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. 2009 Dec;191(24):7520-30.
doi: 10.1128/JB.00937-09. Epub 2009 Oct 16.

Diamide triggers mainly S Thiolations in the cytoplasmic proteomes of Bacillus subtilis and Staphylococcus aureus

Dierk-Christoph Pöther  1 , Manuel Liebeke, Falko Hochgräfe, Haike Antelmann, Dörte Becher, Michael Lalk, Ulrike Lindequist, Ilya Borovok, Gerald Cohen, Yair Aharonowitz, Michael Hecker
Affiliations

Diamide triggers mainly S Thiolations in the cytoplasmic proteomes of Bacillus subtilis and Staphylococcus aureus

Dierk-Christoph Pöther et al. J Bacteriol. 2009 Dec.

Abstract

Glutathione constitutes a key player in the thiol redox buffer in many organisms. However, the gram-positive bacteria Bacillus subtilis and Staphylococcus aureus lack this low-molecular-weight thiol. Recently, we identified S-cysteinylated proteins in B. subtilis after treatment of cells with the disulfide-generating electrophile diamide. S cysteinylation is thought to protect protein thiols against irreversible oxidation to sulfinic and sulfonic acids. Here we show that S thiolation occurs also in S. aureus proteins after exposure to diamide. We further analyzed the formation of inter- and intramolecular disulfide bonds in cytoplasmic proteins using diagonal nonreducing/reducing sodium dodecyl sulfate gel electrophoresis. However, only a few proteins were identified that form inter- or intramolecular disulfide bonds under control and diamide stress conditions in B. subtilis and S. aureus. Depletion of the cysteine pool was concomitantly measured in B. subtilis using a metabolomics approach. Thus, the majority of reversible thiol modifications that were previously detected by two-dimensional gel fluorescence-based thiol modification assay are most likely based on S thiolations. Finally, we found that a glutathione-producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show enhanced oxidative stress resistance compared to the wild type.

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Figures

FIG. 1.
FIG. 1.
Monitoring of S thiolations in S. aureus UAMS-1. In brief, protein synthesis was inhibited and subsequently [35S]Cys was supplied. Proteins of exponentially grown S. aureus cells (control [ctrl]) and those exposed to 2 mM diamide were isolated, and the bound radioactivity was measured (gray bars). To prove reversible S thiolations, parallel aliquots of the protein extracts were reduced prior to radioactivity measurement (white bars).
FIG. 2.
FIG. 2.
Effect of diamide on intra- and intermolecular disulfide bond formation in B. subtilis 168. Gel images of protein extracts separated by diagonal gel electrophoresis are shown. Protein extracts of exponentially grown B. subtilis cells harvested before (A and C) or after (B and D) exposure to diamide were separated by nonreducing and subsequent reducing SDS-PAGE. Afterwards, proteins were stained with SYPRO Ruby (A and B). In a modified approach, protein thiols were labeled with BODIPY FL C1-IA instead of IAM (C and D) prior to the second separation and the fluorescence pattern was scanned. Underlined proteins were also identified as reversibly oxidized by Hochgräfe et al. (32). ctrl, control.
FIG. 3.
FIG. 3.
Effect of diamide on intra- and intermolecular disulfide bond formation of S. aureus UAMS-1. Gel images of protein extracts separated by diagonal gel electrophoresis are shown. Protein extracts from exponentially grown S. aureus cells (A and C) and those exposed to diamide (B and D) were separated by nonreducing and subsequent reducing SDS-PAGE. Afterwards, proteins were stained with SYPRO Ruby (A and B). In a modified approach, protein thiols were labeled with BODIPY FL C1-IA instead of IAM (C and D) prior to the second separation and the fluorescence pattern was scanned. DeoD*, purine-nucleoside phosphorylase, a DeoD homologue. SAR2275† contains 0 cysteines and was also detected at the same position in a reducing/reducing diagonal gel electrophoresis. Underlined proteins were also identified as reversibly oxidized by Wolf et al. (64). ctrl, control.
FIG. 4.
FIG. 4.
Global metabolomic changes in the cellular amounts of different metabolites of B. subtilis after exposure to diamide. Cytoplasmic extracts of exponentially grown B. subtilis cells (0 min) and those exposed to 1 mM diamide (30, 60, and 120 min) were measured by GC-MS or LC-MS as described in Materials and Methods. Control values are set to 1, and ratios of the diamide samples are shown at their sample points, except for ppGpp. The pathways of biosynthesis of cysteine (A) and branched-chain amino acids (B) are depicted with metabolites as dots and enzyme reactions as lines.
FIG. 5.
FIG. 5.
Growth curves and survival ratios of B. subtilis gshFLm strain cells exposed to diamide. (A) Growth curve of B. subtilis gshFLm strain without (empty squares) and with (filled squares) 40 μM IPTG. Ctrl, control. (B) The MetE region is shown as section of the dual-channel images of protein extracts stained with SYPRO Ruby from the noninduced (green) and IPTG-induced B. subtilis gshFLm strain (red). (C) Growth curves (lines) and survival ratios (bars) of the B. subtilis gshFLm strain exposed to 1 mM diamide with (filled triangles and bars) or without (empty triangles and bars) 40 μM IPTG. As a control, B. subtilis gshFLm strain cells without IPTG were grown (filled squares).
FIG. 6.
FIG. 6.
Analysis of LMW and protein thiols of the B. subtilis gshFLm strain. LMW thiols were separated as their bimane derivatives of the IPTG-induced (B) or noninduced (D) B. subtilis gshFLm strain cells. (The gshFLm strain is indicated as “+gshF” on the figure.) For control reasons, NEM derivatives of equally treated cells were separated (C and E). Peaks were compared with a chromatogram of a standard mixture of thiols (A) containing cysteine (cys), penicillamine (pen [internal standard in all samples]), GSH, γ-glutamyl-cysteine (γ-GC), N-acetylcysteine (NAC), sulfide (H2S) and CoA. The concentrations of different thiols of the samples treated with mBBr were calculated using dilutions of the standard mixture (F) of noninduced (gray bars) and induced (white bars) cultures. LMW thiols (G) were extracted from noninduced (−IPTG) and induced (+IPTG) B. subtilis gshFLm strain cells before (gray bars) and after (white bars) exposure to diamide. LMW thiols were quantified by DTNB. The remaining cell pellets of noninduced (−IPTG) and induced (+IPTG) B. subtilis gshFLm strain cells before (gray bars) and after (white bars) exposure to diamide were resuspended in denaturing buffer, and (H) protein thiols were quantified using DTNB. (I) The ratio of GSH (gray bars) and GSSG (white bars) was analyzed in induced B. subtilis gshFLm strain cells under control (ctrl) and disulfide stress (diamide) conditions.
FIG. 7.
FIG. 7.
Monitoring of S thiolations in the B.subtilis gshFLm strain. In brief, protein synthesis was inhibited and subsequently [35S]Cys was supplied. Proteins of exponentially grown B. subtilis gshFLm strain cells (control [ctrl]) and those exposed to 1 mM diamide (diamide) without (A) or with (B) 40 μM IPTG were isolated, and the bound radioactivity was measured (gray bars). To prove reversible S thiolations, parallel aliquots of the protein extracts were reduced prior to radioactivity measurement (white bars).
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