Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells.
Oxidative stress causes damage to nucleic acids, membrane lipids and proteins. One striking effect is the metal-catalyzed, site-specific carbonylation of proteins. In the gram-positive soil bacterium Bacillus subtilis, the PerR-dependent specific stress response and the sigmaB-dependent general stress response act together to make cells more resistant to oxidative stress. In this study, we analyzed the carbonylation of cytoplasmic proteins in response to hydrogen peroxide stress in B. subtilis. Furthermore, we asked whether the sigmaB-dependent response to oxidative stress also confers protection against protein carbonylation. To monitor the amount and specificity of protein damage, carbonyls were derivatized with 2,4-dinitrophenylhydrazine, and the resulting stable hydrazones were detected by immunoanalysis of proteins separated by one- or two-dimensional gel electrophoresis. The overall level of protein carbonylation increased strongly in cells treated with hydrogen peroxide. Several proteins, including the elongation factors EF-G, TufA and EF-Ts, were found to be highly carbonylated. Induction of the peroxide specific stress response by treatment with sub-lethal peroxide concentrations, prior to exposure to otherwise lethal levels of peroxide, markedly reduced the degree of protein carbonylation. Cells starved for glucose also showed only minor amounts of peroxide-mediated protein carbonylation compared to exponentially growing cells. We could not detect any differences between wild-type and deltasigB cells starved for glucose or preadapted by heat treatment with respect to the amount or specificity of protein damage incurred upon subsequent exposure to peroxide stress. However, artificial preloading with proteins that are normally induced by sigmaB-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.
Mostertz
J
J
Institut für Mikrobiologie, Ernst-Moritz-Arndt-Universität Greifswald, F.-L.-Jahnstr. 15, 17489 Greifswald, Germany.
Hecker
M
M
D001412:Bacillus subtilis / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D002244:Carbon / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D009154:Mutation; D018384:Oxidative Stress; D012808:Sigma Factor / Q000235:genetics*
D001426:Bacterial Proteins; C079011:SigB protein, Bacteria; D012808:Sigma Factor; D002244:Carbon; D006861:Hydrogen Peroxide