Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Three Non-Aspartate Amino Acid Mutations in the ComA Response Regulator Receiver Motif Severely Decrease Surfactin Production, Competence Development, and Spore Formation in Bacillus subtilis

  • Wang, Xiaoyu (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) ;
  • Luo, Chuping (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) ;
  • Liu, Youzhou (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) ;
  • Nie, Yafeng (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) ;
  • Liu, Yongfeng (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) ;
  • Zhang, Rongsheng (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences) ;
  • Chen, Zhiyi (Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences)
  • Published : 2010.02.28
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Abstract

Bacillus subtilis strains produce a broad spectrum of bioactive peptides. The lipopeptide surfactin belongs to one well-known class, which includes amphiphilic membrane-active biosurfactants and peptide antibiotics. Both the srfA promoter and the ComP-ComA signal transduction system are an important part of the factor that results in the production of surfactin. Bs-M49, obtained by means of low-energy ion implantation in wild-type Bs-916, produced significantly lower levels of surfactin, and had no obvious effects against R. solani. Occasionally, we found strain Bs-M49 decreased spore formation and the development of competence. Blast comparison of the sequences from Bs-916 and M49 indicate that there is no difference in the srfA operon promoter PsrfA, but there are differences in the coding sequence of the comA gene. These differences result in three missense mutations within the M49 ComA protein. RT-PCR analyses results showed that the expression levels of selected genes involved in competence and sporulation in both the wild-type Bs-916 and mutant M49 strains were significantly different. When we integrated the comA ORF into the chromosome of M49 at the amyE locus, M49 restored hemolytic activity and antifungal activity. Then, HPLC analyses results also showed the comA-complemented strain had a similar ability to produce surf actin with wild-type strain Bs-916. These data suggested that the mutation of three key amino acids in ComA greatly affected the biological activity of Bacillus subtilis. ComA protein 3D structure prediction and motif search prediction indicated that ComA has two obvious motifs common to response regulator proteins, which are the N-terminal response regulator receiver motif and the C-terminal helix-turn-helix motif. The three residues in the ComA N-terminal portion may be involved in phosphorylation activation mechanism. These structural prediction results implicate that three mutated residues in the ComA protein may play an important role in the formation of a salt-bridge to the phosphoryl group keeping active conformation to subsequent regulation of the expression of downstream genes.

Keywords

References

  1. Ames, S. K., N. Frankema, and L. J. Kenney. 1999. C-Terminal DNA binding stimulates N-terminal phosphorylation of the outer membrane protein regulator OmpR from Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 96: 11792-11797. https://doi.org/10.1073/pnas.96.21.11792
  2. Ansaldi, M., D. Marolt, T. Stebe, I. M. Mulec, and D. Dubnau. 2002. Specific activation of the Bacillus quorum-sensing systems by isoprenylated pheromone variants. Mol. Microbiol. 44: 1561-1573. https://doi.org/10.1046/j.1365-2958.2002.02977.x
  3. Arima, K., A. Kakinuma, and G. Tamura. 1968. Surfactin, a crystalline peptidelipid surfactant produced by Bacillus subtilis: Isolation, characterization and its inhibition of fibrin clot formation. Biochem. Biophys. Res. Commun. 31: 488-494. https://doi.org/10.1016/0006-291X(68)90503-2
  4. Baikalov, I., I. Schroder, M. Kaczor-Grzeskowiak, K. Grzeskowiak, R. P. Gunsalus, and R. E. Dickerson. 1996. Structure of the Escherichia coli response regulator NarL. Biochemistry 35: 11053-11061. https://doi.org/10.1021/bi960919o
  5. Berka, R. M., J. Hahn, M. Albano, I. Draskovic, M. Persuh, X. Cui, A. Sloma, and W. Widner, D. Dubnau. 2002. Microarray analysis of the Bacillus subtilis K-state: Genome-wide expression changes dependent on ComK. Mol. Microbiol. 43: 1331-1345. https://doi.org/10.1046/j.1365-2958.2002.02833.x
  6. Besson, F., F. Peypoux, G. Michel, and L. 1978. Identification of antibiotics of iturin group in various strains of Bacillus subtilis. J. Antibiot. (Tokyo) 31: 284-288. https://doi.org/10.7164/antibiotics.31.284
  7. Birck, C., L. Mourey, P. Gouet, B. Fabry, J. Schumacher, P. Rousseau, D. Kahn, and J. P. Samama. 1999. Conformational changes induced by phosphorylation of the FixJ receiver domain. Structure 7: 1505-1515. https://doi.org/10.1016/S0969-2126(00)88341-0
  8. Bourret, R. B., J. F. Hess, K. A. Borkovich, A. A. Pakula, and M. I. Simon. 1989. Protein phosphorylation in chemotaxis and two-component regulatory systems of bacteria. J. Biol. Chem. 264: 7085-7088.
