The paralogous MarR/DUF24-family repressors YodB and CatR control expression of the catechol dioxygenase CatE in Bacillus subtilis

Abstract

The redox-sensing MarR/DUF24-type repressor YodB controls expression of the azoreductase AzoR1 and the nitroreductase YodC that are involved in detoxification of quinones and diamide in Bacillus subtilis. In the present paper, we identified YodB and its paralog YvaP (CatR) as repressors of the yfiDE (catDE) operon encoding a catechol-2,3-dioxygenase that also contributes to quinone resistance. Inactivation of both CatR and YodB is required for full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N(5)-GTAA overlapping the -35 promoter region (BS1) and the transcriptional start site (TSS) (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue, which is required for redox sensing of CatR in vivo as shown by Cys-to-Ser mutagenesis. Our data suggest that CatR is modified by intermolecular disulfide formation in response to diamide and quinones in vitro and in vivo. Redox regulation of CatR occurs independently of YodB, and no protein interaction was detected between CatR and YodB in vivo using protein cross-linking and mass spectrometry.

FIG. 1.

(A) Transcription factor arrays identify CatR as a repressor of the catDE operon. The individual colonies are transformants of transcription factor deletion mutants carrying the catE-lacZ fusion which were plated on X-Gal plates. The deletion of the regulatory gene is indicated. (B to E) Dual control of catDE transcription by the paralogous MarR/DUF24-family repressors CatR and YodB. Northern blot analysis of catDE transcription was performed using RNA isolated from the B. subtilis wild type (WT) and ΔcatR, ΔyodB, ΔcatR ΔyodB, and ΔyodB ΔazoR1 mutants before (co) and 10 min after treatment with 1 mM MHQ (M), 2 mM MHQ (M2), 2.5 mM MHQ (M3), 2.4 mM catechol (cat), 12 mM catechol (cat2), 1 mM diamide (D), or 2 mM diamide (D2). The arrow points toward the size of the catDE-specific transcript. (E) The quantification of catDE transcription ratios was performed using ImageJ from three independent Northern blot experiments. (F) Primer extension analysis was performed to determine the 5′ end of the catDE-specific transcript that is marked by an arrow, and the −10 promoter sequence is labeled with a solid line. For sequencing, the dideoxynucleotide added in each reaction is indicated above the corresponding lane.

FIG. 2.

The conserved Cys7 residue of CatR is required for redox sensing in vivo. (A) Northern blot analysis of catDE transcription was performed using RNA isolated from the B. subtilis wild type (WT) and ΔcatR and catRC7S mutant cells before (co) and 10 min after treatment with 1 mM MHQ (M), 2.4 mM catechol (cat), and 1 mM diamide (D). The arrow points toward the size of the catDE-specific transcript. (B) The quantification of the catDE transcription ratios was performed from three independent experiments using ImageJ. (C) Western blot analysis shows that similar CatR and CatRC7S proteins are produced in the wild type and the catRC7S point mutant, respectively. Cells were harvested before and 15 min after 1 mM of diamide stress, and 25 μg of protein extracts was subjected to CatR-specific Western blot analysis.

FIG. 3.

The ΔcatR and ΔyodB mutants are resistant and the ΔcatR yodB mutant is hyper-resistant to catechol and MHQ stress. B. subtilis wild type (WT) and the ΔcatR and ΔcatR ΔyodB mutants were grown in minimal medium to an OD₅₀₀ of 0.4 and treated with 4.8, 7, 12, and 20 mM catechol and 1, 1.5, and 2.5 mM MHQ at the time points that were set to zero, indicated by an arrow.

FIG. 4.

