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Chapter 53 : Methods of Soil Microbial Community Analysis

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Abstract:

Soil microbial communities probably are the most complex of natural communities, and one study estimated that there may be as many as 1,000,000 “distinct genomes” per g of soil. This chapter is divided into two sections, the first covering approaches that require laboratory cultivation or incubation and the second addressing those based on analysis of indicator molecules that have been extracted directly from soil communities. A commonly used approach for identification of environmental isolates is based on growth on specific substrates and fermentative abilities. A more precise method of determining the phylogenetic affiliation of an isolate is by characterization of its rRNA. The technique is based on the concept that rRNA molecules, particularly the 16S and 23S rRNA molecules of prokaryotes, are highly conserved throughout evolution and are therefore useful as specific indicators of the phylogenetic affiliations of environmental isolates. A variety of DNA fingerprinting techniques can be used to rapidly differentiate closely related environmental strains. Regardless of the specific technique, the data obtained are DNA fragments that, when separated on agarose or polyacrylamide gels, yield a banding pattern specific to the genome under investigation. Rapid screening methods are of three categories that include: denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment length polymorphism (T-RFLP) analysis. The chapter talks about automated ribosomal intergenic spacer analysis (ARISA). An alternative approach to soil microbial community analysis is cloning of the soil “metagenome,” defined as the genomes of the total microbiota found in soils.

Citation: Ogram A, Castro H, Chauhan A. 2007. Methods of Soil Microbial Community Analysis, p 652-662. In Hurst C, Crawford R, Garland J, Lipson D, Mills A, Stetzenbach L (ed), Manual of Environmental Microbiology, Third Edition. ASM Press, Washington, DC. doi: 10.1128/9781555815882.ch53

Key Concept Ranking

Denaturing Gradient Gel Electrophoresis
0.48627925
Microbial Ecology
0.48170897
Environmental Microbiology
0.48079145
Restriction Fragment Length Polymorphism
0.4793324
Reverse Transcriptase PCR
0.41564605
0.48627925
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1. Altschul, S. F.,, W. Gish,, W. Miller,, E. W. Myers, and, D. J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215:403410.
2. 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.
3. Atlas, R. M.,, and R. Bartha. 1993. Microbial Ecology: Fundamentals and Applications, 3rd ed. Benjamin/Cummings Co., New York, N.Y.
4. Bakken, L. R.,, and V. Lindahl. 1995. Recovery of bacterial cells from soil, p. 9–27. In J. D. van Elsas and, J. T. Trevors (ed.), Nucleic Acids in the Environment: Methods and Applications. Springer-Verlag, Heidelberg, Germany.
5. Bligh, E. G.,, and W. J. Dyer. 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37:911917.
6. Bobbie, R. J.,, and D. C. White. 1980. Characterization of benthic microbial community structure by high-resolution gas chromatography of fatty acid methyl esters. Appl. Environ. Microbiol. 39:12121222.
7. Boschker, H. T. S.,, S. C. Nold,, P. Wellsbury,, D. Bos,, W. de Graaf,, R. Pel,, R. J. Parkes, and, T. E. Cappenberg. 1998. Direct linking of microbial populations to specific biogeochemical processes by 13C-labelling of biomarkers. Nature 392:801804.
8. Buyer, J. S. 1995. A soil and rhizosphere microorganism isolation and enumeration medium that inhibits Bacillus mycoides. Appl. Environ. Microbiol. 61:18391842.
9. Cano, R. J.,, M. J. Torres,, R. E. Klem,, J. C. Palomares, and, J. Casadesus. 1992. Detection of salmonellas by DNA hybridization with a fluorescent alkaline phosphatase substrate. J. Appl. Bacteriol. 72:393399.
10. Castro, H.,, S. Newman,, K. R. Reddy, and, A. V. Ogram. 2005. Distribution and stability of sulfate reducing prokaryotic and hydrogenotrophic methanogenic assemblages in nutrient-impacted regions of the Florida Everglades. Appl. Environ. Microbiol. 71:26952704.
11. Castro, H.,, K. R. Reddy, and, A. Ogram. 2002. Composition and function of sulfate-reducing prokaryotes in eutrophic and pristine areas of the Florida Everglades. Appl. Environ. Microbiol. 68:61296137.
12. Castro, H. F.,, A. Ogram, and, K. R. Reddy. 2004. Phylogenetic characterization of methanogenic assemblages in eutrophic and oligotrophic areas of the Florida Everglades. Appl. Environ. Microbiol. 70:65596568.
