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Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.

Eiamphungporn W, Soonsanga S, Lee JW, Helmann JD - Nucleic Acids Res. (2009)

Bottom Line: Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids.We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo.The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.

ABSTRACT
Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

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Peroxide-responsiveness of scOhrR in vivo. β-Galactosidase and northern analyses of strains expressing scOhrR and containing an ohrA-cat-lacZ fusion. (A) Cells were either untreated (empty bars) treated (filled bars) with 100 μM CHP for 15 min. (B) Cells were either untreated (empty bars) treated (filled bars) with 5 μM LHP for 15 min. Error bars represent the SD (n = 3). (C) Northern blot analysis of the ohrA transcript in cells with and without CHP treatment, as in (A).
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Figure 2: Peroxide-responsiveness of scOhrR in vivo. β-Galactosidase and northern analyses of strains expressing scOhrR and containing an ohrA-cat-lacZ fusion. (A) Cells were either untreated (empty bars) treated (filled bars) with 100 μM CHP for 15 min. (B) Cells were either untreated (empty bars) treated (filled bars) with 5 μM LHP for 15 min. Error bars represent the SD (n = 3). (C) Northern blot analysis of the ohrA transcript in cells with and without CHP treatment, as in (A).

Mentions: To monitor the activity of the scOhrR proteins, we used an integrated ohrA-lacZ reporter fusion as described (3,16). All four scOhrR repressors were fully functional as repressors as judged by complementation of the ohrR mutant strain (Figure 2). The WT-WT scOhrR responded normally to organic peroxides in vivo as compared to the monomeric OhrR (expressed from the integrational plasmid pDG1731-OhrR). In contrast, those scOhrR variants that retained only a single active site cysteine were significantly reduced in their ability to respond to CHP. This relatively poor responsiveness was observed regardless of which monomer domain contained the active site C15 residue (Figure 2A) and was observed with both the 5 and 10 amino-acid linkers (data not shown). The scOhrR with the active site within the C-terminal domain responded better to oxidant under all conditions tested. When cells were treated with LHP, a similar pattern of responsiveness was observed, and in this case the C15S-WT scOhrR variant allowed approximately 50% derepression relative to the WT-WT scOhrR and monomeric WT OhrR controls (Figure 2B). This pattern of responsiveness was further confirmed by using northern blot analyses to directly monitor expression of the ohrA mRNA (Figure 2C).Figure 2.


Oxidation of a single active site suffices for the functional inactivation of the dimeric Bacillus subtilis OhrR repressor in vitro.

Eiamphungporn W, Soonsanga S, Lee JW, Helmann JD - Nucleic Acids Res. (2009)

Peroxide-responsiveness of scOhrR in vivo. β-Galactosidase and northern analyses of strains expressing scOhrR and containing an ohrA-cat-lacZ fusion. (A) Cells were either untreated (empty bars) treated (filled bars) with 100 μM CHP for 15 min. (B) Cells were either untreated (empty bars) treated (filled bars) with 5 μM LHP for 15 min. Error bars represent the SD (n = 3). (C) Northern blot analysis of the ohrA transcript in cells with and without CHP treatment, as in (A).
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Figure 2: Peroxide-responsiveness of scOhrR in vivo. β-Galactosidase and northern analyses of strains expressing scOhrR and containing an ohrA-cat-lacZ fusion. (A) Cells were either untreated (empty bars) treated (filled bars) with 100 μM CHP for 15 min. (B) Cells were either untreated (empty bars) treated (filled bars) with 5 μM LHP for 15 min. Error bars represent the SD (n = 3). (C) Northern blot analysis of the ohrA transcript in cells with and without CHP treatment, as in (A).
Mentions: To monitor the activity of the scOhrR proteins, we used an integrated ohrA-lacZ reporter fusion as described (3,16). All four scOhrR repressors were fully functional as repressors as judged by complementation of the ohrR mutant strain (Figure 2). The WT-WT scOhrR responded normally to organic peroxides in vivo as compared to the monomeric OhrR (expressed from the integrational plasmid pDG1731-OhrR). In contrast, those scOhrR variants that retained only a single active site cysteine were significantly reduced in their ability to respond to CHP. This relatively poor responsiveness was observed regardless of which monomer domain contained the active site C15 residue (Figure 2A) and was observed with both the 5 and 10 amino-acid linkers (data not shown). The scOhrR with the active site within the C-terminal domain responded better to oxidant under all conditions tested. When cells were treated with LHP, a similar pattern of responsiveness was observed, and in this case the C15S-WT scOhrR variant allowed approximately 50% derepression relative to the WT-WT scOhrR and monomeric WT OhrR controls (Figure 2B). This pattern of responsiveness was further confirmed by using northern blot analyses to directly monitor expression of the ohrA mRNA (Figure 2C).Figure 2.

Bottom Line: Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids.We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo.The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Cornell University, Ithaca, NY 14853-8101, USA.

ABSTRACT
Bacillus subtilis OhrR is a dimeric repressor that senses organic peroxides and regulates the expression of the OhrA peroxiredoxin. Derepression results from oxidation of an active site cysteine which ultimately results in formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to the sulfinic or sulfonic acids. We expressed a single-chain OhrR (scOhrR) in which the two monomers were connected by a short amino-acid linker. scOhrR variants containing only one active site cysteine were fully functional as repressors and still responded, albeit with reduced efficacy, to organic peroxides in vivo. Biochemical analyses indicate that oxidation at a single active site is sufficient for derepression regardless of the fate of the active site cysteine. scOhrR with only one active site cysteine in the amino-terminal domain is inactivated at rates comparable to wild-type whereas when the active site is in the carboxyl-terminal domain the protein is inactivated much more slowly. The incomplete derepression noted for single active site variants of scOhrR in vivo is consistent with the hypothesis that protein reduction regenerates active repressor and that, in the cell, oxidation of the second active site may also contribute to derepression.

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