YlaC is an Extracytoplasmic Function (ECF) Sigma Factor Contributing to Hydrogen Peroxide Resistance in Bacillus subtilis

  • Ryu Han-Bong (School of Biological Sciences, and Institute of Microbiology, Seoul National University) ;
  • Shin In-Ji (School of Biological Sciences, and Institute of Microbiology, Seoul National University) ;
  • Yim Hyung-Soon (School of Biological Sciences, and Institute of Microbiology, Seoul National University) ;
  • Kang Sa-Ouk (School of Biological Sciences, and Institute of Microbiology, Seoul National University)
  • Published : 2006.04.01

Abstract

In this study, we have attempted to characterize the functions of YlaC and YlaD encoded by ylaC and ylaD genes in Bacillus subtilis. The GUS reporter gene, driven by the yla operon promoter, was expressed primarily during the late exponential and early stationary phase, and its expression increased as the result of hydrogen peroxide treatment. Northern and Western blot analyses revealed that the level of ylaC transcripts and YlaC increased as the result of challenge with hydrogen peroxide. A YlaC-overexpressing strain evidenced hydrogen peroxide resistance and a three-fold higher peroxidase activity as compared with a deletion mutant. YlaC-overexpressing and YlaD-disrupted strains evidenced higher sporulation rates than were observed in the YlaC-disrupted and YlaD-overexpressing strains. Analyses of the results of native polyacrylamide gel electrophoresis of recombinant YlaC and YlaD indicated that interaction between YlaC and YlaD was regulated by the redox state of YlaD in vitro. Collectively, the results of this study appear to suggest that YlaC regulated by the YlaD redox state, contribute to oxidative stress resistance in B. subtilis.