13. Connon, S. A.,, and S. J. Giovannoni. 2002. High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl. Environ. Microbiol. 68:38783885.
14. de Bruijn, F. J. 1992. Use of repetitive (repetitive extra-genic palindromic and enterobacterial repetitive intergenic consensus) sequences and the polymerase chain reaction to fingerprint the genomes of Rhizobium meliloti isolates and other soil bacteria. Appl. Environ. Microbiol. 58:21802187.
15. Dembitsky, V. M.,, E. E. Shubina, and, A. G. Kashin. 1992. Phospholipid and fatty acid composition of some basidiomycetes. Phytochemistry 31:845849.
16. Dunbar, J.,, L. O. Ticknor, and, C. R. Kuske. 2000. Assessment of microbial diversity in four southwestern United States soils by 16S rRNA gene terminal restriction fragment analysis. Appl. Environ. Microbiol. 66:29432950.
17. Farrelly, V.,, F. A. Rainey, and, E. Stackebrandt. 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. 61:27982801.
18. Felske, A.,, A. D. L. Akkermans, and, W. M. De Vos. 1998. Quantification of 16S rRNAs in complex bacterial communities by multiple competitive reverse transcription PCR in temperature gradient gel electrophoresis fingerprints. Appl. Environ. Microbiol. 64:45814587.
19. 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.
20. Frostegard, A.,, E. Baath, and, A. Tunlid. 1993. Shift in the structure of soil microbial communities in limed forests as revealed by phospholipid fatty acid analysis. Soil Biol. Biochem. 25:723730.
21. Gans, J.,, M. Wolinsky, and, J. Dunbar. 2005. Computational improvements reveal great bacterial diversity and high metal toxicity in soil. Science 309:13871390.
22. Ginige, M. P.,, P. Hugenholtz,, H. Daims,, M. Wagner,, J. Keller, and, L. L. Blackall. 2004. Use of stable-isotope probing, full-cycle rRNA analysis, and fluorescence in situ hybridization-microautoradiography to study a methanolfed denitrifying microbial community. Appl. Environ. Microbiol. 70:588596.
23. Giovannoni, S. J.,, T. B. Britschgi,, C. L. Moyer, and, K. G. Field. 1990. Genetic diversity in Sargasso Sea bacterioplankton. Nature (London) 345:6063.
24. Guckert, J. B.,, M. A. Hood, and, D. C. White. 1986. Phospholipid ester-linked fatty acid profile changes during nutrient deprivation of Vibrio cholerae: increases in the trans/cis ratio and proportions of cyclopropyl fatty acids. Appl. Environ. Microbiol. 52:794801.
25. Haack, S. K.,, H. Garchow,, D. A. Odelson,, L. J. Forney, and, M. J. Klug. 1994. Accuracy, reproducibility, and interpretation of fatty acid methyl ester profiles of model bacterial communities. Appl. Environ. Microbiol. 60:24832493.
26. Hadrys, H.,, M. Balick, and, B. Schierwater. 1992. Applications of random amplified polymorphic DNA (RAPD) in molecular ecology. Mol. Ecol. 1:5563.
27. Haldeman, D. L.,, and P. S. Amy. 1993. Diversity within a colony morphotype: implications for ecological research. Appl. Environ. Microbiol. 59:933935.
28. Handelsman, J. 2004. Metagenomics: application of genomics to uncultured microorganisms. Microbiol. Mol. Biol. Rev. 68:669685.
29. 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.
30. Heck, K. L.,, G. van Belle, and, D. Simberloff. 1975. Explicit calculation of the rarefaction diversity measurement and the determination of sufficient sample size. Ecology 56:14591461.
31. Hutchens, E.,, S. Radajewski,, M. G. Dumont,, I. R. McDonald, and, J. C. Murrell. 2004. Analysis of methanotrophic bacteria in Movile Cave by stable isotope probing. Environ. Microbiol. 6:111120.
32. Janssen, P. H.,, P. Yates,, B. Grinton,, P. Taylor, and, M. Sait. 2003. Improved culturability of soil bacteria and isolation in pure culture of novel members of the divisions Acidobacteria, Actinobacteria, Proteobacteria, and Verrucomicrobia. Appl. Environ. Microbiol. 68:23912396.
