Genetic structure and stability of a copy number mutant of IncFI group plasmid ColV-K94 in Escherichia coli K-12.

Mutants pWS10, pWS11 and pWS12 were derived from an IncFI group plasmid ColV-K94 by the insertion of a transposon Tn903 (Kmr). These plasmids were all approximately 130 kb in length. The plasmid pWS12 resembled the wild type ColV-K94 in transmissibility, incompatibility and stable maintenance. Cells harboring pWS11 were poor conjugal donors but resistant to the same level of kanamycin as pWS12 containing hosts. In contrast, pWS10 conferred a higher resistance to kanamycin and exhibited reduced incompatibility properties in comparison with pWS12. The higher drug resistance associated with pWS10 appeared to be a consequence of an increase in its copy number and the generation of miniplasmids of varying sizes. Electron microscope analysis of the copy mutant pWS10 revealed that Tn903 was located at a site adjacent to a region 32.6F to 35.3F. The latter region appears to be the primary replicon of ColV-K94 and is homologous with the secondary replicon of F. The insertional mutagenesis with Tn903 brought about an extensive DNA rearrangement including the duplication and translocation of the stems of two inverted repeat structures. The DNA alterations of pWS10 were distinguishable through comparison of its EcoRI digestion patterns with those of pWS11 and pWS12.