Keywords

References

  1. Antelmann, H., S. Engelmann, R. Schmid, and M. Hecker. 1996. General and oxidative stress response in Bacillus subtilis: cloning, expression, and mutation of the alkyl hydroperoxide reductase operon. J. Bacteriol. 178, 6571- 6578 https://doi.org/10.1128/jb.178.22.6571-6578.1996
  2. Bae, J.B., J.H. Park, M.Y. Hahn, M.S. Kim, and J.H. Roe. 2004. Redox-dependent changes in RsrA, an anti-sigma factor in Streptomyces coelicolor: zinc release and disulfide bond formation. J. Mol. Biol. 335, 425-435 https://doi.org/10.1016/j.jmb.2003.10.065
  3. Bol, D.K. and R.E. Yasbin. 1990. Characterization of an inducible oxidative stress system in Bacillus subtilis. J. Bacteriol. 172, 3503-3506 https://doi.org/10.1128/jb.172.6.3503-3506.1990
  4. Braun, V. 1997. Surface signaling: novel transcription initiation mechanism starting from the cell surface. Arch. Microbiol. 167, 325-331 https://doi.org/10.1007/s002030050451
  5. Brown, K.L. and K.T. Hughes. 1995. The role of anti-sigma factors in gene regulation. Mol. Microbiol. 16, 397-404 https://doi.org/10.1111/j.1365-2958.1995.tb02405.x
  6. Bsat, N., L. Chen, and J.D. Helmann. 1996. Mutation of the Bacillus subtilis alkyl hydroperoxide reductase (ahpCF) operon reveals compensatory interactions among hydrogen peroxide stress genes. J. Bacteriol. 178, 6579-6586 https://doi.org/10.1128/jb.178.22.6579-6586.1996
  7. Cole, S.T., R. Brosch, J. Parkhill, T. Garnier, C. Churcher, D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, A. Krogh, J. Mclean, S. Moule, L. Murphy, K. Oliver, J. Osborne, M. A. Quail, M.A. Rajandream, J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares, S. Squares, J. E. Sulston, K. Taylor, S. Whitehead, and B. G. Barrell. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537-544 https://doi.org/10.1038/31159
  8. Chen, L. and J.D. Helmann. 1995. Bacillus subtilis MrgA is a Dps (PexB) homologue: evidence for metalloregulation of an oxidative-stress gene. Mol. Microbiol. 18, 295-300 https://doi.org/10.1111/j.1365-2958.1995.mmi_18020295.x
  9. Chen, L., L. Keramati, and J.D. Helmann. 1995. Coordinate regulation of Bacillus subtilis peroxide stress genes by hydrogen peroxide and metal ions. Proc. Natl. Acad. Sci. USA 92, 8190-8194
  10. Dowds, B.C.A., P. Murphy, D.J. McConnell, and K.M. Devine. 1987. Relationship among oxidative stress, growth cycle, and sporulation in Bacillus subtilis. J. Bacteriol. 169, 5771-5775 https://doi.org/10.1128/jb.169.12.5771-5775.1987
  11. Dowds, B.C.A. 1994. The oxidative stress response in Bacillus subtilis. FEMS Microbiol. Lett. 124, 255-263 https://doi.org/10.1111/j.1574-6968.1994.tb07294.x
  12. Dubnau, D. and R. Davidoff-Abelson. 1971. Fate of transforming DNA following uptake by competent Bacillus subtilis, I. Formation and properties of the donor-recipient complex. J. Mol. Biol. 56, 209-221 https://doi.org/10.1016/0022-2836(71)90460-8
  13. Gorham, H.C., S.J. McGowan, P.R.H. Robson, and D.A. Hodgson. 1996. Light-induced carotenogenesis in Myxococcus xanthus: light-dependent membrane sequestration of ECF sigma factor CarQ by anti-sigma factor CarR. Mol. Microbiol. 19, 171-186 https://doi.org/10.1046/j.1365-2958.1996.360888.x
  14. Haldenwang, W.G. 1995. The sigma factor of Bacillus subtilis. Microbiol. Rev. 59, 1-30
  15. Harwood, C.R. and S.M. Cutting. 1990. Molecular Biological Methods for Bacillus
  16. Helmann, J.D. 1999. Anti-sigma factors. Curr. Opin. Microbiol. 2, 135-141 https://doi.org/10.1016/S1369-5274(99)80024-1
  17. Horsburgh, M.J. and A. Moir. 1999. $\sigma^M$, an ECF RNA polymerase sigma factor of Bacillus subtilis 168, is essential for growth and survival in high concentration of salt. Mol. Microbiol. 32, 41-50 https://doi.org/10.1046/j.1365-2958.1999.01323.x
  18. Huang, X., A. Decatur, A. Sorokin, and J.D. Helmann. 1997. The Bacillus subtilis sigma(X) protein is an extracytoplasmic function sigma factor contributing to survival at high-temperature stress. J. Bacteriol. 179, 2915-2921 https://doi.org/10.1128/jb.179.9.2915-2921.1997
  19. Huang, X. and J.D. Helmann. 1998. Identification of target promoters for the Bacillus subtilis sigmaX factor using a consensus-directed search. J. Mol. Biol. 279, 165-173 https://doi.org/10.1006/jmbi.1998.1765
  20. Huang, X., K.L. Fredrick and J.D. Helmann. 1998. Promoter recognition by Bacillus subtilis sigmaW: autoregulation and partial overlap with the sigX regulon. J. Bacteriol. 180, 3765-3770
  21. Kang, J.G., M.S.B. Paget, Y.J. Seok, M.Y. Hahn, J.B. Bae, J.S. Hahn, C. Kleanthous, M.J. Buttner, and J.H. Roe. 1999. RsrA, an anti-sigma factor regulated by redox change. EMBO J. 18, 4292-4298 https://doi.org/10.1093/emboj/18.15.4292
  22. Karow, M.L. and P.J. Piggot. 1995. Construction of gusA transcriptional fusion vectors for Bacillus subtilis and their utilization for studies of spore formation. Gene 163, 69-74 https://doi.org/10.1016/0378-1119(95)00402-R
