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. 1996 Nov;178(22):6571–6578. doi: 10.1128/jb.178.22.6571-6578.1996

General and oxidative stress responses in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon.

H Antelmann 1, S Engelmann 1, R Schmid 1, M Hecker 1
PMCID: PMC178544  PMID: 8932314

Abstract

The AhpC subunit of the Bacillus subtilis alkyl hydroperoxide reductase was identified as a general stress protein induced in response to heat or salt stress or after entry of the organism into the stationary phase. The ahp operon, encoding the two subunits AhpC and AhpF, was cloned and localized between the gntRKPZ operon and the bglA locus. Two-dimensional gel analyses revealed an especially strong induction of AhpC and AhpF in cells subjected to oxidative stress. Transcriptional studies showed a 3- to 4-fold induction of ahp mRNA after heat or salt stress or starvation for glucose and a 20-fold induction by oxidative stress, thus confirming the protein induction data for AhpC and AhpF. Stress induction occurred at a sigmaA-dependent promoter that overlaps with operator sites similar to the per box. Compared with the wild type, the ahpC mutant was resistant to hydrogen peroxide because of the derepression of the peroxide regulon (N. Bsat, L. Chen, and J. D. Helmann, J. Bacteriol. 178:6579-6586, 1996) but more sensitive to cumene hydroperoxide (CHP) during exponential growth. In contrast, stationary-phase wild-type and ahpC mutant cells displayed complete resistance to treatment with 1 mM CHP. Moreover, a sigmaB mutant was found to be extremely sensitive to CHP during vegetative growth and in stationary phase, which indicates that sigmaB-dependent general stress proteins are involved in the protection of cells against oxidative stress.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Anagnostopoulos C., Spizizen J. REQUIREMENTS FOR TRANSFORMATION IN BACILLUS SUBTILIS. J Bacteriol. 1961 May;81(5):741–746. doi: 10.1128/jb.81.5.741-746.1961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Antelmann H., Bernhardt J., Schmid R., Hecker M. A gene at 333 degrees on the Bacillus subtilis chromosome encodes the newly identified sigma B-dependent general stress protein GspA. J Bacteriol. 1995 Jun;177(12):3540–3545. doi: 10.1128/jb.177.12.3540-3545.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Armstrong-Buisseret L., Cole M. B., Stewart G. S. A homologue to the Escherichia coli alkyl hydroperoxide reductase AhpC is induced by osmotic upshock in Staphylococcus aureus. Microbiology. 1995 Jul;141(Pt 7):1655–1661. doi: 10.1099/13500872-141-7-1655. [DOI] [PubMed] [Google Scholar]
  5. Binnie C., Lampe M., Losick R. Gene encoding the sigma 37 species of RNA polymerase sigma factor from Bacillus subtilis. Proc Natl Acad Sci U S A. 1986 Aug;83(16):5943–5947. doi: 10.1073/pnas.83.16.5943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bol D. K., Yasbin R. E. The isolation, cloning and identification of a vegetative catalase gene from Bacillus subtilis. Gene. 1991 Dec 20;109(1):31–37. doi: 10.1016/0378-1119(91)90585-y. [DOI] [PubMed] [Google Scholar]
  7. Boylan S. A., Redfield A. R., Brody M. S., Price C. W. Stress-induced activation of the sigma B transcription factor of Bacillus subtilis. J Bacteriol. 1993 Dec;175(24):7931–7937. doi: 10.1128/jb.175.24.7931-7937.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Boylan S. A., Redfield A. R., Price C. W. Transcription factor sigma B of Bacillus subtilis controls a large stationary-phase regulon. J Bacteriol. 1993 Jul;175(13):3957–3963. doi: 10.1128/jb.175.13.3957-3963.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Bsat N., Chen L., Helmann J. D. Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes. J Bacteriol. 1996 Nov;178(22):6579–6586. doi: 10.1128/jb.178.22.6579-6586.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chae H. Z., Chung S. J., Rhee S. G. Thioredoxin-dependent peroxide reductase from yeast. J Biol Chem. 1994 Nov 4;269(44):27670–27678. [PubMed] [Google Scholar]
