MOLECULAR MICROBIAL DIVERSITY OF AN AGRICULTURAL SOIL IN WISCONSIN

A culture-independent survey of the soil microbial diversity in a clov er-grass pasture in southern Wisconsin was conducted by sequence analy sis of a universal clone library of genes coding for small-subunit rRN A (rDNA), A rapid and efficient method for extraction of DNA from soil s which resulted in highly purified DNA with minimal shearing was deve loped, Universal small-subunit-rRNA primers were used to amplify DNA e xtracted 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 fr om the domain Bacteria, Two of the rDNA sequences were derived from eu karyotic organelles, Two of the 124 sequences were of nuclear origin, one being fungal and the other a plant sequence, No sequences of the d omain Archaea were found, Within the domain Bacteria, three kingdoms w ere highly represented: the Proteobacteria (16.1%), the Cytophaga-Flex ibacter-Bacteroides group (21.8%), and the low-G+C-content gram-positi ve 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 calcu lated to display the enormous microbial diversity found in this soil i n two ways, as phylogenetic trees and as multidimensional-scaling plot s.