Chapter 18 : Molecular Methods To Study Complex Microbial Communities

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This chapter provides a survey of established molecular approaches as well as emerging ones for studying microbial communities in gastrointestinal systems. Whole-genome typing methods are cost-effective alternatives for assessing genetic relatedness. These molecular techniques provide more specificity than phenotypic identification of culturable microbes and include pulsed-field gel electrophoresis (PFGE), rep-PCR, multilocus sequence typing (MLST), and octamer-based genome scanning (OGBS). The genotyping method used in recent years for has been a PCR-based technique using primers targeting repetitive sequences (rep-PCR), which also produces numerous DNA fragments that are separable by electrophoresis. MLST is another PCR-enabled genotyping method that has found widespread use for subtyping a variety of pathogens. The technique is an extension of multilocus enzyme electrophoresis and was first described in 1998 using as an example. OBGS, a powerful method enabled by genome sequence information, was developed as a result of determining the complete genome sequence of. Researchers using molecular community fingerprinting methods can rapidly infer the genetic composition or community structure following the analysis of a region of amplified DNA. All community fingerprinting methods begin with extraction of total genomic DNA from the sample. Direct DNA extraction followed by PCR amplification and subsequent restriction digestion is the basis for (terminal restriction fragment length polymorphism (T-RFLP). The greatest challenge to microbial community analysis is linking the community structure at the phylogenetic level with its metabolic potential and subsequent functionality in the environment being studied.

Citation: Antonopoulos D, Brulc J, Yannarell A, White B. 2009. Molecular Methods To Study Complex Microbial Communities, p 325-345. In Jaykus L, Wang H, Schlesinger L (ed), Food-Borne Microbes. ASM Press, Washington, DC. doi: 10.1128/9781555815479.ch18

Key Concept Ranking

Denaturing Gradient Gel Electrophoresis
Restriction Fragment Length Polymorphism
Random Amplified Polymorphic DNA
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1. Akopyants, N. S.,, A. Fradkov,, L. Diatchenko,, J. E. Hill,, P. D. Siebert,, S. A. Lukyanov,, E. D. Sverdlov, and, D. E. Berg. 1998. PCR-based subtractive hybridization and differences in gene content among strains of Helicobacter pylori. Proc. Natl. Acad. Sci. USA 95:1310813113.
2. Amann, R.,, J. Snaidr,, M. Wagner,, W. Ludwig, and, K. H. Schleifer. 1996. In situ visualization of high genetic diversity in a natural microbial community. J. Bacteriol. 178:34963500.
3. Amann, R. I. 1995. Fluorescently labelled, rRNA-targeted oligonucleotide probes in the study of microbial ecology. Mol. Ecol. 4:543554.
4. Amann, R. I.,, L. Krumholz, and, D. A. Stahl. 1990. Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J. Bacteriol. 172:762770.
5. Amann, R. I.,, C. Lin,, R. Key,, L. Montgomery, and, D. A. Stahl. 1992. Diversity among Fibrobacter isolates: towards a phylogenetic classification. Syst. Appl. Microbiol. 15:2331.
6. Amann, R. I.,, W. Ludwig, and, K. H. Schleifer. 1994. Identification of uncultured bacteria: a challenging task for molecular taxonomists. ASM News 60:360365.
7. Amann, R. I.,, W. Ludwig, and, K. H. Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143169.
8. Anderson, I. C., and, J. W. Cairney. 2004. Diversity and ecology of soil fungal communities: increased understanding through the application of molecular techniques. Environ. Microbiol. 6:769779.
9. Ashelford, K. E.,, N. A. Chuzhanova,, J. C. Fry,, A. J. Jones, and, A. J. Weightman. 2005. At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl. Environ. Microbiol. 71:77247736.
10. Beja, O.,, L. Aravind,, E. V. Koonin,, M. T. Suzuki,, A. Hadd,, L. P. Nguyen,, S. B. Jovanovich,, C. M. Gates,, R. A. Feldman,, J. L. Spudich,, E. N. Spudich, and, E. F. DeLong. 2000. Bacterial rhodopsin: evidence for a new type of phototrophy in the sea. Science 289:19021906.
11. Beja, O.,, E. V. Koonin,, L. Aravind,, L. T. Taylor,, H. Seitz,, J. L. Stein,, D. C. Bensen,, R. A. Feldman,, R. V. Swanson, and, E. F. DeLong. 2002. Comparative genomic analysis of archaeal genotypic variants in a single population and in two different oceanic provinces. Appl. Environ. Microbiol. 68:335345.
12. Beja, O.,, M. T. Suzuki,, E. V. Koonin,, L. Aravind,, A. Hadd,, L. P. Nguyen,, R. Villacorta,, M. Amjadi,, C. Garrigues,, S. B. Jovanovich,, R. A. Feldman, and, E. F. De-Long. 2000. Construction and analysis of bacterial artificial chromosome libraries from a marine microbial assemblage. Environ. Microbiol. 2:516529.
13. Bensch, S., and, M. Akesson. 2005. Ten years of AFLP in ecology and evolution: why so few animals? Mol. Ecol. 14:28992914.
14. Beyer, J.,, D. Fichtner,, H. Schirrmeier,, H. Granzow,, U. Polster,, E. Weiland,, A. Berndt, and, H. Wege. 1998. Arterivirus PRRSV. Experimental studies on the pathogenesis of respiratory disease. Adv. Exp. Med. Biol. 440:593599.
15. Bonaldo, M. F.,, G. Lennon, and, M. B. Soares. 1996. Normalization and subtraction: two approaches to facilitate gene discovery. Genome Res. 6:791806.