  9. Chen, Z. and Z. Xu. 2000. Evaluation and utilization of antagonistic bacteria against rice sheath blight. Chinese J. Rice Sci. 14: 98-102.
  10. Cho, H. S., S. Y. Lee, D. Yan, X. Pan, J. S. Parkinson, S. Kustu, D. E. Wemmer, and J. G. Pelton. 2000. NMR structure of activated CheY. J. Mol. Biol. 297: 543-551. https://doi.org/10.1006/jmbi.2000.3595
  11. Cho, H. S., J. G. Pelton, D. Yan, S. Kustu, and D. E. Wemmer. 2001. Phosphoaspartates in bacterial signal transduction. Curr. Opin. Struc. Biol. 11: 679-684. https://doi.org/10.1016/S0959-440X(01)00271-8
  12. Comella, N. and A. D. Grossman. 2005. Conservation of genes and processes controlled by the quorum response in bacteria: Characterization of genes controlled by the quorum-sensing transcription factor ComA in Bacillus subtilis. Mol. Microbiol. 57: 1159-1174. https://doi.org/10.1111/j.1365-2958.2005.04749.x
  13. Core, L. and M. Perego. 2003. TPR-mediated interaction of RapC with ComA inhibits response regulator-DNA binding for competence development in Bacillus subtilis. Mol. Microbiol. 49: 1509-1522. https://doi.org/10.1046/j.1365-2958.2003.03659.x
  14. Cutting, S. M. and C. R. Harwood. 1990. Molecular Biological Methods for Bacillus. John Wiley and Sons, Sussex, England.
  15. Gardino, A. K., B. F. Volkman, H. S. Cho, S. Y. Lee, D. E. Wemmer, and D. Kern. 2003. The NMR solution structure of BeF3-activated Spo0F reveals the conformational switch in a phosphorelay system. J. Mol. Biol. 331: 245-254. https://doi.org/10.1016/S0022-2836(03)00733-2
  16. Gouet, P., B. Fabry, V. Guillet, C. Birck, L. Mourey, D. Kahn, and J. P. Samama. 1999. Structural transitions in the FixJ receiver domain. Structure 7: 1517-1526. https://doi.org/10.1016/S0969-2126(00)88342-2
  17. Grossman, A. D. 1995. Genetic networks controlling the initiation of sporulation and the development of genetic competence in Bacillus subtilis. Annu. Rev. Genet. 29: 477-508. https://doi.org/10.1146/annurev.ge.29.120195.002401
  18. Guerout-Fleury, A.-M., N. Frandsen, and P. Stragier. 1996. Plasmids for ectopic integration in Bacillus subtilis. Gene 180:57-61. https://doi.org/10.1016/S0378-1119(96)00404-0
  19. Guillen, N., Y. Weinrauch, and D. A. Dubnau. 1989. Cloning and characterization of the regulatory Bacillus subtilis competence genes comA and comB. J. Bacteriol. 171: 5354-5361.
  20. Hamoen, L. W., H. Eshuis, J. Jongbloed, G. Venema, and D. van Sinderen. 1995. A small gene, designated comS, located within the coding region of the fourth amino acid activation domain of srfA, is required for competence development in Bacillus subtilis. Mol. Microbiol. 15: 55-64. https://doi.org/10.1111/j.1365-2958.1995.tb02220.x
  21. Hosono, K. and H. Suzuki. 1983. Acylpeptides, the inhibitors of cyclic adenosine 3',5'-monophosphate phosphodiesterase. I. Purification, physicochemical properties and structures of fatty acid residues. J. Antibiot. (Tokyo) 36: 667-673. https://doi.org/10.7164/antibiotics.36.667
  22. Jarmer, H., T. S. Larsen, A. Krogh, H. H. Saxild, S. Brunak, and S. Knudsen. 2001. Sigma A recognition sites in the Bacillus subtilis genome. Microbiology 147: 2417-2424.
