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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 paralogs, CatR and YodB, results in full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N5-GTAA overlapping the -35 promoter region (BS1) and the transcriptional start site (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue that is essential 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-crosslinking and mass spectrometry.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE22603 | GEO | 2010/06/29

SECONDARY ACCESSION(S): PRJNA128355

REPOSITORIES: GEO

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

Project description: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 paralogs, CatR and YodB, results in full derepression of catDE transcription. DNA-binding assays and promoter mutagenesis studies showed that CatR protects two inverted repeats with the consensus sequence TTAC-N5-GTAA overlapping the -35 promoter region (BS1) and the transcriptional start site (BS2). The BS1 operator was required for binding of YodB in vitro. CatR and YodB share the conserved N-terminal Cys residue that is essential 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-crosslinking and mass spectrometry. Control of Bacillus subtilis 168 wild type (Ko_WT) vs. DyvaP mutant (Ko_DyvaP). The experiment was conducted in duplicates using two independent total RNA preparations. For all datasets used for final analysis, wild-type samples were labeled with Cy3 and DyvaP mutant samples were labeled with Cy5.

2010-06-29 | E-GEOD-22603 | biostudies-arrayexpress

RNA-seq of Mycobacterium smegmatis wild type and deletion mutant of the redox-sensing MarR-type repressor HypS revelas HypS function in hypochlorite and antimicrobial resistance.

Project description:Illumina TruSeq stranded mRNA RNA-seq including Illumina ribezeo rRNA depletion of Mycobacterium smegmatis wild type and deletion mutant of the redox-sensing MarR-type repressor HypS in three bilogical replicates prior and after 30 min after exposure to 500 µM NaOCl stress revealed that hypS is autoregulated and represses transcription of the co-transcribed hypO gene which encodes a multidrug efflux pump. DNA binding activity of HypS to the 8-5-8 bp inverted repeat sequence upstream of the hypSO operon was inhibited under NaOCl stress. HypS was identified as a novel redox-sensitive repressor that contributes to mycobacterial resistance towards HOCl stress and antibiotics.

2019-11-29 | E-MTAB-8534 | biostudies-arrayexpress

S-bacillithiolation of the OhrR repressor and the methionine synthase MetE protect against hypochlorite stress in Bacillus subtilis

Project description:Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In B. subtilis, the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate) termed as S-bacillithiolation. We have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid (NaOCl), the active component of house-hold bleach. The OhrR-controlled peroxiredoxin OhrA was most strongly up-regulated by NaOCl stress and conferred specific protection against NaOCl. Inactivation of the OhrR repressor was caused by S-bacillithiolation of the redox-sensing Cys15 residue in response to NaOCl. Two cobalamin-independent methionine synthases MetE and YxjG were identified as S-bacillithiolated at their essential active site cysteines resulting in hypochlorite-induced methionine limitation. In summary, our studies show that S-bacillithiolation of OhrR and the methionine synthases is the major mechanism in protection against hypochlorite stress in B. subtilis. The B. subtilis 168 wild type strain was grown in minimal medium to OD500 of 0.4 and harvested before and 10 minutes after exposure to 50 µM NaOCl. Microarray hybridizations were performed in triplicate using RNA isolated from independent cultures.

2011-07-04 | E-GEOD-27637 | biostudies-arrayexpress

The metalloregulatory site in Streptococcus pneumoniae AdcR, a zinc-activated MarR-family repressor

2010-10-01 | GSE21506 | GEO

The metalloregulatory site in Streptococcus pneumoniae AdcR, a zinc-activated MarR-family repressor

