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Updated: May 15, 2025
Inducing a Site Specific Replication Blockage in E. coli Using a Fluorescent Repressor Operator System
Published on: August 21, 2016
Activation, incompatibility, and displacement of FIB replicons in E. coli
Georgina S Lloyd1, Elton R Stephens1, Alessandro Di Maio1
1School of Biosciences and Institute of Microbiology and Infection, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
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.
Insights
Researchers found that the anti-FIB segment is key to efficiently displacing large plasmids like sex-factor F in bacteria. Optimizing this segment enhances antimicrobial resistance plasmid curing, crucial for combating infections.
Area of Science:
- Molecular Biology
- Microbiology
- Plasmid Biology
Background:
- Large plasmids, such as sex-factor F, are significant vectors for antimicrobial resistance (AMR) and virulence factors in clinical Enterobacteriaceae.
- Current methods for plasmid displacement (curing) using high-copy-number vectors are effective but can be inefficient for certain plasmids.
Purpose of the Study:
- To investigate the inefficiency of displacing the F'prolac plasmid using a conjugative IncP-1 plasmid RK2 vector carrying an anti-F cassette.
- To identify the specific genetic elements responsible for the reduced displacement efficiency and determine methods for potentiation.
Main Methods:
- Constructed and tested an anti-F cassette on an IncP-1 plasmid RK2 vector.
- Manipulated the copy number of the curing plasmid and analyzed the role of the anti-FIB segment and its ribosome-binding site (rbs).
- Compared the displacement efficiency of different FIB replicons and their interactions.
Main Results:
- The anti-FIB segment, derived from plasmid pO157, was identified as crucial for potentiation, requiring increased vector copy number for efficient F plasmid curing.
- A defective ribosome-binding site (rbs) in the F-FIB replicon was found to limit its activity, which could be overcome by expressing FIB-Rep from the anti-FIB segment.
- Deleting the FIB-rep from the anti-F cassette eliminated the need for increased vector copy number, and sequence divergence between F and pO157 FIB replicons affected cross-reactivity.
Conclusions:
- The anti-FIB segment's sequence and its interaction with the target replicon's rbs are critical for efficient plasmid curing.
- Slight increases in vector copy number can enhance the displacement of plasmids with sequences similar, but not identical, to those in the curing cassette.
- Understanding these interactions provides a basis for developing more effective strategies to combat AMR and virulence factor spread via plasmids.
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