Allele dynamics in conjugative plasmids: horizontal transfer promotes allele segregation in multicopy plasmids

The emergence of multidrug resistance and the spread of resistance genes among bacteria pose a major threat to human health. Plasmids, particularly through horizontal transfer, are considered key drivers of antimicrobial resistance spread because they can disseminate resistance alleles between bacterial lineages and, in some cases, even across species boundaries. As plasmids often reside within prokaryotic cells in multiple copies, intracellular allele diversity (heteroplasmy) can arise upon plasmid mutation. The segregation of heteroplasmic intracellular plasmid pools depends on the modes of plasmid replication and partitioning. Conjugation may additionally alter plasmid allele composition; however, its impact on plasmid allele dynamics remains poorly understood.

Here, conjugative plasmid transfer is shown to accelerate the segregation of plasmid heteroplasmy by promoting the emergence of homoplasmic hosts, while vertical inheritance maintains genetic diversity over prolonged timescales. A quantitative experimental system was established to track plasmid allele dynamics in Acinetobacter baylyi under non-selective conditions. It enabled separate monitoring of a novel antibiotic resistance allele, introduced into an ancestral donor population, in donors and recipients. Comparing a narrow-host-range and a broad-host-range plasmid as well as different plasmid copy numbers revealed that alleles in heteroplasmic donors segregated over time in a plasmid-specific manner, but not as rapidly as current mathematical models suggest. Transient plasmid fusion and fission, and non-random plasmid segregation were identified as potential contributors to this observation.

In contrast to vertical inheritance, conjugation produced almost exclusively homoplasmic recipients whose allele composition closely mirrored that of the donor plasmid pool across all plasmid systems. Thus, conjugation accelerates plasmid allele segregation, with effects scaling with plasmid copy number. Our findings identify horizontal transfer as a previously unrecognized segregation pathway shaping the evolution of mobile genetic elements and improve our understanding of how newly emerged resistance alleles become established and spread.