33. Joseph, S. J.,, P. Hugenhotlz,, P. Sangwan,, C. Osborne, and, P. Janssen. 2003. Laboratory cultivation of widespread and previously uncultured soil bacteria. Appl. Environ. Microbiol. 69:72107215.
34. Kaeberlein, T.,, K. Lewis, and, S. S. Epstein. 2002. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296:11271129.
35. Kennedy, A. C. 1994. Carbon utilization and fatty acid profiles for characterization of bacteria, p. 543–553. In R. W. Weaver,, S. Angle,, P. Bottomley,, D. Bezdicek,, S. Smith,, A. Tabatabai, and, A. Wollum (ed.), Methods of Soil Analysis, part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, Wis.
36. Kieft, T. L.,, D. B. Ringelberg, and, D. C. White. 1994. Changes in ester-linked phospholipid fatty acid profiles of subsurface bacteria during starvation and desiccation in a porous medium. Appl. Environ. Microbiol. 60:32923299.
37. Lane, D. J. 1991. 16S/23SrRNA sequencing, p. 115–175. In E. Stackebrandt and, M. Goodfellow (ed.), Nucleic Acid Techniques in Bacterial Systematics. John Wiley & Sons, New York, N.Y.
38. Liesack, W.,, and E. Stackebrandt. 1992. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J. Bacteriol. 174:50725078.
39. Lin, J. L.,, S. Radajewski,, B. T. Eshinimaev,, Y. A. Trotsenko,, I. R. McDonald, and, J. C. Murrell. 2004. Molecular diversity of methanotrophs in Transbaikal soda lake sediments and identification of potentially active populations by stable isotope probing. Environ. Microbiol. 6:10491060.
40. 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.
41. 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.
42. Lu, Y. H.,, T. Lueders,, M. W. Friedrich, and, R. Conrad. 2005. Detecting active methanogenic populations on rice roots using stable isotope probing. Environ. Microbiol. 7:326336.
43. Ludwig, W.,, O. Strunk,, R. Westram,, L. Richter,, H. Meier,, Yadhukumar,, A. Buchner,, T. Lai,, S. Steppi,, G. Jobb,, W. Forster,, I. Brettske,, S. Gerber,, A. W. Ginhart,, O. Gross,, S. Grumann,, S. Hermann,, R. Jost,, A. Konig,, T. Liss,, R. Lussmann,, M. May,, B. Nonhoff,, B. Reichel,, R. Strehlow,, A. Stamatakis,, N. Stuckmann,, A. Vilbig,, M. Lenke,, T. Ludwig,, A. Bode, and, K. H. Schleifer. 2004. ARB: a software environment for sequence data. Nucleic Acids Res. 32:13631371.
44. Lueders, T.,, B. Pommerenke, and, M. W. Friedrich. 2004. Stable-isotope probing of microorganisms thriving at thermodynamic limits: syntrophic propionate oxidation in flooded soil. Appl. Environ. Microbiol. 70:57785786.
45. Malik, M.,, J. Kain,, C. Pettigrew, and, A. Ogram. 1994. Purification and molecular analysis of microbial DNA from compost. J. Microbiol. Methods 20:183196.
46. 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.
47. Manefield, M.,, A. S. Whiteley,, N. Ostle,, P. Ineson, and, M. J. Bailey. 2002. Technical considerations for RNA-based stable isotope probing: an approach to associating microbial diversity with microbial community function. Rapid Commun. Mass Spectrom. 16:21792183.
48. Mayr, C.,, A. Winding, and, N. B. Hendriksen. 1999. Community level physiological profile of soil bacteria unaffected by extraction method. J. Microbiol. Methods 36:2933.
49. McSpadden Gardener, B. B.,, K. L. Schroeder,, S. E. Kalloger,, J. M. Raaijmakers,, L. S. Thomashow, and, D. M. Weller. 2000. Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat. Appl. Environ. Microbiol. 66:19391946.
50. Moss, C. W. 1981. Gas-liquid chromatography as an analytical tool in microbiology. J. Chromatogr. 203:337347.
51. Moyer, C. L.,, J. M. Tiedje,, F. C. Dobbs, and, D. M. Karl. 1996. A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: efficacy of selected tetrameric restriction enzymes for studies of microbial diversity in nature. Appl. Environ. Microbiol. 62:25012507.
52. Muyzer, G.,, E. C. de Waal, and, A. G. Uitterlinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of poly-merase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695700.
