Plasmids drive the dissemination and diversification 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. Analyzing 43,213 plasmids and 29,161 chromosomes, we reveal plasmids as an underestimated reservoir and vehicle for T6SS diversification. We identified 405 complete plasmidencoded T6SSs and 929 orphan islands containing hcp, vgrG, and/or PAAR genes, often independent of full systems. Plasmidencoded 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 rely on partition and toxin-antitoxin maintenance. Phylogenomic analyses show some plasmid lineages stably integrating T6SSs as core traits, while others undergo recurrent acquisition and diversification. Comparative and ancestral analyses indicate pervasive bidirectional transfers between plasmids and chromosomes, mediated by insertion sequences. The presence of nearidentical 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--short-term vectors that enable rapid horizontal spread, and long-term reservoirs that foster stabilization and adaptive diversification.