16. Borneman, J., and, E. W. Triplett. 1997. Molecular microbial diversity in soils from eastern Amazonia: evidence for unusual microorganisms and microbial population shifts associated with deforestation. Appl. Environ. Microbiol. 63:26472653.
17. Brodie, E. L.,, T. Z. Desantis,, D. C. Joyner,, S. M. Baek,, J. T. Larsen,, G. L. Andersen,, T. C. Hazen,, P. M. Richardson,, D. J. Herman,, T. K. Tokunaga,, J. M. Wan, and, M. K. Firestone. 2006. Application of a high-density oligonucleotide microarray approach to study bacterial population dynamics during uranium reduction and reoxidation. Appl. Environ. Microbiol. 72:62886298.
18. Bruce, K. D. 1997. Analysis of mer gene subclasses within bacterial communities in soils and sediments resolved by fluorescent-PCR–restriction fragment length polymorphism profiling. Appl. Environ. Microbiol. 63:49144919.
19. Burtscher, M. M.,, K. E. Kollner,, R. Sommer,, K. Keiblinger,, A. H. Farnleitner, and, R. L. Mach. 2006. Development of a novel amplified fragment length polymorphism (AFLP) typing method for enterococci isolates from cattle faeces and evaluation of the single versus pooled faecal sampling approach. J. Microbiol. Methods 67:281293.
20. Castro-Gonzalez, M.,, G. Braker,, L. Farias, and, O. Ulloa. 2005. Communities of nirS-type denitrifiers in the water column of the oxygen minimum zone in the eastern South Pacific. Environ. Microbiol. 7:12981306.
21. Chandler, D. P.,, G. J. Newton,, J. A. Small, and, D. S. Daly. 2003. Sequence versus structure for the direct detection of 16S rRNA on planar oligonucleotide microarrays. Appl. Environ. Microbiol. 69:29502958.
22. Cole, J. R.,, B. Chai,, R. J. Farris,, Q. Wang,, S. A. Kulam,, D. M. McGarrell,, G. M. Garrity, and, J. M. Tiedje. 2005. The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res. 33:D294D296.
23. Colombi, M.,, L. Moro,, N. Zoppi, and, S. Barlati. 1993. Quantitative evaluation of mRNAs by in situ hybridization and image analysis: principles and applications. DNA Cell Biol. 12:629636.
24. Curtis, T. P.,, W. T. Sloan, and, J. W. Scannell. 2002. Estimating prokaryotic diversity and its limits. Proc. Natl. Acad. Sci. USA 99:1049410499.
25. de Bruijn, F. J. 1992. Use of repetitive (repetitive extragenic palindromic and enterobacterial repetitive intergeneric consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl. Environ. Microbiol. 58:21802187.
26. DeLong, E. F. 2002. Microbial population genomics and ecology. Curr. Opin. Microbiol. 5:520524.
27. DeLong, E. F. 2004. Microbial population genomics and ecology: the road ahead. Environ. Microbiol. 6:875878.
28. Deng, W.,, S. R. Liou,, G. Plunkett III,, G. F. Mayhew,, D. J. Rose,, V. Burland,, V. Kodoyianni,, D. C. Schwartz, and, F. R. Blattner. 2003. Comparative genomics of Salmonella enterica serovar Typhi strains Ty2 and CT18. J. Bacteriol. 185:23302337.
29. Dennis, P.,, E. A. Edwards,, S. N. Liss, and, R. Fulthorpe. 2003. Monitoring gene expression in mixed microbial communities by using DNA microarrays. Appl. Environ. Microbiol. 69:769778.
30. Deplancke, B.,, K. R. Hristova,, H. A. Oakley,, V. J. McCracken,, R. Aminov,, R. I. Mackie, and, H. R. Gaskins. 2000. Molecular ecological analysis of the succession and diversity of sulfate-reducing bacteria in the mouse gastrointestinal tract. Appl. Environ. Microbiol. 66:21662174.
31. DeSantis, T. Z.,, P. Hugenholtz,, N. Larsen,, M. Rojas,, E. L. Brodie,, K. Keller,, T. Huber,, D. Dalevi,, P. Hu, and, G. L. Andersen. 2006. Greengenes, a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl. Environ. Microbiol. 72:50695072.
32. Dicksved, J.,, H. Floistrup,, A. Bergstrom,, M. Rosenquist,, G. Pershagen,, A. Scheynius,, S. Roos,, J. S. Alm,, L. Engstrand,, C. Braun-Fahrlander,, E. von Mutius, and, J. K. Jansson. 2007. Molecular fingerprinting of the fecal microbiota of children raised according to different lifestyles. Appl. Environ. Microbiol. 73:22842289.
33. Edwards, J. E.,, N. R. McEwan,, A. J. Travis, and, R. J. Wallace. 2004. 16S rDNA library-based analysis of ruminal bacterial diversity. Antonie van Leeuwenhoek 86:263281.
34. Edwards, R. A.,, B. Rodriguez-Brito,, L. Wegley,, M. Haynes,, M. Breitbart,, D. M. Peterson,, M. O. Saar,, S. Alexander,, E. C. Alexander, and, F. Rohwer. 2006. Using pyrosequencing to shed light on deep mine microbial ecology. BMC Genomics 7:5769.
35. El Fantroussi, S.,, H. Urakawa,, A. E. Bernhard,, J. J. Kelly,, P. A. Noble,, H. Smidt,, G. M. Yershov, and, D. A. Stahl. 2003. Direct profiling of environmental microbial populations by thermal dissociation analysis of native rRNAs hybridized to oligonucleotide microarrays. Appl. Environ. Microbiol. 69:23772382.
36. Fierer, N., and, R. B. Jackson. 2006. The diversity and biogeography of soil bacterial communities. Proc. Natl. Acad. Sci. USA 103:626631.
