Molecular microbial diversity of an agricultural soil in Wisconsin (original) (raw)

Abstract

A culture-independent survey of the soil microbial diversity in a clover-grass pasture in southern Wisconsin was conducted by sequence analysis of a universal clone library of genes coding for small-subunit rRNA (rDNA). A rapid and efficient method for extraction of DNA from soils which resulted in highly purified DNA with minimal shearing was developed. Universal small-subunit-rRNA primers were used to amplify DNA extracted from the pasture soil. The PCR products were cloned into pGEM-T, and either hypervariable or conserved regions were sequenced. The relationships of 124 sequences to those of cultured organisms of known phylogeny were determined. Of the 124 clones sequenced, 98.4% were from the domain Bacteria. Two of the rDNA sequences were derived from eukaryotic organelles. Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence. No sequences of the domain Archaea were found. Within the domain, Bacteria, three kingdoms were highly represented: the Proteobacteria (16.1%), the Cytophaga-Flexibacter-Bacteroides group (21.8%), and the low G+C-content gram-positive group (21.8%). Some kingdoms, such as the Thermotogales, the green nonsulfur group, Fusobacteria, and the Spirochaetes, were absent. A large number of the sequences (39.4%) were distributed among several clades that are not among the major taxa described by Olsen et al. (G.J. Olsen, C.R. Woese, and R. Overbeek, J. Bacteriol., 176:1-6, 1994). From the alignments of the sequence data, distance matrices were calculated to display the enormous microbial diversity found in this soil in two ways, as phylogenetic trees and as multidimensional-scaling plots.