53. Muyzer, G.,, and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie Leeuwenhoek 73:127141.
54. Myers, R. M.,, T. Maniatis, and, L. S. Lerman. 1987. Detection and localization of single base changes by denaturing gradient gel electrophoresis. Methods Enzymol. 155:501527.
55. Nei, M.,, and W. H. Li. 1979. Mathematical model for studying genetic variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. USA 76:52695273.
56. Nunan, N.,, K. Wu,, I. M. Young,, J. W. Crawford, and, K. Ritz. 2003. Spatial distribution of bacterial communities and their relationships with the micro-architecture of soil. FEMS Microbiol. Ecol. 44:203215.
57. Ogram, A. 2000. Soil molecular microbial ecology at age 20: methodological challenges for the future. Soil Biol. Biochem. 32:14991504.
58. Ogram, A. V.,, and D. F. Bezdicek. 1994. Nucleic acid probes, p. 543–553. In R. W. Weaver,, S. Angle,, P. Bottomley,, D. Bezdicek,, S. Smith,, A. Tabatabai, and, A. Wollum (ed.), Methods of Soil Analysis, part 2. Microbiological and Biochemical Properties. Soil Science Society of America, Madison, Wis.
59. Oline, D. K.,, and M. C. Grant. 2002. Scaling patterns of biomass and soil properties: an empirical analysis. Landscape Ecol. 17:1326.
60. Osborn, A. M.,, E. R. B. Moore, and, K. N. Timmis. 2000. An evaluation of terminal-restriction fragment length polymorphism (T-RFLP) analysis for the study of microbial community structure and dynamics. Environ. Microbiol. 2:3950.
61. Penner, G. A.,, A. Bush,, R. Wise,, W. Kim,, L. Domier,, K. Kasha,, A. Laroche,, G. Scoles,, S. J. Molnar, and, G. Fedak. 1993. Reproducibility of random amplified polymorphic DNA (RAPD) analysis among laboratories. PCR Methods Appl. 2:341345.
62. Radajewski, S.,, P. Ineson,, N. R. Parekh, and, J. C. Murrell. 2000. Stable-isotope probing as a tool in microbial ecology. Nature 403:646649.
63. Radajewski, S.,, I. R. McDonald, and, J. C. Murrell. 2003. Stable-isotope probing of nucleic acids: a window to the function of uncultured microorganisms. Curr. Opin. Biotechnol. 14:296302.
64. Rainey, F. A.,, N. L. Ward-Rainey,, P. H. Janssen,, H. Hippe, and, E. Stackebrandt. 1996. Clostridium paradoxum DSM 7308T contains multiple 16S rRNA genes with heterogeneous intervening sequences. Microbiology 142:20872095.
65. Raskin, L.,, W. C. Capman,, M. D. Kane,, B. E. Rittmann, and, D. A. Stahl. 1996. Critical evaluation of membrane supports for use in quantitative hybridizations. Appl. Environ. Microbiol. 62:300303.
66. Robison-Cox, J. F.,, M. M. Bateson, and, D. M. Ward. 1995. Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences. Appl. Environ. Microbiol. 61:12401245.
67. Sait, M.,, P. Hugenholtz, and, P. Janssen. 2002. Cultivation of globally distributed soil bacteria from phylogenetic lineages previously only detected in cultivation-independent surveys. Environ. Microbiol. 4:654666.
68. Sambrook, J.,, E. Fritsch, and, T. Maniatis. 1989. Molecular Cloning: a Laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
69. Sayler, G. S.,, and A. C. Layton. 1990. Environmental application of nucleic acid hybridization. Annu. Rev. Microbiol. 44:625648.
70. Sayler, G. S.,, M. S. Shields,, E. Tedford,, A. Breen,, S. Hooper,, K. Sirotkin, and, J. Davis. 1985. Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples. Appl. Environ. Microbiol. 49:12951303.
71. Seeley, H. W.,, and P. VanDemark. 1981. Microbes in Action: a Laboratory Manual of Microbiology, 3rd ed. W. H. Freeman and Co., New York, N.Y.
72. Stackebrandt, E.,, and B. M. Goebel. 1994. Taxonomic note: a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol. 44:846849.
73. Stevenson, B. S.,, S. A. Eichorst,, J. T. Wertz,, T. M. Schmidt, and, J. A. Breznak. 2004. New strategies for cultivation and detection of previously uncultured microbes. Appl. Environ. Microbiol. 70:47484755.
