Oxidative phosphorylation by isolated membrane vesicles from Bacillus megaterium and its uncoupler-resistant mutant...

ATP synthesis was studied in ADP + Pi-loaded, right-side-out membrane vesicles from Bacillus megaterium and its uncoupler-resistant mutant strain, C8. Upon energization with ascorbate/phenazine methosulfate, more ATP synthesis was observed in C8 vesicles than in those from the wild type. ATP synthesis by C8 vesicles was more resistant to low levels (0.5-1.0 microM) of carbonyl cyanide m-chlorophenylhydrazone than was synthesis by wild type vesicles, whereas synthesis by both preparations was completely inhibited by N,N'-dicyclohexylcarbodiimide. Upon energization by a valinomycin-induced potassium diffusion potential, vesicles from the wild type strain synthesized more ATP than vesicles from C8, but that synthesis was still lower than observed with electron donors. The results indicate that the characteristic bioenergetic properties exhibited by whole cells of C8 are retained in a vesicle system and thus cannot be attributed to a cytoplasmic, substrate level activity. Interestingly, lipophilic cations that were efficacious in measuring the transmembrane electrical potential of whole cells appeared to accurately measure artificially generated potentials across vesicle membranes, but were not taken up upon addition of ascorbate/phenazine methosulfate.