Isolation and characterization of Tn 5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

Uwe K.K Hilgert, J. Schell, F. J. de Bruijn

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Random Tn 5 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 Tn 5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn 5-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.

Original languageEnglish (US)
Pages (from-to)195-202
Number of pages8
JournalMGG Molecular & General Genetics
Volume210
Issue number2
DOIs
StatePublished - Dec 1987
Externally publishedYes

Fingerprint

Azorhizobium
Glutamate Synthase
NADP
Organism Cloning
Escherichia coli
Nitrogen Fixation
Ammonium Compounds
Mutagenesis
Bacteriophages
Nitrogen
Clone Cells
Chromosomes
Phenotype
Mutation
DNA

Keywords

  • Azorhizobium sesbaniae ORS571
  • Glutamate synthase
  • Nitrogen fixation/assimilation
  • Transposon Tn 5 mutagenesis

ASJC Scopus subject areas

  • Genetics

Cite this

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title = "Isolation and characterization of Tn 5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus",
abstract = "Random Tn 5 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 Tn 5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn 5-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.",
keywords = "Azorhizobium sesbaniae ORS571, Glutamate synthase, Nitrogen fixation/assimilation, Transposon Tn 5 mutagenesis",
author = "Hilgert, {Uwe K.K} and J. Schell and {de Bruijn}, {F. J.}",
year = "1987",
month = "12",
doi = "10.1007/BF00325684",
language = "English (US)",
volume = "210",
pages = "195--202",
journal = "Molecular Genetics and Genomics",
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T1 - Isolation and characterization of Tn 5-induced NADPH-glutamate synthase (GOGAT-) mutants of Azorhizobium sesbaniae ORS571 and cloning of the corresponding glt locus

AU - Hilgert, Uwe K.K

AU - Schell, J.

AU - de Bruijn, F. J.

PY - 1987/12

Y1 - 1987/12

N2 - Random Tn 5 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 Tn 5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn 5-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.

AB - Random Tn 5 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 Tn 5 insertions ca. 1.5 kb apart in the ORS571 chromosome. The Tn 5-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.

KW - Azorhizobium sesbaniae ORS571

KW - Glutamate synthase

KW - Nitrogen fixation/assimilation

KW - Transposon Tn 5 mutagenesis

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