Cyanobacterial Diversity Analysis Using cpcBA-Intergenic Spacer Region

cpcBA-Intergenic Spacer Region을 이용한 Cyanobacteria의 다양성 분석

  • Choi Gang-Guk (Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Park Yong-Ha (Biological Resource Center, Korea Research Institute of Bioscience and Biotechnology) ;
  • Ahn Chi-Yong (Environmental Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Bae Myoung-Sook (Environmental Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology) ;
  • Oh Hee-Mock (Environmental Biotechnology Laboratory, Korea Research Institute of Bioscience and Biotechnology)
  • 최강국 (한국생명공학연구원 생물자원센터) ;
  • 박용하 (한국생명공학연구원 생물자원센터) ;
  • 안치용 (한국생명공학연구원 환경생명공학연구실) ;
  • 배명숙 (한국생명공학연구원 환경생명공학연구실) ;
  • 오희목 (한국생명공학연구원 환경생명공학연구실)
  • Published : 2005.12.01

Abstract

The cyanobacterial diversity was analyzed by restriction fragment length polymorphism (RFLP) of PCR-amplified rpcBA-Intergenic Spacer (IGS) genes and cpcBA-IGS gene sequencing with a sample collected at Chuso-ri in Daechung Reservoir on March 15, 2005, The Shannon-Weiner diversity index was 0.65, indicating that the cyanobacterial community structure was simple. PCR-RFLP profiles obtained were Phormidium spp. (58 clones), Anabaena spp. (14 clones), Microcystis spp. (4 clones), Spirulina sp. (1 clone) and uncultured cyanobacteria (2 clones). The PCR-RFLP of cpcBA-IGS revealed that Phormidium spp. and Anabaena spp. dominated in the invested sample. As a consequence, it seems that the analysis of functional genes such as cpcBA-IGS can be used for the species identification and community analysis of cyanobacteria.

대청호에서 수화 발생이 빈번한 추소리 수역에서 2005년 3월 15일에 채취한 시료를 대상으로 유전자 분식에 의한 cyanobacteria의 다양성을 조사하였다. rpcBA-Intergenic Spacer (IGS)는 cyanobacteria에 특징적 색소인 phycocyanin을 합성하는 유전자와 유전자 사이의 부분으로, 환경시료에서 cyanobacteria의 다양성을 조사하기에 매우 유용한 기능 유전자이다. cpcBA-IGS를 이용하여 restriction fragment length polymorphism (RELP)으로 cyanobacteria의 다양성을 분석한 결과 Phomidium 속은 58 clones, Anabaena 속은 14 clones, Microcyxtis 속은 4 clones, Spirulina 속은 1 clone 그리고 uncultured cyanobacteria 2 clones가 존재하였다. 전반적으로 Phormidium 속이 우점하였으며, 여름철에 수화를 일으키는 Anabaena 속과 Microcystis 속도 많이 분포하였다. 따라서 cyanobacteria는 cpcBA-IGS와 같은 기능 유전자에 의한 종 동정 및 군집분석이 가능함을 보였다.

