Analysis of establishment phase replication of the plasmid ColE1

The replication regulatory mechanisms by which the small, multicopy plasmid ColE1 maintains a constant steady-state copy number have been extensively characterized by a combination of in vivo genetics and in vitro biochemistry. We have extended the analysis of replication control into the "establishment" phase of replication, when ColE1-directed replicons replicate more than once per cell generation and the intracellular concentrations of plasmid-encoded replication regulatory elements are changing. To study establishment phase replication, in which plasmid-directed replicons amplify from an initially low concentration to the characteristic, steady-state concentration, bacteriophage-plasmid hybrids, termed phasmids, were constructed. Phasmids were shown to exhibit stability, segregation, and incompatibility properties similar to those of the parent plasmid. Establishment phase replication was analyzed by measuring the number of phasmids per cell as a function of time after infection. We observed a linear increase in phasmid concentration until the steady-state concentration characteristic of the ColE1 plasmid component of the hybrid was reached. The number of cell doublings required for the phasmid concentration to reach steady-state was inversely related to cell growth rate. The observed amplification kinetics imply that the frequency of replication initiation per phasmid continually decreases until steady-state is reached. Kinetics of establishment phase amplification were sensitive to rate of expression of RNA II. A phasmid containing an up mutation in the RNA II promoter amplified at a 15-fold faster rate than the wild-type phasmid. Concentration of the ColE1 replication negative regulator (RNA I) was proportional to phasmid concentration throughout the amplification phase. These results suggest that the same elements that regulate steady-state replication also control establishment phase replication.