Molec. gen. Genet. 178, 233 235 (1980) © by Springer-Verlag 1980 Short Communication Curing of Escherichia coli K12 Plasmids by Coumermycin O.N. Danilevskaya and A.I. Gragerov Institute of Molecular Genetics, USSR Academy of Sciences, Kurchatov Sq. 46, Moscow 123182, USSR Summary. Low concentrations of the antibiotic cou- mermycin At, the inhibitor of bacterial DNA gyrase, effectively eliminate pBR322, pMB9 and other ColE1 related plasmids from E. coli K12 strains. The curing action of antibiotic seems to result from the plasmid degradation and not just from the inhibition of repli- cation. Coumermycin inhibits the supercoiling activity of DNA gyrase (Gellert et al., 1976) by binding itself to the product of the cou gene, the enzyme's B-subunit (Sugino et al., 1978; Mizuuchi et al., 1978). DNA gy- rase is required for replication of the E. coli chromo- some, a number of phages and the small plasmids RSF1030 and ColE1 (Gellert et al., 1976; Tomizawa, 1978; Gragerov and Mirkin, 1980). In some cases DNA supercoiling is necessary for initiation of repli- cation, in others for elongation. In this paper we report the effective curing action of coumermycin on E. coli K12 cells harboring relaxed plasmids, which are not eliminated by agents affecting the F and R factors (Clowes, 1972). Curing action or coumermycin is clearly seen when C600 rk mk cells harboring the tetracycline re- sistant pMB9 plasmid are treated with the drug at various concentrations. Viable counts of tetracycline- resistant and sensitive (that is plasmidless)survivors were checked in overnight cultures. Representative results are shown in Fig. 1. One can see that plasmid elimination occurs at coumermycin concentrations 1-7 gg/ml and the maximum effect is observed at the highest drug concentration (5 pg/ml) which does not block cell growth. Other derivatives of Cole 1-pBR322 (Bolivar et al., 1977) and the pOD162 hybrid plasmid derived from For offprints contact. O.N. Danilevskaya pMB9 (Bass et al., 1979) - and an unrelated plasmid pSC101 originating from the R factor (Cohen and Chang, 1973) also are effectively cured by coumermy- cin (Table 1). The effect of coumermycin is based on an inhibition of DNA gyrase since the drug does not affect the plasmid in a E. coli cou-rl mutant (Table 1) which has the coumermycin resistant DNA gyrase (Mirkin et al., 1979). Nalidixic acid was also tested because it inhibits DNA replication by -1 '°° r E 8 o .--,0t / -u] i/ / 6 I E._ I / ~ 14°~ I 3 5 7 9 || 20 Cournermycin pg/mL Fig. 1. Effect of coumermycin at various concentrations on the cell survival and plasmid elimination. Coumermycin was dissolved in dimethylsulfoxide (1 mg/ml) and kept for no more than 3 days at -10 ° C. An overnight culture grown in LB medium (Miller, 1976) was diluted 105-fold in the same medium and after adding coumermycin was aerated overnight at 37 ° C. Appropriate dilu- tions of grown cultures were plated onto 2% nutrient agar without the antibiotic and incubated overnight at 37 ° C. Colonies were tested for tetracycline resistance character by replica-plating onto nutrient agar with the antibiotic (20 gg/ml) and without it. The absence of plasmids in tetracycline-sensitive derivatives was confirmed by electrophoresis of crude lysates in agarose gel (see legend to Fig. 2). The plasmid elimination varies from 20 to 100%. The best effect is observed with a freshly prepared drug solution. Symbols: --e viable counts (cells/ml); --.--A--- fre- quency of plasmidless colonies among survivors (in %) 0026-8925/80/0178/0233/$01.00