title: The gene encoding dinitrogenase reductase 2 is required for expression of the second alternative nitrogenase from Azotobacter vinelandii.
abstract: Under diazotrophic conditions in the absence of molybdenum (Mo) and vanadium (V), Azotobacter vinelandii reduces N2 to NH4+ by using nitrogenase 3 (encoded by anfHDGK). However, dinitrogenase reductase 2 (encoded by vnfH) is also expressed under these conditions even though this protein is a component of the V-containing alternative nitrogenase. Mutant strains that lack dinitrogenase reductase 2 (VnfH-) grow slower than the wild-type strain in N-free, Mo-, and V-deficient medium. In this medium, these strains synthesize dinitrogenase reductase 1 (a component of the Mo-containing nitrogenase encoded by nifH), even though this component is not normally synthesized in the absence of Mo. Strains that lack both dinitrogenase reductases 1 and 2 (NifH-VnfH-) are unable to grow diazotrophically in Mo- and V-deficient medium. In this medium, NifH- VnfH- strains containing an anfH-lacZ transcriptional fusion exhibited less than 3% of the beta-galactosidase activity observed in the wild type with the same fusion. Beta-Galactosidase activity expressed by VnfH- mutants containing the anfH-lacZ fusion ranged between 57 and 78% of that expressed by the wild type containing the same fusion. Thus, expression of dinitrogenase reductase 2 seems to be required for transcription of the anfHDGK operon, although, in VnfH-mutants, dinitrogenase reductase 1 appears to serve this function. Active dinitrogenase reductase 1 or 2 is probably required for this function since a nifM deletion mutant containing the anfH-lacZ fusion was unable to synthesize beta-galactosidase above background levels. An anfA deletion strain containing the anfH-lacZ fusion exhibited beta-galactosidase activity at 16% of that of the wild type containing the same fusion. However, in the presence of NH4+, the beta-galactosidase activity expressed by this strain more than doubled. This indicates that AnfA is required not only for normal levels of anfHDGK transcription but also for NH4+ -and, to a lesser extent, Mo-mediated repression of this transcription.
authors.affiliation: Agricultural Research Service, U.S. Department of Agriculture, Raleigh, North Carolina.
authors.forename: R D
authors.initials: RD
authors.lastname: Joerger
authors.forename: E D
authors.initials: ED
authors.lastname: Wolfinger
authors.forename: P E
authors.initials: PE
authors.lastname: Bishop
mesh_terms: D001395:Azotobacter / Q000201:enzymology / Q000235:genetics*; D017037:Dinitrogenase Reductase; D004926:Escherichia coli / Q000235:genetics; D005288:Ferredoxins / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D005798:Genes, Bacterial; D005838:Genotype; D007527:Isoenzymes / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009591:Nitrogenase / Q000235:genetics*; D010641:Phenotype; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D017037:Dinitrogenase Reductase; D005288:Ferredoxins; D007527:Isoenzymes; D009591:Nitrogenase; D001616:beta-Galactosidase