Full Text

The Full Text of this article is available as a PDF (453.1 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Amann R. I., Ludwig W., Schleifer K. H. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol Rev. 1995 Mar;59(1):143–169. doi: 10.1128/mr.59.1.143-169.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barns S. M., Fundyga R. E., Jeffries M. W., Pace N. R. Remarkable archaeal diversity detected in a Yellowstone National Park hot spring environment. Proc Natl Acad Sci U S A. 1994 Mar 1;91(5):1609–1613. doi: 10.1073/pnas.91.5.1609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boivin-Jahns V., Bianchi A., Ruimy R., Garcin J., Daumas S., Christen R. Comparison of phenotypical and molecular methods for the identification of bacterial strains isolated from a deep subsurface environment. Appl Environ Microbiol. 1995 Sep;61(9):3400–3406. doi: 10.1128/aem.61.9.3400-3406.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Choi B. K., Paster B. J., Dewhirst F. E., Göbel U. B. Diversity of cultivable and uncultivable oral spirochetes from a patient with severe destructive periodontitis. Infect Immun. 1994 May;62(5):1889–1895. doi: 10.1128/iai.62.5.1889-1895.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. DeLong E. F., Wu K. Y., Prézelin B. B., Jovine R. V. High abundance of Archaea in Antarctic marine picoplankton. Nature. 1994 Oct 20;371(6499):695–697. doi: 10.1038/371695a0. [DOI] [PubMed] [Google Scholar]
  6. Embley T. M., Hirt R. P., Williams D. M. Biodiversity at the molecular level: the domains, kingdoms and phyla of life. Philos Trans R Soc Lond B Biol Sci. 1994 Jul 29;345(1311):21–33. doi: 10.1098/rstb.1994.0083. [DOI] [PubMed] [Google Scholar]
  7. Farrelly V., Rainey F. A., Stackebrandt E. 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. 1995 Jul;61(7):2798–2801. doi: 10.1128/aem.61.7.2798-2801.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kopczynski E. D., Bateson M. M., Ward D. M. Recognition of chimeric small-subunit ribosomal DNAs composed of genes from uncultivated microorganisms. Appl Environ Microbiol. 1994 Feb;60(2):746–748. doi: 10.1128/aem.60.2.746-748.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Lane D. J., Pace B., Olsen G. J., Stahl D. A., Sogin M. L., Pace N. R. Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci U S A. 1985 Oct;82(20):6955–6959. doi: 10.1073/pnas.82.20.6955. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Leff L. G., Dana J. R., McArthur J. V., Shimkets L. J. Comparison of methods of DNA extraction from stream sediments. Appl Environ Microbiol. 1995 Mar;61(3):1141–1143. doi: 10.1128/aem.61.3.1141-1143.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Liesack W., Stackebrandt E. Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment. J Bacteriol. 1992 Aug;174(15):5072–5078. doi: 10.1128/jb.174.15.5072-5078.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Moré M. I., Herrick J. B., Silva M. C., Ghiorse W. C., Madsen E. L. Quantitative cell lysis of indigenous microorganisms and rapid extraction of microbial DNA from sediment. Appl Environ Microbiol. 1994 May;60(5):1572–1580. doi: 10.1128/aem.60.5.1572-1580.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Neefs J. M., Van de Peer Y., Hendriks L., De Wachter R. Compilation of small ribosomal subunit RNA sequences. Nucleic Acids Res. 1990 Apr 25;18 (Suppl):2237–2317. doi: 10.1093/nar/18.suppl.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Olsen G. J., Woese C. R., Overbeek R. The winds of (evolutionary) change: breathing new life into microbiology. J Bacteriol. 1994 Jan;176(1):1–6. doi: 10.1128/jb.176.1.1-6.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Reysenbach A. L., Giver L. J., Wickham G. S., Pace N. R. Differential amplification of rRNA genes by polymerase chain reaction. Appl Environ Microbiol. 1992 Oct;58(10):3417–3418. doi: 10.1128/aem.58.10.3417-3418.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Robison-Cox J. F., Bateson M. M., Ward D. M. Evaluation of nearest-neighbor methods for detection of chimeric small-subunit rRNA sequences. Appl Environ Microbiol. 1995 Apr;61(4):1240–1245. doi: 10.1128/aem.61.4.1240-1245.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Roszak D. B., Colwell R. R. Survival strategies of bacteria in the natural environment. Microbiol Rev. 1987 Sep;51(3):365–379. doi: 10.1128/mr.51.3.365-379.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  19. Schmidt T. M., DeLong E. F., Pace N. R. Analysis of a marine picoplankton community by 16S rRNA gene cloning and sequencing. J Bacteriol. 1991 Jul;173(14):4371–4378. doi: 10.1128/jb.173.14.4371-4378.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Stackebrandt E., Liesack W., Goebel B. M. Bacterial diversity in a soil sample from a subtropical Australian environment as determined by 16S rDNA analysis. FASEB J. 1993 Jan;7(1):232–236. doi: 10.1096/fasebj.7.1.8422969. [DOI] [PubMed] [Google Scholar]
  21. Staley J. T., Konopka A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu Rev Microbiol. 1985;39:321–346. doi: 10.1146/annurev.mi.39.100185.001541. [DOI] [PubMed] [Google Scholar]
  22. Torsvik V., Goksøyr J., Daae F. L. High diversity in DNA of soil bacteria. Appl Environ Microbiol. 1990 Mar;56(3):782–787. doi: 10.1128/aem.56.3.782-787.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ueda T., Suga Y., Yahiro N., Matsuguchi T. Genetic diversity of N2-fixing bacteria associated with rice roots by molecular evolutionary analysis of a nifD library. Can J Microbiol. 1995 Mar;41(3):235–240. doi: 10.1139/m95-032. [DOI] [PubMed] [Google Scholar]
  24. Ward D. M., Weller R., Bateson M. M. 16S rRNA sequences reveal numerous uncultured microorganisms in a natural community. Nature. 1990 May 3;345(6270):63–65. doi: 10.1038/345063a0. [DOI] [PubMed] [Google Scholar]
  25. Woese C. R. Bacterial evolution. Microbiol Rev. 1987 Jun;51(2):221–271. doi: 10.1128/mr.51.2.221-271.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]