37. Fisher, M. M., and, E. W. Triplett. 1999. Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl. Environ. Microbiol. 65:46304636.
38. Fouts, D. E.,, E. F. Mongodin,, R. E. Mandrell,, W. G. Miller,, D. A. Rasko,, J. Ravel,, L. M. Brinkac,, R. T. DeBoy,, C. T. Parker,, S. C. Daugherty,, R. J. Dodson,, A. S. Durkin,, R. Madupu,, S. A. Sullivan,, J. U. Shetty,, M. A. Ayodeji,, A. Shvartsbeyn,, M. C. Schatz,, J. H. Badger,, C. M. Fraser, and, K. E. Nelson. 2005. Major structural differences and novel potential virulence mechanisms from the genomes of multiple campylobacter species. PLoS Biol. 3:e15.
39. Fox, G. E.,, E. Stackebrandt,, R. B. Hespell,, J. Gibson,, J. Maniloff,, T. A. Dyer,, R. S. Wolfe,, W. E. Balch,, R. S. Tanner,, L. J. Magrum,, L. B. Zablen,, R. Blakemore,, R. Gupta,, L. Bonen,, B. J. Lewis,, D. A. Stahl,, K. R. Luehrsen,, K. N. Chen, and, C. R. Woese. 1980. The phylogeny of prokaryotes. Science 209:457463.
40. Fox, J. L. 2005. Ribosomal gene milestone met, already left in dust. ASM News 71:67.
41. Franklin, R. B.,, D. R. Taylor, and, A. L. Mills. 1999. Characterization of microbial communities using randomly amplified polymorphic DNA (RAPD). J. Microbiol. Methods 35:225235.
42. Galbraith, E. A.,, D. A. Antonopoulos, and, B. A. White. 2004. Suppressive subtractive hybridization as a tool for identifying genetic diversity in an environmental metagenome: the rumen as a model. Environ. Microbiol. 6:928937.
43. Garaizar, J.,, N. Lopez-Molina,, I. Laconcha,, D. Lau Baggesen,, A. Rementeria,, A. Vivanco,, A. Audicana, and, I. Perales. 2000. Suitability of PCR fingerprinting, infrequent-restriction-site PCR, and pulsed-field gel electrophoresis, combined with computerized gel analysis, in library typing of Salmonella enterica serovar Enteritidis. Appl. Environ. Microbiol. 66:52735281.
44. Garcia-Martinez, J.,, S. G. Acinas,, A. I. Anton, and, F. Rodriguez-Valera. 1999. Use of the 16S–23S ribosomal genes spacer region in studies of prokaryotic diversity. J. Microbiol. Methods 36:5564.
45. Gillespie, D. E.,, S. F. Brady,, A. D. Bettermann,, N. P. Cianciotto,, M. R. Liles,, M. R. Rondon,, J. Clardy,, R. M. Goodman, and, J. Handelsman. 2002. Isolation of antibiotics turbomycin A and B from a metagenomic library of soil microbial DNA. Appl. Environ. Microbiol. 68:43014306.
46. Gilmore, M. S., and, J. J. Ferretti. 2003. Microbiology. The thin line between gut commensal and pathogen. Science 299:19992002.
47. Gray, N. D.,, R. Howarth,, R. W. Pickup,, J. G. Jones, and, I. M. Head. 2000. Use of combined microautoradiography and fluorescence in situ hybridization to determine carbon metabolism in mixed natural communities of un-cultured bacteria from the genus Achromatium. Appl. Environ. Microbiol. 66:45184522.
48. Gurtler, V., and, B. C. Mayall. 2001. Genomic approaches to typing, taxonomy and evolution of bacterial isolates. Int. J. Syst. Evol. Microbiol. 51:316.
49. Guschin, D. Y.,, B. K. Mobarry,, D. Proudnikov,, D. A. Stahl,, B. E. Rittmann, and, A. D. Mirzabekov. 1997. Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology. Appl. Environ. Microbiol. 63:23972402.
50. Hamady, M.,, J. J. Walker,, J. K. Harris,, N. J. Gold, and, R. Knight. 10 February 2008, posting date. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat. Methods doi:10.1038/nmeth.1182.
51. Handelsman, J.,, M. R. Rondon,, S. F. Brady,, J. Clardy, and, R. M. Goodman. 1998. Molecular biological access to the chemistry of unknown soil microbes: a new frontier for natural products. Chem. Biol. 5:R245R249.
52. Helmus, M. R.,, T. J. Bland,, C. K. Williams, and, A. R. Ives. 2007. Phylogenetic measures of biodiversity. Am. Nat. 169:E68E83.
53. Henne, A.,, R. Daniel,, R. A. Schmitz, and, G. Gottschalk. 1999. Construction of environmental DNA libraries in Escherichia coli and screening for the presence of genes conferring utilization of 4-hydroxybutyrate. Appl. Environ. Microbiol. 65:39013907.
54. Hespell, R. B.,, D. E. Akin, and, B. A. Dehority. 1997. Bacteria, fungi, and protozoa of the rumen, p. 59141. In R. I. Mackie,, B. A. White, and, R. E. Isaacson (ed.), Gastrointestinal Microbes and Host Interactions, vol. 2. Chapman and Hall, New York, NY.
55. Hiraishi, A.,, M. Iwasaki, and, H. Shinjo. 2000. Terminal restriction pattern analysis of 16S rRNA genes for the characterization of bacterial communities of activated sludge. J. Biosci. Bioeng. 90:148156.
