Universal rules govern plasmid copy number

Plasmids –autonomously replicating DNA molecules– are pervasive in the microbial world, where they fuel evolution as key vehicles for gene transfer. Plasmids are as diverse as their hosts and exhibit a broad range of replication and mobility strategies, genetic repertoires, host ranges, sizes, and copies per cell. While most of these features have been extensively characterized, the determinants of plasmid copy number (PCN) remain poorly understood. Here, we leveraged extensive DNA sequencing data to comprehensively analyze the copy number of thousands of diverse bacterial plasmids. We found that the copy number of natural plasmids is highly variable, spanning nearly three orders of magnitude, and that it is intrinsically robust against changes in genomic context. We further show that PCN variability is tightly associated with plasmid lifestyles and develop the concept of replicon dominance to explain interactions in widespread multi-replicon plasmids. Finally, we uncover a universal scaling law that links copy number and plasmid size across phylogenetic boundaries, indicating that pervasive constraints orchestrate the PCN-size trade-off. Altogether, our work pioneers large-scale analyses of PCN while uncovering the fundamental rules that govern plasmid biology.