Molecular Genetics and Genomics

, Volume 269, Issue 5, pp 640–648 | Cite as

Patterns of protein carbonylation following oxidative stress in wild-type and sigB Bacillus subtilis cells

  • J. Mostertz
  • M. HeckerEmail author
Original Paper


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 σB-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 σB-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 ΔsigB 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 σB-dependent mechanisms resulted in a lower level of protein carbonylation when cells were later subjected to oxidative stress.


General stress response Oxidative stress Protein carbonylation Cross adaptation  sigB mutant 



We thank Gerhard Mittenhuber and Finn Viehberg for carefully reading the manuscript. Birgit Voigt, Haike Henkel and Björn Maul are gratefully acknowledged for providing plasmids for antibody generation, purified GsiB and anti-GsiB antibody. This work was supported by grants from the Deutsche Forschungsgemeinschaft, the BMBF, the Bildungsministerium Land Mecklenburg-Vorpommern and the Fonds der Chemischen Industrie to M.H.


  1. Almirón M, Link A, Furlong D, Kolter R (1992) A novel DNA binding protein with regulatory and protective roles in starved Escherichia coli cells. Genes Dev 6:2646–2654PubMedGoogle Scholar
  2. Antelmann H, Engelmann S, Schmid R, Hecker M (1996) General and oxidative stress responses in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon. J Bacteriol 178:6571–6578PubMedGoogle Scholar
  3. Antelmann H, Engelmann S, Schmid R, Sorokin A, Lapidus A, Hecker M (1997) Expression of a stress- and starvation-induced dps/pexB -homologous gene is controlled by the alternative sigma factor σB in Bacillus subtilis. J Bacteriol 179:7251–7256.PubMedGoogle Scholar
  4. Berlett BS, Stadtman ER (1997) Protein oxidation in aging, disease and oxidative stress. J Biol Chem 272:20313–20316PubMedGoogle Scholar
  5. Bernhardt J, Völker U, Völker A, Antelmann H, Schmid R, Mach H, Hecker M (1997) Specific and general stress proteins in Bacillus subtilis --a two-dimensional protein electrophoresis study. Microbiology 143:999–1017PubMedGoogle Scholar
  6. Bol DK, Yasbin RE (1990) Characterization of an inducible oxidative stress system in Bacillus subtilis. J Bacteriol 172:3503–3506PubMedGoogle Scholar
  7. Boylan SA, Redfield AR, Brody MS, Price CW (1993) Stress-induced activation of the σB transcription factor of Bacillus subtilis. J Bacteriol 175:7931–7937PubMedGoogle Scholar
  8. Bsat N, Chen L, Helmann JD (1996) Mutation of the Bacillus subtilis alkyl hydroperoxide reductase ( ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes. J Bacteriol 178:6579–6586PubMedGoogle Scholar
  9. Bsat N, Herbig A, Casillas-Martinez L, Setlow P, Helmann JD (1998) Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 29:189–198CrossRefPubMedGoogle Scholar
  10. Büttner K, Bernhardt J, Scharf C, Schmid R, Mader U, Eymann C, Antelmann H, Völker A, Völker U, Hecker M (2001) A comprehensive two-dimensional map of cytosolic proteins of Bacillus subtilis. Electrophoresis 22:2908–2935CrossRefPubMedGoogle Scholar
  11. Chen L, Keramati L, Helmann JD (1995) Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions. Proc Natl Acad Sci USA 92:8190–8194PubMedGoogle Scholar
  12. Davies KJA (1987) Protein damage and degradation by oxygen radicals. I. General aspects. J Biol Chem 262:9895–9901PubMedGoogle Scholar
  13. Dowds BC, Murphy P, McConnell DJ, Devine KM (1987) Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis. J Bacteriol 169:5771–5775PubMedGoogle Scholar
  14. Dukan S, Nyström T (1998) Bacterial senescence: stasis results in increased and differential oxidation of cytoplasmic proteins leading to developmental induction of the heat shock regulon. Genes Dev 12:3431–3441Google Scholar
  15. Dukan S, Nyström T (1999) Oxidative stress defense and deterioration of growth-arrested Escherichia coli cells. J Biol Chem 274:26027–26032PubMedGoogle Scholar
  16. Engelmann S, Hecker M (1996) Impaired oxidative stress resistance of Bacillus subtilis sigB mutants and the role of katA and katE. FEMS Microbiol Lett 145:63–69CrossRefPubMedGoogle Scholar
  17. Engelmann S, Lindner C, Hecker M (1995) Cloning, nucleotide sequence, and regulation of katE encoding a σB-dependent catalase in Bacillus subtilis. J Bacteriol 177:5598–5605PubMedGoogle Scholar
