Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

Abstract

A mutant temperature-sensitive for R-plasmid replication, Rms201 ts 14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201 ts 14) + was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201 ts 14) + in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201 ts 14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201 ts 14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201 ts 14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201 ts 14. Temperature-sensitive miniplasmid pMS ts 214, which has a molecular weight of 5.3 × 10 6 and encodes ampicillin resistance, was isolated from Rms201 ts 14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10 6 . These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.