56. Hodson, R. E.,, W. A. Dustman,, R. P. Garg, and, M. A. Moran. 1995. In situ PCR for visualization of microscale distribution of specific genes and gene products in prokaryotic communities. Appl. Environ. Microbiol. 61:40744082.
57. Holder, M., and, P. O. Lewis. 2003. Phylogeny estimation: traditional and Bayesian approaches. Nat. Rev. Genet. 4:275284.
58. Hongoh, Y.,, P. Deevong,, T. Inoue,, S. Moriya,, S. Trakulnaleamsai,, M. Ohkuma,, C. Vongkaluang,, N. Noparatnaraporn, and, T. Kudo. 2005. Intra- and interspecific comparisons of bacterial diversity and community structure support coevolution of gut microbiota and termite host. Appl. Environ. Microbiol. 71:65906599.
59. Hugenholtz, P.,, B. M. Goebel, and, N. R. Pace. 1998. Impact of culture-independent studies on the emerging phylogenetic view of bacterial diversity. J. Bacteriol. 180:47654774.
60. Hulton, C. S.,, C. F. Higgins, and, P. M. Sharp. 1991. ERIC sequences: a novel family of repetitive elements in the genomes of Escherichia coli, Salmonella typhimurium and other entero-bacteria. Mol. Microbiol. 5:825834.
61. Huse, S. M.,, J. A. Huber,, H. G. Morrison,, M. L. Sogin, and, D. M. Welch. 2007. Accuracy and quality of massively parallel DNA pyrosequencing. Genome Biol. 8:R143.
62. Jongman, R. H. G.,, C. J. F. ter Braak, and, O. F. R. van Tongeren (ed.). 1995. Data Analysis in Community and Landscape Ecology. Cambridge University Press, New York, NY.
63. Kiehnbaum, L. A.,, A. Amonsin,, S. J. Wells, and, V. Kapur. 2005. Amplified fragment length polymorphism to detect clonal diversity and distribution of Mycobacterium avium subspecies paratuberculosis in selected Minnesota dairy cattle. J. Vet. Diagn. Investig. 17:311315.
64. Kim, J.,, J. Nietfeldt, and, A. K. Benson. 1999. Octamer-based genome scanning distinguishes a unique subpopulation of Escherichia coli O157:H7 strains in cattle. Proc. Natl. Acad. Sci. USA 96:1328813293.
65. Kim, J.,, J. Nietfeldt,, J. Ju,, J. Wise,, N. Fegan,, P. Desmarchelier, and, A. K. Benson. 2001. Ancestral divergence, genome diversification, and phylogeographic variation in subpopulations of sorbitol-negative, beta-glucuronidase-negative enterohemorrhagic Escherichia coli O157. J. Bacteriol. 183:68856897.
66. Klappenbach, J. A.,, P. R. Saxman,, J. R. Cole, and, T. M. Schmidt. 2001. rrndb: the ribosomal RNA operon copy number database. Nucleic Acids Res. 29:181184.
67. Kocherginskaya, S. A.,, R. I. Aminov, and, B. A. White. 2001. Analysis of the rumen bacterial diversity under two different diet conditions using denaturing gradient gel electrophoresis, random sequencing, and statistical ecology approaches. Anaerobe 7:119134.
68. Kocherginskaya, S. A.,, I. K. O. Cann, and, R. I. Mackie. 2005. Denaturing gradient gel electrophoresis, p. 119128. In H. P. S. Makkar and, C. S. McSweeney (ed.), Methods in Gut Microbial Ecology for Ruminants. Springer, Dordrecht, The Netherlands.
69. Koizumi, Y.,, J. J. Kelly,, T. Nakagawa,, H. Urakawa,, S. El-Fantroussi,, S. Al-Muzaini,, M. Fukui,, Y. Urushigawa, and, D. A. Stahl. 2002. Parallel characterization of anaerobic toluene- and ethylbenzene-degrading microbial consortia by PCR-denaturing gradient gel electrophoresis, RNA-DNA membrane hybridization, and DNA microarray technology. Appl. Environ. Microbiol. 68:32153225.
70. Krause, D. O.,, B. P. Dalrymple,, W. J. Smith,, R. I. Mackie, and, C. S. McSweeney. 1999. 16S rDNA sequencing of Ruminococcus albus and Ruminococcus flavefaciens: design of a signature probe and its application in adult sheep. Microbiology 145(Pt 7):17971807.
71. Kysela, D. T.,, C. Palacios, and, M. L. Sogin. 2005. Serial analysis of V6 ribosomal sequence tags (SARST-V6): a method for efficient, high-throughput analysis of microbial community composition. Environ. Microbiol. 7:356364.
72. Larue, R.,, Z. Yu,, V. A. Parisi,, A. R. Egan, and, M. Morrison. 2005. Novel microbial diversity adherent to plant biomass in the herbivore gastrointestinal tract, as revealed by ribosomal intergenic spacer analysis and rrs gene sequencing. Environ. Microbiol. 7:530543.
73. Reference deleted.
74. Legendre, P., and, L. Legendre. 1998. Numerical Ecology, 2nd English ed. Elsevier Science BV, Amsterdam, The Netherlands.
75. Leser, T. D.,, J. Z. Amenuvor,, T. K. Jensen,, R. H. Lindecrona,, M. Boye, and, K. Moller. 2002. Culture-independent analysis of gut bacteria: the pig gastrointestinal tract microbiota revisited. Appl. Environ. Microbiol. 68:673690.
76. Reference deleted.
77. Liebana, E. 2002. Molecular tools for epidemiological investigations of S. enterica subspecies enterica infections. Res. Vet. Sci. 72:169175.
78. Liles, M. R.,, B. F. Manske,, S. B. Bintrim,, J. Handelsman, and, R. M. Goodman. 2003. A census of rRNA genes and linked genomic sequences within a soil metagenomic library. Appl. Environ. Microbiol. 69:26842691.
