Use of chimeric type IV secretion systems to define contributions of outer membrane subassemblies for contact-dependent translocation

Recent studies have shown that conjugation systems of Gram-negative bacteria are composed of distinct inner and outer membrane core complexes (IMCs and OMCCs, respectively). Here, we functionally characterized the OMCC, focusing first on a cap domain that forms a channel across the outer membrane. Strikingly, the OMCC caps of the Escherichia coli pKM101 Tra and Agrobacterium tumefaciens VirB/VirD4 systems are completely dispensable for substrate transfer, but required for formation of conjugative pili. The pKM101 OMCC cap and extended pilus also are dispensable for activation of a Pseudomonas aeruginosa type VI secretion system (T6SS). Chimeric conjugation systems composed of the IMCpKM101 joined to OMCCs from the A. tumefaciens VirB/VirD4, E. coli R388 Trw, and Bordetella pertussis Ptl systems support conjugative DNA transfer in E. coli and trigger P. aeruginosa T6SS killing, but not pilus production. The A. tumefaciens VirB/VirD4 OMCC, solved by transmission electron microscopy, adopts a cage structure similar to the pKM101 OMCC. Our findings establish that OMCCs are highly structurally and functionally conserved - but also intrinsically conformationally flexible - scaffolds for translocation channels. Furthermore, the OMCC cap and a pilus tip protein coregulate pilus extension but are not required for channel assembly or function.