Plasmids serve as vehicles and reservoirs of type VI secretion systems

The Type VI secretion system (T6SS) is a major determinant of bacterial competition, yet its dissemination across lineages remains unclear. By analyzing 43 213 plasmids and 29 161 chromosomes, we reveal plasmids as an underestimated reservoir and vehicle for T6SS diversification. We identified 405 complete plasmid-encoded T6SSs and 929 orphan islands containing hcp, vgrG, and/or PAAR genes, often independent of full systems. Plasmid-encoded T6SSs are biased toward large replicons, frequently megaplasmids, with distinct stability and mobility traits: orphan island plasmids are enriched in conjugation modules, whereas complete systems are associated with partition and toxin-antitoxin maintenance systems. Phylogenomic analyses show that some plasmid lineages stably integrate T6SSs as core traits, while others undergo recurrent acquisition and diversification. Comparative and ancestral analyses indicate pervasive bidirectional transfers between plasmids and chromosomes, with insertion sequences frequently detected in their vicinity. The presence of near-identical homologs across compartments underscores the capacity of plasmids to transcend phylogenetic barriers and propagate these nanoweapons. Together, our results identify plasmids as dual evolutionary actors in T6SS ecology, functioning as short-term vectors for rapid horizontal spread and as long-term reservoirs that foster stabilization and adaptive diversification.