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Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments

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Abstract

Terminal Restriction Fragment Length Polymorphism (T-RFLP) or Fluorescent Polymerase Chain Reaction/Restriction Fragment Length Polymorphism (FluRFLP) have made a significant impact on the way in which PCR products amplified from mixed community DNA extracts have been assessed. Technically, these approaches are essentially the same. PCR products are generated that contain at one 5′ end label, typically a fluorescent moiety, that will be detected by a DNA sequencing machine. Upon digestion using a specific restriction endonuclease, labeled and unlabeled fragments are generated. This restriction endonuclease is chosen such that following this digestion, each labeled fragment corresponds to a different sequence variant. During electrophoretic separation, the DNA sequencing machine detects only these labeled fragments and therefore detects only the sequence variants. The aim of this article is to describe the protocois and demonstrate that this profiling can be performed using different DNA sequencing machines. The analysis and applications of this approach are also discussed.

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References

  1. Liu, W. T., Marsh, T. L., Cheng, H., and Forney, L. J. (1997) Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63, 4516–4522.

    PubMed  CAS  Google Scholar 

  2. Bruce, K. D. (1997) Analysis of mer gene sub-classes within bacterial communities in soils and sediments resolved by fluorescent PCR/restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 63, 4914–4919.

    PubMed  CAS  Google Scholar 

  3. Bruce, K. D. and Hughes, M. R. (1999) Fluorescent polymerase chain reaction/restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments, in Environmental Monitoring of Bacteria (Edwards, C., ed.), Humana, Towota, NJ, pp. 127–138.

    Chapter  Google Scholar 

  4. Amann, R. I., Ludwig, W., and Schleifer, K. H. (1995) Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59, 143–169.

    PubMed  CAS  Google Scholar 

  5. McDonald, I. R., Upton, M., Hall, G., Pickup, R. W., Edwards, C., Saunders, J. R., Ritchie, D. A., and Murrell, J. C. (1999) Molecular ecological analysis of methanogens and methanotrophs in blanket bog peat. Microb. Ecol. 38, 225–233.

    Article  PubMed  CAS  Google Scholar 

  6. Head, I. M., Saunders, J. R., and Pickup, R. W. (1998) Microbial evolution, diversity, and ecology: A decade of ribosomal RNA analysis of uncultivated microorganisms. Microb. Ecol. 35, 1–21.

    Article  PubMed  CAS  Google Scholar 

  7. van Elsas, J. D., Duarte, G. F., Rosado, A. S., and Smalla, K. (1998) Microbiological and molecular biological methods for monitoring microbial inoculants and their effects in the soil environment. J. Microbiol. Meth. 32, 133–154.

    Article  Google Scholar 

  8. Van de Peer, Y., Chapelle, S., and De Wachter, R. (1996) A quantitative map of nucleotide substitution rates in bacterial rRNA. Nucleic Acid Res. 24, 3381–3391.

    Article  PubMed  Google Scholar 

  9. Hiorns, W. D., Hastings, R. C., Head, I. M., McCarthy, A. J., Saunders, J. R., Pickup, R. W., and Hall, G. H. (1995) Amplification of 16S ribosomal RNA genes of autotrophic ammonia-oxidizing bacteria demonstrates the ubiquity of Nitrosospiras in the environment. Microbiol. 141, 2793–2800.

    Google Scholar 

  10. Degrange, V. and Bardin, R. (1995) Detection and counting of Nitrobacter populations in soil by PCR. Appl. Environ. Microbiol. 61, 2093–2098.

    PubMed  CAS  Google Scholar 

  11. Liesack, W. and Stackebrandt, E. (1992) Occurrence of novel groups of the domain bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J. Bacteriol. 174, 5072–5078.

    PubMed  CAS  Google Scholar 

  12. Muyzer, G., De Waal, E. C., and Uitterlinden, A. G. (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, 695–701.

    PubMed  CAS  Google Scholar 

  13. Muyzer, G. and 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, 127–141.

    Article  CAS  Google Scholar 

  14. Muyzer, G. (1999) DGGE/TGGE a method for identifying genes from natural ecosystems. Curr. Opin. Microbiol. 2, 317–322.

    Article  PubMed  CAS  Google Scholar 

  15. Heuer, H., Krsek, M., Baker, P., Smalla, K., and Wellington, E. M. H. (1997) Analysis of actinomycete communities by specific amplification of genes encoding 16S rRNA and gel-electrophoretic separation in denaturing gradients. Appl. Environ. Microbiol. 63, 3233–3241.

    PubMed  CAS  Google Scholar 

  16. Wise, M. G., McArthur, J. V., and Shimkets, L. J. (1999) Methanotroph diversity in landfill soil: Isolation of novel type I and type II methanotrophs whose presence was suggested by culture-independent 16S ribosomal DNA analysis. Appl. Environ. Microbiol. 65, 4887–4897.