  11. Chae H. Z., Robison K., Poole L. B., Church G., Storz G., Rhee S. G. Cloning and sequencing of thiol-specific antioxidant from mammalian brain: alkyl hydroperoxide reductase and thiol-specific antioxidant define a large family of antioxidant enzymes. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7017–7021. doi: 10.1073/pnas.91.15.7017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chae H. Z., Uhm T. B., Rhee S. G. Dimerization of thiol-specific antioxidant and the essential role of cysteine 47. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):7022–7026. doi: 10.1073/pnas.91.15.7022. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Chen L., Helmann J. D. Bacillus subtilis MrgA is a Dps(PexB) homologue: evidence for metalloregulation of an oxidative-stress gene. Mol Microbiol. 1995 Oct;18(2):295–300. doi: 10.1111/j.1365-2958.1995.mmi_18020295.x. [DOI] [PubMed] [Google Scholar]
  14. Chen L., James L. P., Helmann J. D. Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions. J Bacteriol. 1993 Sep;175(17):5428–5437. doi: 10.1128/jb.175.17.5428-5437.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Chen L., Keramati L., Helmann J. D. Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions. Proc Natl Acad Sci U S A. 1995 Aug 29;92(18):8190–8194. doi: 10.1073/pnas.92.18.8190. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Deuerling E., Paeslack B., Schumann W. The ftsH gene of Bacillus subtilis is transiently induced after osmotic and temperature upshift. J Bacteriol. 1995 Jul;177(14):4105–4112. doi: 10.1128/jb.177.14.4105-4112.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Dowds B. C., Murphy P., McConnell D. J., Devine K. M. Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis. J Bacteriol. 1987 Dec;169(12):5771–5775. doi: 10.1128/jb.169.12.5771-5775.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Dowds B. C. The oxidative stress response in Bacillus subtilis. FEMS Microbiol Lett. 1994 Dec 15;124(3):255–263. doi: 10.1111/j.1574-6968.1994.tb07294.x. [DOI] [PubMed] [Google Scholar]
  19. Duncan M. L., Kalman S. S., Thomas S. M., Price C. W. Gene encoding the 37,000-dalton minor sigma factor of Bacillus subtilis RNA polymerase: isolation, nucleotide sequence, chromosomal locus, and cryptic function. J Bacteriol. 1987 Feb;169(2):771–778. doi: 10.1128/jb.169.2.771-778.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Engelmann S., Lindner C., Hecker M. Cloning, nucleotide sequence, and regulation of katE encoding a sigma B-dependent catalase in Bacillus subtilis. J Bacteriol. 1995 Oct;177(19):5598–5605. doi: 10.1128/jb.177.19.5598-5605.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ferrari F. A., Nguyen A., Lang D., Hoch J. A. Construction and properties of an integrable plasmid for Bacillus subtilis. J Bacteriol. 1983 Jun;154(3):1513–1515. doi: 10.1128/jb.154.3.1513-1515.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Fujita Y., Fujita T., Miwa Y., Nihashi J., Aratani Y. Organization and transcription of the gluconate operon, gnt, of Bacillus subtilis. J Biol Chem. 1986 Oct 15;261(29):13744–13753. [PubMed] [Google Scholar]
  23. Hartford O. M., Dowds B. C. Isolation and characterization of a hydrogen peroxide resistant mutant of Bacillus subtilis. Microbiology. 1994 Feb;140(Pt 2):297–304. doi: 10.1099/13500872-140-2-297. [DOI] [PubMed] [Google Scholar]
  24. Hecker M., Heim C., Völker U., Wölfel L. Induction of stress proteins by sodium chloride treatment in Bacillus subtilis. Arch Microbiol. 1988;150(6):564–566. doi: 10.1007/BF00408250. [DOI] [PubMed] [Google Scholar]
  25. Hecker M., Schumann W., Völker U. Heat-shock and general stress response in Bacillus subtilis. Mol Microbiol. 1996 Feb;19(3):417–428. doi: 10.1046/j.1365-2958.1996.396932.x. [DOI] [PubMed] [Google Scholar]
