Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
권호 표지

Isolation and Phylogenetic Characterization of Chitinase Producing Oligotrophic Bacteria

Chitinase생산 저영양세균의 분리 및 계통분류학적 특성

  • Kim Soo-Jin (Microbial Function Team, National Institute of Agricultural Biotechnology(NIAB)) ;
  • Kim Min-Young (Microbial Function Team, National Institute of Agricultural Biotechnology(NIAB)) ;
  • Koo Bon-Sung (Microbial Function Team, National Institute of Agricultural Biotechnology(NIAB)) ;
  • Yoon San-Hong (Microbial Function Team, National Institute of Agricultural Biotechnology(NIAB)) ;
  • Yeo Yun-Soo (Microbial Function Team, National Institute of Agricultural Biotechnology(NIAB)) ;
  • Park In-Cheol (Microbial Function Team, National Institute of Agricultural Biotechnology(NIAB)) ;
  • Kim Yoon-Ji (Dept. of Biotechnology, Mokwon University) ;
  • Lee Jong-Wha (Inst. of Microbial Ecology and Resources) ;
  • Whang Kyung-Sook (Dept. of Biotechnology, Inst. of Microbial Ecology and Resources, Mokwon University)
  • 김수진 (농촌진흥청 농업생명공학연구원 미생물기능팀) ;
  • 김민영 (농촌진흥청 농업생명공학연구원 미생물기능팀) ;
  • 구본성 (농촌진흥청 농업생명공학연구원 미생물기능팀) ;
  • 윤상홍 (농촌진흥청 농업생명공학연구원 미생물기능팀) ;
  • 여윤수 (농촌진흥청 농업생명공학연구원 미생물기능팀) ;
  • 박인철 (농촌진흥청 농업생명공학연구원 미생물기능팀) ;
  • 김윤지 (목원대학교 생명산업학부) ;
  • 이종화 (미생물생태자원연구소) ;
  • 황경숙 (목원대학교 생명산업학부, 미생물생태자원연구소)
  • Published : 2005.12.01
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Abstract

Many isolates from soil of Korean ginseng rhizosphere did not show remarkable growth on full strength of the conventional nutrient broth (NB medium) but grew on its 100-fold dilution (DNB medium). Six hundred-forty strains were isolated as oligotrophic bacteria. In the course of screening for new bioactive compounds from oligotrophic bacteria from soil, 8 strains which had appeared to form of clear zone on a medium containing colloidal chitin as a sole carbon source were selected for further studies. Strain CR42 hydrolyzed a fluorogenic analogue of chitin, 4-methylumbelliferyl-D-glucosaminide (MUF-NAG) . Mo st of the culture supernatant of these isolates hydrolyzed 4-methylumbelliferyl-D-N,N'-diacetylchitobioside (MUF-diNAG). The isolates were heterogeneous and categorized to gamma- and beta-proteobacteria, Bacillaceae, Actinobactepia, and Bacteroides by 16S rRNA analysis. Two strains, WR164 and CR18, had a 16S rRNA sequence of $95-96\%$ identical to uncultured bacteria. It was observed that CR2 and CR75 could inhibit the growth of Colletotrichum gloeosporioides with hyphal extention-inhibition assay on PDA plate supplemented with $1\%$ colloidal chitin.

인삼근권토양으로부터 분리된 총640 저영양세균 중 유일한 탄소원으로 colloidal chitin을 첨가한 배지에서 투명환을 나타낸 8균주를 선발하였다. 대부분의 균주가 chitin의 형광성 유사체인 4-methylumbelliferyl D-N,N'-diacetylchitobioside (MUF-diNAG)을 분해하였고, CR-42균주의 경우 4-methylumbelliferyl-D-glucosaminide (MUF-NAG)를 분해하였다. 이들 chitinase 생산균주의 16S rDNA 염기서열을 결정하여 계통학적 위치를 확인한 결과 5개의 주요한 계통군: proteobacteria $\gamma-subdivision$ (3균주), proteobacteria $\beta-subdivision$ (1 균주), Actinobacteriaceae (1 균주), Bacillaceae (1 균주) 그리고 Bacteriodetes (2 균주)로 분류되었다. 이들 분리균주 중 WR164와 CR18 균주는 16S rDNA염기서열의 유사도가 미배양 및 미동정 등록균주와 $97\%$ 미만으로 나타나 신규미생물로 제안할 수 있는 균주로 예상되었다. 한편 CR2와 CR75 chitinase 생산균주는 인삼 탄저 병원균인 Colletotrichum gloeosporioides의 생장을 저해하는 것으로 나타났다.

