Molecular microbial diversity of an agricultural soil in Wisconsin

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, the 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.