  26. Hoch J. A. Genetic analysis in Bacillus subtilis. Methods Enzymol. 1991;204:305–320. doi: 10.1016/0076-6879(91)04015-g. [DOI] [PubMed] [Google Scholar]
  27. Igo M., Lampe M., Ray C., Schafer W., Moran C. P., Jr, Losick R. Genetic studies of a secondary RNA polymerase sigma factor in Bacillus subtilis. J Bacteriol. 1987 Aug;169(8):3464–3469. doi: 10.1128/jb.169.8.3464-3469.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jacobson F. S., Morgan R. W., Christman M. F., Ames B. N. An alkyl hydroperoxide reductase from Salmonella typhimurium involved in the defense of DNA against oxidative damage. Purification and properties. J Biol Chem. 1989 Jan 25;264(3):1488–1496. [PubMed] [Google Scholar]
  29. Krüger E., Msadek T., Hecker M. Alternate promoters direct stress-induced transcription of the Bacillus subtilis clpC operon. Mol Microbiol. 1996 May;20(4):713–723. doi: 10.1111/j.1365-2958.1996.tb02511.x. [DOI] [PubMed] [Google Scholar]
  30. Krüger E., Völker U., Hecker M. Stress induction of clpC in Bacillus subtilis and its involvement in stress tolerance. J Bacteriol. 1994 Jun;176(11):3360–3367. doi: 10.1128/jb.176.11.3360-3367.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Li M., Wong S. L. Cloning and characterization of the groESL operon from Bacillus subtilis. J Bacteriol. 1992 Jun;174(12):3981–3992. doi: 10.1128/jb.174.12.3981-3992.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Loewen P. C., Hengge-Aronis R. The role of the sigma factor sigma S (KatF) in bacterial global regulation. Annu Rev Microbiol. 1994;48:53–80. doi: 10.1146/annurev.mi.48.100194.000413. [DOI] [PubMed] [Google Scholar]
  33. Majumdar D., Avissar Y. J., Wyche J. H. Simultaneous and rapid isolation of bacterial and eukaryotic DNA and RNA: a new approach for isolating DNA. Biotechniques. 1991 Jul;11(1):94–101. [PubMed] [Google Scholar]
  34. Mathieu I., Meyer J., Moulis J. M. Cloning, sequencing and expression in Escherichia coli of the rubredoxin gene from Clostridium pasteurianum. Biochem J. 1992 Jul 1;285(Pt 1):255–262. doi: 10.1042/bj2850255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Maul B., Völker U., Riethdorf S., Engelmann S., Hecker M. sigma B-dependent regulation of gsiB in response to multiple stimuli in Bacillus subtilis. Mol Gen Genet. 1995 Jul 22;248(1):114–120. doi: 10.1007/BF02456620. [DOI] [PubMed] [Google Scholar]
  36. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Murphy P., Dowds B. C., McConnell D. J., Devine K. M. Oxidative stress and growth temperature in Bacillus subtilis. J Bacteriol. 1987 Dec;169(12):5766–5770. doi: 10.1128/jb.169.12.5766-5770.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Niimura Y., Ohnishi K., Yarita Y., Hidaka M., Masaki H., Uchimura T., Suzuki H., Kozaki M., Uozumi T. A flavoprotein functional as NADH oxidase from Amphibacillus xylanus Ep01: purification and characterization of the enzyme and structural analysis of its gene. J Bacteriol. 1993 Dec;175(24):7945–7950. doi: 10.1128/jb.175.24.7945-7950.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Petricek M., Rutberg L., Schröder I., Hederstedt L. Cloning and characterization of the hemA region of the Bacillus subtilis chromosome. J Bacteriol. 1990 May;172(5):2250–2258. doi: 10.1128/jb.172.5.2250-2258.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Riethdorf S., Völker U., Gerth U., Winkler A., Engelmann S., Hecker M. Cloning, nucleotide sequence, and expression of the Bacillus subtilis lon gene. J Bacteriol. 1994 Nov;176(21):6518–6527. doi: 10.1128/jb.176.21.6518-6527.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Schmidt A., Schiesswohl M., Völker U., Hecker M., Schumann W. Cloning, sequencing, mapping, and transcriptional analysis of the groESL operon from Bacillus subtilis. J Bacteriol. 1992 Jun;174(12):3993–3999. doi: 10.1128/jb.174.12.3993-3999.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Smith I., Paress P., Cabane K., Dubnau E. Genetics and physiology of the rel system of Bacillus subtilis. Mol Gen Genet. 1980;178(2):271–279. doi: 10.1007/BF00270472. [DOI] [PubMed] [Google Scholar]