Project description:Streptococcus pneumoniae D39 AdcR (adhesion competence repressor) is the first metal-sensing member of the MarR (multiple antibiotic resistance repressor) family to be characterized. Expression profiling of a âadcR strain grown in liquid culture under microaerobic conditions revealed that transcript amounts for 13 genes were up-regulated relative to the wild-type strain, among them adcR, adcCBA, encoding a high affinity ABC uptake system for zinc, and genes encoding cell-surface zinc-binding pneumococcal histidine triad (pht) and adcII (lmb, laminin binding) proteins. Down-regulated transcripts included those encoding two putative zinc-containing alcohol dehydrogenases. Bacterial strains were grown exponentially in rich (BHI) media at 37C and an atmosphere of 5% CO2 to OD620~0.2, and were processed as described in the related Sample records. Samples were collected from three independent biological replicates and included one dye swap. Data were normalized using the Lowess (block) method without background subtraction. Changes in relative transcript amounts of positive or negative 2-fold with Bayesian P value of <0.001 were considered significant, and were included as supplementary material for the accompanying manuscript (The metalloregulatory site in Streptococcus pneumoniae AdcR, a zinc-activated MarR-family repressor; Reyes-Caballero, H. et al, manuscript in preparation).

2010-10-01 | E-GEOD-21506 | biostudies-arrayexpress

S-bacillithiolation protects against hypochlorite stress in Bacillus subtilis as revealed by transcriptomics and redox proteomics

Project description:Protein S-thiolation is a post-translational thiol-modification that controls redox-sensing transcription factors and protects active site cysteine residues against irreversible oxidation. In Bacillus subtilis the MarR-type repressor OhrR was shown to sense organic hydroperoxides via formation of mixed disulfides with the redox buffer bacillithiol (Cys-GlcN-Malate, BSH), termed as S-bacillithiolation. Here, we have studied changes in the transcriptome and redox proteome caused by the strong oxidant hypochloric acid in B. subtilis. The expression profile of NaOCl stress is indiciative of disulfide stess as shown by the induction of the thiol- and oxidative stress-specific Spx, CtsR and PerR regulons. Thiol redox proteomics identified only few cytoplasmic proteins with reversible thiol-oxidations in response to NaOCl stress that include GapA and MetE. Shotgun-LC-MS/MS analyses revealed that GapA, Spx and PerR are oxidized to intramolecular disulfides by NaOCl stress. Furthermore, we identified six S-bacillithiolated proteins in NaOCl-treated cells, including the OhrR repressor, two methionine synthases MetE and YxjG, the inorganic pyrophosphatase PpaC, the 3-D-phophoglycerate dehydrogenase SerA and the putative bacilliredoxin YphP. S-bacillithiolation of the OhrR repressor leads to up-regulation of the OhrA peroxiredoxin that confers together with BSH specific protection against NaOCl. S-bacillithiolation of MetE, YxjG, PpaC and SerA causes hypochlorite-induced methionine starvation as supported by the induction of the S-box regulon. The mechanism of S-glutathionylation of MetE has been described in Escherichia coli also leading to enzyme inactivation and methionine auxotrophy. In summary, our studies discover an important role of the BSH redox buffer in protection against hypochloric acid by S-bacillithiolation of the redox-sensing regulator OhrR and of four enzymes of the methionine biosynthesis pathway.

2011-07-04 | GSE27637 | GEO

ChIP-seq of the multiple antibiotic resistance repressor (MarR) protein of Enterotoxigenic Escherichia coli strain H10407

Project description:The experiment contains ChIP-seq data for Enterotoxigenic Escherichia coli strain H10407 transformed with plasmids pAMNF (encoding an N-terminal MarR-3xFLAG fusion) or pAMNM (encoding an N-terminal MarR-8xmyc fusion). The cells was cultured at 37 degrees in LB medium and crosslinked with 1 % (v/v) formaldehyde. After sonication, to break open cells and fragment DNA, immunoprecipitations were done using an anti-FLAG antibody (i.e. the strain encoding the MarR-8xmyc fusion was used as a control). Libraries were prepared using DNA remaining after immunoprecipitation.

