Determination of microbial diversity in environmental samples: Pitfalls of PCR-based rRNA analysis

FEMS Microbiology Reviews

Volumn 21, Issue 3, 1997, Pages 213-229

  Wintzingerode, Friedrich V ^a   Göbel, Ulf B ^a   Stackebrandt, Erko ^b  

^a CHARITÉ UNIVERSITÄTSMEDIZIN BERLIN   (Germany)

^b LEIBNIZ INSTITUTE DSMZ GERMAN COLLECTION OF MICROORGANISMS AND CELL CULTURES   (Germany)

Author keywords

16S rRNA gene; Microbial diversity; Nucleic acid; Phylogenetic analysis; Polymerase chain reaction

Indexed keywords

RIBOSOME RNA;

BIODIVERSITY; DNA DETERMINATION; ENVIRONMENT; NONHUMAN; PHYLOGENY; POLYMERASE CHAIN REACTION; PROKARYOTE; REVIEW; RNA ANALYSIS; RNA SEQUENCE;

PROKARYOTA;

EID: 0345195995     PISSN: 01686445     EISSN: None     Source Type: Journal    
DOI: 10.1016/S0168-6445(97)00057-0     Document Type: Article

References (123)

1. Stahl, D.A., Flesher, B., Mansfield, H.R. and Montgomery, L. (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54, 1079-1084.
2. Pace, N.R., Stahl, D.A., Lane, D.J. and Olsen, G.J. (1986) The analysis of natural microbial populations by ribosomal RNA sequences. Adv. Microb. Ecol. 9, 1-55.
3. Seewaldt, E. and Stackebrandt, E. (1982) Partial sequence of 16S ribosomal RNA and the phylogeny of Prochloron. Nature 295, 618-620.
4. Schmidt, T.M., De Long, E.F. and Pace, N.R. (1991) Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J. Bacteriol. 173, 4371-4378.
5. Ward, D.M., Weller, R. and Bateson, M.M. (1990) 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature 345, 63-65.
6. Giovannoni, S.J., Britschgi, T.B., Moyer, C.L. and Field, K. G. (1990) Genetic diversity in Sargasso Sea bacterioplankton. Nature 345, 60-63.
7. 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, 695-700.
8. Nübel, U., Engelen, B., Felske, A., Snaidr, J., Wieshuber, A., Amann, R.I., Ludwig, W. and Backhaus, H. (1996) Sequence heterogeneities of genes encoding 16S rRNAs in Paenibacillus polymyxa detected by temperature gradient gel electrophoresis. J. Bacteriol. 178, 5636-5643.
9. Liesack, W. and Stackebrandt, E. (1992) Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material from an Australian terrestrial environment. J. Bacteriol. 174, 5072-5078.
10. Köhler, G., Ludwig, W. and Schleifer, K.H. (1991) Differentiation of lactococci by rRNA gene restriction analysis. FEMS Microbiol. Lett. 84, 307-312.
11. Weidner, S., Arnold, W. and Pühler, A. (1996) Diversity of uncultured microorganisms associated with the seagrass Halophila stipulacea estimated by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes. Appl. Environ. Microbiol. 62, 766-771.
12. Ohkuma, M. and Kudo, T. (1996) Phylogenetic diversity of the intestinal bacterial community in the Termite Reticulitermes speratus. Appl. Environ. Microbiol. 62, 461-468.
13. Choi, B.K., Paster, B.J., Dewhirst, F.E. and Göbel, U.B. (1994) Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis. Infect. Immun. 62, 1889-1895.
14. Mirza, M.S., Hameed, S. and Akkermans, A.D.L. (1994) Genetic diversity of Datisca cannabina-compatible Frankia strains as determined by sequence analysis of the PCR-amplified 16S rRNA gene. Appl. Environ. Microbiol. 60, 2371-2376.
15. Choi, B.K., Nattermann, H., Grund, S., Haider, W. and Göbel, U.B. (1997) Spirochetes from digital dermatitis lesions in cattle are closely related to treponemes associated with human periodontitis. Int. J. Syst. Bacteriol. 47, 175-181.
