Isolation and characterization of Tn5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus |
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Authors: | U. Hilgert J. Schell F. J. de Bruijn |
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Affiliation: | (1) Max-Planck-Institut für Züchtungsforschung, D-5000 Köln 30, Germany |
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Abstract: | Summary Random Tn5 mutagenesis was used to isolate two independent Azorhizobium sesbaniae ORS571 mutants disturbed in ammonium assimilation (Asm-). Both Asm- mutant 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 pACYC184 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 homology with 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 ORS571 NADPH-GOGAT activity was shown to be independent of the nitrogen regulation (ntr) system. |
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Keywords: | Transposon Tn5 mutagenesis Azorhizobium sesbaniae ORS571 Nitrogen fixation/assimilation Glutamate synthase |
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