79. Lin, C.,, B. Flesher,, W. C. Capman,, R. I. Amann, and, D. Stahl. 1994. Taxon specific hybridization probes for fiber-digesting bacteria suggest novel gut-associated Fibrobacter. Syst. Appl. Microbiol. 17:418424.
80. Lin, C., and, D. A. Stahl. 1995. Taxon-specific probes for the cellulolytic genus Fibrobacter reveal abundant and novel equine-associated populations. Appl. Environ. Microbiol. 61:13481351.
81. Lin, C. Z.,, L. Raskin, and, D. A. Stahl. 1997. Microbial community structure in gastrointestinal tracts of domestic animals: comparative analyses using rRNA-targeted oligonucleotide probes. FEMS Microbiol. Ecol. 22:281294.
82. Liu, W. T.,, T. L. Marsh,, H. Cheng, and, L. J. Forney. 1997. Characterization of microbial diversity by determining terminal restriction fragment length polymorphisms of genes encoding 16S rRNA. Appl. Environ. Microbiol. 63:45164522.
83. Liu, Z. Z.,, C. Lozupone,, M. Hamady,, F. D. Bushman, and, R. Knight. 2007. Short pyro-sequencing reads suffice for accurate microbial community analysis. Nucleic Acids Res. 35:110.
84. Lopez-Garcia, P.,, C. Brochier,, D. Moreira, and, F. Rodriguez-Valera. 2004. Comparative analysis of a genome fragment of an uncultivated mesopelagic crenarchaeote reveals multiple horizontal gene transfers. Environ. Microbiol. 6:1934.
85. Louws, F. J.,, D. W. Fulbright,, C. T. Stephens, and, F. J. de Bruijn. 1994. Specific genomic fingerprints of phytopathogenic Xanthomonas and Pseudomonas pathovars and strains generated with repetitive sequences and PCR. Appl. Environ. Microbiol. 60:22862295.
86. Lozupone, C., and, R. Knight. 2005. UniFrac: a new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 71:82288235.
87. Lu, J.,, U. Idris,, B. Harmon,, C. Hofacre,, J. J. Maurer, and, M. D. Lee. 2003. Diversity and succession of the intestinal bacterial community of the maturing broiler chicken. Appl. Environ. Microbiol. 69:68166824.
88. Lu, J.,, S. Sanchez,, C. Hofacre,, J. J. Maurer,, B. G. Harmon, and, M. D. Lee. 2003. Evaluation of broiler litter with reference to the microbial composition as assessed by using 16S rRNA and functional gene markers. Appl. Environ. Microbiol. 69:901908.
89. Maiden, M. C. 2006. Multilocus sequence typing of bacteria. Annu. Rev. Microbiol. 60:561588.
90. Maiden, M. C.,, J. A. Bygraves,, E. Feil,, G. Morelli,, J. E. Russell,, R. Urwin,, Q. Zhang,, J. Zhou,, K. Zurth,, D. A. Caugant,, I. M. Feavers,, M. Achtman, and, B. G. Spratt. 1998. Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms. Proc. Natl. Acad. Sci. USA 95:31403145.
91. Majernik, A.,, G. Gottschalk, and, R. Daniel. 2001. Screening of environmental DNA libraries for the presence of genes conferring Na+(Li+)/H+ antiporter activity on Escherichia coli: characterization of the recovered genes and the corresponding gene products. J. Bacteriol. 183:66456653.
92. Manefield, M.,, A. S. Whiteley,, R. I. Griffiths, and, M. J. Bailey. 2002. RNA stable isotope probing, a novel means of linking microbial community function to phylogeny. Appl. Environ. Microbiol. 68:53675373.
93. Margulies, M.,, M. Egholm,, W. E. Altman,, S. Attiya,, J. S. Bader,, L. A. Bemben,, J. Berka,, M. S. Braverman,, Y. J. Chen,, Z. Chen,, S. B. Dewell,, L. Du,, J. M. Fierro,, X. V. Gomes,, B. C. Godwin,, W. He,, S. Helgesen,, C. H. Ho,, G. P. Irzyk,, S. C. Jando,, M. L. Alenquer,, T. P. Jarvie,, K. B. Jirage,, J. B. Kim,, J. R. Knight,, J. R. Lanza,, J. H. Leamon,, S. M. Lefkowitz,, M. Lei,, J. Li,, K. L. Lohman,, H. Lu,, V. B. Makhijani,, K. E. McDade,, M. P. McKenna,, E. W. Myers,, E. Nickerson,, J. R. Nobile,, R. Plant,, B. P. Puc,, M. T. Ronan,, G. T. Roth,, G. J. Sarkis,, J. F. Simons,, J. W. Simpson,, M. Srinivasan,, K. R. Tartaro,, A. Tomasz,, K. A. Vogt,, G. A. Volkmer,, S. H. Wang,, Y. Wang,, M. P. Weiner,, P. Yu,, R. F. Begley, and, J. M. Rothberg. 2005. Genome sequencing in microfabricated high-density picolitre reactors. Nature 437:376380.
94. Martin, A. P. 2002. Phylogenetic approaches for describing and comparing the diversity of microbial communities. Appl. Environ. Microbiol. 68:36733682.
95. Massol-Deya, A.,, R. Weller,, L. Rios-Hernandez,, J. Z. Zhou,, R. F. Hickey, and, J. M. Tiedje. 1997. Succession and convergence of biofilm communities in fixed-film reactors treating aromatic hydrocarbons in groundwater. Appl. Environ. Microbiol. 63:270276.