    PubMed  CAS  Google Scholar 

  17. Kowalchuk, G. A., Stienstra, A. W., Heilig, G. H. J., Stephen, J. R., and Woldendorp, J. W. (2000) Molecular analysis of ammonia-oxidizing bacteria in soil of successional grasslands of the Drentsche A (The Netherlands). FEMS Microbiol. Ecol. 31, 207–215.

    Article  PubMed  CAS  Google Scholar 

  18. Overmann, J., Coolen, M. J. L., and Tuschak, C. (1999) Specific detection of different phylogenetic groups of chemocline bacteria based on PCR and denaturing gradient gel electrophoresis of 16S rRNA gene fragments. Arch. Microbiol. 172, 83–94.

    Article  PubMed  CAS  Google Scholar 

  19. Henckel, T., Friedrich, M., and Conrad, R. (1999) Molecular analyses of the methane-oxidizing microbial community in rice field soil by targeting the genes of the 16S rRNA, particulate methane monooxygenase, and methanol dehydrogenase. Appl. Environ. Microbiol. 65, 1980–1990.

    PubMed  CAS  Google Scholar 

  20. Felske, A. and Akkermans, A. D. L. (1998) Spatial homogeneity of abundant bacterial 16S rRNA molecules in grassland soils. Microb. Ecol. 36, 31–36.

    Article  PubMed  CAS  Google Scholar 

  21. Porteous, L. A., Armstrong, J. L., Seidler, R. J., and Watrud, L. S. (1994) An effective method to extract DNA from environmental samples for Polymerase Chain Reaction amplification and DNA fingerprint analysis. Curr. Microbiol. 29, 301–307.

    Article  PubMed  CAS  Google Scholar 

  22. Martinez-Murcia, A. J., Acinas, S. G., and Rodriguez-Valera, F. (1995) Evaluation of prokaryotic diversity by restriction digestion of 16S rDNA directly amplified from hypersaline environments. FEMS Microbiol. Ecol. 17, 247–256.

    CAS  Google Scholar 

  23. Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, WI.

  24. Levitt, R. C., Kiser, M. B., Dragwa, C., Jeducka, A. E., Xu, J., Meyers, D. A., and Hudson, J. R. (1994) Fluorescence-based resource for semiatutomated genomic analyses using microsatellite markers. Genomics 24, 361–365.

    Article  PubMed  CAS  Google Scholar 

  25. Suzuki, M. T. and Giovannoni, S. J. (1996) Bias caused by template annealing in the amplification of mixtures of 16S ribosomal RNA genes by PCR. Appl. Environ. Microbiol. 62, 625–630.

    PubMed  CAS  Google Scholar 

  26. Trevors, J. T. (1996) DNA in soil-adsorption, genetic transformation, molecular evolution and genetic microchip. Antonie van Leeuwenhoek Int. J. Gen. Mol. Microbiol. 70, 1–10.

    Article  CAS  Google Scholar 

  27. Wang, G. C. Y. and Wang, Y. (1996) The frequency of chimeric molecules as a consequence of PCR coamplification of 16S ribosomal RNA genes from different bacterial species. Microbiol. 142, 1107–1114.

    Article  CAS  Google Scholar 

  28. Marsh, T. L. (1999) Terminal restriction fragment length polymorphism (T-RFLP): an emerging method for characterizing diversity among homologous populations of amplification products. Curr. Opin. Microbiol. 2, 323–327.

    Article  PubMed  CAS  Google Scholar 

  29. Osborn, A. M., Moore, E. R. B., and Timmis, K. N. (2000) An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol. 2, 39–50.

    Article  PubMed  CAS  Google Scholar 

  30. Moeseneder, M. M., Arrieta, J. M., Muyzer, G., Winter, C., and Herndl, G. J. (1999) Optimization of terminal-restriction fragment length polymorphism analysis for complex marine bacterioplankton communities and comparison with denaturing gradient gel electrophoresis. Appl. Environ. Microbiol. 65, 3518–3525.

    PubMed  CAS  Google Scholar 

  31. Flynn, S. J., Loffler, F. E., and Tiedje, J. M. (2000) Microbial community changes associated with a shift from reductive dechlorination of PCE to reductive dechlorination of cis-DCE and VC. Environ. Sci. Technol. 34, 1056–1061.

    Article  CAS  Google Scholar 

  32. Kerkhof, L., Santoro, M., and Garland, J. (2000) Response of soybean rhizosphere communities to human hygiene water addition as determined by community level physiological profiling (CLPP) and terminal restriction fragment length polymorphism (T-RFLP) analysis. FEMS Microbiol. Lett. 184, 95–101.

