Scaling laws of plasmids across the microbial tree of life

Plasmids play a critical role in shaping the dynamics and evolution of microbial communities. The capacity of a plasmid to express genes is constrained by two parameters: length and copy number. However, the interplay between these parameters and their constraints on plasmid evolution have remained elusive due to the absence of comprehensive quantitative analyses. To address this gap, we developed Probabilistic Iterative Read Assignment (PIRA), a new computational method that overcomes previous computational bottlenecks, enabling rapid and accurate determination of plasmid copy numbers at an unprecedented scale. Applying PIRA to all microbial genomes in the NCBI RefSeq database with linked short-read sequencing data in the Sequencing Read Archive (SRA), we analyzed 4,317 bacterial and archaeal genomes encompassing 11,338 plasmids, spanning the microbial tree of life. Our analysis reveals three scaling laws of plasmids: first, an inverse power-law correlation between plasmid copy number and plasmid length; second, a positive linear correlation between protein-coding genes and plasmid length; and third, a positive correlation between metabolic genes per plasmid and plasmid length, particularly for large plasmids. These scaling laws imply fundamental constraints on plasmid evolution and functional organization, indicating that as plasmids increase in length, they converge toward chromosomal characteristics in copy number and functional content. Our findings not only advance the understanding of plasmid dynamics but also have implications for microbial evolution, biotechnology, and the design of synthetic plasmids.