  23. Kenney, T.J. and C.P. Jr. Moran. 1987. Organization and regulation of an operon that encodes a sporulationessential sigma factor in Bacillus subtilis. J. Bacteriol. 169, 3329-3339 https://doi.org/10.1128/jb.169.7.3329-3339.1987
  24. Kunst, F., N. Ogasawara, I. Moszer, A.M. Albertini, G. Alloni, V. Azevedo, M.G. Bertero, P. Bessieres, A. Bolotin, S. Borchert, R. Borriss, L. Boursier, A. Brans, M. Braun, S.C. Brignell, S. Bron, S. Brouillet, C.V. Bruschi, B. Caldwell, V. Capuano, N.M. Carter, S.K. Choi, J.J. Codani, I.F. Connerton, A. Danchin, et al. 1997. The complete genome sequence of the Gram-positive Bacillus subtilis. Nature 390, 249-256 https://doi.org/10.1038/36786
  25. Liesegang, H., K. Lemke, R.A. Siddiqui, and H.G. Schlegel. 1993. Characterization of the inducible nickel and cobalt resistance determinant cnr from pMOL28 of Alcaligenes eutrophus CH34. J. Bacteriol. 175, 767-778 https://doi.org/10.1128/jb.175.3.767-778.1993
  26. Li, W., A.R. Bottrill, M.J. Bibb, M.J. Buttner, and C. Kleanthous. 2003. The role of zinc in the disulphide stress-regulated anti-sigma factor RsrA form Steptomyces coelicolor. J. Mol. Biol. 333, 461-472 https://doi.org/10.1016/j.jmb.2003.08.038
  27. Li, W., C.E. Stevenson, N. Burton, P. Jakimowicz, M.S.B. Paget, M.J. Buttner, D.M. Lawson, and C. Kleanthous. 2002. Identification and structure of the anti-sigma factor-binding domain of the disulphide-stress regulated sigma factor sigma(R) from Streptomyces coelicolor. J. Mol. Biol. 323, 225-236 https://doi.org/10.1016/S0022-2836(02)00948-8
  28. Lonetto, M.A., K.L. Brown, K.E. Rudd, and M.J. Buttner. 1994. Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic function. Proc. Natl. Acad. Sci. USA 91, 7573-7577
  29. Matsumoto, T., K. Nakanishi, K. Asai, and Y. Sadaie. 2005. Transcriptional analysis of the ylaABCD operon of Bacillus subtilis encoding a sigma factor of extracytoplasmic function family. Genes Genet. Syst. 80, 385-393 https://doi.org/10.1266/ggs.80.385
  30. Milhaud, P. and G. Balassa. 1973. Biochemical genetics of bacterial sporulation. IV. Sequential development of resistances to chemical and physical agents during sporulation of Bacillus subtilis. Mol. Gen. Genet. 125, 241-250 https://doi.org/10.1007/BF00270746
  31. Missiakas, D. and S. Raina. 1998. The extracytoplasmic function sigma factors: role and regulation. Mol. Microbiol. 28, 1059-1066 https://doi.org/10.1046/j.1365-2958.1998.00865.x
  32. Newman, J.D., J.R. Anthony, and T.J. Donohue. 2001. The importance of zinc-binding to the function of Rhodobacter sphaeroides ChrR as an anti-sigma factor. J. Mol. Biol. 313, 485-499 https://doi.org/10.1006/jmbi.2001.5069
  33. Nunoshiba, T., F. Obata, A.C. Boss, S. Oikawa, T. Mori, S. Kawanishi, and K. Yamamoto. 1999. Role of iron and superoxide for generation of hydroxyl radical, oxidative DNA lesions, and mutagenesis in Escherichia coli. J. Biol. Chem. 274, 34832-34837 https://doi.org/10.1074/jbc.274.49.34832
  34. Paget, M.S.B., J.B. Bae, M.Y. Hahn, W. Li, C. Kleanthous, J.H. Roe, and M.J. Buttner. 2001. Mutational analysis of RsrA, zinc-binding anti-sigma factor with a thioldisulphide redox switch. Mol. Microbiol. 39, 1036-1047 https://doi.org/10.1046/j.1365-2958.2001.02298.x
  35. Paget, M.S.B., J.G. Kang, J.H. Roe, and M.J. Butter. 1998. $\sigma^R$, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2). EMBO J. 17, 5776-5 https://doi.org/10.1093/emboj/17.19.5776
  36. Paget, M.S.B., V. Molle, G. Cohen, Y. Aharonowitz, and M.J. Buttner. 2001. Defining the disulphide stress response in Streptomyces coelicolor A3(2): identification of the $\sigma^R$ regulon. Mol. Microbiol. 42, 1007-1020 https://doi.org/10.1046/j.1365-2958.2001.02675.x
  37. Reinhold Bruckner. 1992. A series of shuttle vectors for Bacillus subtilis and Escherichia coli. Gene 122, 187-192 https://doi.org/10.1016/0378-1119(92)90048-T
  38. Phillips, Z.E.V. and M.A. Strauch. 2002. Bacillus subtilis sporulation and stationary phase gene expression. Cell Mol. Life Sci. 59, 392-402 https://doi.org/10.1007/s00018-002-8431-9
  39. Scharf C., S. Riethdorf, H. Ernst, S. Engelmann, U. Volker, and M. Hecker. 1998. Thioredoxin is an essential protein induced by multiple stresses in Bacillus subtilis. J. Bacteriol. 180, 1869-1877
  40. Shin, I., H.B. Ryu, H.S. Yim, and S.O. Kang. 2005. Cytochrome $c_{550}$ is related to initiation of sporulation in Bacillus subtilis. J. Microbiol. 43, 244-250
  41. Stragier, P. and R. Losick. 1996. Molecular genetics of sporulation in Bacillus subtilis. Annu. Rev. Genet. 30, 297-341 https://doi.org/10.1146/annurev.genet.30.1.297
  42. Van Dessel, W., L. Van Mellaert, N. Geukens, E. Lammertyn, and J. Anne. 2004. Isolation of high quality RNA from Streptomyces. J. Microbiol. Methods 58, 135-137 https://doi.org/10.1016/j.mimet.2004.03.015
  43. Yoshimura, M., K. Asai, Y. Sadaie, and H. Yoshikawa. 2004. Interaction of Bacillus subtilis extracytoplasmic function(ECF) sigma factors with the N-terminal regions of their potential anti-sigma factors. Microbiology 150, 591-599 https://doi.org/10.1099/mic.0.26712-0