  18. Farr SB, Kogoma T (1991) Oxidative stress responses in Escherichia coli and Salmonella typhimurium. Microbiol Rev 55:561–585PubMedGoogle Scholar
  19. Fuangthong M, Atichartpongkul S, Mongkolsuk S, Helmann JD (2001) OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant in Bacillus subtilis. J Bacteriol 183:4134–4141PubMedGoogle Scholar
  20. Hecker M, Völker U (1998) Non-specific, general and multiple stress resistance of growth-restricted Bacillus subtilis cells by the expression of the σB regulon. Mol Microbiol 29:1129–1136CrossRefPubMedGoogle Scholar
  21. Hecker M, Völker U (2001) General stress response of Bacillus subtilis and other bacteria. Adv Microb Physiol 44:35–91PubMedGoogle Scholar
  22. Igo M, Lampe M, Ray C, Schafer W, Moran CP Jr, Losick R (1987) Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis. J Bacteriol 169:3464–3469PubMedGoogle Scholar
  23. Imlay JA, Fridovich I (1991) Assay of metabolic superoxide production in Escherichia coli. J. Biol. Chem. 266:6957–6965Google Scholar
  24. Leichert LIO, Scharf C, Hecker M (2003) Global characterization of disulfide stress in Bacillus subtilis. J Bacteriol 185:1967–1975CrossRefPubMedGoogle Scholar
  25. Levine RL (1983a) Oxidative modification of glutamine synthetase. I. Inactivation is due to loss of one histidine residue. J Biol Chem 258:11823–11827PubMedGoogle Scholar
  26. Levine RL (1983b) Oxidative modification of glutamine synthetase. II. Characterization of the ascorbate model system. J Biol Chem 258:11828–11833PubMedGoogle Scholar
  27. Levine RL, Williams JA, Stadtman ER, Shacter E (1994) Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233:346–357PubMedGoogle Scholar
  28. Murphy P, Dowds BC, McConnell DJ, Devine KM (1987) Oxidative stress and growth temperature in Bacillus subtilis. J Bacteriol 169:5766–5770PubMedGoogle Scholar
  29. Petersohn A, Brigulla M, Haas S, Hoheisel JD, Völker U, Hecker M (2001) Global analysis of the general stress response of Bacillus subtilis. J Bacteriol 183:5617–5631CrossRefPubMedGoogle Scholar
  30. Price CW (2000) Protective function and regulation of the general stress response in B. subtilis and related Gram-positive bacteria. In: Storz G, Hengge-Aronis R (eds) Bacterial stress responses. ASM Press, Washington D.C., pp 179–198Google Scholar
  31. Price CW (2002) General stress response. In: Sonenshein AL, Hoch JA, Losick R (eds) Bacillus subtilis and its closest relatives (from genes to cells). ASM Press, Washington, D.C., pp 369–384Google Scholar
  32. Reinheckel T, Korn S, Mohring S, Augustin W, Halangk W, Schild L (2000) Adaptation of protein carbonyl detection to the requirements of proteome analysis demonstrated for hypoxia/reoxygenation in isolated rat liver mitochondria. Arch Biochem Biophys 376:59–65CrossRefPubMedGoogle Scholar
  33. Rivett AJ (1986) Regulation of intracellular protein turnover: covalent modification as a mechanism of marking proteins for degradation. Curr Top Cell Regul 28:291–PubMedGoogle Scholar
  34. Shacter E, Williams JA, Lim M, Levine RL (1994) Differential susceptibility of plasma proteins to oxidative modification: examination by Western blot immunoassay. Free Radic Biol Med 17:429–437CrossRefPubMedGoogle Scholar
  35. Stadtman ER (1993) Oxidation of free amino acids and amino acid residues in proteins by radiolysis and by metal-catalyzed reactions. Annu Rev Biochem 62:797–821PubMedGoogle Scholar
  36. Stülke J, Hanschke R, Hecker M (1993) Temporal activation of betaglucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol 139:2041–2045PubMedGoogle Scholar
  37. Völker U, Engelmann S, Maul B, Riethdorf S, Völker A, Schmid R, Mach H, Hecker M (1994) Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology 140:741–752PubMedGoogle Scholar
  38. Völker U, Dufour A, Haldenwang WG (1995) The Bacillus subtilis rsbU gene product is necessary for RsbX-dependent regulation of σB. J Bacteriol 177:114–122PubMedGoogle Scholar
  39. Völker U, Maul B, Hecker M (1999) Expression of the σB-dependent general stress regulon confers multiple stress resistance in Bacillus subtilis. J Bacteriol 181:3942–3948PubMedGoogle Scholar
  40. Von Ossowski I, Melvey MR, Leco PA, Borys A, Loewen PC (1991) Nucleotide sequence of Escherichia coli katE, which encodes catalase HPII. J Bacteriol 173:514–520PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  1. 1.Institut für MikrobiologieErnst-Moritz-Arndt-Universität GreifswaldGreifswaldGermany

Personalised recommendations