The Plant Pathology Journal

The Korean Society of Plant Pathology (한국식물병리학회)
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Characterization of a Chitinase Gene Exhibiting Antifungal Activity from a Biocontrol Bacterium Bacillus licheniformis N1

  • Published : 2009.12.01
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Abstract

A biocontrol bacterium Bacillus licheniformis N1 grown in nutrient broth showed no chitinolytic activity, while its genome contains a gene which encodes a chitinase. The gene for chitinase from B. licheniformis N1 was amplified by PCR and the deduced amino acid sequence analysis revealed that the chitinase exhibited over 95% identity with chitinases from other B. licheniformis strains. Escherichia coli cells carrying the recombinant plasmid displayed chitinase activity as revealed by the formation of a clear zone on chitin containing media, indicating that the gene could be expressed in E. coli cells. Chitinase gene expression in B. licheniformis N1 was not detected by RT-PCR analysis. The protein was over-expressed in E. coli BL21 (DE3) as a glutathione S-transferase fusion protein. The protein could also be produced in B. subtilis 168 strain carrying the chitinase gene of N1 strain. The crude protein extract from E. coli BL21 carrying GST fusion protein or culture supernatant of B. subtilis carrying the chitinase gene exhibited enzyme activity by hydrolyzing chitin analogs, 4-methylumbelliferyl-$\beta$-D-N,N'-diacetylchitobioside and 4-methylumbelliferyl-$\beta$-D-N,N',N"-triacetylchitotrioside. These results indicated that even though the chitinase gene is not expressed in the N1 strain, the coding region is functional and encodes an active chitinase enzyme. Furthermore, B. subtilis 168 transformants expressing the chitinase gene exhibited antifungal activity against Fulvia fulva by suppressing spore germination. Our results suggest that the proper engineering of the expression of the indigenous chitinase gene, which will lead to its expression in the biocontrol strain B. licheniformis N1, may further enhance its biocontrol activity.