96. Mau, M., and, K. N. Timmis. 1998. Use of subtractive hybridization to design habitat-based oligonucleotide probes for investigation of natural bacterial communities. Appl. Environ. Microbiol. 64:185191.
97. Meudt, H. M., and, A. C. Clarke. 2007. Almost forgotten or latest practice? AFLP applications, analyses and advances. Trends Plant Sci. 12:106117.
98. Mohamed, M. A. N.,, L. Ranjard,, C. Catroux,, G. Catroux, and, A. Hartmann. 2005. Effect of natamycin on the enumeration, genetic structure and composition of bacterial community isolated from soils and soybean rhizosphere. J. Microbiol. Methods 60:3140.
99. 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. 59:695700.
100. Muyzer, G., and, K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek 73:127141.
101. Nelson, K. E.,, S. H. Zinder,, I. Hance,, P. Burr,, D. Odongo,, D. Wasawo,, A. Odenyo, and, R. Bishop. 2003. Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche. Environ. Microbiol. 5:12121220.
102. Neppelenbroek, K. H.,, N. H. Campanha,, D. M. P. Spolidorio,, L. C. Spolidorio,, R. S. Seó, and, A. C. Pavarina. 2006. Molecular fingerprinting methods for the discrimination between C. albicans and C. dubliniensis. Oral Dis. 12:242253.
103. Nielsen, J. L.,, D. Christensen,, M. Kloppenborg, and, P. H. Nielsen. 2003. Quantification of cell-specific substrate uptake by probe-defined bacteria under in situ conditions by microautoradiography and fluorescence in situ hybridization. Environ. Microbiol. 5:202211.
104. Noll, M.,, D. Matthies,, P. Frenzel,, M. Derakshani, and, W. Liesack. 2005. Succession of bacterial community structure and diversity in a paddy soil oxygen gradient. Environ. Microbiol. 7:382395.
105. Olsen, J. E.,, M. N. Skov,, E. J. Threlfall, and, D. J. Brown. 1994. Clonal lines of Salmonella enterica serotype Enteritidis documented by IS200-, ribo-, pulsed-field gel electrophoresis and RFLP typing. J. Med. Microbiol. 40:1522.
106. On, S. L., and, D. L. Baggesen. 1997. Determination of clonal relationships of Salmonella typhimurium by numerical analysis of macro-restriction profiles. J. Appl. Microbiol. 83:699706.
107. Ouverney, C. C., and, J. A. Fuhrman. 1999. Combined microautoradiography-16S rRNA probe technique for determination of radioisotope uptake by specific microbial cell types in situ. Appl. Environ. Microbiol. 65:17461752.
108. Pace, N. R. 1997. A molecular view of microbial diversity and the biosphere. Science 276:734740.
109. Reference deleted.
110. Peplies, J.,, F. O. Glockner, and, R. Amann. 2003. Optimization strategies for DNA microarray-based detection of bacteria with 16S rRNA-targeting oligonucleotide probes. Appl. Environ. Microbiol. 69:13971407.
111. Punia, P.,, M. D. Hampton,, A. M. Ridley,, L. R. Ward,, B. Rowe, and, E. J. Threlfall. 1998. Pulsed-field electrophoretic fingerprinting of Salmonella indiana and its epidemiological applicability. J. Appl. Microbiol. 84:103107.
112. Quaiser, A.,, T. Ochsenreiter,, H. P. Klenk,, A. Kletzin,, A. H. Treusch,, G. Meurer,, J. Eck,, C. W. Sensen, and, C. Schleper. 2002. First insight into the genome of an uncultivated crenarchaeote from soil. Environ. Microbiol. 4:603611.
113. Ranjard, L.,, F. Poly, and, S. Nazaret. 2000. Monitoring complex bacterial communities using culture-independent molecular techniques: application to soil environment. Res. Microbiol. 151:167177.
114. Raskin, L.,, W. C. Capman,, R. Sharp,, L. K. Poulsen, and, D. A. Stahl. 1997. Molecular ecology of gastrointestinal ecosystems, p. 243298. In R. I. Mackie,, B. A. White, and, R. E. Isaacson (ed.), Gastrointestinal Microbes and Host Interactions, vol. 2. Chapman and Hall, New York, NY.
115. Rhee, S. K.,, X. Liu,, L. Wu,, S. C. Chong,, X. Wan, and, J. Zhou. 2004. Detection of genes involved in biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide microarrays. Appl. Environ. Microbiol. 70:43034317.
116. Riesenfeld, C. S.,, R. M. Goodman, and, J. Handelsman. 2004. Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environ. Microbiol. 6:981989.
117. Roesch, L. F. W.,, R. R. Fulthorpe,, A. Riva,, G. Casella,, A. K. M. Hadwin,, A. D. Kent,, S. H. Daroub,, F. A. O. Camargo,, W. G. Farmerie, and, E. W. Triplett. 2007. Pyrosequencing enumerates and contrasts soil microbial diversity. ISME J. 1:283290.
118. Rondon, M. R.,, P. R. August,, A. D. Bettermann,, S. F. Brady,, T. H. Grossman,, M. R. Liles,, K. A. Loiacono,, B. A. Lynch,, I. A. MacNeil,, C. Minor,, C. L. Tiong,, M. Gilman,, M. S. Osburne,, 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. 66:25412547.
119. Saikaly, P. E.,, P. G. Stroot, and, D. B. Oerther. 2005. Use of 16S rRNA gene terminal restriction fragment analysis to assess the impact of solids retention time on the bacterial diversity of activated sludge. Appl. Environ. Microbiol. 71:58145822.
120. Salamon, H.,, M. A. Behr,, J. T. Rhee, and, P. M. Small. 2000. Genetic distances for the study of infectious disease epidemiology. Am. J. Epidemiol. 151:324334.