  23. Jiang, M., R. Grau, and M. Perego. 2000. Differential processing of propeptide inhibitors of Rap phosphatases in Bacillus subtilis. J. Bacteriol. 182: 303-310. https://doi.org/10.1128/JB.182.2.303-310.2000
  24. Kakinuma, A., M. Hori, M. Isono, G. Tamura and K. Arima. 1969. Determination of amino acid sequence in surfactin, a crystalline peptide lipid surfactant produced by Bacillus subtilis. Agric. Biol. Chem. 33: 971-972. https://doi.org/10.1271/bbb1961.33.971
  25. Kleerebezem, M., L. E. Quadri, O. P. Kuipers, and V. W. de Vos. 1997. Quorum sensing by peptide pheromones and twocomponent signal-transduction systems in Gram-positive bacteria. Mol. Microbiol. 24: 895-904. https://doi.org/10.1046/j.1365-2958.1997.4251782.x
  26. Levitt, M. 1992. Accurate modeling of protein conformation by automatic segment matching. J. Mol. Biol. 226: 507-533. https://doi.org/10.1016/0022-2836(92)90964-L
  27. Levitt, M., M. Hirshberg, R. Sharon, and V. Daggett. 1995. Potential energy function and parameters for simulations of the molecular dynamics of proteins and nucleic acids in solution. Comput. Phys. Commun. 91: 215-231. https://doi.org/10.1016/0010-4655(95)00049-L
  28. Li, D., F. Nie, L. Wei, B. Wei, and Z. Chen. 2007. Screening of high-yielding biocontrol bacterium Bs-916 mutant by ion implantation. Appl. Microbiol. Biol. 75: 1401-1408. https://doi.org/10.1007/s00253-007-0951-7
  29. Magnuson, R., J. Solomon, and A. D. Grossman. 1994. Biochemical and genetic characterization of a competence pheromone from B. subtilis. Cell 77: 207-216. https://doi.org/10.1016/0092-8674(94)90313-1
  30. Msadek, T., F. Kunst, A. Klier, and G. Rapoport. 1991. DegSDegU and ComP-ComA modulator-effector pairs control expression of the Bacillus subtilis pleiotropic regulatory gene degQ. J. Bacteriol. 173: 2366-2377.
  31. Mueller, J. P., G. Bukusoglu, and A. L. Sonenshein. 1992. Transcriptional regulation of Bacillus subtilis glucose starvationinducible genes: Control of gsiA by the ComP-ComA signal transduction system. J. Bacteriol. 174: 4361-4373.
  32. Nakano, M. M., R. Magnuson, A. Myers, J. Curry, A. D. Grossman, and P. Zuber. 1991. srfA is an operon required for surfactin production, competence development, and efficient sporulation in Bacillus subtilis. J. Bacteriol. 173: 1770-1778.
  33. Nakano, M. M., M. A. Marahiel, and P. Zuber. 1988. Identification of a genetic locus required for biosynthesis of the lipopeptide antibiotic surfactin in Bacillus subtilis. J. Bacteriol. 170: 5662-5668.
  34. Nakano, M. M., L. A. Xia, and P. Zuber. 1991. Transcription initiation region of the srfA operon, which is controlled by the comP-comA signal transduction system in Bacillus subtilis. J. Bacteriol. 173: 5487-5493.
  35. Nakano, M. M. and P. Zuber. 1993. Mutational analysis of the regulatory region of the srfA operon in Bacillus subtilis. J. Bacteriol. 175: 3188-3191.
  36. Ogura, M., H. Yamaguchi, K. Kobayashi, N. Ogasawara, Y. Fujita, and T. Tanaka. 2002. Whole-genome analysis of genes regulated by the Bacillus subtilis competence transcription factor ComK. J. Bacteriol. 184: 2344-2351. https://doi.org/10.1128/JB.184.9.2344-2351.2002
  37. Ogura, M., H. Yamaguchi, K. Yoshida, Y. Fujita, and T. Tanaka. 2001. DNA microarray analysis of Bacillus subtilis DegU, ComA and PhoP regulons: An approach to comprehensive analysis of B. subtilis two-component regulatory systems. Nucl. Acids Res. 29: 3804-3813. https://doi.org/10.1093/nar/29.18.3804
  38. Roggiani, M. and D. Dubnau. 1993. ComA, a phosphorylated response regulator protein of Bacillus subtilis, binds to the promoter region of srfA. J. Bacteriol. 175: 3182-3187.
  39. Sambrook, J. and D. W. Russell. 2001. Molecular Cloning: A Laboratory Manual, 3rd Ed. Cold Spring Harbor Laboratory Press, New York.
