Plasmids promote bacterial evolution through a copy number-driven increase in mutation rate

Plasmids are autonomously replicating DNA molecules that stably coexist with chromosomes in bacterial cells. These genetic elements drive horizontal gene transfer and play a fundamental role in bacterial ecology and evolution. Theory suggests that plasmids might evolve faster than chromosomes, as the mutation rate per gene should proportionally increase with plasmid copy number. However, the segregation of plasmid copies to daughter cells is random, introducing an additional layer of genetic drift, known as segregational drift, that might delay plasmid evolution. The interplay between plasmid mutational supply and segregational drift determines the evolutionary rate of plasmid-encoded genes, yet the relative contribution of these opposite forces in plasmid evolution remains unclear. Here, we took a classical population genetics framework to devise a mathematical approximation that predicts the fate of plasmid mutations in bacterial populations. We then validate these predictions by integrating computational, experimental, and bioinformatic approaches. Our findings show that plasmid mutation rates scale logarithmically with copy number: while increasing copy number elevates the mutation rate, the effect diminishes at higher copy numbers, where additional copies yield only marginal increases. Nonetheless, the supply of new mutations consistently surpasses the impact of segregational drift across all copy number levels. These results underscore plasmids as powerful platforms for bacterial evolvability and help explain their remarkable prevalence across microbial phylogeny.