EVALUATION OF THE DNA-FINGERPRINTING METHOD AFLP AS A NEW TOOL IN BACTERIAL TAXONOMY

We investigated the usefulness of a novel DNA fingerprinting technique , AFLP, which is based on the selective amplification of genomic restr iction fragments by PCR, to differentiate bacterial strains at the sub generic level. In total, 147 bacterial strains were subjected to AFLP fingerprinting: 36 Xanthomonas strains, including 23 pathovars of Xant homonas axonopodis and six pathovars of Xanthomonas vasicola, one stra in of Stenotrophomonas, 90 genotypically characterized strains compris ing all 14 hybridization groups currently described in the genus Aerom onas, and four strains of each of the genera Clostridium, Bacillus, Ac inetobacter, Pseudomonas and Vibrio. Depending on the genus, total gen omic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were liga ted to restriction halfsite-specific adaptors. These adaptors served a s primer-binding sites allowing the fragments to be amplified by selec tive PCR primers that extend beyond the adaptor and restriction site s equences. Following electrophoretic separation on 5% (w/v) polyacrylam ide/8.3 M urea, amplified products could be visualized by autoradiogra phy because one of the selective primers was radioactively labelled. T he resulting banding patterns, containing approximately 30-50 visualiz ed PCR products in the size range 80-550 bp, were captured by a high-r esolution densitoscanner and further processed for computer-assisted a nalysis to determine band-based similarity coefficients. This study re veals extensive evidence for the applicability of AFLP in bacterial ta xonomy through comparison of the newly obtained data with results prev iously obtained by well-established genotypic and chemotaxonomic metho ds such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to charac terize strains at the infrasubspecific level), highlighting the potent ial of this novel fingerprinting method in epidemiological and evoluti onary studies.