Rapid identification of Rhizobium strains by targeted PCR fingerprinting

Numerous polymerase chain reaction (PCR) based procedures are routinely used to produce genomic fingerprints of prokaryotes. Many of them have drawbacks however such as sensitivity to experimental variation, lack of reproducibility, poor resolution and the inability to distinguish between closely related strains. To overcome these difficulties, we developed an alternative procedure, Targeted PCR Fingerprinting (TPF) which is based upon the amplification of few but carefully selected markers, followed by high resolution RFLP analysis of the amplified DNA fragments. In contrast to most fingerprinting protocols that use low resolution agarose gels, TPF patterns are produced on denaturing polyacrylamide gels which allow the precise recording of the genomic fingerprints. TPF analysis, which can simultaneously process 96 samples in less than 12 h and remains unaffected by slight experimental variations, is particularly adapted for the rapid identification of target strains amongst many field isolates. Using PCR primers specific for the nifH and recA genes, this procedure was also sufficiently sensitive to discriminate between Rhizobium species NGR234 and R. fredii USDA257, two closely related bacteria in which the symbiotic loci are 98% homologous. Interestingly, comparison of several of their symbiotic genes as well as the partial DNA sequences of their 16S rDNA and recA genes suggest that chromosomes and symbiotic plasmids did not co-evolve, but that symbiotic functions were acquired by lateral gene transfer long after NGR234 and USDA257 diverged from their common ancestors. In this respect, TPF fingerprints produced with distinct chromosomal and plasmid born markers, such as the recA and the nifH genes in NGR234 and USDA257, are probably more Likely to detect lateral transfer of genes in bacterial field-populations than procedures relying on the amplification of numerous fragments of unknown genomic position and biological function.