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

Abstract

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.

Incompatibility repressor in a RepA-like replicon of the IncFI plasmid ColV2-K94

“…Although ColV2-K94 is a member of the IncFI incompatibility group and contains the secondary replicon (Rep2) and partitioning genes of the F plasmid (10,21), it also has a replicon (Repl) homologous to the RepA replicon of IncFII plasmids and a transfer (tra) operon similar to that of the IncFII plasmid R538 (22). The relationship between ColV2-K94 and R538 is also evident from studies which produced a ColV2-K94-like plasmid through recombination between R538 and a small, nontransmissible IncFI ColV plasmid (4).…”

“…The procedures for incompatibility testing and copy number determinations by both dye-buoyant density centrifugation and gel densitometry have been described previously (10,21).…”

Second replicon in ColV2-K94 mediates the stable coexistence of two incompatible plasmids

“…Cloning the replication regions of ColV2-K94. Self-replicating restriction fragments of the ColV plasmid pWS12 were obtained as described previously (12). Plasmid pWS12 was digested with EcoRI or BamHI, and the resulting fragments were ligated with T4 ligase.…”

“…The resulting plasmids contained the Repl replicon of ColV, as will be discussed later. The cloning of the Rep2 replicon of CoIV in pWS14 has already been described (12).…”

“…Mating time was 1 h, and single selection for the incoming plasmid only was performed. Incompatibility testing was done in strain C600 as described previously (12). To ensure that recombination between plasmids was not occurring in this strain, the presence of two separate plasmids was verified by screening five colonies for coexisting plasmids by a rapid alkaline isolation procedure (2); isolated DNA was observed on 0.7% agarose gels stained with ethidium bromide.…”