Analysis of Bacterial Community Composition by Oligonucleotide Fingerprinting of rRNA Genes

Abstract

ABSTRACT One of the first steps in characterizing an ecosystem is to describe the organisms inhabiting it. For microbial studies, experimental limitations have hindered the ability to depict diverse communities. Here we describe oligonucleotide fingerprinting of rRNA genes (OFRG), a method that permits identification of arrayed rRNA genes (rDNA) through a series of hybridization experiments using small DNA probes. To demonstrate this strategy, we examined the bacteria inhabiting two different soils. Analysis of 1,536 rDNA clones revealed 766 clusters grouped into five major taxa: Bacillus , Actinobacteria , Proteobacteria , and two undefined assemblages. Soil-specific taxa were identified and then independently confirmed through cluster-specific PCR of the original soil DNA. Near-species-level resolution was obtained by this analysis as clones with average sequence identities of 97% were grouped in the same cluster. A comparison of these OFRG results with the results obtained in a denaturing gradient gel electrophoresis analysis of the same two soils demonstrated the significance of this methodological advance. OFRG provides a cost-effective means to extensively analyze microbial communities and should have applications in medicine, biotechnology, and ecosystem studies.