  44. Storz G., Jacobson F. S., Tartaglia L. A., Morgan R. W., Silveira L. A., Ames B. N. An alkyl hydroperoxide reductase induced by oxidative stress in Salmonella typhimurium and Escherichia coli: genetic characterization and cloning of ahp. J Bacteriol. 1989 Apr;171(4):2049–2055. doi: 10.1128/jb.171.4.2049-2055.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Stülke J., Hanschke R., Hecker M. Temporal activation of beta-glucanase synthesis in Bacillus subtilis is mediated by the GTP pool. J Gen Microbiol. 1993 Sep;139(9):2041–2045. doi: 10.1099/00221287-139-9-2041. [DOI] [PubMed] [Google Scholar]
  46. Tai S. S., Zhu Y. Y. Cloning of a Corynebacterium diphtheriae iron-repressible gene that shares sequence homology with the AhpC subunit of alkyl hydroperoxide reductase of Salmonella typhimurium. J Bacteriol. 1995 Jun;177(12):3512–3517. doi: 10.1128/jb.177.12.3512-3517.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Tartaglia L. A., Storz G., Brodsky M. H., Lai A., Ames B. N. Alkyl hydroperoxide reductase from Salmonella typhimurium. Sequence and homology to thioredoxin reductase and other flavoprotein disulfide oxidoreductases. J Biol Chem. 1990 Jun 25;265(18):10535–10540. [PubMed] [Google Scholar]
  48. Völker U., Engelmann S., Maul B., Riethdorf S., Völker A., Schmid R., Mach H., Hecker M. Analysis of the induction of general stress proteins of Bacillus subtilis. Microbiology. 1994 Apr;140(Pt 4):741–752. doi: 10.1099/00221287-140-4-741. [DOI] [PubMed] [Google Scholar]
  49. Wada K., Wada Y., Doi H., Ishibashi F., Gojobori T., Ikemura T. Codon usage tabulated from the GenBank genetic sequence data. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):1981–1986. doi: 10.1093/nar/19.suppl.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Wetzstein M., Völker U., Dedio J., Löbau S., Zuber U., Schiesswohl M., Herget C., Hecker M., Schumann W. Cloning, sequencing, and molecular analysis of the dnaK locus from Bacillus subtilis. J Bacteriol. 1992 May;174(10):3300–3310. doi: 10.1128/jb.174.10.3300-3310.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Xu X. M., Koyama N., Cui M., Yamagishi A., Nosoh Y., Oshima T. Nucleotide sequence of the gene encoding NADH dehydrogenase from an alkalophile, Bacillus sp. strain YN-1. J Biochem. 1991 May;109(5):678–683. doi: 10.1093/oxfordjournals.jbchem.a123440. [DOI] [PubMed] [Google Scholar]
  52. Yuan G., Wong S. L. Isolation and characterization of Bacillus subtilis groE regulatory mutants: evidence for orf39 in the dnaK operon as a repressor gene in regulating the expression of both groE and dnaK. J Bacteriol. 1995 Nov;177(22):6462–6468. doi: 10.1128/jb.177.22.6462-6468.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yuan G., Wong S. L. Regulation of groE expression in Bacillus subtilis: the involvement of the sigma A-like promoter and the roles of the inverted repeat sequence (CIRCE). J Bacteriol. 1995 Oct;177(19):5427–5433. doi: 10.1128/jb.177.19.5427-5433.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Zhang J., Aronson A. A Bacillus subtilis bglA gene encoding phospho-beta-glucosidase is inducible and closely linked to a NADH dehydrogenase-encoding gene. Gene. 1994 Mar 11;140(1):85–90. doi: 10.1016/0378-1119(94)90735-8. [DOI] [PubMed] [Google Scholar]
  55. Zuber U., Schumann W. CIRCE, a novel heat shock element involved in regulation of heat shock operon dnaK of Bacillus subtilis. J Bacteriol. 1994 Mar;176(5):1359–1363. doi: 10.1128/jb.176.5.1359-1363.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]

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