Mechanism of increased kanamycin-resistance generated by protoplast regeneration of Streptomyces griseus. II. Mutational gene alteration and gene amplification.

Genetic mechanisms involved in the kanamycin (KM)-hyper-resistance of Streptomyces griseus SS-1198PR and NP1-1PR generated by protoplast regeneration were investigated. Southern hybridization of Sph I-digested genomic DNA with a KM-resistance gene (kan) probe revealed that the strain SS-1198PR and its KM-sensitive parent (SS-1198) had the same copy number of a 4.2-kb Sph I fragment hybridizing to the kan probe, while the kan gene of the strain NP1-1PR was located on a highly amplified DNA sequence (100-200 copies/chromosome) consisting of the 6.7 kb amplifiable unit with Sph I site at the junction site region. There was no difference in the restriction endonuclease cleavage map of the kan gene region of the Sph I fragments from the three strains. However, the level (50 micrograms/ml) of KM-resistance conferred by the cloned NP1-1PR kan gene was much lower than that (1,000 micrograms/ml) conferred by the cloned SS-1198PR kan gene in Streptomyces lividans TK21 although the strain TK21 harboring these kan genes produced an aminoglycoside acetyltransferase, AAC(3)-V, with the same substrate specificity. It seemed, therefore, likely that a mutational alteration of the kan gene and a DNA amplification involving the kan region played major roles for the KM-hyper-resistance depending on AAC(3)-V in the strains SS-1198PR and NP1-1PR, respectively.