Plasmid copy number control: isolation and characterization of high-copy-number mutants of plasmid pE194

Abstract

A plasmid, pE194, obtained from Staphylococcus aureus confers resistance to macrolide, lincosamide and streptogramin type B (MLS) antibiotics. For full expression, the resistance phenotype requires a period of induction by subinhibitory concentrations of erythromycin. A copy number in the range of 10-25 copies/cell is maintained during cultivation at 32.degree. C. It is possible to transfer pE194 to Bacillus subtilis by transformation. In B. subtilis, the plasmid is maintained at a copy number of approximately 10/cell at 37.degree. C, and resistance is inducible. Tylosin, a macrolide antibiotic which resembles erythromycin structurally and to which erythromycin induces resistance, lacks inducing activity. Two types of plasmid mutants were obtained and characterized after selection on medium containing 10 .mu.g of tylosin/ml. One mutant class appeared to express resistance constitutively and maintained a copy number indistinguishable from that of the parent plasmid. The other mutant type had a 5- to 10-fold-elevated plasmid copy number (i.e., 50-100 copies/cell) and expressed resistance inducibly. Both classes of tylosin-resistant mutants were due to alterations in the plasmid and not to modifications of the host genome.