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Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation.In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

ABSTRACT
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

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HypO induction in the proteome (A), transcriptional induction of hypO and hypR in the transcriptome (B) and northern blot analysis of hypO and hypR transcription (C) under thiol-specific stress conditions. (A) Cytoplasmic proteins were labelled with 35S-methionine before (control) and after stress exposure and separated by 2D–PAGE as described (36). Close-ups of the overlay proteome images are shown for the wild-type before (green images) and 10 min after exposure to 1 mM diamide (left, red image) or 0.5 mM MHQ (right, red image). Proteins with increased protein synthesis ratios after MHQ and diamide stress including HypO are labelled that were identified from Coomassie-stained 2D gels as described (36). (B) The values represent fold-changes of hypR and hypO induction ratios in two biological replicates of transcriptome experiments of cells treated with 1 mM diamide, 0.5 mM MHQ, 50 µM NaOCl and 100 µM chromanon according to previously published transcriptome data (17,26,44). (C) Northern blot analysis was performed using RNA isolated from B. subtilis wild-type, the ΔhypR mutant and the ΔhypR mutant complemented with hypR, hypRC14S, hypRC49S and hypRC14,49S before (co) and 10 min after treatment with 0.5 mM MHQ, 1 mM diamide and 50 µM NaOCl. The arrows point toward the hypO and hypR specific transcripts. The methylen-blue stained northern blot is shown below as RNA loading control and the 16S and 23S rRNAs are labelled.
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gkr1316-F1: HypO induction in the proteome (A), transcriptional induction of hypO and hypR in the transcriptome (B) and northern blot analysis of hypO and hypR transcription (C) under thiol-specific stress conditions. (A) Cytoplasmic proteins were labelled with 35S-methionine before (control) and after stress exposure and separated by 2D–PAGE as described (36). Close-ups of the overlay proteome images are shown for the wild-type before (green images) and 10 min after exposure to 1 mM diamide (left, red image) or 0.5 mM MHQ (right, red image). Proteins with increased protein synthesis ratios after MHQ and diamide stress including HypO are labelled that were identified from Coomassie-stained 2D gels as described (36). (B) The values represent fold-changes of hypR and hypO induction ratios in two biological replicates of transcriptome experiments of cells treated with 1 mM diamide, 0.5 mM MHQ, 50 µM NaOCl and 100 µM chromanon according to previously published transcriptome data (17,26,44). (C) Northern blot analysis was performed using RNA isolated from B. subtilis wild-type, the ΔhypR mutant and the ΔhypR mutant complemented with hypR, hypRC14S, hypRC49S and hypRC14,49S before (co) and 10 min after treatment with 0.5 mM MHQ, 1 mM diamide and 50 µM NaOCl. The arrows point toward the hypO and hypR specific transcripts. The methylen-blue stained northern blot is shown below as RNA loading control and the 16S and 23S rRNAs are labelled.

Mentions: Previous transcriptome analyses revealed that the unknown DUF24-type regulator encoded by yybR responds strongly to diamide, quinones and hypochlorite (Figure 1B) (17,43,44). In addition, several FMN-dependent NAD(P)H oxidoreductase-encoding genes were up-regulated, including yfmJ, ytkL, ywnB, yqiG, yqjM, ywrO, ydeQ, yugJ and yfkO (Figure 1A and B) (43). Some of these redox enzymes are members of the Spx regulon, such as yqiG and yugJ suggesting that these could function in maintenance of the thiol-redox homeostasis (45,46). We were interested if the DUF24-family regulator YybR controls one of these oxidoreductases. Using northern blot analysis we found that the NAD(P)H-flavin oxidoreductase encoded by yfkO was strongly induced in the wild-type but not in the yybR mutant in response to diamide, NaOCl and MHQ stress (Figure 1C). This suggests that yybR positively controls yfkO transcription in response to diamide and NaOCl stress. In addition, yybR transcription is probably also strongly autoregulated by thiol-specific stress conditions. Hence, yybR was renamed as hypochlorite-responsive regulator hypR and yfkO was renamed as hypO. The northern blot analysis further revealed that hypR and hypO are transcribed monocistronically. Using primer extension, the 5′-ends of the hypO and hypR specific transcripts were mapped at T and A, respectively, located 31 and 55 bp upstream of the start codon (Supplementary Figure S2).Figure 1.


Structural insights into the redox-switch mechanism of the MarR/DUF24-type regulator HypR.

Palm GJ, Khanh Chi B, Waack P, Gronau K, Becher D, Albrecht D, Hinrichs W, Read RJ, Antelmann H - Nucleic Acids Res. (2012)

