A runaway-replication mutant of plasmid R1drd-19: Temperature-dependent loss of copy number control

Abstract

A two-step mutant plasmid, originating from the resistance plasmid R1drd-19, was shown to replicate without control of the plasmid copy number when the host bacteria were grown at temperatures above 35°C. The uncontrolled plasmid replication, so called runaway-replication, eventually inhibits the host cell growth and results in a conditionally lethal phenotype. As much as 75% of the total DNA in the cells was found to be covalently closed circular plasmid DNA at the stage when the cell growth stopped. At 30°C the mutant plasmid had a strictly controlled copy number and the onset and kinetics of runaway-replication was studied by temperatureshift experiments. The synthesis of plasmid DNA after a temperature shift from 30°C to 40°C (or 37°C) was found to be exponential with a doubling time of about half the doubling time of the host bacteria, in broth medium as well as in minimal salt-glucose medium. In broth medium the plasmid population had a doubling time of about 12 min which is longer than expected if it is assumed that the rate of elongation during the DNA polymerization is the only limitation on the uncontrolled replication. The results indicate that the minimum time required between successive replications of a plasmid copy is determined by some rate-limiting step(s) presumably after the duplication process. The uncontrolled plasmid replication was shown to be inhibited by the protein synthesis inhibitor chloramphenicol. The plasmid synthesis stopped within 20 min after the addition of the drug and the plasmid DNA increment was less than 50%, suggesting that the uncontrolled plasmid replication requires de novo protein synthesis. Similarly, the RNA polymerase inhibitor rifampicin did inhibit the plasmid replication. The mutational alteration could be reversed, and the copy number control restored, by lowering the temperature before the host cell growth was inhibited. This type of plasmid mutant offers new possibilities in studies of host cell-plasmid interactions.