Microbial Ecology

, Volume 51, Issue 4, pp 479–486 | Cite as

Denaturing Gradient Gel Electrophoresis Can Rapidly Display the Bacterial Diversity Contained in 16S rDNA Clone Libraries

  • M. D. Burr
  • S. J. Clark
  • C. R. Spear
  • A. K. Camper
Article

Abstract

Two different strategies for molecular analysis of bacterial diversity, 16S rDNA cloning and denaturing gradient gel electrophoresis (DGGE), were combined into a single protocol that took advantage of the best attributes of each: the ability of cloning to package DNA sequence information and the ability of DGGE to display a community profile. In this combined protocol, polymerase chain reaction products from environmental DNA were cloned, and then DGGE was used to screen the clone libraries. Both individual clones and pools of randomly selected clones were analyzed by DGGE, and these migration patterns were compared to the conventional DGGE profile produced directly from environmental DNA. For two simple bacterial communities (biofilm from a humics-fed laboratory reactor and planktonic bacteria filtered from an urban freshwater pond), pools of 35–50 clones produced DGGE profiles that contained most of the bands visible in the conventional DGGE profiles, indicating that the clone pools were adequate for identifying the dominant genotypes. However, DGGE profiles of two different pools of 50 clones from a lawn soil clone library were distinctly different from each other and from the conventional DGGE profile, indicating that this small number of clones poorly represented the bacterial diversity in soil. Individual clones with the same apparent DGGE mobility as prominent bands in the humics reactor community profiles were sequenced from the clone plasmid DNA rather than from bands excised from the gel. Because a longer fragment was cloned (∼1500 bp) than was actually analyzed in DGGE (∼350 bp), far more sequence information was available using this approach that could have been recovered from an excised gel band. This clone/DGGE protocol permitted rapid analysis of the microbial diversity in the two moderately complex systems, but was limited in its ability to represent the diversity in the soil microbial community. Nonetheless, clone/DGGE is a promising strategy for fractionating diverse microbial communities into manageable subsets consisting of small pools of clones.