HypO induction in the proteome (A), transcriptional induction of hypO and hypR in the transcriptome (B) and northern blot analysis of hypO and hypR transcription (C) under thiol-specific stress conditions. (A) Cytoplasmic proteins were labelled with 35S-methionine before (control) and after stress exposure and separated by 2D–PAGE as described (36). Close-ups of the overlay proteome images are shown for the wild-type before (green images) and 10 min after exposure to 1 mM diamide (left, red image) or 0.5 mM MHQ (right, red image). Proteins with increased protein synthesis ratios after MHQ and diamide stress including HypO are labelled that were identified from Coomassie-stained 2D gels as described (36). (B) The values represent fold-changes of hypR and hypO induction ratios in two biological replicates of transcriptome experiments of cells treated with 1 mM diamide, 0.5 mM MHQ, 50 µM NaOCl and 100 µM chromanon according to previously published transcriptome data (17,26,44). (C) Northern blot analysis was performed using RNA isolated from B. subtilis wild-type, the ΔhypR mutant and the ΔhypR mutant complemented with hypR, hypRC14S, hypRC49S and hypRC14,49S before (co) and 10 min after treatment with 0.5 mM MHQ, 1 mM diamide and 50 µM NaOCl. The arrows point toward the hypO and hypR specific transcripts. The methylen-blue stained northern blot is shown below as RNA loading control and the 16S and 23S rRNAs are labelled.
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gkr1316-F1: HypO induction in the proteome (A), transcriptional induction of hypO and hypR in the transcriptome (B) and northern blot analysis of hypO and hypR transcription (C) under thiol-specific stress conditions. (A) Cytoplasmic proteins were labelled with 35S-methionine before (control) and after stress exposure and separated by 2D–PAGE as described (36). Close-ups of the overlay proteome images are shown for the wild-type before (green images) and 10 min after exposure to 1 mM diamide (left, red image) or 0.5 mM MHQ (right, red image). Proteins with increased protein synthesis ratios after MHQ and diamide stress including HypO are labelled that were identified from Coomassie-stained 2D gels as described (36). (B) The values represent fold-changes of hypR and hypO induction ratios in two biological replicates of transcriptome experiments of cells treated with 1 mM diamide, 0.5 mM MHQ, 50 µM NaOCl and 100 µM chromanon according to previously published transcriptome data (17,26,44). (C) Northern blot analysis was performed using RNA isolated from B. subtilis wild-type, the ΔhypR mutant and the ΔhypR mutant complemented with hypR, hypRC14S, hypRC49S and hypRC14,49S before (co) and 10 min after treatment with 0.5 mM MHQ, 1 mM diamide and 50 µM NaOCl. The arrows point toward the hypO and hypR specific transcripts. The methylen-blue stained northern blot is shown below as RNA loading control and the 16S and 23S rRNAs are labelled.
Mentions: Previous transcriptome analyses revealed that the unknown DUF24-type regulator encoded by yybR responds strongly to diamide, quinones and hypochlorite (Figure 1B) (17,43,44). In addition, several FMN-dependent NAD(P)H oxidoreductase-encoding genes were up-regulated, including yfmJ, ytkL, ywnB, yqiG, yqjM, ywrO, ydeQ, yugJ and yfkO (Figure 1A and B) (43). Some of these redox enzymes are members of the Spx regulon, such as yqiG and yugJ suggesting that these could function in maintenance of the thiol-redox homeostasis (45,46). We were interested if the DUF24-family regulator YybR controls one of these oxidoreductases. Using northern blot analysis we found that the NAD(P)H-flavin oxidoreductase encoded by yfkO was strongly induced in the wild-type but not in the yybR mutant in response to diamide, NaOCl and MHQ stress (Figure 1C). This suggests that yybR positively controls yfkO transcription in response to diamide and NaOCl stress. In addition, yybR transcription is probably also strongly autoregulated by thiol-specific stress conditions. Hence, yybR was renamed as hypochlorite-responsive regulator hypR and yfkO was renamed as hypO. The northern blot analysis further revealed that hypR and hypO are transcribed monocistronically. Using primer extension, the 5′-ends of the hypO and hypR specific transcripts were mapped at T and A, respectively, located 31 and 55 bp upstream of the start codon (Supplementary Figure S2).Figure 1.

Bottom Line: HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress.The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation.In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'.

View Article: PubMed Central - PubMed

Affiliation: Institute for Biochemistry, Ernst-Moritz-Arndt-University of Greifswald, D-17487 Greifswald, Germany.

ABSTRACT
Bacillus subtilis encodes redox-sensing MarR-type regulators of the OhrR and DUF24-families that sense organic hydroperoxides, diamide, quinones or aldehydes via thiol-based redox-switches. In this article, we characterize the novel redox-sensing MarR/DUF24-family regulator HypR (YybR) that is activated by disulphide stress caused by diamide and NaOCl in B. subtilis. HypR controls positively a flavin oxidoreductase HypO that confers protection against NaOCl stress. The conserved N-terminal Cys14 residue of HypR has a lower pK(a) of 6.36 and is essential for activation of hypO transcription by disulphide stress. HypR resembles a 2-Cys-type regulator that is activated by Cys14-Cys49' intersubunit disulphide formation. The crystal structures of reduced and oxidized HypR proteins were resolved revealing structural changes of HypR upon oxidation. In reduced HypR a hydrogen-bonding network stabilizes the reactive Cys14 thiolate that is 8-9 Å apart from Cys49'. HypR oxidation breaks these H-bonds, reorients the monomers and moves the major groove recognition α4 and α4' helices ∼4 Å towards each other. This is the first crystal structure of a redox-sensing MarR/DUF24 family protein in bacteria that is activated by NaOCl stress. Since hypochloric acid is released by activated macrophages, related HypR-like regulators could function to protect pathogens against the host immune defense.

Show MeSH
Related in: MedlinePlus