Comparison of RNA- and DNA-based species diversity investigations in rhizoplane bacteriology with respect to chloroplast sequence exclusion.

Abstract

Comparative sequence analysis of 16S rDNA genes is a popular method of investigating microbial communities but problems arise when the subjects are rhizoplane consortia. The culture independent direct isolation of DNA from root sample results in huge amounts of plant DNA, and the universal primers designed for the domain Bacteria will amplify chloroplast ribosomal genes as well. A clone library generated from such a PCR product will be dominated by chloroplast, and the emulation of numerous chloroplasts and rhizoplane bacterial 16S rDNA for primers also distorts the results of different fingerprinting analyses. To resolve this problem, a new approach has been developed. The ribosome content is correlated with the metabolic activity of cells; therefore, RNA-based methods seem to be appropriate to exclude cell organelles (e.g. chloroplast) and dormant bacterial cells. A rapid RNA isolation and a reliable reverse transcription (RT)-PCR method were developed to investigate rhizoplane bacterial community and the results were compared with a total DNA isolation-based method of the same sample. 16S rRNA and DNA PCR products were cloned and screened by restriction analysis. The relative abundance of chloroplast amplicons in DNA and RNA clone libraries was compared and a significant decrease was detected (from 63% and 71% to 1% and 7%, respectively).