Cell extract preparations of alder nodules require ATP and reducing equivalents for nitrogenase activity (3). The sources of energy and reductant for nitrogen fixation by Frankia spp. in the nodules are not known. Akkermans et al.(1, 2) showed that enzymes associated with the malateaspartate shuttle system were present in vesicle cluster preparations from nodules and proposed that this system provides the reducing equivalents for nitrogenase. We previously reported (29) that isolated vesicles are capable of nitrogen fixation when they are incubated in the presence of dithionite and Mg-ATP. This observation suggests that vesicles may take up intact ATP to supply energy for nitrogenase activity. This is an interesting idea in light of reports that plant mitochondria are in close association with vesicle clusters within the root nodules (1, 2, 14).The excretion of ATP coupled with ADP uptake is catalyzed by an adenylate nucleotide transport system (termed ATP-ADP translocase) located in the inner membranes of mitochondria and chloroplasts (10,34,38). Translocase activity is involved in ATP uptake by the intracellular parasites Rickettsia (36,37) and Chlamydia (9) cells. Intracellular membranes of Methanobacterium thermoautotrophicum were reported to contain ATP-ADP translocase activity and were postulated to function as "methanochondria" in providing ATP to the cell (5, 6). Kramer and Schonheit (12) MATERIALS AND METHODS Organism and culture conditions. Frankia strain EAN (Frankia registry number ULQ13100144 [13]) was obtained from M. Lalonde, Laval University, Quebec, Canada. Cultures were grown and maintained in basal growth medium under nitrogen-repressed conditions with NH4Cl as the nitrogen source, as described previously (28). Large-scale batch cultures of cells derepressed for nitrogenase were obtained by growing cells in a carboy with 15 liters of medium as described previously (29). Under these conditions, the cells depleted their supply of NH4 after 7 to 8 days of growth and were growing with N2 as the nitrogen source when they were harvested.Vesicle isolation and purification. Cells were incubated under nitrogen-depressed conditions with N2 as the nitrogen source to induce vesicle development and nitrogenase activity. Cells grown for 14 days in medium containing NH4Cl as the nitrogen source were harvested and washed three times with MP buffer (20 mM morpholinepropanesulfonic acid [MOPS] and 10 mM KH2PO4 buffer at [pH 6.8]). The cells were then inoculated into succinate growth medium with N2 as the sole nitrogen source and incubated for 4 days at 25°C before harvest.Vesicles were isolated from the N2-grown cells and purified by the procedure of Tisa and Ensign (29). Cells were passed through a French pressure cell at 10,000 to 12,000 lb/ in2 at 4°C to disrupt mycelia and separate vesicles. The vesicles were purified from cellular debris by a series of low-speed centrifugations at 20°C. For some experiments, stringent anaerobic techniques were used to ensure that there was no exposure to oxygen.Nitroge...