OXYGEN PROTECTION OF NITROGEN-FIXATION IN FREE-LIVING AZORHIZOBIUM-CAULINODANS - THE ROLE OF CYTOCHROME AA(3)

The growth properties of Azorhizobium caulinodans wild-type and a cyto chrome aa(3) mutant strain, both growing with N-2 as N source at fixed dissolved partial oxygen pressures in the range 0.5-4.0 kPa, were stu died by making use of continuous cultures (chemostats and pH-auxostats ) and transient cultures. In succinate-limited chemostats, the wild-ty pe exhibited a higher growth yield than the aa(3) mutant at every diss olved oxygen tension tested, indicating activity of cytochrome aa(3) i n this entire oxygen regime. The growth yield of both the wild-type an d the aa(3) mutant declined when the dissolved oxygen tension was rais ed. In contrast, for growth on ammonia at the same dilution rate, the wild-type showed an increase in growth yield with increasing dissolved oxygen tension, whereas the growth yield of the aa(3) mutant remained constant. The transient changes in growth properties observed in chem ostat cultures after pulsing with succinate pointed to a negative effe ct of oxygen on the maximum specific growth rate. This was studied fur ther in steady-state pH-auxostat cultures. The specific growth rate of both strains decreased with increasing dissolved oxygen tension. The less steep decline in growth rate of the wild-type compared to the aa( 3) mutant confirmed that cytochrome aa(3) is active in the wild-type. Again, the growth yield of both strains decreased with the dissolved o xygen tension, but in contrast to the results obtained with chemostats , no difference in growth yield was observed between wild-type and mut ant at any oxygen tension. In either type of continuous culture a decr ease in the overall P/O ratio with increasing dissolved oxygen tension is improbable for the wild-type, and even more so for the aa(3) mutan t. Therefore, the adverse effects of oxygen on the growth of A. caulin odans are not readily explained by respiratory protection; alternative ly, it is proposed that the catalytic oxidation of nitrogen-fixation-s pecific redox enzymes by oxygen (auto-protection) enables the bacteriu m to deal with intracellular oxygen at the expense of reducing equival ents and free energy. to compensate for the loss of free energy, respi ration increases and an active cytochrome aa(3) contributes to this by keeping the P/O ratio high.