Rapid identification of Rhizobium strains by targeted PCR fingerprinting

Numerous polymerase chain reaction (PCR) based procedures are routinely use d to produce genomic fingerprints of prokaryotes. Many of them have drawbac ks however such as sensitivity to experimental variation, lack of reproduci bility, poor resolution and the inability to distinguish between closely re lated strains. To overcome these difficulties, we developed an alternative procedure, Targeted PCR Fingerprinting (TPF) which is based upon the amplif ication of few but carefully selected markers, followed by high resolution RFLP analysis of the amplified DNA fragments. In contrast to most fingerpri nting protocols that use low resolution agarose gels, TPF patterns are prod uced on denaturing polyacrylamide gels which allow the precise recording of the genomic fingerprints. TPF analysis, which can simultaneously process 9 6 samples in less than 12 h and remains unaffected by slight experimental v ariations, is particularly adapted for the rapid identification of target s trains amongst many field isolates. Using PCR primers specific for the nifH and recA genes, this procedure was also sufficiently sensitive to discrimi nate between Rhizobium species NGR234 and R. fredii USDA257, two closely re lated bacteria in which the symbiotic loci are 98% homologous. Interestingl y, comparison of several of their symbiotic genes as well as the partial DN A sequences of their 16S rDNA and recA genes suggest that chromosomes and s ymbiotic plasmids did not co-evolve, but that symbiotic functions were acqu ired by lateral gene transfer long after NGR234 and USDA257 diverged from t heir common ancestors. In this respect, TPF fingerprints produced with dist inct chromosomal and plasmid born markers, such as the recA and the nifH ge nes in NGR234 and USDA257, are probably more Likely to detect lateral trans fer of genes in bacterial field-populations than procedures relying on the amplification of numerous fragments of unknown genomic position and biologi cal function.