  40. Schneider, K. B., T. M. Palmer, and A. D. Grossman. 2002. Characterization of comQ and comX, two genes required for production of ComX pheromone in Bacillus subtilis. J. Bacteriol. 184: 410-419. https://doi.org/10.1128/JB.184.2.410-419.2002
  41. Simonovic, M. and K. Volz. 2001. A distinct meta-active conformation in the 1.1-A resolution structure of wild-type ApoCheY. J. Biol. Chem. 276: 28637-28640. https://doi.org/10.1074/jbc.C100295200
  42. Sola, M., E. Lopez-Hernández, P. Cronet, E. Lacroix, L. Serrano, M. Coll, and A. Párraga. 2000. Towards understanding a molecular switch mechanism: Thermodynamic and crystallographic studies of the signal transduction protein CheY. J. Mol. Biol. 303: 213-225. https://doi.org/10.1006/jmbi.2000.4507
  43. Solomon, J. M., B. A. Lazazzera, and A. D. Grossman. 1996. Purification and characterization of an extracellular peptide factor that affects two different developmental pathways in Bacillus subtilis. Genes Dev. 10: 2014-2024. https://doi.org/10.1101/gad.10.16.2014
  44. Souza, C. D., M. M. Nakano, and P. Zuber. 1994. Identification of comS, a gene of the srfA operon that regulates the establishment of genetic competence in Bacillus subtilis. Proc. Natl. Acad. Sci. U.S.A. 91: 9397-9401. https://doi.org/10.1073/pnas.91.20.9397
  45. Tortosa, P., L. Logsdon, B. Kraigher, Y. Itoh, I. Mandic-Mulec, and D. Dubnau. 2001. Specificity and genetic polymorphism of the Bacillus competence quorum-sensing system. J. Bacteriol. 183: 451-460. https://doi.org/10.1128/JB.183.2.451-460.2001
  46. Tortosa, P. and D. Dubnau. 1999. Competence for transformation: A matter of taste. Curr. Opin. Microbiol. 2: 588-592. https://doi.org/10.1016/S1369-5274(99)00026-0
  47. Turgay, K., J. Hahn, J. Burghoorn, and D. Dubnau. 1998. Competence in Bacillus subtilis is controlled by regulated proteolysis of a transcription factor. EMBO J. 17: 6730-6738. https://doi.org/10.1093/emboj/17.22.6730
  48. Vagner, V., E. Dervyn, and S. D. Ehrlich. 1998. A vector for systematic gene inactivation in Bacillus subtilis. Microbiology 144: 3097-3104. https://doi.org/10.1099/00221287-144-11-3097
  49. van Sinderen, D., S. Withoff, H. Boels, and G. 1990. Isolation and characterization of comL, a transcription unit involved in competence development of Bacillus subtilis. Mol. Gen. Genet. 224: 396-404.
  50. Vanittanakom, N., W. Loeffler, and U. J. G. Koch. 1986. Fengycin - a novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J. Antibiot. (Tokyo) 39: 888-901. https://doi.org/10.7164/antibiotics.39.888
  51. Volkman, B. F., D. Lipson, D. E. Wemmer, and D. Kern. 2001. Two-state allosteric behavior in a single-domain signaling protein. Science 291: 2429-2433. https://doi.org/10.1126/science.291.5512.2429
  52. Weinrauch, Y., N. Guillen, and D. A. Dubnau. 1989. Sequence and transcription mapping of Bacillus subtilis competence genes comB and comA, one of which is related to a family of bacterial regulatory determinants. J. Bacteriol. 171: 5362-5375.
  53. Weinrauch, Y., R. Penchev, E. Dubnau, I. Smith, and D. Dubnau. 1990. A Bacillus subtilis regulatory gene product for genetic competence and sporulation resembles sensor protein members of the bacterial two-component signal-transduction systems. Genes Dev. 4: 860-872. https://doi.org/10.1101/gad.4.5.860
  54. Welch, M., K. Oosawa, S. Aizawa, and M. Eisenbach. 1993. Phosphorylation-dependent binding of a signal molecule to the flagellar switch of bacteria. Proc. Natl. Acad Sci. U.S.A. 90: 8787-8791. https://doi.org/10.1073/pnas.90.19.8787
  55. Wisedchaisri, G., M. Wu, D. R. Sherman, and W. G. Hol. 2008. Crystal structures of the response regulator DosR from Mycobacterium tuberculosis suggest a helix rearrangement mechanism for phosphorylation activation. J. Mol. Biol. 378: 227-242. https://doi.org/10.1016/j.jmb.2008.02.029

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