Co-option of a degenerated polysaccharide transporter for type VI protein secretion across diverse α-Proteobacteria

Type VI secretion system (T6SS) is a widespread bacterial nanomachine that mediates interbacterial antagonism and ecological competitiveness. Thirteen core type six secretion (Tss) proteins, including the outer membrane-anchoring protein TssJ, constitute the canonical T6SS. Yet, TssJ is absent from T6SSs in several lineages, raising the question of how their systems assemble and function without it. Using the TssJ-lacking T6SS of Agrobacterium C58 as a model, we describe a form of co-optive evolution in which a degenerated outer membrane polysaccharide transporter, TssX, functionally replaces TssJ. We delineated the assembly pathway of the agrobacterial T6SS and uncovered an assembly order in which TssX acts downstream of the membrane complex and prior to contractile sheath polymerization. Comparative genomic analyses of 32,414 complete proteobacterial genomes revealed that tssX defines a distinct T6SS subtype and is mutually exclusive with tssJ, indicating functional compensation through evolutionary replacement. Phylogenetic and synteny analyses revealed multiple independent losses of tssJ and subsequent co-option of tssX in α-proteobacterial lineages. Our work uncovers a novel T6SS outer membrane protein and shows that essential nanomachine elements can be replaced. The variation in both composition and assembly mechanisms exemplifies the remarkable flexibility in the evolution of protein complexes that are both structurally and functionally conserved.