    Article  PubMed  CAS  Google Scholar 

  33. Tiedje, J. M., Asuming-Brempong, S., Nusslein, K., Marsh, T. L., and Flynn, S. J. (1999) Opening the black box of soil microbial diversity. Appl. Soil Ecol. 13, 109–122.

    Article  Google Scholar 

  34. Maidak, B. L., Cole, J. R., Lilburn, T. G., Parker, C. T., Saxman, P. R., Stredwick, J. M., et al. (2000) The RDP (Ribosomal Database Project) continues. Nucleic Acid Res. 28, 173, 174.

    Article  PubMed  CAS  Google Scholar 

  35. Barkay, T., Liebert, C., and Gillman, M. (1989) Hybridization of DNA probes with whole-community genome for detection of genes that encode microbial responses to pollutants: mer genes and Hg2+ resistance. Appl. Environ. Microbiol. 55, 1574–1577.

    PubMed  CAS  Google Scholar 

  36. Rochelle, P. A., Wetherbee M. K., and Olson, B. H. (1991) Distribution of DNA sequences encoding narrow- and broad-spectrum resistance mercury resistance. Appl. Environ. Microbiol. 57, 1581–1589.

    PubMed  CAS  Google Scholar 

  37. Hobman, J. L. and Brown, N. L. (1996) Bacterial mercury resistance genes, in Metal ions in Biological Systems (Sigel, H. and Sigel, A. eds.), Marcel Dekker, New York, pp. 527–568.

    Google Scholar 

  38. Brown, N. L., Ford, S. J., Pridmore, R. D., and Fritzinger, D. C. (1983) Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercury resistance. Biochem. 22, 4089–4095.

    Article  CAS  Google Scholar 

  39. Barrineau, P., Gilbert, P., Jackson, W. J., Jones, C. S., Summers, A. O., and Wisdom, S. (1984) The DNA sequence of the mercury resistance operon of the Inc EII plasmid NR1. J. Mol. Appl. Genet. 2, 601–619.

    PubMed  CAS  Google Scholar 

  40. Kholodii, G. Y., Gorlenko, Z. M., Lomovskaya, O. L., Mindlin, S. Z., Yurieva, O. V., and Nikiforov, V. G. (1993) Molecular characterisation of an aberrant mercury resistance transposable element from an environmental Acinetobacter strain. Plasmid 30, 303–308.

    Article  PubMed  CAS  Google Scholar 

  41. Griffin, H. G., Foster, T. J., Silver, S., and Misra, T. K. (1987) Cloning and DNA sequence of the mercuric- and organomercurial resistance determinants of plasmid pDU1358. Proc. Natl. Acad. Sci. USA 84, 3112–3116.

    Article  PubMed  CAS  Google Scholar 

  42. Osborn, A. M., Bruce, K. D., Strike, P., and Ritchie, D. A. (1995) Sequence conservation between regulatory mercury resistance genes in bacteria from mercury polluted and pristine environments. Syst. Appl. Microbiol. 18, 1–6.

    CAS  Google Scholar 

  43. Bruce, K. D., Osborn, A. M., Pearson, A. J., Strike, P., and Ritchie, D. A. (1995) Genetic diversity within mer genes directly amplified from communites of non-cultivated soil and sediment bacteria. Mol. Ecol. 4, 605–612.

    PubMed  CAS  Google Scholar 

  44. Brown, D. J. S. and Brown, T. (1995) In PCR: essential data (Newton, C. R., ed.) Wiley, New York, pp. 57–70.

    Google Scholar 

  45. Grzybowski, J., McPhilips, F., and Brown, T. (1995) In PCR: essential data (Newton, C. R., ed.) Wiley, New York, pp. 93–98.

    Google Scholar 

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Author notes

  1. Kenneth D. Bruce & Mark R. Hughes

    Present address: Department of Biological Sciences, Donnan Labs, University of Liverpool, L69 7ZD, Liverpool, UK

Authors and Affiliations

  1. Division of Life Sciences, Franklin-Wilkins Building, 150 Stamford Street, King’s College, SE1 8WA, London, UK

    Kenneth D. Bruce

  2. MWG-BIOTECH UK Ltd, Waterside House, Peartree Bridge, MK6 3BY, Milton Keynes, UK

    Mark R. Hughes

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  1. Kenneth D. Bruce
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Correspondence to Kenneth D. Bruce.

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Bruce, K.D., Hughes, M.R. Terminal restriction fragment length polymorphism monitoring of genes amplified directly from bacterial communities in soils and sediments. Mol Biotechnol 16, 261–269 (2000). https://doi.org/10.1385/MB:16:3:261

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