The Korean Journal of Pesticide Science (농약과학회지)

The Korean Society of Pesticide Science (한국농약과학회)
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Characterization of antimicrobial proteins produced by Bacillus sp. N32

Bacillus sp. N32 균주가 생산하는 항균 단백질 특성

  • Lee, Mi-Hye (Microbial Genetics Division, National Institute of Agricultural Biotechnology (NIAB)) ;
  • Park, In-Cheol (Microbial Genetics Division, National Institute of Agricultural Biotechnology (NIAB)) ;
  • Yeo, Yun-Soo (Microbial Genetics Division, National Institute of Agricultural Biotechnology (NIAB)) ;
  • Kim, Soo-Jin (Microbial Genetics Division, National Institute of Agricultural Biotechnology (NIAB)) ;
  • Yoon, Sang-Hong (Microbial Genetics Division, National Institute of Agricultural Biotechnology (NIAB)) ;
  • Lee, Suk-Chan (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Chung, Tae-Young (Department of Genetic Engineering, Sungkyunkwan University) ;
  • Koo, Bon-Sung (Microbial Genetics Division, National Institute of Agricultural Biotechnology (NIAB))
  • 이미혜 (농촌진흥청 농업생명공학연구원 미생물유전과) ;
  • 박인철 (농촌진흥청 농업생명공학연구원 미생물유전과) ;
  • 여윤수 (농촌진흥청 농업생명공학연구원 미생물유전과) ;
  • 김수진 (농촌진흥청 농업생명공학연구원 미생물유전과) ;
  • 윤상홍 (농촌진흥청 농업생명공학연구원 미생물유전과) ;
  • 이석찬 (성균관대학교 유전공학과) ;
  • 정태영 (성균관대학교 유전공학과) ;
  • 구본성 (농촌진흥청 농업생명공학연구원 미생물유전과)
  • Published : 2006.03.31
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Abstract

An antagonistic bacterial isolate, that inhibits the growth of plant pathogens, was selected and identified from 5,000 isolates screened from the rhizosphere of various crop plants. An isolate Bacillus sp. N32, tested against Colletotrichum gloeosporioides causing anthracnose disease in hot pepper, produced both a heat resistant antifungal protein and a heat sensitive antifungal protein. The heat resistant protein was partially purified by Ammonium sulfate fractionation and gel filtration chromatography. The bioautography showed that the proteins possessed high antifungal activity. The biosynthetic gene cluster responsible for the heat resistant antifungal protein was cloned from cosmid library using DNA probe obtained from PCR product with the primers targeting the conserved nucleotide sequence of the synthetic genes reported earlier, Most of the clones obtained showed higher homology to fengycin antibiotic synthetic gene family reported earlier. On the other hand, the heat sensitive protein was isolated from SDS-PAGE and electroblotting to determine the N-terminal amino acid sequences. The heat sensitive antifungal protein gene was cloned from the ${\lambda}-ZAP$ libraries using a DNA probe based on the N-terminal amino acid sequences of the heat sensitive protein. We are contemplating to clone and sequence the whole gene cluster encoding the heat sensitive protein for further analysis.

작물 근권 토양으로부터 분리한 5,000여 길항 균주로부터 Erwinia 및 Pseudomonas등의 세균과 Trichoderma, Colletotrichum 등 곰팡이의 성장을 동시에 억제하는 Bacillus sp. N 32 균주를 선발 동정 하였다. 특히 Bacillus sp. N32 균주는 고추 탄저병균인 Colletotrichum gloeosporioides에 대하여 열에 저항성이 있는 단백질과 열에 민감한 단백질의 2종류의 항균 단백질을 동시에 생산함을 단백질 침전과 활성 검정을 통하여 확인하였다. 이 항균 단백질들을 FPLC를 이용한 gel filtration chromatography방법으로 분리한 후 SDS-PAGE와 bioautography로 항균력을 확인하였다. 또한 이 항균 단백질의 유전자들을 선발하기 위하여 기존의 알려진 그람양성 세균의 대표적인 열 저항성 항균 펩타이드 생합성 유전자 서열을 primer로 이용한 PCR 방법으로 fengycin의 생합성 유전자 단편을 분리하고 이 PCR 산물을 이용하여 Bacillus sp. N32 균주의 cosmid library로부터 fengycin의 생합성 유전자 cluster중 일부를 분리하여 염기서열을 분석하였다. 또한 열에 민감한 항균 단백질 생산 유전자는 이 항균 단백질을 SDS-PAGE 및 electroblotting으로 분리한 뒤 N-terminal 부위의 15개의 아미노산 서열을 분석하고 이를 DNA 염기배열로 치환한 다음 probe로 이용하여 ${\lambda}-ZAP$ library로부터 항균 단백질 생산 유전자가 포함된 다수의 clone을 선발하였다.

