Browse > Article
http://dx.doi.org/10.5657/kfas.2007.40.6.356

Diversity of Marine Microbes by PCR-DGGE  

Kim, Yeong-Jin (Department of Microbiology and Immunology, College of Medicine, Pusan National University)
Cho, Hyo-Jin (Department of Microbiology and Immunology, College of Medicine, Pusan National University)
Yu, Sun-Nyoung (Department of Microbiology and Immunology, College of Medicine, Pusan National University)
Kim, Kwang-Youn (Department of Microbiology and Immunology, College of Medicine, Pusan National University)
Kim, Hyeung-Rak (Division of Food Science and Biotechnology, Pukyung National University)
Ahn, Soon-Cheol (Department of Microbiology and Immunology, College of Medicine, Pusan National University)
Publication Information
Korean Journal of Fisheries and Aquatic Sciences / v.40, no.6, 2007 , pp. 356-361 More about this Journal
Abstract
Recently, the development of various culture-independent identification techniques for environmental microbes has greatly enhanced our knowledge of microbial diversity. In particular, denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments, generated using the polymerase chain reaction (PCR) is frequently used to examine the diversity of environmental bacterial populations. This method consists of direct extraction of the environmental DNA, amplification of the 200-600 bp 16S rDNA fragments with universal primers, and separation of the fragments according to their melting point on a denaturing gradient gel. In this study, we investigated the seaside microbial community in coastal areas of Busan, Korea, using culture-independent techniques. First, marine genomic DNA was extracted from seawater samples collected at Songjeong, Gwangahn, and Songdo Beaches. Then, PCR was used to amplify the bacterial 16S rDNA using universal primers, and DGGE was used to separate the amplified 500 bp 16S rDNA fragments. Finally, the tested 16S rDNA genes were further analyzed by sequencing. Based on these experiments, we found that DGGE analysis clearly showed variation among the regional groups. It can be used to monitor rapid changes in the bacterial diversity of various environments. In addition, the sequence analysis indicated the existence of many unculturable bacteria, in addition to Arcobacter, Pseudoaltermonas, and Vibrio species.
Keywords
Culture-independent; 16S rDNA; PCR; DGGE;
Citations & Related Records
연도 인용수 순위
  • Reference
1 Famteitner, AH., F. Zibuschka, M.M. Burtscher, G. Lindner, G. Reischer and R.L. Mach. 2004. Eubacterial 16S-rDNA amplicon profiling: a rapid technique for comparison and differentiation of heterotrophic plate count communities from drinking water. Int. J. Food Microbiol., 92, 333-375   DOI   ScienceOn
2 Muyzer, G., 1999. DGGE/TGGE a method for identifying genes from natural ecosystems. Curr. Opin. in Microbiol., 2, 317-322   DOI   ScienceOn
3 Nakagawa, T., S. Sato, Y. Yamamoto and M. Fukui. 2002. Successive changes in community structure of an ethylbenzene-degrading sulfate-reducing consortium. Water Res., 36, 2813-2823   DOI   ScienceOn
4 Reinheimer, G. 1985. Aquatic Microbiology, 3nd ed., Wiley and Sons. London, UK, 158-159
5 Thompson, J.D., D.G. Higgins and T.J. Gibson. 1994. CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res., 22, 4673-4680   DOI
6 Rodriguez-Valera, F. 2002. Approaches to prokaryotic biodiversity: a population genetics perspective. Environ. Microbiol., 4, 628-633   DOI   ScienceOn
7 Altschul, S.F., T.L. Madden, AA Schaffer, J. Zhang, Z. Zhang, W. Miller and D.J. Lipman. 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucl. Acids Res., 25, 3398-3402
8 Muyzer, G., E.C. de Waal and A.G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol., 59, 695-700
9 Rondon, M.R., P.R. August, AD. Bettermann, S.F. Brady, T.H. Grossman, M.R. Liles, K.A. Loiacono, B.A Lynch, LA MacNeil, C. Minor, C.L. Tiong, M. Gilman, M.S. Osbume, J. Clardy, J. Handelsman and R.M. Goodman. 2000. Cloning the soil metagenome: a strategy for accessing the genetic and functional diversity of uncultured microorganisms. Appl. Environ. Microbiol. 66, 2541-2547   DOI
10 Wimpenny, J.W.T., W.L. Robert and C. Philip. 1983. Laboratory model systems for the investigation of specialty and temporally organized microbial eco systems. In: Microbes in Their Natural Environments, Society for General Microbiology, ed. Cambridge University Press, New York, USA, 67-117
11 Novitsky, J.A. 1983. Starvation-survival of heterotrphs in the marine environments. Adv. Microbiol. Ecol., 6, 171-198
12 Watanabe, K., Y. Kodama and S. Harayama. 2001. Design and evaluation of PCR primers to amplify bacterial 16S ribosomal DNA fragments used for community fingerprinting. J. Microbiol. Meth., 44, 253-262   DOI   ScienceOn
13 Cole, J.R., B. Chai, T.L. Marsh, R.J. Farris, Q. Wang, S.A Kulam, S. Chandra, D.M. McGarrell, T.M. Schmidt, G.M. Garrity and J.M. Tiedje. 2003. The Ribosomal Database Project (RDP-II): preview a new autoaligner that allows regular updates and the new prokaryotic taxonomy. Nucl. Acids Res., 31, 442-443   DOI   ScienceOn
14 Muyzer, G., T. Brinkhoff, U. Niibel, C. Santegoeds, H. Schafer and C. Wawer. 1998. Denaturing gradient gel electrophoresis (DGGE) in microbial ecology. In: Molecular Microbial Ecology Manual, Vol. 3.4.4. Akkermans, A.D.L., J.D. van Elsas and F.J. de Bruijn, eds. Kluwer Academic Publishers, Dordrecht, The Netherlands. 1-27