Relaxation complexes and mobilisation properties of COLE1 and related plasmids
In this thesis the role of plasmid relaxation complexes was investigated. A correlation between the levels of mobilisation and relaxation was demonstrated for the wild type COLE1 plasmid. A previously undiscovered cryptic plasmid (pLG500) was found to be present in the CoIK+ clinical isolate of E. coli - K216. This plasmid was shown to be a 1 megadalton autonomous replicon, which was non-mobilisable (by R64drd11 and RP4) and non-relaxable. However, in the presence of either of the two closely related plasmids Co1K and COLE1, pLG5OO was rendered relaxable, and mobilisable by the appropriate sex factors. Following the comobilisation of pLG5OO and COLE1, or pLG5OO and Co1K, 2-5% of the Col+ transconjugants were found to contain novel plasmids which were about 1 megadalton larger than COLE1 or Co1K. Restriction analysis demonstrated that these novel plasmids were formed by site-specific recombination between pLG500 and COLE1 or Co1K, and further mating experiments showed that such recombination occurred independently of the recA gene product. These composite plasmids apparently possessed higher copy numbers and produced more colicin than COLE1 or Co1K, yet were less relaxable and less efficiently mobilised than the parental Col plasmids. Whilst no evidence for recombination (leading either to the formation or dissociation of these cointegrate plasmids) could be found in vegetatively growing cells, some recipients containing the two original plasmids could be found following the mobilisation of cointegrates. The unique sites within the C0IEI and Co1K plasmids at which recombination with pLG5OO occurred were mapped using restriction endonucleases. In the case of C0IEI this site was mapped to within 47 base pairs of the site at which relaxation nicking occurs. For Co1K the recombination site was localised to within the same 350 base pair fragment (the HaeII E fragment) as is know to contain a sequence essential for Co1K mobilisation. An explanation for this novel recombination phenomenon is presented which strongly suggests that single-stranded DNA transfer from, and strand recircularisation at, the relaxation, nicking site, is the underlying process in the mobilisation of COLE1 and related plasmids.