2017-12-01 | E-MTAB-5591 | biostudies-arrayexpress

NTRC controls chloroplast redox homeostasis through the regulation of the redox balance of 2-Cys Prxs in Arabidopsis

Project description:Thiol-dependent redox regulation is essential for the rapid adaptation of chloroplast metabolism to unpredictable changes of light intensity. The disulfide reductase activity of thioredoxins (Trxs), which relies on photo-reduced ferredoxin (Fdx) and a Fdx-dependent Trx reductase (FTR), constitutes the Fdx-FTR-Trxs system, which links chloroplast redox regulation to light. In addition, chloroplasts harbor an NADPH-dependent Trx reductase (NTR) with a joint Trx domain, NTRC. The activity of these two redox systems is integrated by the balance of the hydrogen peroxide scavenging enzyme 2-Cys peroxiredoxin (2-Cys Prx), which thus plays a key role in maintaining the reducing capacity of chloroplast Trxs in response to light intensity. Based on the severe phenotype of mutant lines lacking NTRC, it is clear that this enzyme plays an essential role in chloroplast redox homeostasis. However, whether the function of NTRC depends on its capacity of reduce 2-Cys Prxs or has additional targets remains unknown. Here, we have addressed this issue by a comparative analysis of the triple mutant of Arabidopsis thaliana, ntrc-2cpab, simultaneously lacking 2-Cys Prxs and NTRC, and the double mutant 2cpab lacking 2-Cys Prxs. The phenotype of the ntrc-2cpab mutant is indistinguishable of the 2cpab mutant, as shown by growth rate, photosynthesis performance, light-dependent redox regulation of enzyme activity and comparative transcriptomics based RNA-Seq. Based on these results, we propose that the function of NTRC in chloroplast redox homeostasis is exerted by the regulation of the redox balance of 2-Cys Prxs rather than by the direct reduction of additional targets.

2021-09-09 | GSE147793 | GEO

Cyanophage MarR-type transcription factor regulates host RNase E expression

Project description:The marine picocyanobacterium Prochlorococcus contributes significantly to global primary production, and its abundance and diversity is shaped in part by viral infection. Here, we identified a cyanophage-encoded MarR-type transcription factor that induces gene expression of host Prochlorococcus MED4 endoribonuclease (RNase) E during phage infection. The increase in rne transcript levels relies on the phage (p)MarR-mediated activation of an alternative promoter that gives rise to a truncated, yet, enzymatically fully functional RNase E isoform. In this study, we demonstrate that pMarR binds to an atypical activator site downstream of the transcriptional start site and that binding is enhanced in the presence of Ca2+ ions. Furthermore, we show that dimeric pMaR interacts with the α subunit of RNA polymerase, and we identified amino acid residues S66, R67 and G106, which are important for Ca2+ binding, DNA binding and dimerization of pMarR, respectively.

2023-03-11 | PXD035458 | Pride

The plant thimet oligopeptidases TOP1 and TOP2 have divergent redox sensitivities and substrate cleavage specificity

Project description:Physiological and pathogen-induced oscillations in the plant cells' redox environment trigger response mechanisms that involve redox-sensitive cysteines and consequent changes in proteins' biochemical and functional characteristics. Arabidopsis thimet oligopeptidases (TOPs) TOP1 and TOP2 are components of the immune signaling and oxidative stress response. Here, TOP1 and TOP2 were evaluated comparatively to understand their role as redox sensors. We show that TOPs catalytic activity is augmented in plants undergoing an immune response and in mutant lines with altered expression of the chloroplastic NTRC impaired in the chloroplast thiol-disulfide regulatory system; ntrc was unable to mount a systemic immune response. Oxidation promoted TOP1 self-interaction to dimers and oligomers; Cys-to-Ala mutagenesis of multiple Cys residues inhibited TOP1 oxidative self-interaction. In contrast, TOP1 lacking the signal peptide (ΔSPTOP1) and TOP2 were detected solely as monomers. Treatment with oxidized glutathione increased the catalytic activities of TOPs; Ala substitution of the unique Cys405 of TOP2 abolished its oxidative activation. The immune regulator ROC1 was identified as a candidate TOPs substrate. TOPs cleaved a ROC1 peptide (ROC1p) at similar and unique cleavage sites; ROC1p concentration helped determine TOPs cleavage specificity. We propose that TOP1 and TOP2 form a proteolysis couple with divergent redox-sensing mechanisms and specificity.

2021-04-19 | PXD024059 | Pride

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