Dynamics and control of bacterial plasmid replication
Plasmid replication during the Escherichia coli division cycle has been investigated using the membrane-elution technique to produce cells labelled at different times during the division cycle and subsequent quantitation of the label incorporated into the plasmids. The results indicate that the naturally-occurring, low-copy F plasmid and P1 prophage replicate in a cell-cycle-specific manner, with replication occurring approximately one-half generation between subsequent rounds of chromosome replication initiation. In contrast, the naturally-occurring, high-copy R6K, pSC101, and ColE1-type plasmids replicate in a cell-cycle-independent manner.
The cell-cycle replication patterns of two mini-F plasmids were investigated in an attempt to determine the genetic locus regulating cell-cycle-specific F plasmid replication. A mini-F plasmid containing the oriV and oriS F plasmid origins replicates in a cell-cycle-specific manner, whereas a mini-F plasmid containing only the oriS F plasmid origin replicates in a cell-cycle-independent manner and is maintained at a higher copy number than the two-origin mini-F plasmid. These results imply that the oriV origin contributes in some way to the cell-cycle-specific replication behavior of the F plasmid.
A conceptual model of cell-cycle-specific plasmid replication has been developed in order to understand the mechanism responsible for the replication pattern. The experimental data support the model prediction that the mechanisms timing low-copy plasmid replication and initiation of chromosome replication are similar: initiation of replication occurs when a constant mass per origin is achieved, but at a different mass per origin for the low-copy plasmids than for the chromosome.
Mathematical modelling of high-copy plasmid replication is simplified by the finding that its replication is cell-cycle independent. A detailed kinetic model of the known molecular biology of the ColE1 plasmid has been developed and the relevant parameters have been extracted from the biology literature. Time constant analysis was used to reduce this complex model to a simple, realistic description of the overall replication kinetics. The analysis leads to the identification of key parameters governing plasmid replication and to the estimation of parameters that are difficult to determine experimentally. The overall kinetics of ColE1-type plasmid replication are zeroth-order in production and first order in dilution. Experimental data confirm these predictions.
0307: Molecular biology