16. Polz, M.F., Distel, D.L., Zarda, B., Amann, R.I., Felbeck, H., Ott, J.A. and Cavanaugh, C.M. (1994) Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode. Appl. Environ. Microbiol. 60, 4461-4467.
17. Goebel, B.M. and Stackebrandt, E. (1996) Molecular analysis of the microbial diversity in a natural acidic environment. In: Biohydrometallurgical Processing (Jerez, C.A., Vargas, T., Toledo, H. and Wiertz, J.V., Eds.), pp. 43-52. University Press, University of Chile, Santiago.
18. Borneman, J., Skroch, P.W., O'Sullivan, K.M., Palus, J.A., Rumjanek, N.G., Jansen, J.L., Nienhuis, J. and Triplett, E.W. (1996) Molecular microbial diversity of an agricultural soil in Wisconsin. Appl. Environ. Microbiol. 62, 1935-1943.
19. Schuppler, M., Mertens, F., Schön, G. and Göbel, U.B. (1995) Molecular characterization of nocardioform actinomycetes in activated sludge by 16S rRNA analysis. Microbiology 141, 513-521.
20. Rheims, H., Rainey, F.A. and Stackebrandt, E. (1996) A molecular approach to search for diversity among bacteria in the environment. J. Ind. Microbiol. 17, 159-169.
21. Zhou, J., Bruns, M.A. and Tiedje, J.M. (1996) DNA recovery from soils of diverse composition. Appl. Environ. Microbiol. 62, 316-322.
22. Bond, P.L., Hugenholtz, P., Keller, J. and Blackall, L.L. (1995) Bacterial community structures of phosphate-removing and non-phosphate-removing activated sludges from sequencing batch reactors. Appl. Environ. Microbiol. 61, 1910-1916.
23. Pedersen, K., Arlinger, J., Ekendahl, S. and Hallbeck, L. (1996) 16S rRNA gene diversity of attached and unattached bacteria in boreholes along the access tunnel to the Aspo hard rock laboratory, Sweden. FEMS Microbiol. Ecol. 19, 249-262.
24. Ferris, M.J., Muyzer, G. and Ward, D.M. (1996) Denaturing gradient gel electrophoresis profiles of 16S rRNA-defined populations inhabiting a hot spring microbial mat community. Appl. Environ. Microbiol. 62, 340-346.
25. Rochelle, P.A., Cragg, B.A., Fry, J.C., Parkes, R.J. and Weightman, A.J. (1994) Effect of sample handling on estimation of bacterial diversity in marine sediments by 16S rRNA gene sequence analysis. FEMS Microbiol. Ecol. 15, 215-225.
26. Gordon, D.A. and Giovannoni, S.J. (1996) Detection of stratified microbial populations related to Chlorobium and Fibrobacter species in the Atlantic and Pacific oceans. Appl. Environ. Microbiol. 62, 1171-1177.
27. Muralidharan, K. and Wemmer, C. (1994) Transporting and storing field-collected specimens for DNA without refrigeration for subsequent DNA extraction and analysis. BioTechniques 17, 420-422.
28. Liesack, W., Weyland, H. and Stackebrandt, E. (1991) Potential risks of gene amplification by PCR as determined by 16S rDNA analysis of a mixed culture of strict barophilic bacteria. Microb. Ecol. 21, 191-198.
29. Leff, L.G., Dana, J.R., McArthur, J.V. and Shimkets, L.J. (1995) Comparison of methods of DNA extraction from stream sediments. Appl. Environ. Microbiol. 61, 1141-1143.
30. Ogram, A., Sayler, G.S. and Barkay, T. (1987) The extraction and purification of microbial DNA from sediments. J. Microbiol. Methods 7, 57-66.
31. Tsai, Y.L. and Olsen, B.H. (1991) Rapid method for direct extraction of DNA from soil and sediments. Appl. Environ. Microbiol. 57, 1070-1074.
