A hypervariable 23S rRNA region provides a discriminating target for specific characterization of uncultured and cultured Frankia
1994
Honerlage, W. | Hahn, D. | Zepp, K. | Zeyer, J. | Normand, P.
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 host infection groups. The Casuarina and Elaeagnus groups were characterized by no or only small sequence variation. The Alnus group could be separated roughly into four subgroups, three containing typical nitrogen-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 Purshia tridentata, respectively, could be aligned. The uncultured nodular endophyte of Coriaria nepalensis appeared well separated from all other sequences. The results of hybridization experiments with in vitro transcripts, PCR amplification products and oligonucleotides indicated a sufficient ratio of variability and stability within this 23S rRNA insertion to serve as target for subgroup-specific detection of Frankia within the Alnus group. Reliable discrimination with large probes like transcripts or PCR products, however, was only achieved between the non-nitrogen fixing Frankia strains and the nitrogen-fixing strains of the Alnus host infection group. The subgroups of the nitrogen-fixing strains were subsequently distinguished by oligonucleotide probes. Uncultured Frankia populations in root nodules of Alnus glutinosa could be characterized by these probes after PCR assisted sequence retrieval of the 23S rRNA insertion and cloning into E. coli.
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