DNase I footprinting experiments of purified CatR to the top strand (A) and bottom strand (B) and footprinting experiments of YodB to the top strand of the catDE promoter (C) and CatR/YodB box alignments (D). The CatR- and YodB-protected sequences in the promoter region are indicated to the right (A to C), and the two TTAC-N₅-GTAA inverted repeats (BS1 and BS2) are labeled by arrows in the sequence alignment (D). The positions relative to the transcriptional start site are shown to the left. The transcriptional start site (+1) is indicated by an asterisk. For dideoxynucleotide sequencing, the dideoxynucleotide added in each reaction mixture is indicated above the corresponding lane. (D) Regions protected by CatR and YodB are shown with gray shading in the catD promoter sequence, including the inverted repeats as putative CatR boxes, which are labeled by arrows.

FIG. 5.

Alignment of the conserved catDE promoter regions and CatR boxes among Bacillus species. (A) The conserved upstream regions of the catDE operons of B. subtilis, B. licheniformis, B. halodurans, and B. amyloliquefaciens were aligned using ClustalW, and the putative CatR boxes are indicated using WebLogo (B). Promoter sequences (−10 and −35) and the transcriptional start site are indicated, and the CatR boxes are indicated by arrows. (C) The conserved YodB box identified in the upstream regions of spx, azoR1, yodC, and catD is displayed using WebLogo.

FIG. 6.

DNA-binding experiments of CatR and YodB to the mutated catDE promoter. Electrophoretic gel mobility shift assays (EMSAs) were applied using purified CatR and YodB proteins and catD promoter probes ranging from −146 to +20 without mutations (+20) and with mutations in one and two repeat elements of BS2 (+20M1 and +20M2), deletions in the upstream binding region (−60 and −40), and mutations of the two repeat elements of BS1 (−60M3).

FIG. 7.

CatR forms intersubunit disulfides by diamide and quinones. (A to C) Purified CatR protein was treated with diamide (A), MHQ (B), or catechol (C) and subsequently either reduced with 10 mM DTT (+ DTT) or left oxidized (- DTT) and subjected to nonreducing SDS-PAGE analysis. (D) Diagonal nonreducing/reducing CatR-specific Western blot analysis of immunoprecipitated CatR protein that was harvested from wild-type (WT) cells and ΔcatR and ΔyodB mutant cells before (co) or after exposure to diamide (Dia), MHQ, and catechol. (E) Protein extracts were harvested from wild-type cells under control conditions and after diamide, MHQ, and catechol stress and alkylated either by IAM or AMS that resulted in a mass shift of 500 Da per modified Cys. AMS- and IAM-treated extracts were separated using reducing SDS-PAGE and subjected to CatR-specific Western blot analysis.

FIG. 8.

Analysis of protein interactions between YodB and CatR in vivo. Soluble fractions were prepared from cells of the wild type (WT) and ΔcatR catR-FLAG and ΔyodB catR-FLAG strains after protein cross-linking with DSP according to the description in Materials and Methods. CatR-FLAG protein was immunoprecipitated using anti-FLAG affinity agarose, and the proteins were eluted using reducing sample buffer containing 5% mercaptoethanol (IP-CatR-FLAG). The protein extracts were separated in lanes 4 to 6 each. Detection of YodB and CatR was performed using the specific antibodies. Note that the FLAG-tag results in a higher molecular mass of CatR-FLAG compared to CatR detected in wild-type extracts.

FIG. 9.

Quinone resistance network of the MhqR, YodB, and CatR regulons of B. subtilis. Three MarR-type repressors, MhqR, YodB, and CatR, respond to diamide and quinone-like electrophiles in B. subtilis. YodB and CatR represent paralogous MarR/DUF24-family repressors that sense electrophiles via the conserved N-terminal Cys residue. The regulatory mechanism of the MhqR repressor is unknown. YodB controls the azoreductase AzoR1, the nitroreductase YodC, and Spx and it also controls together with CatR the DoxX-like oxidoreductase CatD and thiol-dependent dioxygenase CatE. The MhqR repressor regulates paralogous enzymes of the YodB and CatR regulons, including the azoreductase AzoR2, the nitroreductase MhqN, the DoxX-like oxidoreductase MhqP, and three thiol-dependent dioxygenases, MhqA, MhqE, and MhqO. The operator sequences for binding of MhqR, YodB, and CatR are generated using WebLogo.