Functional production of the Na+ F1FO ATP synthase from Acetobacterium woodii in Escherichia coli requires the native AtpI

The Na⁺ F₁F_O ATP synthase of the anaerobic, acetogenic bacterium Acetobacterium woodii has a unique F_OV_O hybrid rotor that contains nine copies of a F_O-like c subunit and one copy of a V_O-like c ₁ subunit with one ion binding site in four transmembrane helices whose cellular function is obscure. Since a genetic system to address the role of different c subunits is not available for this bacterium, we aimed at a heterologous expression system. Therefore, we cloned and expressed its Na⁺ F₁F_O ATP synthase operon in Escherichia coli. A Δatp mutant of E. coli produced a functional, membrane-bound Na⁺ F₁F_O ATP synthase that was purified in a single step after inserting a His₆-tag to its β subunit. The purified enzyme was competent in Na⁺ transport and contained the F_OV_O hybrid rotor in the same stoichiometry as in A. woodii. Deletion of the atpI gene from the A. woodii operon resulted in a loss of the c ring and a mis-assembled Na⁺ F₁F_O ATP synthase. AtpI from E. coli could not substitute AtpI from A. woodii. These data demonstrate for the first time a functional production of a F_OV_O hybrid rotor in E. coli and revealed that the native AtpI is required for assembly of the hybrid rotor.