ClpV recycles VipA / VipB tubules and prevents non‐productive tubule formation to ensure efficient type VI protein secretion

Summary The multicomponent type VI secretion system ( T6SS ) mediates the transport of effector proteins by puncturing target membranes. T6SSs are suggested to form a contractile nanomachine, functioning similar to the cell‐puncturing device of tailed bacteriophages. The T6SS members VipA / VipB form tubular complexes and are predicted to function in analogy to viral tail sheath proteins by providing the energy for secretion via contraction. The ATPase ClpV disassembles VipA / VipB tubules in vitro , but the physiological relevance of tubule disintegration remained unclear. Here, we show that VipA / VipB tubules localize near‐perpendicular to the inner membrane of V ibrio cholerae cells and exhibit repetitive cycles of elongation, contraction and disassembly. VipA / VipB tubules are decorated by ClpV in vivo and become static in Δ clpV cells, indicating that ClpV is required for tubule removal. VipA / VipB tubules mislocalize in Δ clpV cells and exhibit a reduced frequency of tubule elongation, indicating that ClpV also suppresses the spontaneous formation of contracted, non‐productive VipA / VipB tubules. ClpV activity is restricted to the contracted state of VipA / VipB , allowing formation of functional elongated tubules at a T6SS assembly. Targeting of an unrelated ATPase to VipA / VipB is sufficient to replace ClpV function in vivo , suggesting that ClpV activity is autonomously regulated by VipA / VipB conformation.