Depletion of thiol‐containing proteins in response to quinones in Bacillus subtilis

Abstract

SummaryQuinones are highly toxic naturally occurring thiolreactive compounds. We have previously described novel pathways for quinone detoxification in the Gram-positive bacterium Bacillus subtilis. In this study, we have investigated the extent of irreversible and reversible thiol modifications caused in vivo by electrophilic quinones. Exposure to toxic benzoquinone (BQ) concentrations leads to depletion of numerous Cys-rich cytoplasmic proteins in the proteome of B. subtilis. Mass spectrometry and immunoblot analyses demonstrated that these BQ-depleted proteins represent irreversibly damaged BQ aggregates that escape the two-dimensional gel separation. This enabled us to quantify the depletion of thiolcontaining proteins which are the in vivo targets for thiol-(S)-alkylation by toxic quinone compounds. Metabolomic approaches confirmed that protein depletion is accompanied by depletion of the lowmolecular-weight (LMW) thiol cysteine. Finally, no increased formation of disulphide bonds was detected in the thiol-redox proteome in response to sublethal quinone concentrations. The glyceraldehyde-3-phosphate dehydrogenase (GapA) was identified as the only new target for reversible thiol modifications after exposure to toxic quinones. Together our data show that the thiol-(S)-alkylation reaction with protein and non-protein thiols is the in vivo mechanism for thiol depletion and quinone toxicity in B. subtilis and most likely also in other bacteria.

The Paralogous MarR/DUF24-Family Repressors YodB and CatR Control Expression of the Catechol Dioxygenase CatE inBacillus subtilis

“…As electrophiles, quinones can form S adducts with cellular thiols via the thiol-(S)-alkylation chemistry. Recently, we showed that toxic quinones react mainly via S-alkylation with cellular thiol-containing proteins in vivo, which leads to aggregation and depletion of proteins in the proteome (18). However, we also found the glyceraldehyde-3-phosphate dehydrogenase GapA as a target for reversible thiol oxidation by quinones in vivo.…”

“…The polyclonal antisera were used for immunoprecipitation experiments, and the purified antibodies were used at a dilution of 1:200 in the Western blot experiments. Protein amounts of 25 g were loaded onto a 14% SDS-PAGE gel, and the Western blot analysis was performed as described previously (18).…”

“…Quinones can act as oxidants or electrophiles (13,20,25). As oxidants, quinones redox cycle with their semiquinones, producing reactive oxygen species (ROS), such as superoxide anion or hydrogen peroxide (18). The production of ROS could lead in turn to reversible thiol modifications.…”

Diamide Triggers Mainly S Thiolations in the Cytoplasmic Proteomes of Bacillus subtilis and Staphylococcus aureus

“…Analyses of intracellular metabolites by gas chromatography-mass spectrometry (GC-MS) were performed as described by Liebeke et al (46). Briefly, samples were harvested by fast filtration, and subsequently the metabolism was quenched with an ethanol solution and liquid nitrogen.…”

“…RES include different chemical compounds such as aldehydes, quinones, and the azo compound diamide (2,43,45,46,53,66). Quinones and aldehydes have electron-deficient centers that result in thiol-(S) alkylation of cysteine.…”