Engineered RepE Variants Tune Plasmid Copy Number in Bacteria

Researchers have identified two point mutations in the RepE initiator protein of the F plasmid that raise the copy number of a low-copy-number bacterial plasmid, offering a way to adjust gene dosage without altering the plasmid’s architecture. The study, published as an open-access article in Applied Microbiology and Biotechnology, combined random mutagenesis of the repE gene with high-stringency selection to isolate variants that modulate plasmid replication control.

RepE normally performs a dual role in the F-plasmid system. As an initiator protein, it coordinates the start of plasmid replication, while through transcriptional autoregulation it keeps replication in check. This balance depends on the equilibrium between monomeric and dimeric forms of the protein, and the new variants shift that balance in ways that increase copying. The two mutations, A78S and Q45L, were each shown by quantitative PCR and droplet digital PCR to increase the copy number of plasmid pFO1 relative to the parental repE(R118P) allele from which the screen started.

The effects of the two mutations were not identical across growth conditions. A78S had a stronger impact during exponential growth, when cells divide rapidly, whereas Q45L exerted its larger effect during stationary phase. A double mutant carrying both mutations showed a cumulative effect on copy number, particularly in exponential phase, suggesting that the two substitutions act through partly separable mechanisms that can be combined.

The study also addressed regulation. RepE transcription increased in the presence of pFO1 and correlated with plasmid copy number, a result the authors interpret as supporting a titration-based regulatory model, in which plasmid copy number feeds back on the expression of its own replication initiator. The findings indicate that engineering RepE variants enables tunable control of plasmid copy number, which the paper describes as a potentially useful strategy for optimizing gene dosage in synthetic biology and biotechnology. The results remain at the stage of laboratory characterization of specific plasmid systems, and the reported measurements apply to the pFO1 plasmid and the repE alleles tested in the study.

Subject of Research: Engineering RepE initiator protein variants to increase bacterial plasmid copy number

Article Title: Identification and functional characterization of RepE variants that increase plasmid copy number

Article References: Krahulec, J. (2026). Identification and functional characterization of RepE variants that increase plasmid copy number. Applied Microbiology and Biotechnology. https://doi.org/10.1007/s00253-026-14077-5

Image Credits: AI Generated

DOI: 10.1007/s00253-026-14077-5

Keywords: RepE, plasmid copy number, F plasmid, replication initiation, mutagenesis, qPCR, ddPCR, gene dosage, synthetic biology, biotechnology, autoregulation, titration