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

ABSTRACT: 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.

ORGANISM(S): Bacillus subtilis subsp. subtilis str. 168

SUBMITTER: Ulrike Mäder

PROVIDER: E-GEOD-27637 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

Comparative assessment of the physiological and transcriptomic response of Escherichia coli to disinfectants: Ferrate versus hypochlorite

Project description:Disinfection is very critical for the removal of pathogens that constitute public health concerns. However, a rounded understanding of the antimicrobial action of different classes of disinfectants is still lacking. In this work, the physiological and transcriptomic responses of Escherichia coli to two different disinfectants namely: potassium ferrate (K2FeO4) and more conventional disinfectant, sodium hypochlorite (NaOCl), were examined. The study aimed to examine E. coli genetic response to ferrate and compare and contrast it to the genetic response of E. coli to hypochlorite.

2022-02-01 | GSE126176 | GEO

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Mycobacterium smegmatis under hypochlorite stress

Project description:Mycothiol (AcCys-GlcN-Ins, MSH) is the major thiol-redox buffer in Actinomycetes, including Mycobacterium and Corynebacterium species. ). Protein S-mycothiolation controls the activities of several redox enzymes that function in detoxification of ROS and methionine sulfoxides, including the thiol peroxidase Tpx, the mycothiol peroxidase Mpx and the methionine sulfoxide reductase MsrA. Here we investigated the level of protein S-mycothiolation in Mycobacterium smegmatis under oxidative stress as well as its NaOCl stress response.

2017-06-30 | PXD003303 | Pride

Monitoring global protein thiol-oxidation and protein S-mycothiolation in Corynebacterium diphtheriae under hypochlorite stress

Project description:Mycothiol (AcCys-GlcN-Ins, MSH) is the major thiol-redox buffer in Actinomycetes, including Mycobacterium and Corynebacterium species. Protein S-mycothiolation controls the activities of several redox enzymes that function in detoxification of ROS and methionine sulfoxides, including the thiol peroxidase Tpx, the mycothiol peroxidase Mpx and the methionine sulfoxide reductase MsrA. Here we investigated the level of protein S-mycothiolation in Corynebacterium diphtheriae DSM43989 under oxidative stress as well as its NaOCl stress response.

2017-07-17 | PXD003321 | Pride

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

Understanding environmental and biocide survival of Candida auris through transcriptional profiling

Project description:Candida auris is an enigmatic fungal pathogen, recently elevated to the Critical Priority group of fungal pathogens by the World Health Organisation. Of key concern is its ability to cause outbreaks within intensive and chronic care units, which is facilitated through biofilm formation. We investigated the susceptibility of phenotypically-diverse C. auris isolates to sodium hypochlorite (NaOCl) disinfection, and the subsequent role of biofilms in surviving disinfection using a dry-surface biofilm (DSB) model and transcriptomic profiling. Planktonic cells were tested for susceptibility to NaOCl using minimum inhibitory concentration and disinfection protocols. Biofilm formation using the DSB model consisted of consecutive 48 hr cycles with/without media across a 12-day period, assessed using viable counts, biomass assays, and microscopy. Disinfection efficacy was assessed using clinically relevant protocols of 500-1000 ppm for 1-5 min. RNA-sequencing was performed on untreated DSBs in comparison to planktonic cells. Isolates were found to be sensitive to NaOCl planktonically at concentrations ≤62.5 ppm, and grew robust biofilms using the DSB protocol. Biofilms developed tolerance to all NaOCl treatment parameters, with 2-4 log10-reductions in viable cells observed at highest concentrations. Transcriptomics identified ABC transporters and iron acquisition pathways as strongly upregulated in DSBs relative to planktonic cells. We have optimised a DSB protocol in which C. auris biofilms can mediate tolerance to adverse conditions such as NaOCl disinfection, suggesting a lifestyle through which this problematic yeast can environmentally persist and transmit. Mechanistically, the upregulation of small-molecule and iron transport pathways have been identified as potential facilitators of survival.

2023-08-07 | GSE239851 | GEO

Transcription profiling of Burkholderia cenocepacia J2315 sessile cells to treatment with high doses of hydrogen peroxide, sodium hypochlorite and chlorhexidine

Project description:In order to unravel the molecular mechanisms involved in the resistance of biofilm-grown Burkholderia cenocepacia cells against high concentrations of disinfectants, the present study focussed on the transcriptional response in sessile B. cenocepacia J2315 cells following exposure to high levels of H2O2, NaOCl or chlorhexidine. In addition differences in gene expression were examined between untreated B. cenocepacia sessile cells and B. cenocepacia planktonic cells.

2010-03-02 | E-MEXP-2502 | biostudies-arrayexpress

Monitoring of protein thiol-oxidation in S.aureus USA300 under hypochlorite stress using OxICAT approach.

Project description:During infections, S. aureus has to cope with the oxidative burst of activated macrophages and neutrophils, including reactive oxygen and nitrogen species (RNS, ROS) and the strong oxidant hypochloric acid. We aimed to understand the global thiol-redox state in the major pathogen S. aureus and discover new NaOCl-sensitive proteins driven by thiol-switches. Thus, we performed a quantitative redox proteomics approach based on OxICAT and analyzed the percentages of thiol-oxidation levels in S.aureus before and after sub-lethal doses of 150 µM NaOCl stress. In parallel, we searched for protein S-bacillithiolation.

2016-12-22 | PXD004918 | Pride

The regulon of the pleiotropic transcription factor Spx in Bacillus subtilis

Project description:The transcriptional regulator Spx plays a key role in maintaining the redox homeostasis of Bacillus subtilis cells exposed to disulfide stress. Defects in Spx were previously shown to lead to differential expression of numerous genes but direct and indirect regulatory effects could not be distinguished. Wild-type strain and spx mutant cells exposed or not to diamide stress were subjected to tiling array gene expression analysis. To distinguish direct and indirect effects, the genomic sites bound by the Spx-RNAP complex were mapped using a chromatine immunoprecipitation approach. This allowed the identification of 144 transcription units comprising 275 genes under direct Spx regulation. This data set contains 4 samples. Immunoprecipitated (IP) DNA from Bacillus subtilis Bas044 strain (BSB1, a tryptophan-prototrophic derivative 168 strain expressing a SPA-tagged Spx protein) were extracted from bacterial cultures in absence or presence of diamide. IP and whole cell extract DNA were hybridized on tiling array to generate ChIP-on-chip profiles. Two biological replicates were analyzed.

2012-09-06 | E-GEOD-35097 | biostudies-arrayexpress

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