Journal of Life Science (생명과학회지)

Korean Society of Life Science (한국생명과학회)
권호 표지
DOI QR코드

DOI QR Code

Identification and Molecular Characterization of Three Isoforms of Iturin Produced by Endophytic Bacillus sp. CY22

식물 내생균 Bacillus sp. CY22가 생성하는 iturin isoform의 분리 및 특성

  • Cho, Soo-Jeong (Laboratory of Microbial Functions, Bioprocess Engineering Division, Korea Research Institute of Bioscience and Biotechnology) ;
  • Yun-Han-Dae (Division of Applied Life Science, Gyeongsang National University, Research Institute of Agriculture & Life Sciences, Gyeongsang National University)
  • 조수정 (한국생명공학연구원 바이오소재연구부) ;
  • 윤한대 (경상대학교 응용생명과학과 경상대학교 농업생명과학연구원)
  • 발행 : 2005.12.01
Download PDF
⟨ Previous Next ⟩

초록

Endophytic Bacillus sp. CY22 was previously isolated from the interior of balloon flower root and showed strong antifungal activity against phytopathogenic fungi such as Rhizoctonia solnni, Fusarium oxysporum, and Phythium ultimum. Many Bacillus strains produce antifungal compound such as iturin, fengycin, and mycosubtilin. We isolated and identified antifungal compound from cell supernatant of the endophytic strain. By the MALDI-TOF mass result, the antifungal compound was similar to the known antifungal lipopeptide iturin. It was found that the purified iturin had three isoforms with protonated masses of m/z 1,043.39, 1,057.42, and 1,071.42 and different structures in combination with $Na^{+}$ ion using MALDI-TOF MS. The ita22 gene, which transacylase gene is associated with production of antifungal iturin, had an open reading frame (ORF) of 1,200 bp encoding 400 amino acids. Results of deduced amino acids sequence homology search, Ita22 was homologous with FenF (BAB69697) of Bacillus subtilis 168.

식물 내생균 Bacillus sp. CY22는 식물병원균 Rhizoctonia solani, Fusarium oxysporum 및 Phythium ultimum에 대해 강한 항균력을 나타내었다. 일반적으로 많은 Bacillus속 균주들은 iturin, fengycin, mycosubtulin과 같은 항균 물질을 분비한다. 본 연구에서는 식물내생균 Bacillus sp. CY22의 배양액으로부터 항균물질을 분리, 정제하였으며 MALDI-TOF mass로 분자량을 확인하였다. MALDI-TOF mass spectrum분석 결과 분리된 항균물질은 Bacillus 속 균주가 생성하는 항균물질로서 잘 알려져 있는 iturin의 분자량과 거의 일치하였으며, m/z 1043.4, 1057.4, 1071.4에서 molecular ion peak를 나타내었다. 이들은 각각 m/z 14차이를 가진 iturin의 isoform으로 추정되며 이 것은 iturin을 구성하고 있는 지방산의 탄소수 차이로 생각되며 m/z 1065.4, 1079.4 peak는 sodium adduct로서 추정된다. 또한 항균물질 iturin을 생성하는데 관여하는 transacylase 유전자를 크로닝하여 ita22 유전자로 명명하고, 그 특성으로 ita22 유전자는 400 개의 아미노산을 인지하는 1,200 bp의 open reading frame (ORF)을 가지며, 아미노산의 상동성을 조사한 결과 Bacillus subtilis 168의 FenF (BAB69697)와 가장 유사하였다.