32. Jacobsen, C.S. and Rasmussen, O.F. (1992) Development and application of a new method to extract bacterial DNA from soil based on separation of bacteria from soil with cation-exchange resin. Appl. Environ. Microbiol. 58, 2458-2462.
33. Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R. and Stahl, D.A. (1990) Combination of 16S rRNA- targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl. Environ. Microbiol. 56, 1919-1925.
34. Volossiouk, T., Robb, E.J. and Nazar, R.N. (1995) Direct DNA extraction for PCR mediated assays of soil organisms. Appl. Environ. Microbiol. 61, 3972-3976.
35. Picard, C., Ponsonnet, C., Paget, E., Nesme, X. and Simonet, P. (1992) Detection and enumeration of bacteria in soil by direct DNA extraction and polymerase chain reaction. Appl. Environ. Microbiol. 58, 2717-2722.
36. Tsai, Y.-L. and Olson, B.H. (1992) Rapid method for separation of bacterial DNA from humic substances in sediments for PCR. Appl. Environ. Microbiol. 58, 2292-2295.
37. Smalla, K., Cresswell, N., Mendoca-Hagler, L.C., Wolters, A. and van Elsas, J.D. (1993) Rapid DNA extraction protocol from soil for polymerase chain reaction-mediated amplification. J. Appl. Bacteriol. 74, 78-85.
38. Tebbe, C.C. and Vahjen, W. (1993) Interference of humic acids and DNA extracted directly from soil in detection and transformation of recombinant DNA from bacteria and yeast. Appl. Environ. Microbiol. 59, 2657-2665.
39. Porteous, L.A. and Armstrong, J.L. (1991) Recovery of bulk DNA from soil by a rapid, small-scale extraction method. Curr. Microbiol. 22, 345-348.
40. Steffan, R.J. and Atlas, R.M. (1988) DNA amplification to enhance detection of genetically engineered bacteria in environmental samples. Appl. Environ. Microbiol. 54, 2185-2191.
41. More, M.I., Herrick, J.B., Silva, M.C., Ghiorse, W.C. and Madsen, E.L.. (1994) Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Appl. Environ. Microbiol. 60, 1572-1580.
42. Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhl, K. (1990) Current Protocols in Molecular Biology, Vol. 2. Greene/Wiley-Interscience, New York.
43. Holben, W.E., Jansson, J.K., Chelm, B.K. and Tiedje, J.M. (1988) DNA probe method for the detection of specific microorganisms in the soil bacterial community. Appl. Environ. Microbiol. 54, 703-711.
44. Barns, S.M., Fundyga, R.E., Jeffries, M.W. and Pace, N.R. (1994) Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc. Natl. Acad. Sci. USA 91, 1609-1613.
45. Somerville, C.C., Knight, I.T., Straube, W.L. and Colwell, R.R. (1989) Simple, rapid method for direct isolation of nucleic acids from aquatic environments. Appl. Environ. Microbiol. 55, 548-554.
46. Moran, M.A., Torsvik, V.L., Torsvik, T. and Hodson, R.E. (1993) Direct extraction and purification of rRNA for ecological studies. Appl. Environ. Microbiol. 59, 915-918.
47. Ogram, A., Sun, W., Brockman, F.J. and Frederickson, J.K. (1995) Isolation and characterization of RNA from low-biomass deep-subsurface sediments. Appl. Environ. Microbiol. 61, 763-768.
48. Felske, A., Engelen, B., Nübel, U. and Backhaus, H. (1996) Direct ribosome isolation from soil to extract bacterial rRNA for community analysis. Appl. Environ. Microbiol. 62, 4162-4167.
