Bacteroidota assemble a unique type VI secretion system membrane complex

Microbes have evolved elaborate mechanisms to cope with competitors, including the type VIsecretion system (T6SS) of Gram-negative bacteria. The T6SS inhibits target cells through contact-dependent translocation of toxic effector proteins across two cellular membranes via an inverted phage-like apparatus. Proteobacteria accomplish this feat by passage of theT6SS needle through a megadalton-size membrane complex (MC), which is essential for T6SS function. Remarkably, although the phylum Bacteroidota encodes a T6SS, it lacks homologs of the MC. We identified five novel genes, essential for T6SS function, that encode a candidate unique Bacteroidota T6SSMC. We purified the T6SSiii MC and revealed its dimensions using electron microscopy. We identified an intricate protein protein interaction network underlying the assembly of the MC, the stoichiometry of the five TssNQOPR components., tTheir predicted structures were validated using crosslinking mass-spectrometry and we assessed the structural homology with known proteins. Importantly, we determine the connection between the T6SSiii MC and the otherwise conserved baseplate involving the hub protein Tss.Finally, phylogenomic analysis of the distribution of T6SSiii MC genes across the phylum Bacteroidota highlights patterns of conservation including the invariant