Analysis of bacterial community composition by oligonucleotide fingerprinting of rRNA genes - PubMed (original) (raw)

Analysis of bacterial community composition by oligonucleotide fingerprinting of rRNA genes

Lea Valinsky et al. Appl Environ Microbiol. 2002 Jul.

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.

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Figures

FIG. 1.

FIG. 1.

Diagram of OFRG strategy.

FIG. 2.

FIG. 2.

Arrayed bacterial rDNA clones hybridized with 33P-labeled DNA oligonucleotide probes. (A) Discriminating probes 1 to 27 and reference probe 28 hybridized to a common set of clones; (B) discriminating probe 4 hybridized to 1,536 clones.

FIG. 3.

FIG. 3.

Taxonomic depiction of soil bacteria produced by OFRG. The UPGMA tree was constructed from rDNA hybridization fingerprints derived from two soils. (A) Complete UPGMA tree. The numbers indicate cluster designations. (B) Detailed depiction of the Bacillus clade. This tree is composed of three segments; the asterisks and solid circles indicate connection sites. The full-length tree can be obtained from the corresponding author. rDNA clones are designated by a number followed by a space and then either 1 or 2; 1 indicates that the clone was from soil 9C, and 2 indicates that the clone was from soil 9E. Clones whose nucleotide sequences were determined are indicated by the suffix S; identities with GenBank sequences are indicated. Cluster 1 is indicated.

FIG. 3.

FIG. 3.

Taxonomic depiction of soil bacteria produced by OFRG. The UPGMA tree was constructed from rDNA hybridization fingerprints derived from two soils. (A) Complete UPGMA tree. The numbers indicate cluster designations. (B) Detailed depiction of the Bacillus clade. This tree is composed of three segments; the asterisks and solid circles indicate connection sites. The full-length tree can be obtained from the corresponding author. rDNA clones are designated by a number followed by a space and then either 1 or 2; 1 indicates that the clone was from soil 9C, and 2 indicates that the clone was from soil 9E. Clones whose nucleotide sequences were determined are indicated by the suffix S; identities with GenBank sequences are indicated. Cluster 1 is indicated.

FIG. 3.

FIG. 3.

Taxonomic depiction of soil bacteria produced by OFRG. The UPGMA tree was constructed from rDNA hybridization fingerprints derived from two soils. (A) Complete UPGMA tree. The numbers indicate cluster designations. (B) Detailed depiction of the Bacillus clade. This tree is composed of three segments; the asterisks and solid circles indicate connection sites. The full-length tree can be obtained from the corresponding author. rDNA clones are designated by a number followed by a space and then either 1 or 2; 1 indicates that the clone was from soil 9C, and 2 indicates that the clone was from soil 9E. Clones whose nucleotide sequences were determined are indicated by the suffix S; identities with GenBank sequences are indicated. Cluster 1 is indicated.

FIG. 4.

FIG. 4.

Amplification of soil DNA with cluster-specific PCR primers. The numbers at the top are cluster designations (see Fig. 3). C and E indicate soils 9C and 9E, respectively. Clusters 1, 3, and 5 contain only soil 9C clones. Clusters 2, 4, 6, 7, and 8 contain only soil 9E clones. Lane M contained a DNA ladder. The arrows indicate the DNA fragments amplified by the PCR primer pairs.

FIG. 5.

FIG. 5.

DGGE analysis of bacterial rDNA from soils 9C and 9E.

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