Differentiation between mutants of Escherichia coli K₁₂ defective in oxidative phosphorylation

Hybrid membrane particles from two mutants of Escherichia coli K₁₂, B_v4 and K_I1, defective in oxidative phosphorylation, have been prepared, in which ATP-driven membrane energization is restored. A soluble factor of mutant K_I1 was found to have properties similar to parental crude coupling factor, ATPase (EC 3.6.1.3). Membrane particles of this mutant could not be reconstituted by parental coupling factor. Either parental coupling factor, or the soluble factor of mutant K_I1 could reconstitute both respiration-driven and ATP-driven energization to membrane particles of mutant B_V4 or to parental particles depleted of ATPase. Mutant B_V4 was found to be devoid of coupling factor activity, while retaining the ability to hydrolyze ATP. Both mutants possess an ATPase with an altered binding to the membrane. Mutant K_I1 is impaired in respiration-driven amino acid transport, in contrast to mutant B_V4. The three major subunits of parental Escherichia coli ATPase have been isolated and antibodies have been prepared against these subunits. Antibodies against the largestsubunit (α component) or against the intact catalytic subunits (α + β components) inhibit both ATP-P_i exchange in the parent organism as well as ATP hydrolytic activity in parent and mutants. Antibodies against the two other subunits (β or γ components) also inhibit these two reactions, but were found to be less effective. Mutant N_I44, which lacks ATPase activity, shows no precipitin lines with anti-α, anti-β, anti-γ, or anti-(α + β) preparations. In contrast, mutants B_V4 and K_I1, exhibit cross-reactivity with all of the antisera.