Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS 571 mutants disturbed in ammonium assimilation (Asm-). Both Asmmutant strains were shown to lack NADPH-glutamate synthase NADPH-GOGAT) activity and to carry Tn5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn5-containing region of one of the GOGAT- mutant strains was cloned in pACYC 184 and used to identify the wild-type glt (GOGAT) locus in a phage clone bank of ORS571. The cloned region was shown to have DNA homologywith the Escherichia coli glt locus and to complement the Asm-phenotype of E. coli and ORS571 GOGAT strains. The ORS571 GOGAT mutations were found to interfere with free-living as well as symbiotic nitrogen fixation. Expression of ORS 571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation(ntr) system

The nitrogen-fixing bacterial strain Azorhizobium sesbaniae ORS 571 (referred to as ORS 571 hereinafter), isolated from aerial stem nodules of its host plant, the tropical legume Sesbania rostrata (Dreyfus and Dommergues 1981) is unusual in several respects. In addition to being able to induce highly effective stem and root nodules on S. rostrata, it is also capable of fixing nitrogen and of dinitrogen-dependent growth in the free-living state (Elmerich et al. 1982; Dreyfus etal. 1983; Gebhardt etal. 1984). Thus the ORS571/S. rostrata system has facilitated a comparison of the regulation of nitrogen fixation and nitrogen (ammonium) assimilation genes in the free-living versus symbiotic state. Under free-living, nitrogen-fixing conditions ORS571 appears to couple the processes of nitrogen fixation and assimilation in order to allow dinitrogen dependent growth, thus resembling the free-living diazotroph Klebsiella pneumoniae (see Magasanik 1982; Ausubel 1984). In the symbiotic state (in ORS571-induced nodules on S rostrata) these processes appear to be uncoupled, allowing ammonium to be exported by the bacteroids for assimilation utilization by the host plant, as observed in other symbiotic Brady-)Rhizobium plant systems (see Miflin and Cullimore 1984). These observations predict a switch in regulatory circuitry between the free-living and symbiotic nitrogen- fixing state and have prompted us to analyse the induction/repression of nif/fix versus nitrogen assimilation genes in culture and in planta

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