Our findings, together with results of Cys accessibility studies, indicate that VirB10 stably integrates into the IM, extends via its PRR across the periplasm, and interacts via its β-barrel domain with the VirB7–VirB9 channel complex. Another class of Tra + mutations also selectively disrupted pilus biogenesis but caused release of pilin monomers to the milieu these mutations included deletions of α-helical projections extending from the β-barrel domain. Mutations permissive for substrate transfer but blocking pilus production (Tra +, Pil −) included a cytoplasmic domain deletion and TM domain insertion mutations.
Mutations conferring a transfer- and pilus-minus (Tra −, Pil −) phenotype included PRR deletion and β-barrel substitution mutations that prevented VirB10 interaction with the outer membrane (OM) VirB7–VirB9 channel complex. Here, we determined the functional importance of VirB10 domains denoted as the: (i) N-terminal cytoplasmic region, (ii) transmembrane (TM) α-helix, (iii) proline-rich region (PRR) and (iv) C-terminal β-barrel domain. Agrobacterium tumefaciens VirB10 couples inner membrane (IM) ATP energy consumption to substrate transfer through the VirB/D4 type IV secretion (T4S) channel and also mediates biogenesis of the virB-encoded T pilus.
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