49. Stackebrandt, E. (1992) Molecular microbial ecology. In: Encyclopedia of Microbiology (Lederberg, I., Ed.), Vol. 3, pp. 171-179, Academic Press, New York.
50. Stackebrandt, E., Liesack, W. and Witt, D. (1992) Ribosomal RNA and rDNA sequence analyses. Gene 115, 255-260.
51. Lane, D.J., Pace, B., Olsen, G.J., Stahl, D.A., Sogin, M.L. and Pace, N.R. (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc. Natl. Acad. Sci. USA 82, 6955-6959.
52. Weisburg, W.G., Barns, S.M., Pelletier, D.A. and Lane, D.J. (1991) 16S ribosomal DNA amplification for phylogenetic study. J. Bacteriol. 173, 697-703.
53. Jacobsen, C.S. (1995) Microscale detection of specific bacterial DNA in soil with a magnetic capture-hybridization and per amplification assay. Appl. Environ. Microbiol. 61, 3347-3352.
54. Kreader, C.A. (1996) Relief of amplification in PCR with bovine serum albumin or T4 gene 32 protein. Appl. Environ. Microbiol. 62, 1102-1106.
55. McKeown, B.J. (1994) An acetylated (nuclease-free) bovine serum albumin in a PCR buffer inhibits amplification. BioTechniques 2, 247-248.
56. Stinear, T., Matusan, A., Hines, K. and Sandery, M. (1996) Detection of a single viable Cryptosporidium parvum oocyst in environmental water concentrates by reverse transcription-PCR. Appl. Environ. Microbiol. 62, 3385-3390.
57. Suzuki, M.T. and Giovannoni, S.J. (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62, 625-630.
58. Farrelly, V., Rainey, F.A. and Stackebrandt, E. (1995) Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species. Appl. Environ. Microbiol. 61, 2798-2801.
59. Brunk, C.F., Avaniss-Aghajani, E. and Brunk, C.A. (1996) A computer analysis of primer and probe hybridization potential with bacterial small-subunit rRNA sequences. Appl. Environ. Microbiol. 62, 872-879.
60. Larsen, N., Olsen, G.J., Maidak, B.L., McCaughey, M.J., Overbeek, R., Macke, T.J., Marsh, T.L. and Woese, C.R. (1993) The ribosomal database project. Nucleic Acids Res. 21, 3021-3023.
61. Zheng, D., Alm, E.W., Stahl, D.A. and Raskin, L. (1996) Characterization of universal small-subunit rRNA hybridization probes for quantitative molecular microbial ecology studies. Appl. Environ. Microbiol. 62, 4504-4513.
62. Paster, B.J., Pelletier, D.A., Dewhirst, F.E., Weisburg, W.G., Fussing, V., Poulsen, L.K., Dannenberg, S. and Schroeder, I. (1996) Phylogenetic position of the spirochetal genus Cristispira. Appl. Environ. Microbiol. 62, 942-946.
63. Rainey, F.A., Ward, N., Sly, L.I. and Stackebrandt, E. (1994) Dependence on the taxon composition of clone libraries for PCR amplified, naturally occurring 16S rDNA, on the primer pair and the cloning system used. Experientia 50, 796-797.
64. Maidak, B.L., Olsen, G.J., Larsen, N., Overbeek, R., McCaughey, M.J. and Woese, C.R. (1996) The Ribosomal Database Project (RDP). Nucleic Acids Res. 24, 82-85.
65. Strunk, O. and Ludwig, W. (n.d.) ARB Software Package. Technical University, Munich.
66. Reysenbach, A.-L., Giver, L.J., Wickham, G.S. and Pace, N.R. (1992) Differential amplification of rRNA genes by polymerase chain reaction. Appl. Environ. Microbiol. 58, 3417-3418.
67. Baskaran, N., Kandpal, R.P., Bhargava, A.K., Glynn, M.W., Bale, A. and Weissman, S.M. (1996) Uniform amplification of a mixture of deoxyribonucleic acids with varying GC content. Genome Res. 6, 633-638.
