Plasmid-determined tetracycline resistance in Streptococcus faecalis: evidence for gene amplification during growth in presence of tetracycline

Abstract

The tetracycline (TG)-resistant Streptococcus faecalis strain DS-5Cl harbors two plasmids designated alpha and gamma with molecular masses of approximately 6 and 35 million daltons, respectively. TC-sensitive variants were derived by storing cells at 45 degrees for 2-3 weeks. Analysis of covalently closed circular DNA from five such variants (derived independently) revealed that in each variant the alpha-plasmid, which normally sediments at 28 S (supercoiled) in a sucrose density gradient, was replaced by a 22S substance. Growth of DS-5Cl in the presence of 150 mug/ml of TC (minimum inhibitory concentration is 250 mug/ml in liquid broth) for a prolonged period of time (50-60 generations) resulted in the disappearance of 28S DNA and the appearance of a heterogeneous covalently-closed circular DNA sedimenting at about 40-48 S. This phenomenon was accompanied by an increase in the level of bacterial TC-resistance, whereby tells were subsequently grown in the absence of TC for 70-80 generations, the heterogeneous DNA disappeared and a typical 28S alpha-plasmid reappeared. The cells also became less resistant to TC, i.e., the minimum inhibitory concentration returned to 250 mug/ml. These data suggest that bacterial growth in the presence of TC results in a reversible gene amplification with respect to a TC-resistant determinant residing on the alpha-plasmid.