A SIMPLE METHOD FOR QUANTIFICATION OF UNCULTURED MICROORGANISMS IN THE ENVIRONMENT BASED ON IN-VITRO TRANSCRIPTION OF 16S RIBOSOMAL-RNA

A simple method for the quantification of uncultured microorganisms in the environment was developed. In vitro-transcribed 16S rRNA is used as a template for midpoint dissociation temperature (T-d) determinatio ns of specific oligonucleotide probes and as a standard in quantitativ e probing. It replaces the need for total nucleic acids extracted from pure cultures of the organisms to be quantified. A sense RNA of a siz e almost identical to that of native 16S rRNA can be transcribed from ribosomal DNA clones recovered in studies of the phylogenetic diversit y of microbial communities. This in vitro-transcribed rRNA yields diss ociation curves typical of oligonucleotides. They parallel cut-yes det ermined with total nucleic acids but yield slightly higher T-d values. Neither unspecific sticking of the probe nor probe washing off the DN A template at low temperatures fully accounted for the discrepancy in probe release from the two templates. This suggests that the native rR NA itself has melting characteristics different from those of its in v itro-transcribed counterpart. However, this difference does not affect the performance of in vitro-transcribed rRNA compared with total nucl eic acids as a standard in quantitative hybridizations. No difference was found between the estimates of the relative quantity of a single b acterial species in a mixed community obtained with the two standards, regardless of whether DNA was removed from the samples. This protocol will allow the large-scale quantification of the ecological importanc e of uncultured microorganisms in natural environments for the first t ime.