Depletion of thiol-containing proteins in response to quinones in Bacillus subtilis.
Quinones are highly toxic naturally occurring thiol-reactive 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 thiol-containing 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 low-molecular-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.
Liebeke
Manuel
M
Institutes of Pharmaceutical Biology, Ernst-Moritz-Arndt-University of Greifswald, F.-L.-Jahn-Str. 15, D-17487 Greifswald, Germany.
Pöther
Dierk-Christoph
DC
van Duy
Nguyen
N
Albrecht
Dirk
D
Becher
Dörte
D
Hochgräfe
Falko
F
Lalk
Michael
M
Hecker
Michael
M
Antelmann
Haike
H
D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D001412:Bacillus subtilis / Q000187:drug effects*; D001426:Bacterial Proteins / Q000378:metabolism*; D015153:Blotting, Western; D003545:Cysteine / Q000378:metabolism; D003593:Cytoplasm / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism; D013058:Mass Spectrometry; D053858:Metabolic Networks and Pathways; D020543:Proteome / Q000032:analysis; D011809:Quinones / Q000494:pharmacology*; D013438:Sulfhydryl Compounds / Q000378:metabolism*
D000900:Anti-Bacterial Agents; D001426:Bacterial Proteins; D020543:Proteome; D011809:Quinones; D013438:Sulfhydryl Compounds; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D003545:Cysteine