68. Chandler, D.P., Fredrickson, J.K. and Brockman, F.J. (1997) Effect of PCR template concentration on the composition and distribution of total community 16S rDNA clone libraries. Mol. Ecol. 6, 475-482.
69. Waterhouse, R.N. and Glover, L.A. (1993) Differences in the hybridization pattern of Bacillus subtilis genes coding for rRNA depend on the method of DNA preparation. Appl. Environ. Microbiol. 59, 919-921.
70. Shuldiner, A.R., Nirula, A. and Roth, J. (1989) Hybrid DNA artefact from PCR of closely related target sequences. Nucleic Acids Res. 17, 4409.
71. Meyerhans, A., Vartanian, J.-P. and Wain-Hobson, S. (1990) DNA recombination during PCR. Nucleic Acids Res. 18, 1687-1691.
72. Wang, G.C.Y. and Wang, Y. (1996) The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology 142, 1107-1114.
73. Ford, J.E., McHeyzer-Williams, M.G. and Lieber, M.R. (1994) Chimeric molecules created by gene amplification interfere with the analysis of somatic hypermutation of murine immunoglobulin genes. Gene 142, 279-283.
74. Pääbo, S., Irwin, D.M. and Wilson, A.C. (1990) DNA damage promotes jumping between templates during enzymatic amplification. J. Biol. Chem. 265, 4718-4721.
75. Brakenhoff, R.H., Schoenmakers, J.G.G. and Lubsen, N.H. (1991) Chimeric cDNA clones: a novel PCR artifact. Nucleic Acids Res. 19, 1949.
76. Cariello, N.F., Thilly, W.G., Swenberg, J.A. and Skopek, T.R. (1991) Deletion mutagenesis during polymerase chain reaction: dependence on DNA polymerase. Gene 99, 105-108.
77. Pallansch, L., Beswick, H., Talian, J. and Zelenka, P. (1990) Use of an RNA folding algorithm to choose regions for amplification by the polymerase chain reaction. Anal. Biochem. 185, 57-62.
78. Holstege, F.C.P., van der Vliet, P.C. and Timmers, H.T.M. (1994) Instability of human TATA-binding protein CAG triplet repeats during amplification by PCR. Biochim. Biophys. Acta 1219, 157-159.
79. Gutell, R.R., Larsen, N. and Woese, C.R. (1994) Lessons from an evolving rRNA: 16S and 23S rRNA structures from a comparative perspective. Microbiol. Rev. 58, 10-26.
80. Chou, Q. (1992) Minimizing deletion mutagenesis artifact during Taq DNA polymerase PCR by E. coli SSB. Nucleic Acids Res. 20, 4371.
81. Gelfand, D.H. (1992) Taq DNA polymerase, PCR technology. In: Principles and Applications for DNA Amplification (Erlich, H.A., Ed.), pp. 17-22. Freeman and Company, New York.
82. Eckert, K.A. and Kunkel, T.A. (1991) DNA polymerase fidelity and the polymerase chain reaction. PCR Methods Appl. 1, 17-24.
83. Roberts, J.D., Bebenek, K. and Kunkel, T.A. (1988) The accuracy of reverse transcriptase from HIV-1. Science 242, 1171-1173.
84. Stewart, A.C.M., Gravitt, P.E., Cheng, S. and Wheeler, C.M. (1995) Generation of entire human papillomavirus genomes by long PCR : frequency of errors produced during amplification. PCR Methods Appl. 5, 79-88.
85. Lundberg, K.S., Shoemaker, D.D., Adams, M.W.W., Short, J.M., Sorge, J.A. and Mathur, E.J. (1991) High-fidelity amplification using a thermostable DNA polymerase isolated from Pyrococcus furiosus. Gene 108, 1-6.
86. Böttger, E.C. (1990) Frequent contamination of Taq polymerase with DNA. Clin. Chem. 36, 1258-1259.
87. Rand, K.H. and Houck, H. (1990) Taq polymerase contains bacterial DNA of unknown origin. Mol. Cell. Probes 4, 445-450.
88. Schmidt, T.M., Pace, B. and Pace, N.R. (1991) Detection of DNA contamination in Taq polymerase. BioTechniques 11, 176-177.
