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 (N(2)) 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 N(2) fixation, we previously isolated mutants of Frankia casuarinae that cannot fix N(2). 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 N(2) as free-living cells were isolated from the mutant cell population. These restored strains also regained the ability to stimulate nodulation. A comparative ana­lysis 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 N(2) fixation in Frankia.