Typing method for N2-fixing bacteria based on PCR-F — application to the characterization of Frankia strains |
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Authors: | S. JAMANN M. P. FERNANDEZ P. NORMAND |
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Affiliation: | Laboratoire d'Ecologie microbienne du Sol, LIRA CNRS 1450, UniversitéClaude Bernard, 43 Boulevard du 11 November, 69622 Villeurbanne, France |
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Abstract: | 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. |
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Keywords: | Frankia typing method nifD-K intergenic spacer (IGS) nifDK primers polymerase chain reaction (PCR) restriction fragment length polymorphism (RFLP) |
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