89. Maiwald, M., Ditton, H.-J., Sonntag, H.-G. and von Knebel Doeberitz, M. (1994) Characterization of contaminating DNA in Taq polymerase which occurs during amplification with a primer set for Legionella 5S ribosomal RNA. Mol. Cell. Probes 8, 11-14.
90. Niederhauser, C., Höfelein, C., Wegmüller, B. and Candrian, U. (1994) Reliability of PCR decontamination systems. PCR Methods Appl. 4, 117-123.
91. Krawiec, S. and Riley, M. (1990) Organization of the bacterial chromosome. Microbiol. Rev. 54, 502-539.
92. Kündig, C., Beck, C., Hennecke, H. and Göttfert, M. (1995) A single rRNA gene region in Bradyrhizobium japonicum. J. Bacteriol. 177, 5151-5154.
93. La Fontaine, S. and Rood, J.I. (1996) Organization of ribosomal RNA genes from the footrot pathogen Dichelobacter nodosus. Microbiology 142, 889-899.
94. Stewart, G.G., Wilson, F.E. and Bott, K.F. (1982) Detailed physical mapping of the ribosomal RNA genes of Bacillus subtilis. Gene 19, 153-162.
95. Johansen, T. and Kolsto, C.R. (1996) Variable numbers of rRNA gene operons in Bacillus cereus strains. FEMS Microbiol. Lett. 136, 325-328.
96. T contains multiple 16S rRNA genes with heterogenous intervening sequences. Microbiology 142, 2087-2095.
97. Mylvaganam, S. and Dennis, P.P. (1992) Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic Archaebacterium Haloarcula marismortui. Genetics 130, 399-410.
98. Wang, Y., Zhang, Z. and Narendrakumar, R. (1997) The actinomycete Thermobispora bispora contains two distinct types of transcriptionally active 16S rRNA genes. J. Bacteriol. 179, 3270-3276.
99. Ogasawara, N., Nakai, S. and Yoshikawa, H. (1994) Systematic sequencing of the 180 kilobase region of the Bacillus subtilis chromosome containing the replication origin. DNA Res. 1, 1-14.
100. Dryden, S.C. and Kaplan, S. (1990) Localization and structural analysis of the ribosomal RNA operons of Rhodobacter sphaeroides. Nucleic Acids Res. 18, 7267-7277.
101. Fleischmann, R.D., Adams, M.D., White, O., Clayton, R.A., Kirkness, E.F., Kerlavage, A.R., Bult, C.J., Tomb, J.-F., Dougherty, B.A., Merrick, J.M., McKenney, K., Sutton, G., FitzHugh, W., Fields, C., Gocayne, J.D., Scott, J., Shirley, R., Liu, L.I., Glodek, A., Kelley, J.M., Weidman, J.F., Phillips, C.A., Spriggs, T., Hedblom, E., Cotton, M.D., Utterback, T.R., Hanna, M.C., Nguyen, D.T., Saudek, D.M., Brandon, R.C., Fine, L..D., Fritchman, J.L., Fuhrmann, J.L., Geoghagen, N.S.M., Gnehm, C.L., McDonald, L.A., Small, K.V., Fraser, C.M., Smith, H.O. and Venter, J.C. (1995) Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. Science 269, 496-512.
102. Clayton, R.A., Sutton, G., Hinkle J.R., Bult, C. and Fields, C. (1995) Intraspecific variation in small-subunit rRNA sequences in GenBank: why single sequences may not adequately represent prokaryotic taxa. Int. J. Syst. Bacteriol. 45, 595-599.
103. Linton, D., Dewhirst, F.E., Clewley, J.P., Owen, R.J., Burnens, A.P. and Stanley, J. (1994) Two types of 16S rRNA genes are found in Campylobacter helveticus: analysis, applications and characterization of the intervening sequence found in some strains. Microbiology 140, 847-855.
104. Linton, D., Clewley, J.P., Burnens, A., Owen, R.J. and Stanley, J. (1994) An intervening sequence (IVS) in the 16S rRNA gene of the eubacterium Helicobacter canis. Nucleic Acids Res. 22, 1954-1958.
