Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Phylogenetic Analysis of Bacterial Diversity in the Marine Sponge, Asteropus simplex, Collected from Jeju Island

제주도에서 채집한 해양 해면, Asteropus simplex의 공생세균에 관한 계통학적 분석

  • Jeong, In-Hye (Department of Biological Science and Biotechnology, Hannam University) ;
  • Park, Jin-Sook (Department of Biological Science and Biotechnology, Hannam University)
  • 정인혜 (한남대학교 생명시스템과학과) ;
  • 박진숙 (한남대학교 생명시스템과학과)
  • Received : 2012.12.15
  • Accepted : 2012.12.26
  • Published : 2012.12.31
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Abstract

Culture-dependent RFLP and culture-independent DGGE were employed to investigate the bacterial community associated with the marine sponge Asteropus simplex collected from Jeju Island. A total of 120 bacterial strains associated with the sponge were cultivated using modified Zobell and MA media. PCR amplicons of the 16S rDNA from the bacterial strains were digested with the restriction enzymes HaeIII and MspI, and then assigned into different groups according to their restriction patterns. The 16S rDNA sequences derived from RFLP patterns showed more than 94% similarities compared with known bacterial species, and the isolates belonged to five phyla, Alphaproteobacteria, Gammaproteobacteria Actinobacteria, Bacteroidetes, and Firmicutes, of which Gammaproteobacteria was dominant. DGGE fingerprinting of 16S rDNAs amplified from the sponge-derived total gDNA showed 12 DGGE bands, and their sequences showed more than 90% similarities compared with available sequences. The sequences derived from DGGE bands revealed high similarity with the uncultured bacterial clones. DGGE revealed that bacterial community consisted of seven phyla, including Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteira, Chloroflexi, and Nitrospira. Alphaproteobacteria, Gammaproteobacteria, and Actinobacteria were commonly found in bacteria associated with A. simplex by both RFLP and DGGE methods, however, overall bacterial community in the sponge differed depending on the analysis methods. Sponge showed more various bacterial community structures in culture-independent method than in culture-dependent method.

해양 해면 Asteropus simplex를 제주도에서 채집하여 배양에 의한 RFLP와 비배양에 의한 DGGE 분석 방법에 의해 세균군집 구조를 조사하였다. 16S rDNA-RFLP 분석을 위해 변형된 Zobell 배지와 MA를 이용하여 120균주를 선별하고 제한효소, HaeIII와 MspI을 사용하여 각각의 다른 RFLP 패턴으로 구분하였다. RFLP 패턴으로부터 유래한 16S rDNA 염기서열 분석결과, 알려진 세균 종과 94% 이상의 유사도를 나타내었으며 Alphaproteobacteria, Gammaproteobacteria, Actinobacteria, Bacteroidetes, Firmicutes, 5개의 문이 관찰되었다. 그 중 Gammaproteobacteria가 우점하였다. 같은 해면, A. simplex의 DGGE 분석을 위해 total genomic DNA로부터 16S rDNA를 증폭하여 DGGE fingerprinting을 수행한 결과 12개의 서로 다른 밴드가 관찰되었다. 각 밴드의 16S rDNA 염기서열은 알려진 세균의 염기서열과 90% 이상의 유사성을 나타내었으며 대부분의 염기서열은 uncultured bacteria에 속하였다. DGGE 분석으로부터 미생물의 군집은 Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Deltaproteobacteria, Actinobacteria, Chloroflexi, Nitrospira, 7개의 문으로 나타났다. RFLP와 DGGE 방법에 의해 Alphaproteobacteria, Gammaproteobacteria, Actinobacteria가 공통적으로 발견되었으나 전체적인 공생세균의 군집구조는 분석방법에 따른 차이를 나타내었다. 배양에 의한 방법보다 비배양 방법에서 더 다양한 세균군집구조를 나타내었다.

