A System to Explore the Adaptive Dynamics of Multicopy Plasmids: The Role of Copy Number and Mutation Rate in Evolutionary Outcomes

Multicopy plasmids are widespread in nature and compose a common strategy for spreading beneficial traits across microbes. However, the role of plasmids in supporting the evolution of encoded genes remains underexplored due to challenges in experimentally manipulating key parameters such as plasmid copy number and mutation rate. In this work, we developed a strategy for controlling copy number in the plasmid-based continuous evolution system, OrthoRep, and used our resulting capabilities to investigate the evolution of a conditionally essential gene under varying CN and mutation rate conditions. Our results show that low CN facilitated the faster enrichment of beneficial alleles while high CN promoted robustness through the maintenance of allelic diversity. High CN also slowed the removal of deleterious mutations and increased the fraction of non-functional alleles that could hitchhike during evolution. This study highlights the nuanced relationships between plasmid CN, mutation rate, and evolutionary outcomes, providing insights into the adaptive dynamics of genes encoded on multicopy plasmids and nominating OrthoRep as a versatile tool for studying plasmid evolution.