References

  1. 1.
    Altschul, SF, Madden, TL, Schaffer, AA, Zhang, JH, Zhang, Z, Miller, W, Lipman, DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25(17): 3389–3402PubMedCrossRefGoogle Scholar
  2. 2.
    Amann, RI, Ludwig, W, Schleifer, KH (1995) Phylogenetic identification and in-situ detection of individual microbial cells without cultivation. Microbiol Rev 59(1): 143–169PubMedGoogle Scholar
  3. 3.
    Avrhami, S, Conrad, R (2003) Patterns of community change among ammonia oxidizers in meadow soils upon long-term incubation at different temperatures. Appl Environ Microbiol 69(10): 6152–6164CrossRefGoogle Scholar
  4. 4.
    Borneman, J, Triplett, EW (1997) Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl Environ Microbiol 63(7): 2647–2653PubMedGoogle Scholar
  5. 5.
    Dilly, O, Bloem, J, Vos, A, Munch, JC (2004) Bacterial diversity in agricultural soils during litter decomposition. Appl Environ Microbiol 70(1): 468–474PubMedCrossRefGoogle Scholar
  6. 6.
    Ellis, RJ, Morgan, P, Weightman, AJ, Fry, JC (2003) Cultivation-dependent and -independent approaches for determining bacterial diversity in heavy-metal-contaminated soil. Appl Environ Microbiol 69(6): 3223–3230PubMedCrossRefGoogle Scholar
  7. 7.
    Farrelly, V, Rainey, FA, Stackebrandt, E (1995) Effect of genome size and rrn gene copy number on PCR amplification of 16S ribosomal-RNA genes from a mixture of bacterial species. Appl Environ Microbiol 61(7): 2798–2801PubMedGoogle Scholar
  8. 8.
    Ferris, MJ, Muyzer, G, Ward, DM (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl Environ Microbiol 62(2): 340–346PubMedGoogle Scholar
  9. 9.
    Fromin, N, Hamelin, J, Tarnawski, S, Roesti, D, Jourdain-Miserez, K, Forestier, N, Teyssier-Cuvelle, S, Gillet, F, Aragno, M, Rossi, P (2002) Statistical analysis of denaturing gel electrophoresis (DGE) fingerprinting patterns. Environ Microbiol 4(11): 634–643PubMedCrossRefGoogle Scholar
  10. 10.
    Frostegard, A, Courtois, S, Ramisse, V, Clerc, S, Bernillon, D, Le Gall, F, Jeannin, P, Nesme, X, Simonet, P (1999) Quantification of bias related to the extraction of DNA directly from soils. Appl Environ Microbiol 65(12): 5409–5420PubMedGoogle Scholar
  11. 11.
    Girvan, MS, Bullimore, J, Pretty, JN, Osborn, AM, Ball, AS (2003) Soil type is the primary determinant of the composition of the total and active bacterial communities in arable soils. Appl Environ Microbiol 69(3): 1800–1809PubMedCrossRefGoogle Scholar
  12. 12.
    Holben, WE, Feris, KP, Kettunen, A, Apajalahti, JHA (2004) GC fractionation enhances microbial community diversity assessment and detection of minority populations of bacteria by denaturing gradient gel electrophoresis. Appl Environ Microbiol 70(4): 2263–2270PubMedCrossRefGoogle Scholar
  13. 13.
    Ibekwe, AM, Grieve, CM, Lyon, SR (2003) Characterization of microbial communities and composition in constructed dairy wetland wastewater effluent. Appl Environ Microbiol 69(9): 5060–5069PubMedCrossRefGoogle Scholar
  14. 14.
    Kelley, ST, Theisen, U, Angenent, LT, Amand, AS, Pace, NR (2004) Molecular analysis of shower curtain biofilm microbes. Appl Environ Microbiol 70(7): 4187–4192PubMedCrossRefGoogle Scholar
  15. 15.
    Kisand, V, Wikner, J (2003) Combining culture-dependent and -independent methodologies for estimation of richness of estuarine bacterioplankton consuming riverine dissolved organic matter. Appl Environ Microbiol 69(6): 3607–3616PubMedCrossRefGoogle Scholar
  16. 16.
    Lane, DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt, E, Goodfellow, M (Eds.) Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, Inc., New York, NY, pp 115–148Google Scholar
  17. 17.
    Liu, WT, Huang, CL, Hu, JY, Song, LF, Ong, SL, Ng, WJ (2002) Denaturing gradient gel electrophoresis polymorphism for rapid 16S rDNA clone screening and microbial diversity study. J Biosci Bioeng 93(1): 101–103Google Scholar
  18. 18.
    McGarvey, JA, Miller, WG, Sanchez, S, Stanker, L (2004) Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers. Appl Environ Microbiol 70(7): 4267–4275PubMedCrossRefGoogle Scholar
  19. 19.
    Muyzer, G, Dewaal, EC, Uitterlinden, AG (1993) Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S ribosomal-RNA. Appl Environ Microbiol 59(3): 695–700PubMedGoogle Scholar
  20. 20.
    Muyzer, G, Smalla, K (1998) Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Van Leeuwenhoek Int J Gen Mol Microbiol 73(1): 127–141CrossRefGoogle Scholar
  21. 21.
    Ogino, A, Koshikawa, H, Nakahara, T, Uchiyama, H (2001) Succession of microbial communities during a biostimulation process as evaluated by DGGE and clone library analyses. J Appl Microbiol 91(4): 625–635PubMedCrossRefGoogle Scholar
  22. 22.
    Palleroni, NJ (1997) Prokaryotic diversity and the importance of culturing. Antonie Van Leeuwenhoek Int J Gen Mol Microbiol 72(1): 3–19CrossRefGoogle Scholar
  23. 23.
    Polz, MF, Cavanaugh, CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol 64(10): 3724–3730PubMedGoogle Scholar
  24. 24.
    Sekiguchi, H, Tomioka, N, Nakahara, T, Uchiyama, H (2001) A single band does not always represent single bacterial strains in denaturing gradient gel electrophoresis analysis. Biotechnol Lett 23(15): 1205–1208CrossRefGoogle Scholar
  25. 25.
    Sekiguchi, H, Watanabe, M, Nakahara, T, Xu, BH, Uchiyama, H (2002) Succession of bacterial community structure along the Changjiang River determined by denaturing gradient gel electrophoresis and clone library analysis. Appl Environ Microbiol 68(10): 5142–5150PubMedCrossRefGoogle Scholar
  26. 26.
    Sun, HY, Deng, SP, Raun, WR (2004) Bacterial community structure and diversity in a century-old manure-treated agroecosystem. Appl Environ Microbiol 70(10): 5868–5874PubMedCrossRefGoogle Scholar
  27. 27.
    Tiedje, JM, Asuming-Brempong, S, Nusslein, K, Marsh, TL, Flynn, SJ (1999) Opening the black box of soil microbial diversity. Appl Soil Ecol 13(2): 109–122CrossRefGoogle Scholar
  28. 28.
    Torsvik, V, Daae, FL, Sandaa, RA, Ovreas, L (1998) Novel techniques for analysing microbial diversity in natural and perturbed environments. J Biotechnol 64(1): 53–62PubMedCrossRefGoogle Scholar
  29. 29.
    Torsvik, V, Goksoyr, J, Daae, FL (1990) High diversity in DNA of soil bacteria. Appl Environ Microbiol 56(3): 782–787PubMedGoogle Scholar
  30. 30.
    Webster, G, Newberry, CJ, Fry, JC, Weightman, AJ (2003) Assessment of bacterial community structure in the deep sub-seafloor biosphere by 16S rDNA-based techniques: a cautionary tale. J Microbiol Methods 55(1): 155–164PubMedCrossRefGoogle Scholar
  31. 31.
    Williams, MM, Domingo, JWS, Meckes, MC, Kelty, CA, Rochon, HS (2004) Phylogenetic diversity of drinking water bacteria in a distribution system simulator. J Appl Microbiol 96(5): 954–964PubMedCrossRefGoogle Scholar
  32. 32.
    Yang, YJ, Dungan, RS, Ibekwe, AM, Valenzuela-Solano, C, Crohn, DM, Crowley, DE (2003) Effect of organic mulches on soil bacterial communities one year after application. Biol Fertil Soils 38(5): 273–281CrossRefGoogle Scholar
  33. 33.
    Yu, ZT, Morrison, M (2004) Comparisons of different hypervariable regions of rrs genes for use in fingerprinting of microbial communities by PCR-denaturing gradient gel electrophoresis. Appl Environ Microbiol 70(8): 4800–4806PubMedCrossRefGoogle Scholar
  34. 34.
    Zhang, T, Fang, HHP (2000) Digitization of DGGE (denaturing gradient gel electrophoresis) profile and cluster analysis of microbial communities. Biotechnol Lett 22(5): 399–405CrossRefGoogle Scholar
  35. 35.
    Zhou, JZ, Xia, BC, Huang, H, Palumbo, AV, Tiedje, JM (2004) Microbial diversity and heterogeneity in sandy subsurface soils. Appl Environ Microbiol 70(3): 1723–1734PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, Inc. 2006

Authors and Affiliations

  • M. D. Burr
    • 1
  • S. J. Clark
    • 1
  • C. R. Spear
    • 1
  • A. K. Camper
    • 1
  1. 1.Center for Biofilm EngineeringMontana State UniversityBozemanUSA

Personalised recommendations