105. Ralph, D. and McClelland, M. (1993) Intervening sequence with conserved open reading frame in eubacterial 23S rRNA genes. Proc. Natl. Acad. Sci. USA 90, 6864-6868.
106. Rainey, F.A., Ward, N.L., Morgan, H.W., Toalster, R. and Stackebrandt, E. (1993) Phylogenetic analysis of anaerobic thermophilic bacteria : aid for their reclassification. J. Bacteriol. 175, 4772-4779.
107. Ralph, D. and McClelland, M. (1994) Phylogenetic evidence for horizontal transfer of an intervening sequence between species in a spirochete genus. J. Bacteriol. 176, 5982-5987.
108. Mägli, A., Rainey, F.A. and Leisinger, T. (1995) Acetogenesis from dichloromethane by a two-component mixed culture comprising a novel bacterium. Appl. Environ. Microbiol. 61, 2943-2949.
109. Scharf, S.J., Horn, G.T. and Erlich, H.A. (1986) Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science 233, 1076-1078.
110. Aslanidis, C. and de Jong, P.J. (1990) Ligation-independent cloning of PCR products (LIC-PCR). Nucleic Acids Res. 18, 6069-6074.
111. Borriello, F. and Krauter, K.S. (1990) Reactive site polymorphism in the murine protease inhibitor gene family is delineated using a modification of the PCR reaction (PCR+1). Nucleic Acids Res. 18, 5481-5487.
112. Santegoeds, C.M., Nold, S.C. and Ward, D.M. (1996) Denaturing gradient gel electrophoresis used to monitor the enrichment culture of aerobic chemoorganotrophic bacteria from a hot spring cyanobacterial mat. Appl. Environ. Microbiol. 62, 3922-3928.
113. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
114. Ward-Rainey, N., Rainey, F.A. and Schlesner, H. (1995) Assignment of hitherto unidentified 16S rDNA species to a main line of descent within the domain bacteria. Microbiology 141, 3247-3250.
115. Stackebrandt, E., Liesack, W. and Goebel, B.M. (1993) Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. FASEB J. 7, 232-236.
116. Kopczynski, E.D., Bateson, M.M. and Ward, D.M. (1994) Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms. Appl. Environ. Microbiol. 60, 746-748.
117. Robison-Cox, J.F., Bateson, M.M. and Ward, D.M. (1995) Evaluation of nearest neighbor methods for detection of chimeric small-subunit rRNA sequences. Appl. Environ. Microbiol. 61, 1240-1245.
118. Sneath, P.H.A. (1993) Evidence from Aeromonas for genetic crossing-over in ribosomal sequences. Int. J. Syst. Bacteriol. 43, 626-629.
119. 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.
120. Huber, R., Burggraf, S., Mayer, T., Barns, S.M., Rossnagel, P. and Stetter, K.O. (1995) Isolation of a hyperthermophilic archaeum predicted by in situ RNA analysis. Nature 376, 57-58.
121. Wyss, C., Choi, B.K., Schüpbach, P., Guggenheim, B. and Göbel, U.B. (1996) Treponema maltophilum sp. nov., a small oral spirochete isolated from human periodontal lesions. Int. J. Syst. Bacteriol. 46, 745-752.
122. Teske, A., Sigalevich, P., Cohen, Y. and Muyzer, G. (1996) Molecular identification of bacteria from a coculture by denaturating gradient gel electrophoresis of 16S ribosomal DNA fragments as a tool for isolation in pure cultures. Appl. Environ. Microbiol. 62, 4210-4215.
123. Holmes, A.J., Owens, N.J.P. and Murrell, J.C. (1995) Detection of novel marine methanotrophs using phylogenetic and functional gene probes after methane enrichment. Microbiology 141, 1947-1955.