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References

  1. Adams, D. J. 2004. Fungal cell wall chitinases and glucanases. Microbiology 150:2029-2035 https://doi.org/10.1099/mic.0.26980-0
  2. Bartnicki-Garcia, S. 1968. Cell wall chemistry, morphogenesis and taxonomy of fungi. Annu. Rev. Microbiol. 22:87-108 https://doi.org/10.1146/annurev.mi.22.100168.000511
  3. Burkholder, P. R. and Giles, N. H. 1947. Induced biochemical mutation in Bacillus subtilis. Am. J. Bot. 34:345-348 https://doi.org/10.2307/2437147
  4. Chemin, L., Ismailov, Z., Haran, S. and Chet, I. 1995. Chitinolytic Enterobacter agglomerans antagonistic to fungal plant pathogens. Appl. Microbiol. Biotechnol. 61:1720-1726
  5. Cohen-Kupiec, R. and Chet, I. 1998. The molecular biology of chitin digestion. Curr. Opin. Biotechnol. 9:270-277 https://doi.org/10.1016/S0958-1669(98)80058-X
  6. Compant, S., Duffy, B., Nowak, J., Clement, C. and Barka, E. A. 2005. Use of plant growth-promoting bacteria for biocontrol of plant disease: principles, mechanisms of action, and future prospects. Appl. Environ. Microbiol. 71:4951-4959 https://doi.org/10.1128/AEM.71.9.4951-4959.2005
  7. Dahiya, N., Tewari, R. and Hoondal, G. S. 2006. Biotechnological aspects of chitinolytic enzymes: a review. Appl. Microbiol. Biotechnol. 71:773-782 https://doi.org/10.1007/s00253-005-0183-7
  8. Emmert, E. A. B. and Handelsman, J. 1999. Biocontrol of plant disease: a (Gram-) positive perspective. FEMS Microbiol. Lett. 171:1-9 https://doi.org/10.1111/j.1574-6968.1999.tb13405.x
  9. Fravel, D. R. 2005. Commercialization and implementation of biocontrol. Annu. Rev. Phytopathol. 43:337-359 https://doi.org/10.1146/annurev.phyto.43.032904.092924
  10. Fravel, D. R., Connick Jr., W. J. and Lewis, J. A. 1998. Formulation of microorganisms to control plant diseases. In: Formulation of microbial pesticides: Beneficial microorganisms, nematodes and seed treatments, ed. by H. D. Burges, pp. 187-202. Kluwer Academic Publishers, Dordrecht, The Netherlands
  11. Haas, D. and Defago, G. 2005. Biological control of soil-borne pathogens by fluorescent pseudomonads. Nat. Rev. Microbiol. 3:307-319 https://doi.org/10.1038/nrmicro1129
  12. Handelsman, J. and Stabb, E. V. 1996. Biocontrol of soilborne plant pathogens. Plant Cell 8:1855-1869 https://doi.org/10.1105/tpc.8.10.1855
  13. Henrissat, B. and Bairoch, A. 1993. New families in the classification of glycosyl hydro lases based on amino acid sequence similarities. Biochem. J. 293:781-788 https://doi.org/10.1042/bj2930781
  14. Kim, H. J., Lee, S. H., Kim, C. S., Lim, E. K., Choi, K. H., Kong, H. K., Kim, D. W., Lee, S.-W. and Moon, B. J. 2007. Biological control of strawberry gray mold caused by Botrytis cinerea using Bacillus licheniformis N1 formulation. J. Microbiol. Biotechnol. 17:438-444
  15. Kobayashi, D. Y, Reedy, R. M., Bick, J. and Oudemans, P. V. 2002. Characterization of a chitinase gene from Stenotrophomonas maltophilia strain 34S1 and its involvement in biological control. Appl. Environ. Microbiol. 68:1047-1054 https://doi.org/10.1128/AEM.68.3.1047-1054.2002
  16. Lane, L. C. 1978. A simple method for stabilizing protein-sulfhydryl groups during SDS-gel electrophoresis. Anal. Chem. 86:655-664 https://doi.org/10.1016/0003-2697(78)90792-3
  17. Leah, R., Tommerup, H., Svendsen, I. and Mundy, J. 1991. Biochemical and molecular characterization of three barley seed proteins with anti-fungal properties. J. BioI. Chem. 266:1564-1573
  18. Lee, J. P., Lee, S.-W., Kim, C. S., Son, J. H., Song, J. H., Lee, K.Y, Kim, H. J., Jung, S. J. and Moon, B. J. 2006. Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea. BioI. Control 37:329-337 https://doi.org/10.1016/j.biocontrol.2006.01.001
  19. Lorito, M., Pietro, A. D., Hayes, C. K., Woo, S. L. and Harman, G. E. 1993. Antifungal, synergistic interaction between chitinolytic enzyme from Trichoderma hazianum and Enterobacter cloacae. Phytopathology 83:721-728 https://doi.org/10.1094/Phyto-83-721