121. Salzberg, S. L.,, A. J. Salzberg,, A. R. Kerlavage, and, J. F. Tomb. 1998. Skewed oligomers and origins of replication. Gene 217:5767.
122. Savage, D. C. 1977. Microbial ecology of the gastrointestinal tract. Annu. Rev. Microbiol. 31:107133.
123. Savelkoul, P. H.,, H. J. Aarts,, J. de Haas,, L. Dijkshoorn,, B. Duim,, M. Otsen,, J. L. Rademaker,, L. Schouls, and, J. A. Lenstra. 1999. Amplified-fragment length polymorphism analysis: the state of an art. J. Clin. Microbiol. 37:30833091.
124. Schloss, P. D., and, J. Handelsman. 2005. Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl. Environ. Microbiol. 71:15011506.
125. Schloss, P. D., and, J. Handelsman. 2006. Introducing TreeClimber, a test to compare microbial community structures. Appl. Environ. Microbiol. 72:23792384.
126. Schloss, P. D.,, B. R. Larget, and, J. Handelsman. 2004. Integration of microbial ecology and statistics: a test to compare gene libraries. Appl. Environ. Microbiol. 70:54855492.
127. Simpson, J. M.,, V. J. McCracken,, H. R. Gaskins, and, R. I. Mackie. 2000. Denaturing gradient gel electrophoresis analysis of 16S ribosomal DNA amplicons to monitor changes in fecal bacterial populations of weaning pigs after introduction of Lactobacillus reuteri strain MM53. Appl. Environ. Microbiol. 66:47054714.
128. Simpson, J. M.,, V. J. McCracken,, B. A. White,, H. R. Gaskins, and, R. I. Mackie. 1999. Application of denaturant gradient gel electrophoresis for the analysis of the porcine gastrointestinal microbiota. J. Microbiol. Methods 36:167179.
129. Singleton, D. R.,, M. A. Furlong,, S. L. Rathbun, and, W. B. Whitman. 2001. Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples. Appl. Environ. Microbiol. 67:43744376.
130. Small, J.,, D. R. Call,, F. J. Brockman,, T. M. Straub, and, D. P. Chandler. 2001. Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays. Appl. Environ. Microbiol. 67:47084716.
131. Sogin, M. L.,, H. G. Morrison,, J. A. Huber,, D. M. Welch,, S. M. Huse,, P. R. Neal,, J. M. Arrieta, and, G. J. Herndl. 2006. Microbial diversity in the deep sea and the underexplored “rare biosphere.” Proc. Natl. Acad. Sci. USA 103:1211512120.
132. Stahl, D. A.,, B. Flesher,, H. R. Mansfield, and, L. Montgomery. 1988. Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54:10791084.
133. Stern, M. J.,, E. Prossnitz, and, G. F. Ames. 1988. Role of the intercistronic region in post-transcriptional control of gene expression in the histidine transport operon of Salmonella typhimurium: involvement of REP sequences. Mol. Microbiol. 2:141152.
134. Stewart, C. S. 1997. Microorganisms in hindgut fermentors, p. 142186. In R. I. Mackie,, B. A. White, and, R. E. Isaacson (ed.), Gastrointestinal Microbes and Host Interactions, vol. 2. Chapman and Hall, New York, NY.
135. Sun, H. Y.,, S. P. Deng, and, W. R. Raun. 2004. Bacterial community structure and diversity in a century-old manure-treated agroeco-system. Appl. Environ. Microbiol. 70:58685864.
136. Tajima, K.,, R. I. Aminov,, T. Nagamine,, H. Matsui,, M. Nakamura, and, Y. Benno. 2001. Diet-dependent shifts in the bacterial population of the rumen revealed with real-time PCR. Appl. Environ. Microbiol. 67:27662774.
137. Tajima, K.,, R. I. Aminov,, T. Nagamine,, K. Ogata,, M. Nakamura,, H. Matsui, and, Y. Benno. 1999. Rumen bacterial diversity as determined by sequence analysis of 16S rDNA libraries. FEMS Microbiol. Ecol. 29:159169.
138. Tajima, K.,, S. Arai,, K. Ogata,, T. Nagamine,, H. Matsui,, M. Nakamura,, R. I. Aminov, and, Y. Benno. 2000. Rumen bacterial community transition during adaptation to high-grain diet. Anaerobe 6:273284.
139. Tajima, K.,, T. Nagamine,, H. Matsui,, M. Nakamura, and, R. Aminov. 2001. Phylogenetic analysis of archaeal 16S rRNA libraries from the rumen suggests the existence of a novel group of archaea not associated with known methanogens. FEMS Microbiol. Ecol. 200:6772.
140. Tannock, G. W. 1997. Normal microbiota of the gastrointestinal tract of rodents, p. 187215. In R. I. Mackie,, B. A. White, and, R. E. Isaacson (ed.), Gastrointestinal Microbes and Host Interactions, vol. 2. Chapman and Hall, New York, NY.
141. Taroncher-Oldenburg, G.,, E. M. Griner,, C. A. Francis, and, B. B. Ward. 2003. Oligonucleotide microarray for the study of functional gene diversity in the nitrogen cycle in the environment. Appl. Environ. Microbiol. 69:11591171.
142. Tenover, F. C.,, R. D. Arbeit,, R. V. Goering,, P. A. Mickelsen,, B. E. Murray,, D. H. Persing, and, B. Swaminathan. 1995. Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel electrophoresis: criteria for bacterial strain typing. J. Clin. Microbiol. 33:22332239.
