Searching for predominant soil bacteria: 16S rDNA cloning versus strain cultivation.

Abstract

The predominant bacteria in Dutch grassland soils, as identified by direct DNA extraction, PCR amplification of 16S rDNA and subsequent cloning and sequencing, were compared to the most abundant culturable bacteria. The 16S rDNAs of the strains from a comprehensive cultivation campaign were compared to some of the predominant cloned sequences by temperature gradient gel electrophoresis (TGGE). Four ribotypes were selected that were found to be abundant in the clone library: two closely related Bacillus-like sequences, a representative from the Verrucomicrobiales cluster and an uncultured member of the Actinobacteria. Using a variety of cultivation approaches a total of 659 pure cultures were isolated. Initially, approximately 8% of all isolates matched any of these ribotypes by same migration speed of their 16S rDNA amplicons on TGGE. However, sequencing analysis of matching isolates indicated that their 16S rDNA sequences were clearly different from the cloned sequences representing the fingerprint bands. Comparing the cultivation approach and the molecular 16S rDNA analysis from the same soil sample, there was no correlation between the collection of cultured strains and the 16S rDNA clone library.