Keywords

References

  1. 전만식, Y. Watanabe, 김범철. 1998. 하천수중에서 식물 플랑크톤의 증식에 대한 희석율과 온도의 영향. 한국육수학회지 31, 328-336
  2. Ahn, C.-Y., H.-S. Kim, B.-D. Yoon, and H.-M. Oh. 2003. Influence of rainfall on cyanobacterial bloom in Daechung reservoir. Korean J. Limnol. 36, 413-419
  3. Bruns, A., U. Nubel, H. Cypionka, and J. Overmann. 2003. Effect of signal compounds and incubation conditions on the culturability of freshwater bacterioplankton. Appl. Environ. Microbiol. 69, 1980-1989 https://doi.org/10.1128/AEM.69.4.1980-1989.2003
  4. Crosbie, N.D., M. Pockl, and T. Weisse. 2003. Dispersal and phylogenetic diversity of nonmarine picocyanobacteria, inferred from 16S rRNA gene and cpcBA-intergenic spacer sequence analyses. Appl. Environ. Microbiol. 69, 5716-5721 https://doi.org/10.1128/AEM.69.9.5716-5721.2003
  5. Crosbie, N.D., M. Pockl, and T. Weisse. 2003. Rapid establishment of clonal isolates of freshwater autotrophic picoplankton by single-cell and single-colony sorting. J. Microbiol. Methods 55, 361-370 https://doi.org/10.1016/S0167-7012(03)00167-2
  6. Dunbar, J., L.O. Ticknor, and C.R. Kuske. 2000. Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66, 2943-2950 https://doi.org/10.1128/AEM.66.7.2943-2950.2000
  7. Dyble, J., H.W. Paerl, and B.A. Neilan. 2002. Genetic characterization of Cylindrospermopsis raciborskii (cyanobacteria) isolates from diverse geographic origins based on nifH and cpcBA-IGS nucleotide sequence analysis. Appl. Environ. Microbiol. 68, 2567-2571 https://doi.org/10.1128/AEM.68.5.2567-2571.2002
  8. Felsenstein, J. 2004. PHYLIP (Phylogeny Inference Package) version 3.6. The web distributed by the author. Department of Genome Sciences, University of Washington, Seattle
  9. Francois, E., D.Y. Wang, R. Fulthorpe, S.N. Liss, and E.A. Edwards. 2003. DNA microarrays for detecting endocrine-disrupting compounds. Biotechnol. Adv. 22, 17-26 https://doi.org/10.1016/j.biotechadv.2003.08.005
  10. Go, S.-R., S.-J.Park, C.-Y. Ahn, A. Choi, J.-S. Lee, H.-S. Kim, B.-D. Yoon, and H.-M. Oh. 2004. Analysis of microbial communities during cyanobacterial bloom in Daechung Reservoir by DGGE. Kor. J. Microbiol. 40, 205-210
  11. Gonzalez, J.M., A. Ortiz-Martinez, M.A. Gonzalez-delValle, L. Laiz, and C. Saiz-Jimenez. 2003. An efficient strategy for screening large cloned libraries of amplified 16S rDNA sequences from complex environmental communities. J. Microbiol. Methods 55, 459-463 https://doi.org/10.1016/S0167-7012(03)00171-4
  12. Hartmann, M., B. Frey, R. Kolliker, and F. Widmer. 2005. Semiautomated genetic analyses of soil microbial communities: comparison of T-RFLP and RISA based on descriptive and discriminative statistical approaches. J. Microbiol. Methods 61, 349-360 https://doi.org/10.1016/j.mimet.2004.12.011
  13. Horz, H.P., M.T. Yimga, and W. Liesack. 2001. Detection of methanotroph diversity on roots of submerged rice plants by molecular retrieval of pmoA, mmoX, mxaF, and 16S rRNA and ribosomal DNA, including pmoA-based terminal restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 67, 4177-4185 https://doi.org/10.1128/AEM.67.9.4177-4185.2001
  14. Janse, I., M. Meima, W.E. Kardinaal, and G. Zwart. 2003. High-resolution differentiation of cyanobacteria by using rRNA-internal transcribed spacer denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 69, 6634-6643 https://doi.org/10.1128/AEM.69.11.6634-6643.2003
  15. Kim, J.-H. and H.-Y. Jang. 2004. 16S rDNA-PCR and RFLP analysis for rapid indentification of spoilage bacteria from low salt cucumber brine. Korean J. Biotech. Bioeng. 19, 72-77
  16. Lee, H.K., J.H. Kim, C.K. Kim, and D.H. Lee. 2004. Molecular characterization of the bacterial community in activated sludges by PCR-RFLP. Kor. J. Microbiol. 40, 307-312
  17. Lee, S.Y. and S.H. Kim. 2003. Genetic variation and discrimination of Korean arkshell Scapharca species (Bivalvia, Arcoida) based on mitochondrial COI gene sequences and PCR-RFLP. Korean J. Genetics 25, 309-315
  18. Lepp, P.W. and T.M. Schmidt. 2004. Changes in Synechococcus population size and cellular ribosomal RNA content in response to predation and nutrient limitation. Microb. Ecol. 48, 1-9 https://doi.org/10.1007/s00248-003-1041-8
  19. Liu, W.T. and L. Zhu. 2005. Environmental microbiology-on-achip and its future impacts. Trends Biotechnol. 23, 174-179 https://doi.org/10.1016/j.tibtech.2005.02.004
  20. Lyra, C., S. Suomalainen, M. Gugger, C. Vezie, P. Sundman, L. Paulin, and K. Sivonen. 2001. Molecular characterization of planktic cyanobacteria of Anabaena, Aphanizomenon, Microcystis and Planktothrix genera. Int. J. Syst. Evol. Microbiol. 51, 513-526 https://doi.org/10.1099/00207713-51-2-513
  21. Muyzer, G. 1999. DGGE/TGGE a method for identifying genes from natural ecosystems. Curr. Opin. Microbiol. 2, 317-322 https://doi.org/10.1016/S1369-5274(99)80055-1
  22. Muyzer, G. and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek 73, 127-141 https://doi.org/10.1023/A:1000669317571
  23. Robertson, B.R., N. Tezuka, and M.M. Watanabe. 2001. Phylogenetic analyses of Synechococcus strains (cyanobacteria) using sequences of 16S rDNA and part of the phycocyanin operon reveal multiple evolutionary lines and reflect phycobilin content. Int. J. Syst. Evol. Microbiol. 51, 861-871 https://doi.org/10.1099/00207713-51-3-861
  24. Rudi, K., J. Treimo, H. Nissen, and G. Vegarud. 2003. Protocols for 16S rDNA array analyses of microbial communities by sequence-specific labeling of DNA probes. TheScientificWorldJOURNAL 3, 578-584 https://doi.org/10.1100/tsw.2003.44
  25. Saitou, N. and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425
  26. Savin, M.C., J.L. Martin, M. LeGresley, M. Giewat, and J. Rooney- Varga. 2004. Plankton diversity in the Bay of Fundy as measured by morphological and molecular methods. Microb. Ecol. 48, 51-65 https://doi.org/10.1007/s00248-003-1033-8
  27. Suh, M.Y., B.H. Kim, and M.S. Han. 2005. Distribution of cyanotoxin microcystin-LR in Han River system and ecological park in Seoul and Kyunggi districts. Korean J. Limnol. 38, 237-248
  28. Wood, L.W. 1985. Chloroform-methanol extraction of chlorophyll-a. Can. J. Fish. Aquat. Sci. 42, 38-43 https://doi.org/10.1139/f85-005
  29. Zwart, G., M.P. Kamst-van Agterveld, I. van der Werff-Staverman, F. Hagen, H.L. Hoogveld, and H.J. Gons. 2005. Molecular characterization of cyanobacterial diversity in a shallow eutrophic lake. Environ. Microbiol. 7, 365-377 https://doi.org/10.1111/j.1462-2920.2005.00715.x