143. Theron, J., and, T. E. Cloete. 2000. Molecular techniques for determining microbial diversity and community structure in natural environments. Crit. Rev. Microbiol. 26:3757.
144. Tiquia, S. M.,, L. Wu,, S. C. Chong,, S. Passovets,, D. Xu,, Y. Xu, and, J. Zhou. 2004. Evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in environmental samples. Biotechniques 36:664670, 672, 674675.
145. Torsvik, V., and, L. Ovreas. 2002. Microbial diversity and function in soil: from genes to ecosystems. Curr. Opin. Microbiol. 5:240245.
146. Turnbaugh, P. J.,, R. E. Ley,, M. A. Mahowald,, V. Magrini,, E. R. Mardis, and, J. I. Gordon. 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:10271031.
147. Upholt, W. B. 1977. Estimation of DNA sequence divergence from comparison of restriction endonuclease digests. Nucleic Acids Res. 4:12571265.
148. Urakawa, H.,, S. El Fantroussi,, H. Smidt,, J. C. Smoot,, E. H. Tribou,, J. J. Kelly,, P. A. Noble, and, D. A. Stahl. 2003. Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays. Appl. Environ. Microbiol. 69:28482856.
149. Urakawa, H.,, T. Yoshida,, M. Nishimura, and, K. Ohwada. 2000. Characterization of depth-related population variation in microbial communities of a coastal marine sediment using 16S rDNA-based approaches and quinone profiling. Environ. Microbiol. 2:542554.
150. Vaughan, E. E.,, F. Schut,, H. G. Heilig,, E. G. Zoetendal,, W. M. de Vos, and, A. D. Akkermans. 2000. A molecular view of the intestinal ecosystem. Curr. Issues Intest. Microbiol. 1:112.
151. Vergin, K. L.,, E. Urbach,, J. L. Stein,, E. F. DeLong,, B. D. Lanoil, and, S. J. Giovannoni. 1998. Screening of a fosmid library of marine environmental genomic DNA fragments reveals four clones related to members of the order Planctomycetales. Appl. Environ. Microbiol. 64:30753078.
152. Vos, P.,, R. Hogers,, M. Bleeker,, M. Reijans,, T. van de Lee,, M. Hornes,, A. Fritjers,, J. Pot,, J. Paleman,, M. Kuiper, and, M. Zabeau. 1995. AFLP: a new technique for DNA fingerprinting. Nucleic Acids Res. 23:44074414.
153. Webb, C. O.,, D. D. Ackerly,, M. A. Mc-Peek, and, M. J. Donoghue. 2002. Phylogenies and community ecology. Annu. Rev. Ecol. Syst. 33:475505.
154. Weigel, R. M.,, B. Qiao,, B. Teferedegne,, D. K. Suh,, D. A. Barber,, R. E. Isaacson, and, B. A. White. 2004. Comparison of pulsed field gel electrophoresis and repetitive sequence polymerase chain reaction as genotyping methods for detection of genetic diversity and inferring transmission of Salmonella. Vet. Microbiol. 100:205217.
155. Welsh, J., and, M. McClelland. 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res. 18:72137218.
156. Whitford, M. F.,, R. J. Forster,, C. E. Beard,, J. Gong, and, R. M. Teather. 1998. Phylogenetic analysis of rumen bacteria by comparative sequence analysis of cloned 16S rRNA genes. Anaerobe 4:153163.
157. Williams, J. G.,, A. R. Kubelik,, K. J. Livak,, J. A. Rafalski, and, S. V. Tingey. 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res. 18:65316535.
158. Wilson, K. H. 1997. Biota of the human gastrointestinal tract, p. 3958. In R. I. Mackie,, B. A. White, and, R. E. Isaacson (ed.), Gastrointestinal Microbes and Host Interactions, vol. 2. hapman and Hall, New York, NY.
159. Wu, L.,, X. Liu,, C. W. Schadt, and, J. Zhou. 2006. Microarray-based analysis of subnanogram quantities of microbial community DNAs by using whole-community genome amplification. Appl. Environ. Microbiol. 72:49314941.
160. Wu, L.,, D. K. Thompson,, G. Li,, R. A. Hurt,, J. M. Tiedje, and, J. Zhou. 2001. Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol. 67:57805790.
161. Wu, L.,, D. K. Thompson,, X. Liu,, M. W. Fields,, C. E. Bagwell,, J. M. Tiedje, and, J. Zhou. 2004. Development and evaluation of microarray-based whole-genome hybridization for detection of microorganisms within the context of environmental applications. Environ. Sci. Technol. 38:67756782.
162. Yang, Z.,, J. Kovar,, J. Kim,, J. Nietfeldt,, D. R. Smith,, R. A. Moxley,, M. E. Olson,, P. D. Fey, and, A. K. Benson. 2004. Identification of common subpopulations of non-sorbitol-fermenting, beta-glucuronidase-negative Escherichia coli O157:H7 from bovine production environments and human clinical samples. Appl. Environ. Microbiol. 70:68466854.
163. Yu, Z.,, M. Yu, and, M. Morrison. 2006. Improved serial analysis of V1 ribosomal sequence tags (SARST-V1) provides a rapid, comprehensive, sequence-based characterization of bacterial diversity and community composition. Environ. Microbiol. 8:603611.
164. Zhu, X. Y.,, T. Zhong,, Y. Pandya, and, R. D. Joerger. 2002. 16S rRNA-based analysis of microbiota from the cecum of broiler chickens. Appl. Environ. Microbiol. 68:124137.
165. Zoetendal, E. G.,, C. T. Collier,, S. Koike,, R. I. Mackie, and, H. R. Gaskins. 2004. Molecular ecological analysis of the gastrointestinal microbiota: a review. J. Nutr. 134:465472.

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