NAD⁺ Synthetase is Required for Free-living and Symbiotic Nitrogen Fixation in the Actinobacterium Frankia casuarinae

Frankia spp. are multicellular actinobacteria that fix atmospheric dinitrogen (N₂) not only in the free-living state, but also in root-nodule symbioses with more than 200 plant species, called actinorhizal plants. To identify novel Frankia genes involved in N₂ fixation, we previously isolated mutants of Frankia casuarinae that cannot fix N₂. One of these genes, mutant N3H4, did not induce nodulation when inoculated into the host plant Casuarina glauca. Cell lineages that regained the ability to fix N₂ as free-living cells were isolated from the mutant cell population. These restored strains also regained the ability to stimulate nodulation. A comparative ana-lysis of the genomes of mutant N3H4 and restored strains revealed that the mutant carried a mutation (Thr584Ile) in the glutamine-dependent NAD⁺ synthetase gene (Francci3_3146), while restored strains carried an additional suppressor mutation (Asp478Asn) in the same gene. Under nitrogen-depleted conditions, the concentration of NAD(H) was markedly lower in the mutant strain than in the wild type, whereas it was higher in restored strains. These results indicate that glutamine-dependent NAD⁺ synthetase plays critical roles in both free-living and symbiotic N₂ fixation in Frankia.