74. Suzuki, M. T.,, and S. J. Giovannoni. 1996. Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62:625630.
75. Swofford, D. L. 1999. PAUP: Phylogenetic Analysis Using Parsimony, version 4.0. Sinauer Associates, Sunder-land, Mass.
76. Teske, A.,, C. Wawer,, G. Muyzer, and, N. B. Ramsing. 1996. Distribution of sulfate-reducing bacteria in a stratified fjord (Mariager Fjord, Denmark) as evaluated by most-probable-number counts and denaturing gradient gel electrophoresis of PCR-amplified ribosomal DNA fragments. Appl. Environ. Microbiol. 62:14051415.
77. Torsvik, V.,, J. Goksøyr, and, F. L. Daae. 1990. High diversity in DNA of soil bacteria. Appl. Environ. Microbiol. 56:782787.
78. Torsvik, V.,, K. Salte,, R. Sørheim, and, J. Goksøyr. 1990. Comparison of phenotypic diversity and DNA heterogeneity in a population of soil bacteria. Appl. Environ. Microbiol. 56:776781.
79. Uchiyama, T.,, T. Abe,, T. Ikemura, and, K. Watanabe. 2005. Substrate-induced gene-expression screening of environmental metagenome libraries for isolation of catabolic genes. Nat. Biotechnol. 23:8893.
80. Van de Peer, Y.,, and R. Wachter. 1997. Construction of evolutionary distance trees with TREECON for Windows: accounting for variation in nucleotide substitution rate among sites. Comput. Appl. Biosci. 13:227230.
81. Venter, J. C.,, K. Remington,, J. F. Heidelberg,, A. L. Halpern,, D. Rusch,, J. A. Eisen,, D. Y. Wu,, I. Paulsen,, K. E. Nelson,, W. Nelson,, D. E. Fouts,, S. Levy,, A. H. Knap,, M. W. Lomas,, K. Nealson,, O. White,, J. Peterson,, J. Hoffman,, R. Parsons,, H. Baden-Tillson,, C. Pfannkoch,, Y. H. Rogers, and, H. O. Smith. 2004. Environmental genome shotgun sequencing of the Sargasso Sea. Science 304:6674.
82. Vestal, J. R.,, and D. C. White. 1998. Lipid analysis in microbial ecology. Bioscience 39:535541.
83. Vilgalys, R.,, and M. Hester. 1990. Rapid genetic identification and mapping of enzymatically amplified ribosomal DNA from several Cryptococcus species. J. Bacteriol. 172:42384246.
84. Voordouw, G.,, Y. Shen,, C. S. Harrington,, A. J. Telang,, T. R. Jack, and, D. W. S. Westlake. 1993. Quantitative reverse sample genome probing of microbial communities and its application to oil field production waters. Appl. Environ. Microbiol. 59:41014114.
85. Wackett, L. P. 2004. Stable isotope probing in biodegradation research. Trends Biotechnol. 22:153154.
86. Wellington, E. M. H.,, A. Berry, and, M. Krsek. 2003. Resolving functional diversity in relation to microbial community structure in soil: exploiting genomics and stable isotope probing. Curr. Opin. Biotechnol. 6:295301.
87. White, D. C.,, R. J. Bobbie,, J. D. King,, J. Nickels, and, P. Amoe. 1979. Lipid analysis of sediments for microbial biomass and community structure, p. 87–103. In C. D. Litchfield and, P. L. Seyfried (ed.), Methodology for Biomass Determinations and Microbial Activities in Sediments. ASTM STP 673. American Society for Testing and Materials, Philadelphia, Pa.
88. Woese, C. R.,, O. Kandler, and, M. L. Wheelis. 1990. Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc. Natl. Acad. Sci. USA 87:45764579.
89. Yang, J.,, R. D. Hammer, and, R. W. Blanchar. 1995. Microscale pH spatial distribution in the Ap horizon of Mexico silt loam. Soil Sci. 160:371375.
90. Zelles, L.,, and Q. Y. Bai. 1993. Fractionation of fatty acids derived from soil lipids by solid phase extraction and their quantitative analysis by GC-MS. Soil Biol. Biochem. 25:495507.
91. Zengler, K.,, G. Toledo,, M. Rappe,, J. Elkins,, E. J. Mathur,, J. M. Short, and, M. Keller. 2002. Cultivating the uncultured. Proc. Natl. Acad. Sci. USA 99:1568115686.

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