  20. Melent'ev, A. I., Aktuganov, G. E. and Galirnzynova, N. F. 2001. The role of chitinase in the antifungal activity of Bacillus sp. 739. Microbiology 70:548-552 https://doi.org/10.1023/A:1012304004659
  21. Ohno, T., Armand, S., Hata, T., Nikaidou, N., Henrissat, B., Mitsutomi, M. and Watnabe, T. 1996. A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037. J. Bacteriol. 178:5065-5070 https://doi.org/10.1128/jb.178.17.5065-5070.1996
  22. Ongena, M. and Jacques, P. 2008. Bacillus lipopeptides: versatile weapons for plant disease control. Trends Microbiol. 16:115-125 https://doi.org/10.1016/j.tim.2007.12.009
  23. Park, K., Paul, D., Kim, Y. K., Nam, K. W., Lee, Y. K., Choi, H. W. and Lee, S. Y. 2007. Induced systemic resistance by Bacillus vallismortis EXTN-1 suppressed bacterial wilt in tomato caused by Ralstonia solanacearum. Plant Pathol. J. 23:22-25 https://doi.org/10.5423/PPJ.2007.23.1.022
  24. Rey, M. W., Ramaiya, P., Nelson, B. A., Brody-Karpin, S. D., Zaretsky, E. J., Tang, M., Lopez de Leon, A., Xiang, H., Gusti, V., Clausen, I. G., Olsen, P. B., Rasmussen, M. D., Andersen, J. T., Jorgensen, P. L., Larsen, T. S., Sorokin, A., Bolotin, A., Lapidus, A., Galleron, N., Ehrlich, S. D. and Berka, R. M. 2004. Complete genome sequence of the industrial bacterium Bacillus licheniformis and comparisons with closely related Bacillus species. Genome Biol. 5:R 77
  25. Roberts, W. K. and Selitrennikoff, C. P. 1988. Plant and bacterial chitinases differ in antifungal activity. J. Gen. Microbiol. 134:169-176 https://doi.org/10.1099/00221287-134-1-169
  26. Rodriguez-Kabana, R., Godoy, G., Morgan-Jones, G. and Shelby, R. A. 1983. The determination of soil chitinase activity: conditions for assay and ecological studies. Plant Soil 75:95-106 https://doi.org/10.1007/BF02178617
  27. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning; a laboratory manual, 3rd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y
  28. Selitrennikoff, C. P. 2001. Antifungal protein. Appl. Environ. Microbiol. 67:2883-2894 https://doi.org/10.1128/AEM.67.7.2883-2894.2001
  29. Singh, P. P., Shin, Y. C., Park, C. S. and Chung Y. R. 1999. Biological control of Fusarium wilt of cucumber by chitinolytic bacteria. Phytopathology 82:92-99 https://doi.org/10.1094/PHYTO.1999.89.1.92
  30. Tantimavanich, S., Pantuwatana, S., Bhumiratana, A. and Panbangred, W. 1998. Multiple chitinase enzyme from a single gene of Bacillus licheniformis TP-1. J. Ferment. Bioeng. 85:259-265 https://doi.org/10.1016/S0922-338X(97)85672-3
  31. Veith, B., Herzberg, C., Steckel, S., Feesche, J., Maurer, K. H., Ehrenreich, P., Baumer, S., Henne, A., Liesegang, H., Merkl, R., Ehrenreich, A. and Gottschalk, G. 2004. The complete genome sequence of Bacillus licheniformis DSM13, an organism with great industrial potential. J. Mol. Microbiol. Biotechnolo 7:204-211 https://doi.org/10.1159/000079829
  32. Waldeck, J., Daum, G., Bisping, B. and Meinhardt, F. 2006. Isolation and molecular characterization of chitinase-deficient Bacillus licheniformis strains capable of deproteinization of shrimp shell waste to obtain highly viscous chitin. Appl. Environ. Microbiol. 72:7879-7885 https://doi.org/10.1128/AEM.00938-06
  33. Watanabe, T., Kanai, R., Kawase, T., Tanabe, T., Mitsutomi, M., Sakuda, S. and Miyashita, K. 1999. Family 19 chitinases of Streptomyces species: characterization and distribution. Microbiology 145:3353-3363 https://doi.org/10.1099/00221287-145-12-3353
  34. Whipps, J. M. 2001. Microbial interactions and biocontrol in the rhizosphere. J. Exp. Bot. 52:487-511 https://doi.org/10.1093/jexbot/52.suppl_1.487
  35. Xue, G-P., Johnson, J. S. and Dalymple, B. P. 1999. High osmolarity improves the electro-transformation efficiency of the gram-positive bacteria Bacillus subtilis and Bacillus licheniformis. J. Microbiol. Methods 34:183-191 https://doi.org/10.1016/S0167-7012(98)00087-6

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