Typing method for N2-fixing bacteria based on PCR-RFLP--application to the characterization of Frankia strains

DNA sequences of an intergenic spacer (IGS) and parts of genes in the nif cluster were amplified by the polymerase chain reaction (PCR) using two primers derived from nifD- and nifK-conserved sequences. The PCR products were cleaved by ten 4-base cutting restriction enzymes and the restriction patterns were used as fingerprints to type Frankia strains. The feasability of this PCR-RFLP method for typing Frankia strains was investigated on Frankia reference strains belonging mainly to the Elaeagnaceae infectivity group but also on new Frankia isolates and on other N2-fixing microorganisms. By modulating the stringency of the amplifications, we showed the method allowed to target either Frankia strains or the whole N2-fixing microbial community. DNA digestion patterns were used to estimate the sequence divergence between the Frankia nifD-K fragment. The estimated relationships deduced from these genotypic data correlated well with established Frankia taxonomic schemes.