CHARACTERIZATION OF THE NTRBC GENES OF AZOSPIRILLUM-BRASILENSE SP7 - THEIR INVOLVEMENT IN THE REGULATION OF NITROGENASE SYNTHESIS AND ACTIVITY

A 7.1 kb EcoRI fragment from Azospirillum brasilense, that hybridized with a probe carrying the ntrBC genes from Bradyrhizobium japonicum, w as cloned. The nucleotide sequence of a 3.8 kb subfragment was establi shed. This led to the identification of two open reading frames, encod ing polypeptides of 401 and 481 amino acids, that were similar to NtrB and NtrC, respectively. A broad host range plasmid containing the put ative Azospirillum ntrC gene was shown to restore nitrogen fixation un der free-living conditions to a ntrC-Tn5 mutant of Azorhizobium caulin odans. Several Tn5 insertion mutants were isolated in the ntrBC coding region in A. brasilense. These mutants were prototrophic and Nif+. Ho wever, their nitrogenase activity was slightly lower than in the wild type and they were unable to grow on nitrate as sole nitrogen source. Under microaerobiosis and in the absence of ammonia, a nifA-lacZ fusio n was expressed in the mutants at about 60% of the level in the wild t ype. In the presence of ammonia, the fusion was similarly expressed (6 0% of the maximum) both in the wild type and mutants. Addition of ammo nia to a nitrogen-fixing culture of ntrBC mutants did not abolish nitr ogenase activity, in contrast with the wild type. It thus appears that in Azospirillum the ntrBC genes are not essential for nitrogen fixati on, although NtrC controls nifA expression to some extent. They are, h owever, required for the switch-off of nitrogenase activity.