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

Abstract

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.

Graphical Abstract

Figure 1.

The anti-F cassette (A) and our strategy (B) to determine which segments need to be potentiated. The complete anti-F cassette was introduced on the low(ish) copy number Plasmid 1 (IncP-1 plasmid pCT549 with wild-type oriV) while sub-segments were introduced on a medium copy number Plasmid 2 (P15A replicon plasmids derived from pACYC184). The test strain was JM109 with pUC18 which complements the lac deletion in F’prolac to give Lac⁺ (blue) on Amp IPTG X-gal plates.

Figure 2.

Segments needed to potentiate the anti-F cassette (colour-coded as in Fig. 1A). As in Fig. 1B, displacement was tested by co-transforming E. coli JM109 (pUC18) with Plasmid 1 plus anti-F cassette and Plasmid 2 plus sub-segments (control lines A and B; tests lines C–E). Plasmid 1 derivatives constructed to test the effect of inactivating the FIB rep gene were introduced alone (lines F–H). The effect of sub-segments in Plasmid 2 were tested alone (line I–J). Displacement is measured by the % white colonies appearing on Tc Cm IPTG Xgal plates.

Figure 3.

(A). Hybrid F/pO157 FIB replicon analysis. Structure of the FIB replicon is shown at the top. The rep gene is shown flanked by iterons which bind Rep as well as the DnaA binding site. Dual replicon plasmids with P15A and hybrid FIB replicons (pLAZFIB HybridA to H as shown in Supplementary Table S1) were used to map the sequence change in F plasmid DNA resulting in reduced ability (−) for replication (Repⁿ) in E. coli C2110. The discs represent nucleotide differences between F and pO157 DNA. (B). The predicted secondary structure resulting from CTG > GTG as start codon mutation, also showing the C to T mutation that restores gene function.

Figure 4.

Scheme for replication cycle of the FIB replicon and action of pCURE. Expression of Rep is weak and autoregulated by Rep-iteron interaction. Handcuffing mediated by Rep dimers at upstream (U) and downstream (D) iterons can occur both inter- and intra-molecularly. The handcuffed replicon remains inactive until cell growth dilutes Rep and enough monomer accumulates to invade and disrupt the DNA–Rep sandwich. The downstream iterons in pCURE must act by interaction with the upstream iterons in the target plasmid.