The fast growing strain, Azorhizobium caulinodans ORS571, isolated from stem nodules of the tropical legume Sesbania rostrata, can grow in the free-living state at the expense of molecular nitrogen. Five point mutants impaired in nitrogen fixation in the free-living state have-beencomplemented by a plasmid containing the clonedfix ABC region of strain ORS571. Genetic analysis of the mutants showed that one was impaired in fixC, one in fixA and the three others in a new gene, located upstream from fixA and designated nifO. Site-directed Tn5 mutagenesis. was performed to obtain Tn5 insertions in fixB and fixC The four genes are required for nitrogen fixation both in the free-living state and under symbiotic conditions. The nucleotide sequence of nifO was established. The gene is transcribed independently offixA and does not correspond. to fixX, recently identified in Rhizobium meliloti and R. leguminosarum Biochemical analysis of the five point mutants showed that they synthesized normal amounts of nitrogenase components. It is unlikely that fixA, fixC and nifO are involved in electron transport to nitrogenase. FixCcould be required for the formation of a functional nitrogenase.component 2

The fast growing Rh&obium strain ORS571, now called Azorhizobium caulinodans (Dreyfus et al. 1988), was isolated from stem nodules of the tropical legume Sesbania rostrata Dreyfus and Dommergues 1981). The strain can grow in the free-living state at the expense of molecular nitrogen(Elmerich et al. 1982; Dreyfus et al, 1983). The nitrogenase purified from cells grown in a fermenter, was shown to be composed of two components, a MoFe-protein or component 1 and a Fe-protein or component 2 (Kush et al,1985). The enzyme activity was found to be subject to switch-off" when ammonia was added to a Nz-fixing culture
As in photosynthetic bacteria (Ludden et al. 1984;"Vignais et al. 1987), component 2 was inactivated, whereas component 1 remained fully active (Kush et al. 1985,By hybridization with KlebsielIa pneumoniae n/f probes,

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