키워드

참고문헌

  1. Grau, A., A. Ortiz, A. de Godos and J. C. Gomez-Fernandez. 2000. A biophysical study of the interaction of the lipopeptide antibiotic iturin A with aqueous phospholipid bilayers. Arch. Biochem. Biophy. 377(2), 315-323 https://doi.org/10.1006/abbi.2000.1791
  2. Asaka, O. and M. Shoda. 1996. Biocontrol of Rhizoctonia solani damping-off of tomato with Bacillus subtilis RB14. Appl. Environ. Microbiol. 62, 4081-4085
  3. Cho, S. J., S. R. Park, M. G. Kim, W. J. Lim, S. K. Ryu, C. L. An, S. Y. Hong, H. Y. Lee, S. G. Jeong, Y. U. Cho and H. D. Yun. 2002. Endophytic Bacillus sp. isolated from the interior of balloon flower root. Biosci. Biotechnol. Biochem. 66, 1270-1275 https://doi.org/10.1271/bbb.66.1270
  4. Cho, S. J., W. J. Lim, S. Y. Hong, S. R. Park and H. D. Yun. 2003. Endophytic colonization of balloon flower by antifungal strain Bacillus sp. CY22. Biosci. Biotechnol. Biochem. 67, 2132-2138 https://doi.org/10.1271/bbb.67.2132
  5. Cho, S. J., S. Y. Hong, J. Y. Kim, S. R. Park, M. G. Kim, W. J. Lim, E. C. Shin, E. J. Kim, Y. U. Cho and H. D. Yun. 2003. Endophytic Bacillus sp. CY22 from a balloon flower (Platycodon grandiflorum) produces surfactin isoform. J. Microbiol. Biotechnol.13(6), 859-865
  6. Cook, R. J., L. S. Thomashow, D. M. Weller, D. Fujimoto, M. Mazzola, G. Bangera and D.-S. Kim. 1995. Molecular mechanisms of defense by rhizobacteria against root disease. Proc. Natl. Acad. Sci. USA 92, 4197-4201
  7. Cosmina, P., F. Rodriguez, F. de Ferra, G. Grandi, M. Perego, G. Venema and D. van Sinderen. 1993. Sequence and analysis of the genetic locus responsible for surfactin synthesis in Bacillus subtilis. Mol. Microbiol. 8, 821-831 https://doi.org/10.1111/j.1365-2958.1993.tb01629.x
  8. Desai, J. D. and I. M. Banat. 1997. Microbial production of surfactants and their commercial potential. Microbiol. Mol. Bio. Rev. 61, 47-64
  9. Duitman, E. H., L. W. Hamoen, M. Rembold, G. Venema, H. Seitz, W. Saenger, F. Bernhard, R. Reinhardt, M. Schmidt, C. Ullrich, T. Stein, F. Leenders and J. Vater. 1999. The mycosubtilin synthetase of Bacillus subtilis ATCC6633: a multifunctional hybrid between a peptide synthetase, an amino transferase, and a fatty acid synthase. Proc, Natl. Acad. Sci. USA 96, 13294-13299
  10. Kenji, T., T. Akiyama and M. Shoda. 2001. Cloning, sequencing, and characterization of the iturin A operon. J. Bacteriol. 183, 6265-6273 https://doi.org/10.1128/JB.183.21.6265-6273.2001
  11. Kim, P. I., H. Bai, D. Bai, H. Chae, S. Chung, Y. Kim, R. Park and Y.-T. Chi. 2004. Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26. J. Appl. Microbiol. 97, 942-949 https://doi.org/10.1111/j.1365-2672.2004.02356.x
  12. Konz, D., A. Klens, K. Schörgendorfer and M. A. Marahiel. 1997. The bacitracin biosynthesis operon of Bacillus licheniformis ATCC10716: molecular characterization of three multi-modular peptide synthetases. Chem. Biol. 4, 927-937 https://doi.org/10.1016/S1074-5521(97)90301-X
  13. Konz, D., S. Doekel and M. A. Marahiel. 1999. Molecular and biochemical characterization of the protein template controlling biosynthesis of the lipopeptide lichenysin. J. Bacteriol. 181, 133-140
  14. Kowall, M., J. Vater, B. Kluge, T. Stein, P. Franke and D. Ziessow. 1998. Separation and characterization of surfactin isoforms produced by Bacillus subtilis OKB 105. J. Colloid Interface Sci. 204, 1-8 https://doi.org/10.1006/jcis.1998.5558
  15. Lin, T. S., C. L. Chen, L. K. Chang, J. S. Tschen and S. T. Liu. 1999. Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis. J. Bacteriol. 181, 5060-5067
  16. Maget-Dana, R. and F. Peypoux. 1994. Iturins, a special class of poreforming lipopeptides: biological and physicochemical properties. Toxicology 87, 151-174 https://doi.org/10.1016/0300-483X(94)90159-7
  17. Marahiel, M. A., T. Stachelhaus and H. D. Mootz. 1997. Modular peptide synthetases involved in non-ribosomal peptide synthesis. Chem. Rev. 97, 2651-2673 https://doi.org/10.1021/cr960029e
