Oxygen protection of nitrogen fixation in free-living Azorhizobium caulinodans: the role of cytochrome aa.

The growth properties of Azorhizobium caulinodans wild-type and a cytochrome aa mutant strain, both growing with N as N source at fixed dissolved partial oxygen pressures in the range 0.5--4.0 kPa, were studied by making use of continuous cultures (chemostats and pH-auxostats) and transient cultures. In succinate-limited chemostats, the wild-type exhibited a higher growth yield than the aa mutant at every dissolved oxygen tension tested, indicating activity of cytochrome aa in this entire oxygen regime. The growth yield of both the wild-type and the aa mutant declined when the dissolved oxygen tension was raised. 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 mutant remained constant. The transient changes in growth properties observed in chemostat cultures after pulsing with succinate pointed to a negative effect of oxygen on the maximum specific growth rate. This was studied further 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 mutant confirmed that cytochrome aa is active in the wild-type. Again, the growth yield of both strains decreased with the dissolved oxygen tension, but in contrast to the results obtained with chemostats, no difference in growth yield was observed between wild-type and mutant at any oxygen tension. In either type of continuous culture a decrease in the overall P/O ratio with increasing dissolved oxygen tension is improbable for the wild-type, and even more so for the aa mutant. Therefore, the adverse effects of oxygen on the growth of A. caulinodans are not readily explained by respiratory protection; alternatively, it is proposed that the catalytic oxidation of nitrogen-fixation-specific redox enzymes by oxygen (auto-protection) enables the bacterium to deal with intracellular oxygen at the expense of reducing equivalents and free energy. To compensate for the loss of free energy, respiration increases and an active cytochrome aa contributes to this by keeping the P/O ratio high.