were grown to mid-log phase and collected for β-Gal assay. Note that the mntR mutant strain does not grow in concentrations of Mn(II) of 10 μM and greater (27), so no data were obtained for these conditions. WT, wild type.

FIG. 6.

Induction of PerR regulon genes by H₂O₂. Strains containing the indicated reporter fusions were grown in minimal medium with no added Mn(II) or Fe(III) (None) or with 10 μM Fe(III) (Fe), 5 μM Mn(II) (Mn), or both (Fe+Mn) as described in the legend to Fig. 3. At 2 h after resuspension, the cultures were split and 100 μM H₂O₂ was added to one sample. After growth for another 30 min, cells were harvested for β-Gal assay and induced expression (gray bars) was compared to expression in the absence of H₂O₂ addition (white bars). Note that the data represented by the white bars are the same as those for the 2.5-h time point in Fig. 3. Experiments were performed twice; error bars represent the standard error of the mean.