The combination of active partitioning and toxin-antitoxin systems is most advantageous for low-copy plasmid fitness.

Extrachromosomal and mobile genetic elements, including plasmids, and accessory chromosomes, are prevalent in all life domains. Elements integrated into the host chromosome replicate and segregate via the host life cycle. In contrast, the persistence of autonomously replicating elements relies on their ability to remain within the host population. Here, we compare the evolutionary advantage of different persistence strategies found in prokaryotic plasmids. Through intracellular competitions between plasmid genotypes, we find that the combination of active partitioning during cell division with a toxin-antitoxin (TA) system for post-segregational killing increases plasmid fitness more than either strategy alone. Mathematical modeling of long-term plasmid evolution, calibrated with empirical plasmid loss dynamics, further supports these findings. A survey of enterobacterial genomes indicates that partitioning and TA systems are core features of large plasmids. Indeed, we confirm the presence of a previously unrecognized type I TA system in conjugative IncX3 plasmids, which serve as important vectors of antibiotic resistance in human pathogens. These findings suggest that large plasmids - including conjugative and mobilizable types - encode TA systems, some of which have yet to be identified. The combination of TA and partitioning systems emerges as the most effective strategy for the evolutionary success of low-copy extrachromosomal elements.