Activation, incompatibility, and displacement of FIB replicons in E. coli.

Multi-replicon sex-factor F is the archetype of the largest plasmid group in clinical Enterobacteriaceae. Such plasmids spread antimicrobial resistance (AMR) and virulence functions in commensal bacteria of humans and animals. Displacing (curing) these plasmids by blocking replication and neutralizing addiction is successful with the curing cassette on a high-copy-number vector but, with conjugative IncP-1 plasmid RK2 as vector for our "anti-F cassette", displacement of F'prolac is inefficient unless curing-plasmid copy-number is raised 1.5- to 2-fold. Here we report that it is the anti-FIB segment, originating from FIB-FII plasmid pO157, which needs potentiation. We show that the FIB replicon in F (F-FIB) is defective due to a sub-optimal rep ribosome-binding-site (rbs) but can be activated by FIB-Rep protein expressed from our anti-FIB segment joined to RK2. Deleting FIB-rep from the anti-F cassette removed the need for potentiation. A pO157-FIB single-replicon plasmid was displaced efficiently by the complete anti-F cassette without potentiation, but an F-FIB plasmid, mutated to have a pO157-like rep rbs, was not, indicating that sequence divergence between F and pO157 FIB replicons has weakened their negative cross-reactivity. Thus, raising vector copy-number slightly may be sufficient to increase displacement of plasmids similar but not identical to the sequences in the curing cassette.