Keywords

References

  1. Alexandra K. and X. H. Chen (2004) Structural and functional characterization of gene clusters directing nonribosomal synthesis of bioactive cyclic lipopeptides in Bacillus amyloliquefaciens strain FZB42. J. Bacteriol. 184:1084-1096
  2. Alison J. V., W. R. and C. P. Selityennikoff(1991) A new family of plant antifungal proteins. Mol. Plant-Microbe Interact. 4:315-323 https://doi.org/10.1094/MPMI-4-315
  3. Guang H. L., C. L. Chen, J. S. M. Tschen, S. S. Tsay , Y. S. Chang and S. T. Liu.(1998) Molecular cloning and characterization of fengycin synthetase gene fenB from Bacillus subtilis. J. Bacteriol. 180:1338-1341
  4. Handelsman, J., M., R. Rondon, S. P. Brady, J. Clady, and R. M. Goodman. (1998) Molecular biological access to the chemistry of unknown soil microbe : a new frontier for natural products. Chem. Biol. 5:24 5-249 https://doi.org/10.1016/S1074-5521(98)90108-9
  5. Kenji, T., A. Takashi, H. Mitsuyo, N. Yoshiyuki and S. Makoto (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
  6. Kerr, A. (1980) Biological control of crown gall through production of agrocin 84. Plant Dis. 64:25-30
  7. Kleinkauf, A. and V. Dohren (1996) A non-ribpsomal system of peptide biosynthesis. Eur. J. Biochem. 236:335-351 https://doi.org/10.1111/j.1432-1033.1996.00335.x
  8. Koo B. S., S. B. Lee, S. H. Yoon, G. K. Song, D. S. Chung, M. O. Byun and J. C. Ryu(1998) Characterization of a heat resistant antimicrobial peptide secreted by Bacillus subtilis A 405. Kor. J. Pest. Sci. 2(3):28-35
  9. Lee, S. Y. and S. K. Rhee (1998) Rapid and efficient isolation of gene for biosynthesis of peptide antibiotics from gram-positive bacterial Strains. J. Microbiol. Biotechnol. 8(4):310-317
  10. Lee, J. W. (1995) Isolation and characterization of peptide antibiotics synthetase from Bacillus subtilis isolated from Korea soil. Master Thesis. Department of Biological Science, Myongji University, Korea
  11. Maget D. R.(1994) Itruin, a special class of poreforming lipopeptides : biologocal and physicochemical pro-perties. Toxicology 87: 151-174 https://doi.org/10.1016/0300-483X(94)90159-7
  12. Sam brook, J., E. F. Fritsch and J. Maniatis (1989) Bacteriophage vectors and Cosmid vectors, Molecular cloning, pp. 2.2-3.58, Cold Spring Harbor Laboratory Press, USA
  13. Tsuey P. L., C. L. Chen, H. C. Fu, C. H. Lin, G. H. Lin, S. H. Huang, L. K. Chang and S. J. Liu. (2005) Functional analyses of a fengycin synthetase fenD. Biochemica et Biophysica Acta. 1730:159-164 https://doi.org/10.1016/j.bbaexp.2005.02.005
  14. Tsuey P. L., C. L. Chen, L. K. Chang, J. S. M. Tschen and S. T. Liu. (1999) Functional and transcriptional analyses of a fengycin synthetase gene, fenC, from Bacillus subtilis. J. Bacteriol. 181:5060-5067
  15. Valentina T. and M. Alessandra (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
  16. Vanittanakom, N., W. Loeffler, U. Koch and G. Jung (1986) Fengycin, A novel antifungal lipopeptide antibiotic produced by Bacillus subtilis F-29-3. J. Antibiotics. 7:888-901
  17. Varsha K. and L. D. Barnes (1970) Effect of zwitterionic buffer of small masses of protein with bicinichoninic acid. Anal. Biochem 157:291-294 https://doi.org/10.1016/0003-2697(86)90629-9
  18. Whitman, W. B., D. C. Coleman and W. J. Wiebe (1998) Prokaryotes: the unseen majority. Proc. Natl. Acad. Sci. 95:6578-6583
  19. Zuber, P., M. M. Nakano and M. A. Marahiel (1993) Peptide antibiotics. In Bacillus subtilis and other gram-positive bacteria : Biochemistry, Physiology, and Molecular Genetics. pp.897-916 American Society for Microbiology. Washington DC, USA