Using a method known as low-frequency restriction fragment analysis (LFRFA) (M. L. Beyazova and M. P. Lechevalier, Int. J. Syst. Bacteriol. 42:422-433, 1992), we determined the molecular weights of I restriction fragments of DNAs by pulsed-field gel electrophoresis. The levels of similarity of fragment patterns among strains were determined by using the simple matching coefficient, and clustering was performed by using the unweighted pair group with mathematical average algorithm. A total of 59 strains representing eight species and the numerically classified taxon group A18 (S. T. Williams, M. Goodfellow, G. Alderson, E. M. H. Wellington, P. H. A. Sneath, and M. J. Sackin, J. Gen. Microbiol. 129:1743-1813, 1983) were studied. Forty-two strains (six species) formed eight clusters at levels of similarity of more than 80%; 17 strains (including the entire group) were unclustered. Cluster 1 contained all of the strains studied plus two strains of and two strains of Cluster 2 contained 8 of the 12 strains examined plus one strain each of and Cluster 3 was heterogeneous in terms of species. Cluster 4 contained two strains; cluster 5 contained three strains; cluster 6 contained three strains; and clusters 7 and 8 contained seven and three strains, respectively. The group strains exhibited no clustering among themselves or with the other species examined. Some species which exhibited high levels of similarity (85 to 95%) in physiological tests (e.g., and the strains in cluster 2) exhibited high levels of similarity in the LFRFA (84 and 81%, respectively). Other taxa ( group) which exhibited equally high levels of physiological similarity (90%) appeared to be unrelated as determined by the LFRFA. Species with lower levels of physiological similarity (e.g., [75%], [63%], and [68%]) exhibited low levels of LFRFA similarity (75, 64, and 54%, respectively). High levels of DNA-DNA relatedness (≫90%) () were reflected in high levels of similarity as determined by the LFRFA (75 to 100%); lower levels of DNA-DNA relatedness (ca. 70%) ( group) were reflected in low levels of LFRFA similarity (strains not clustered). We concluded that the presently used physiological tests reflect too small a portion of the genome to be universally useful in streptomycete species characterization. In contrast, high levels of DNA-DNA relatedness (≫90%) and high LFRFA similarity values will probably both be valuable in species delineation in actinomycetes.


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