Keywords

References

  1. 오승환, 유연현, 김영호, 김기광, 이일호. 1988. 인삼의 재배법 변화에 따른 병해충 발생생태 및 방제연구. 인삼 연구보고서(재배분야, 환경 및 육종분야), p. 3-137. 인삼연초연구소
  2. 오승환, 유연현. 1996. 인삼의 병. 최신고려인삼(재배편), p. 197-241. 한국인삼연초연구소
  3. 이강표, 김창남, 유주현, 오두환. 1990. Aeromonas salmonicida YA7-625에 의한 chitinase의 생산 및 정제. 한국산업미생물학회지. 18, 599-606
  4. 中神照太, 外村健三. 1966. 放線菌の生産するキチィン分 解酵素に關する硏究(第1報)分解菌の 檢索と培養條件につ いて. 工業技術院醱酵硏究所報告. 30, 19-26
  5. Chernin, L.S., M.K. Winson, J.M. Thompson, S. Haran, B.W. Bycroft, I. Chet, P. Williams, and G.S.A.B. Stewart. 1998. Chitinolytic activity in Chromobacterium violaceum: Substrate analysis and regulation by quorum sensing. J. Bacteriol. 180, 4435-4441
  6. Chernin, L.S., L. Fuente, V. Sobolev, S. Haran, C.E. Vorgias, A.B. Oppenheim, and I. Chet. 1997. Molecular cloning, structural analysis, and expression in Escherichia coli of a chitinase gene from Enterobacter agglomerans. Appl. Environ. Microbiol. 63, 834-839
  7. Clarke, P.H. and M.V. Tracey. 1956. The occurrence of chitinase in some bacteria. J. Gen. Microbiol. 14, 188-196 https://doi.org/10.1099/00221287-14-1-188
  8. Dempsey, D.M.A., H. Silva, and D.F. Klessig. 1998. Engineering disease and pest resistance in plants. Trends Microbiol. 6, 54-61 https://doi.org/10.1016/S0966-842X(97)01186-4
  9. Downing, K.J., G. Leslie, and J.A. Thomson. 2000. Biocontrol of the sugarcane borer Eldana saccharina by expression of the Bacillus thuringiensis cry1Ac7 and Serratia marcescens chiA genes in sugarcane-associated bacteria. Appl. Environ. Microbiol. 66, 2804-2810 https://doi.org/10.1128/AEM.66.7.2804-2810.2000
  10. Driss, F., M. Kallassy-Awad, N. Zouari, and S. Jaoua. 2005. Molecular characterization of a novel chitinase from Bacillus thuringiensis subsp. kurstaki. J. Appl. Microbiol. 99, 945-953 https://doi.org/10.1111/j.1365-2672.2005.02639.x
  11. Felse, P.A. and T. Panda. 1999. Regulation and cloning of microbial chitinase genes. Appl. Microbiol. Biotechnol. 51, 141-151 https://doi.org/10.1007/s002530051374
  12. Hoster, F., J.E. Schmitz, and R. Daniel. 2005. Enrichment of chitinolytic microorganism: isolation and characterization of a chitinase exhibiting antifungal activity against phytopathogenic fungi from a novel Streptomyces strain. Appl. Micriobiol. Biotechnol. 66, 434-442 https://doi.org/10.1007/s00253-004-1664-9
  13. Hsu, S.C. and J.L. Lockwood. 1975. Powdered chitin agar as a selective medium for enumeration of Actinomycetes in water and soil. Appl. Microbiol. 29, 422-426
  14. Ishida, Y. and H. Kadota. 1979. A new method for enumeration of oligotrophic bacteria in lake water. Arch. Hydrobiol. 12, 77-85
  15. Johnson, J.L. 1994. Simility analysis of rRNAs, In: Gerhardt, P., R.G.E. Murray, W.A. Wood, N.R. Krirg (Eds.), Methods for general and molecular bacteriology. American Society for Microbiology, Washington, DC. pp.683-700
  16. Kuzunetsov, S. I., G.A. Dubinnina, and N.A. Lapteva, 1979. Biology of oligotrophic bacteria. Ann. Rev. Microbiol. 33, 377-387 https://doi.org/10.1146/annurev.mi.33.100179.002113
  17. Lane, D.J. 1991. 16S/23S rRNA sequencing, In: Stackebrandt, E., M. Goodfellow (Eds.), Nucleic acid techniques in bacterial systematics, John Wiley and Sons, Chichester. pp.115-175
  18. Metcalfe, A.C., M. Krsek, G.W. Gooday, J.I. Prosser, and E.M.H. Wellington. 2002. Molecular analysis of a bacterial chitinolytic community in an upland pasture. Appl. Environ. Microbiol. 68, 5042-5050 https://doi.org/10.1128/AEM.68.10.5042-5050.2002
