A HYPERVARIABLE 23S RIBOSOMAL-RNA REGION PROVIDES A DISCRIMINATING TARGET FOR SPECIFIC CHARACTERIZATION OF UNCULTURED AND CULTURED FRANKIA

A highly variable part in domain III of the 23S rRNA specific for Gram -positive bacteria with a high DNA G+C content was evaluated as probe/ target system for the specific detection of the actinomycete Frankia. Comparative sequence analysis of PCR amplified and cloned inserts from 35 Frankia strains confirmed the separation of these strains into hos t infection groups. The Casuarina and Elaeagnus groups were characteri zed by no or only small sequence variation. The Alnus group could be s eparated roughly into four subgroups, three containing typical nitroge n-fixing strains and a fourth one containing only non-nitrogen-fixing strains. To the latter group, two other non-infective and non-nitrogen -fixing strains, Cn3 and PtI4 isolated from Coriaria nepalensis and Pu rshia tridentata, respectively, could be aligned. The uncultured nodul ar endophyte of Coriaria nepalensis appeared well separated from all o ther sequences. The results of hybridization experiments with in vitro transcripts, PCR amplification products and oligonucleotides indicate d a sufficient ratio of variability and stability within this 23S rRNA insertion to serve as target for subgroup-specific detection of Frank ia within the Alnus group. Reliable discrimination with large probes l ike transcripts or PCR products, however, was only achieved between th e non-nitrogen fixing Frankia strains and the nitrogen-fixing strains of the Alnus host infection group. The subgroups of the nitrogen-fixin g strains were subsequently distinguished by oligonucleotide probes. U ncultured Frankia populations in root nodules of Alnus glutinosa could be characterized by these probes after PCR assisted sequence retrieva l of the 23S rRNA insertion and cloning into E. coli.