CHARACTERIZATION OF A SPONTANEOUS THIOSTREPTON-RESISTANT FRANKIA-ALNI INFECTIVE ISOLATE USING PCR-RFLP OF NIF AND GLNII GENES

A spontaneous thiostrepton-resistant isolate of Frankia alni strain ACN14a is described. This isolate, named ACN14tsr, is infective and effective when in association with Alnus glutinosa. The resistance to thiostrepton is stable and maintained after growth in the absence of the antibiotic. The isolate was shown to be related to its parent strain by a three step PCR (polymerase chain reaction)-based method using nif and gln II genes, Frankia-specific PCR amplification of the gln II gene was used as a taxonomic marker of the genus. The defined PCR primers allowed amplification of the gln II gene from all Frankia strains tested so far but did not detect gln II in Geodermatophilus obscurus, Blastococcus aggregatus and the Actinoplanetes. This approach discriminated the thiostrepton-resistant Frankia isolate from closely related genera of the Actinomycetes. RFLP (restriction fragment length polymorphism) analysis of PCR fragments from the nif HD operon of various Frankia species then reliably related the isolate to Frankia alni. Finally, RFLP analysis of PCR fragments from the gln II gene allowed identification of the parent strain of the thiostrepton-resistant isolate. This is the first description of a subspecific characterization of strains of Frankia alni using amplified genes and RFLP patterns.