  19. Mattew, T.C., A.M. Jessica, and L.K. David. 1999. Chitinase from uncultured marine microorganisms. Appl. Environ. Microbiol. 65, 2553-2557
  20. Mattew, T.C., N.W. Daniel, Y. Liying, and L.K. David. 2000. Selected chitinase genes in cultured and uncultured marine bacteria in the ${\alpha}$- and ${\beta}$-subclasses of the proteobacteria. Appl. Environ. Microbiol. 66, 1195-1201 https://doi.org/10.1128/AEM.66.3.1195-1201.2000
  21. Monreal, J. and E. T. Reese. 1969. The chitinase of Serratia marcescens. Can. J. Microbiol. 15, 689-696 https://doi.org/10.1139/m69-122
  22. Nikitin, D.I. and K.V. Chumakov. 1985. The functional role of oligotrophic microorganisms. In V. Jensen (ed.), Microbial communities in soil. FEMS Symposium. 33, 177-189
  23. Ohta, H. and T. Hattori. 1980. Bacteria sensitive to nutrient broth medium in terrestrial environments. Soil Sci. Plant Nutr. 26, 99-107 https://doi.org/10.1080/00380768.1980.10433216
  24. Ohtakara, A. (1971) The chitinase system of a strain of Streptomyces griceus. Bull. Hiroshima Women's Univ. 6, 1-12
  25. Omera, C., B.A. Horwitz, and I. Chet. 2001. A convenient fluorometric method for the detection of extracellular N-acetylglucosaminidase production by filamentous fungi. J. Microbiol. Methods. 43, 165-169 https://doi.org/10.1016/S0167-7012(00)00213-X
  26. Radjacommare, R., A. Ramanathan, A. Kandan, G.V. Sible, S. Harish, and R. Samiyappan. 2004. Purification and anti-fungal activity of chitinase against Pyricularia grisea in finger millet. World J. of Microbiol. Biotechnol. 20, 251-256 https://doi.org/10.1023/B:WIBI.0000023829.98282.0f
  27. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  28. Sakai, D., A. Yokota, H. Kurokawa, M. Wakayama, and M. Moriguchi. 1998. Purification and characterization of three thermostable endochitinases of a noble Bacillus strain, MH-1, isolated from chitin-containing compost. Appl. Environ. Microbiol. 64, 3397-3402
  29. Thompson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTALW: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  30. Tominaga, Y. and Y. Tsujisaka, 1976. Purification and some properties of two chitinase from Streptomyces orientalis which lyse Rhizopus cell wall. Agric. Biol. Chem. 40, 2325-2333 https://doi.org/10.1271/bbb1961.40.2325
  31. Wang S.L. and W.T. Chang. 1997. Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium. Appl. Environ. Microbiol. 63, 380-386
  32. Watanabe, T., R. Kanai, T. Tanabe, M. Mitsutomi, S. Sakuda, and K. Miyashita. 1999. Family 19 chitinases of streptomyces species: characterization and distribution. Microbiology 145, 3353-3363 https://doi.org/10.1099/00221287-145-12-3353
  33. Whang, K. and T. Hattori. 1988. Oligotrophic bacteria in rendzina a forest soil. Antonie van Leewenhoek J. Microbiol. 54, 19-37 https://doi.org/10.1007/BF00393955
  34. Whang, K. and S. Yu. 1995. Growth patterns of soil bacteria in different organic concentrations, and isolation of facultative and obligate oligotrophic bacteria. The Microorganisms & Industry 21, 319-324
  35. Wortman, A.T., C.C. Somerville, and R.R. Cotwell. 1986. Chitinase determinants of Vibrio vulnificus: gene cloning and applications of a chitinase probe. Appl. Environ. Microbiol. 52, 142-145
  36. Zhu, H., F. Qu, and L. Zhu. 1993. Isolation of genomic DNAs from plants, fungi and bacteria using benzyl chloride. Nucleic Acids Res. 21, 5279-5280 https://doi.org/10.1093/nar/21.22.5279