The proton‐motive force is required for translocation of CDI toxins across the inner membrane of target bacteria

Contact‐dependent growth inhibition (CDI) is a mode of bacterial competition orchestrated by the CdiB/CdiA family of two‐partner secretion proteins. The CdiA effector extends from the surface of CDI⁺ inhibitor cells, binds to receptors on neighbouring bacteria and delivers a toxin domain derived from its C‐terminal region (CdiA‐CT). Here, we show that CdiA‐CT toxin translocation requires the proton‐motive force (pmf) within target bacteria. The pmf is also critical for the translocation of colicin toxins, which exploit the energized Ton and Tol systems to cross the outer membrane. However, CdiA‐CT translocation is clearly distinct from known colicin‐import pathways because ΔtolA ΔtonB target cells are fully sensitive to CDI. Moreover, we provide evidence that CdiA‐CT toxins can be transferred into the periplasm of de‐energized target bacteria, indicating that transport across the outer membrane is independent of the pmf. Remarkably, CDI toxins transferred under de‐energized conditions remain competent to enter the target‐cell cytoplasm once the pmf is restored. Collectively, these results indicate that outer‐ and inner‐membrane translocation steps can be uncoupled, and that the pmf is required for CDI toxin transport from the periplasm to the target‐cell cytoplasm.