Intracellular competition shapes plasmid population dynamics

Abstract Conflicts between levels of biological organization are central to evolution, from populations of multicellular organisms to selfish genetic elements in microbes. Plasmids are extrachromosomal, self-replicating genetic elements that underlie much of the evolutionary flexibility of bacteria. Evolving plasmids face selective pressures on their hosts, but also compete within the cell for replication, making them an ideal system for studying the joint dynamics of multilevel selection. While theory indicates that within-cell selection should matter for plasmid evolution, experimental measurement of within-cell plasmid fitness and its consequences has remained elusive. Here we measure the within-cell fitness of competing plasmids and characterize drift and selective dynamics. We achieve this by the controlled splitting of synthetic plasmid dimers to create balanced competition experiments. We find that incompatible plasmids co-occur for longer than expected due to methylation-based plasmid eclipsing. During this period of co-occurrence, less transcriptionally active plasmids display a within-cell selective advantage over their competing plasmids, leading to preferential fixation of silent plasmids. When the transcribed gene is beneficial to the cell, for example an antibiotic resistance gene, there is a cell-plasmid fitness tradeoff mediated by the dominance of the beneficial trait. Surprisingly, more dominant plasmid-encoded traits are less likely to fix but more likely to initially invade than less dominant traits. Taken together, our results show that plasmid evolution is driven by dynamics at two levels, with a transient, but critical, contribution of within-cell fitness.