  18. Menkhaus, M., C. Ullrich, B. Kluge, J. Vater, D. Vollenbroich and R. M. Kamp. 1993. Structural and functional organization of the surfactin synthetase multienzyme system. J. Biol. Chem. 268, 7678-7684
  19. Muller-Hurtig, R., R. Blaszczyk, F. Wagner and N. Kosaric. 1993. Biosurfactants for environmental control. In Biosurfactants: Production, Properties, Applications. pp. 447-470. New York: Marcel Dekker
  20. Munimbazi, C. and L. B. Bullerman. 1998. Isolation and partial characterization of antifungal metabolites of Bacillus pumilus. J. Appl. Microbiol. 84, 959-968 https://doi.org/10.1046/j.1365-2672.1998.00431.x
  21. Peypoux, F., J. M. Bonmatin and J. Wallach. 1999. Recent trends in the biochemistry of surfactin. Appl. Miccrobiol. Biotechnol. 51, 553-563 https://doi.org/10.1007/s002530051432
  22. Sambrook, J, and D. W. Russell. 2001. Molecular Cloning: A laboratory Manual. 3rd eds., Cold Spring Habor Laboratory Press, Cold Spring Harbor, NY, USA
  23. Schlumbohm, B., T. Stein, C. Ullrich, J. Vater, M. Krause, M. A. Marahiel, V. Kruft and B. Wittmann-Liebold. 1991. An active serine is invoved in covalent substrate amino acid binding at each reaction center of gramicidin S synthetase. J. Biol. Chem. 266, 23135-23141
  24. Stachelhaus, T. and M. A. Marahiel. 1995. Modular structure of peptide synthetases revealed by dissection of the multifunctional enzyme GrsA. J. Biol. Chem. 270, 6163-6169 https://doi.org/10.1074/jbc.270.11.6163
  25. Stein, T., B. Kluge, J. Vater, P. Franke, A. Otto and B. Wittmann-Liebold. 1995. Biochemistry 34, 4633-4642 https://doi.org/10.1021/bi00014a017
  26. Stein, T., J. Vater, V. Kruft, A. Otto, B. Wiimann-Liebold, P. Franke, M. Panico, R. McDowell and H. R. Morris. 1996. The multiple carrier model of nonribosomal peptide biosynthesis at modular multienzymatic templates. J. Biol. Chem. 271, 15428-15435 https://doi.org/10.1074/jbc.271.26.15428
  27. Thimon, L., F. Peypoux, J. Wallach and G. Michel. 1995. Effect of lipopeptide antibiotic, iturin A, on morphology and membrane ultrastructure of yeast cells. FEMS Microbiol. Lett. 128, 101-106 https://doi.org/10.1111/j.1574-6968.1995.tb07507.x
  28. Tognoni, A., E. Franchi, C. Magistrelli, E. Colombo, P. Cosmina and G. Grandi. 1995. A putative new peptide synthase operon in Bacillus subtilis: partial characterization. Microbiology 141, 645-648 https://doi.org/10.1099/13500872-141-3-645
  29. Tosato, V., A. M. Albertini, M. Zotti, S. Sonda and C. V. Bruschi. 1997. Sequence completion, identification and definition of the fengycin operon in Bacillus subtilis 168. Microbiology 143, 3443-3450 https://doi.org/10.1099/00221287-143-11-3443
  30. Tsuge, K., T. Ano, M. Hirai, Y. Nakamura and M. Shoda. 1999. The genes degQ, pps, and lpa-8 (sfp) are responsible for conversion of Bacillus subtilis 168 to plipastatin production. Antimicrob. Agents Chemother. 43, 2183-2192
  31. Turgay, K., M. Krause and M. A. Marahiel. 1992. Four homologous domains in the primary structure of GrsB are related to domains in a superfamily of adenylate-forming enzymes. Mol. Microbiol. 6, 529-546 https://doi.org/10.1111/j.1365-2958.1992.tb01498.x
  32. Umezawa, H., T. Aoyagi, T. Nishikiori, A. Okuyama, Y. Yamagishi, M. Hamada and T. Takeuchi. 1986. Plipastatins: new inhibitors of phospholipase A2, produced by Bacillus cereus BMG202-fF67. I. Taxonomy, production, isolation and preliminary characterization. J. Antibiot. 39, 737-744 https://doi.org/10.7164/antibiotics.39.737
  33. Vanittanakom, N., W. Loeffler, U. Koch and G. Jung. 1986. Fengycin-a novel antifungal lipopeptide antibiotic produced by Bacillus subtiis F-29-3. J. Antibiot. 39, 888-901 https://doi.org/10.7164/antibiotics.39.888
  34. Zhang, Y. and R. M. Miller. 1992. Enhanced octadecane dispersion and biodegradation by a Pseudomonas rhamnolipid surfactant (biosurfactant). Appl. Environ. Microbiol. 58, 3276-3282

피인용 문헌

  1. Chemical and Biological Controls of Balloon Flower Stem Rots Caused by Rhizoctonia solani and Sclerotinia sclerotiorum vol.28, pp.2, 2012, https://doi.org/10.5423/PPJ.2012.28.2.156