Replication functions of the F plasmid of Escherichia coli

Abstract

The work in this thesis concerns the replication of the F plasmid of Escherichia coli and is addressed to the question: how many plasmid genes are involved in replication and partition of F, and what are their function? The techniques principally employed were the cloning of restriction fragments containing parts of the F genome, and transposition mutagenesis of mini-F derivatives. The results obtained can be summarised as follows: (1) The F plasmid contains a second, previously undiscovered, replicon which is located within a 3 kilobase segment of EcoRI fragment f7. The f7 replicon was shown to take over replication of the intact F plasmid when the primary (f5) replication system was inhibited. Replication of f7 plasmids was found to be partly defective in E. coli, especially in rich media. (2) Several temperature-sensitive mutations of F were mapped within the essential replication region (F43.8-47.0kb) of -EcoRI fragment f5. The isolation of temperature-sensitive mini-plasmids should greatly facilitate study of the nature of these lesions. (3) A mini-F plasmid was obtained which constitutes the smallest F derivative so far reported. This plasmid is deleted for the two known origins of replication, oriV and oriS, which suggests the existence of a third origin of replication within the f5 fragment. (4) A locus required for stability of f5 mini-plasmids was mapped to the F46.35-49.25kb region. Two possible functions are suggested for this locus. (5) The part of f5 that is responsible for the incompatibility reaction shown between IncFI group plasmids was found to map in the F47.6-49.25kb region. Therefore this area constitutes a third incompatibility region of F, distinct from the incA and incB regions defined by Kahn et al. (1978).