Selection and timing of replication of plasmids R1 drd-19 and F′ lac in Escherichia coli

Abstract

The selection and timing of plasmid replication was studied in exponentially growing cultures of Escherichia coli K-12 carrying the plasmid R1 drd-19 and E. coli strains B/r A and B/r F carrying the plasmid F′ lac. In all cases plasmid replication was studied by analysis of covalently closed circular (CCC) DNA. The turnover time of replicating plasmid DNA into CCC-DNA was found to be less than 4 min. Density shift experiments (from 15NH 4 +, D 2O to 14NH 4 +, H 2O) showed that plasmids R1 drd-19 and F′ lac are selected randomly for replication. This means that one of the plasmid copies in a cell is selected and replicated. There is no further plasmid replication in the cell until all plasmid copies, including the newly formed ones, have the same probability of being selected for replication. The early kinetics of the appearance of light plasmid DNA after the density shift showed that the time interval between successive replications of plasmids R1 drd-19 and F′ lac is τ n , where τ is the generation time and n is the average number of plasmid replications per cell and cell cycle. In a second type of experiment, exponentially growing cells were separated into a series of size classes by low-speed centrifugation in sucrose step gradients. Replication of plasmids R1 drd-19 and F′ lac was equally frequent in all size classes. This result is in accordance with the results of the density shift experiment. It can therefore be concluded that replication of plasmids R1 drd-19 and F′ lac is evenly spread over the whole cell cycle, which means that one plasmid replication occurs every time the cell volume has increased by one initiation mass.