JB Accepts, published online ahead of print on 16 October 2009

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J. Bacteriol. doi:10.1128/JB.00937-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Diamide triggers mainly S-thiolations in the cytoplasmic proteome of Bacillus subtilis and Staphylococcus aureus

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

Institute of Microbiology and Institute of Pharmaceutical Biology, Ernst-Moritz-Arndt-University of Greifswald, F.-L.-Jahnstr. 15, D-17487 Greifswald, Germany; Department of Molecular Microbiology and Biotechnology, George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv 69978, Israel

^* To whom correspondence should be addressed. Email: hecker{at}uni-greifswald.de.

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 non-reducing/reducing SDS gel electrophoresis. However, only 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 using the 2D gel fluorescence-based thiol-modification assay (FALKO-Assay) are most likely based on S-thiolations. Finally, we found that a GSH producing B. subtilis strain which expresses the Listeria monocytogenes gshF gene did not show an enhanced oxidative stress resistance compared to the wild type.