Keywords

References

  1. Bergman, O., Haber, M., Mayzel, B., Anderson, M.A., Shpigel, M., Hill, R.T., and Ilan, M. 2011. Marine-based cultivation of Diacarnus sponges and the bacterial community composition of wild and maricultured sponges and their larvae. Mar. Biotechnol. 13, 1169- 1182. https://doi.org/10.1007/s10126-011-9391-6
  2. Botstein, D., White, R.L., Skolnick, M., and Davis, R.W. 1980. Construction of a genetic linkage map in man using restriction fragment length polymorphisms. Am. J. Hum. Genet. 32, 314-331.
  3. Erwin, P.M., Olson, J.B., and Thacker, R.W. 2011. Phylogenetic diversity, host-specificity and community profiling of sponge-associated bacteria in the Northern Gulf of Mexico. PLoS. 6, e26806. doi:10.1371/journal.pone.0026806.
  4. Fischer, S.G. and Lerman, L.S. 1983. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proc. Natl. Acad. Sci. USA 80, 1579-1583. https://doi.org/10.1073/pnas.80.6.1579
  5. Hentschel, U., Fieseler, L., Wehrl, M., Gernert, C., Steinert, M., Hacker, J., and Horn, M. 2003. Microbial diversity of marine sponges. Prog. Mol. Subcell. Biol. 37, 59-88. https://doi.org/10.1007/978-3-642-55519-0_3
  6. Jackson, S.A., Kennedy, J., Morrissey, J.P., O'Gara, F., and Dobson, A.W. 2012. Pyrosequencing reveals diverse and distinct sponge-specific microbial communities in sponges from a single geographical location in Irish waters. Microb. Ecol. 64, 105-116. https://doi.org/10.1007/s00248-011-0002-x
  7. Jeong, E.J., Im, C.S., and Park, J.S. 2010. A comparison of bacterial diversity associated with the sponge Spirastrella abata depending on RFLP and DGGE. Kor. J. Microbiol. 46, 366-374.
  8. Kennedy, J., Baker, P., Piper, C., Cotter, P.D., Walsh, M., Mooij, M.J., Bourke, M.B., Rea, M.C., O'Connor, P.M., Ross, R.P., and et al. 2009. Isolation and analysis of bacteria with antimicrobial activities from the marine sponge Haliclona simulans collected from Irish waters. Mar. Biotechnol. 11, 384-396. https://doi.org/10.1007/s10126-008-9154-1
  9. Li, Z., He, L., and Miao, X. 2007. Cultivable bacterial community from south china sea sponge as revealed by DGGE fingerprinting and 16S rDNA phylogenetic analysis. Curr. Microbiol. 55, 465-472. https://doi.org/10.1007/s00284-007-9035-2
  10. Mohamed, N.M., Rao, V., Hamann, M.T., Kelly, M., and Hill, R.T. 2008. Monitoring bacterial diversity of the marine sponge Ircinia strobilina upon transfer into aquaculture. Appl. Environ. Microbiol. 74, 4133- 4143. https://doi.org/10.1128/AEM.00454-08
  11. Murayama, S., Nakao, Y., and Matsunaga, S. 2008. Asteropterin, an inhibitor of cathepsin B, from the marine sponge Asteropus simplex. Tetrahedron Lett. 49, 4186-4188. https://doi.org/10.1016/j.tetlet.2008.04.043
  12. Park, S.H., Kwon, K.K., Lee, D.S., and Lee, H.K. 2002. Morphological diversity of marine microorganisms on different media. J. Microbiol. 40, 161-165.
  13. Saitou, N. and Nei, M. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406-425.
  14. Sipkema, D., Schippers, K., Maalcke, W.J., Yang, Y., Salim, S., and Blanch, H.W. 2011. Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp. Appl. Environ. Microbiol. 77, 2130-2140. https://doi.org/10.1128/AEM.01203-10
  15. Tamura, K., Dudley, J., Nei, M., and Kumar, S. 2007. MEGA4: molecular evolutionary genetics analysis (MEGA) software version 4.0. Mol. Biol. Evol. 24, 1596-1599. https://doi.org/10.1093/molbev/msm092
  16. Taylor, M.W., Hill, R., and Hentschel, U. 2011. Meeting report: 1st international symposium on sponge microbiology. Mar. Biotechnol. 13, 1057-1061. https://doi.org/10.1007/s10126-011-9397-0
  17. Thomas, T.R., Kavlekar, D.P., and LokaBharathi, P.A. 2010. Marine drugs from sponge-microbe association-a review. Mar. Drugs 8, 1417 -1468. https://doi.org/10.3390/md8041417
  18. Thompson, F.L., Thompson, C.C., Li, Y., Gomez-Gil, B., Vandenberghe, J., Hoste, B., and Swings, J. 2003. Vibrio kanaloae sp. nov., Vibrio pomeroyi sp. nov. and Vibrio chagasii sp. nov., from sea water and marine animals. Int. J. Syst. Evol. Microbiol. 53, 753-759. https://doi.org/10.1099/ijs.0.02490-0
  19. Thompson, J.D., Higgins, D.G., and Gibson, T.J. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 22, 4673-4680. https://doi.org/10.1093/nar/22.22.4673
  20. Webster, N.S., Cobb, R.E., Soo, R., Anthony, S.L., Battershill, C.N., Whalan, S., and Evans-Illidge, E. 2011. Bacterial community dynamics in the marine sponge Rhopaloeides odorabile under in situ and ex situ cultivation. Mar. Biotechnol. 13, 296-304. https://doi.org/10.1007/s10126-010-9300-4
  21. Webster, N.S., Negri, A.P., Munro, M.M., and Battershill, C.N. 2004. Diverse microbial communities inhabit Antarctic sponges. Environ. Microbiol. 6, 288-300. https://doi.org/10.1111/j.1462-2920.2004.00570.x
  22. Zhang, H., Lee, Y.K., Zhang, W., and Lee, H.K. 2006. Culturable actinobacteria from the marine sponge Hymeniacidon perleve: isolation and phylogenetic diversity by 16S rRNA gene-RFLP analysis. Antonie van Leeuwenhoek 90, 159-169. https://doi.org/10.1007/s10482-006-9070-1

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