A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.

Two Azotobacter vinelandii strains capable of growing on N2 (Nif+) were isolated from two different mutant strains that lacked dinitrogenase activity (Nif-). Extracts of N2-grown cells of the two Nif+ strains lacked significant amounts of the "conventional" dinitrogenase protein subunits, as determined by two-dimensional gel electrophoresis. Instead, the extracts contained at least four new proteins that appeared to be ammonia-repressible (i.e., they were not detected in extracts of ammonia-grown cells). Based on the results of genetic backcrosses, the two Nif+ strains were shown to be pseudorevertants. Both Nif+ pseudorevertant strains were able to grow in N-free media lacking molybdenum but containing tungsten (conditions that prevented growth of the wild-type strain The "conventional" nitrogenase system in Azotobacter vinelandii, which is responsible for the reduction of dinitrogen (N2) to ammonia, consists of dinitrogenase, a protein that contains both molybdenum and iron, and dinitrogenase reductase, an iron-containing protein.[This nomenclature is consistent with that proposed by Hageman and Burris (1). ] Dinitrogenase is a tetramer that has a molecular weight of t245,000 and consists of two pairs of dissimilar subunits, each having a molecular weight of su61,000 (2). Dinitrogenase reductase is dimer that has a molecular weight of 60,500 and two identical subunits, each having a molecular weight of 30,000 (3).Mutant strains of A. vinelandii that are unable to fix N2 (Nif-) have been isolated and characterized with respect to electron paramagnetic resonance signals, activities for dinitrogenase and dinitrogenase reductase, and antigenic crossreactive material (4, 5). The nif mutations carried by these strains have been genetically mapped by using recombination index values (6).In this paper, we present evidence that A. vinelandii possesses an alternative N2-fixation system that is expressed under conditions of molybdenum deficiency.The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 7342MATERIALS AND METHODS Bacterial Strains and Media. The A. vinelandii strains used in this study (Table 1) were cultured in Burk's medium (8) and modification thereof. When it was necessary to include fixed nitrogen in the medium, NH4OAc was added to a concentration of 400 Atg of N/ml. The solid Burk's medium contained 1.5% purified agar (Difco), whereas the medium containing solid NH+ contained 1.5% Bacto agar (Difco). Precautions were taken to minimize contamination by molybdenum (Mo) when tungsten (final concentration of 1 mM Na2WO4-2H20) was added to the medium and when Mo-deficient medium was used. All glassware was base-and acid-washed as described by Benemann et al. (9). Chemical components used to prepare the media containing W and lacking Mo were from the following sources: KH2PO4 (ultrex, ultrapure) and sucrose (ultrex, ultrapure) were purchased fro...

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The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

The nucleotide sequence (6,559 base pairs) of the genomic region containing the structural genes for nitrogenase 2 (V nitrogenase) from Azotobacter vinelandii was determined. The open reading frames present in this region are organized into two transcriptional units. One contains vnJH (encoding dinitrogenase reductase 2) and a ferredoxinlike open reading frame (Fd). The second one includes vnJf) (encoding the a subunit of dinitrogenase 2), vnfG (encoding a product similar to the 8 subunit of dinitrogenase 2 from A. chroococcum), and vnfK (encoding the 3 subunit of dinitrogenase 2). The 5'-flanking regions of vnfH and vnJf) contain sequences similar to ntrA-dependent promoters. This gene arrangement allows independent expression of vn.f-Fd and vnfDGK. Mutant strains (CA80 and CA11.80) carrying an insertion in vnjf are still able to synthesize the a and 1 subunits of dinitrogenase 2 when grown in N-free, Mo-deficient, V-containing medium.A strain (RP1.1l) carrying a deletion-plus-insertion mutation in the vnfDGK region produced only dinitrogenase reductase 2.

The introduction briefly reviews some of the salient features of the well-characterized conventional molybdo-enzyme system for N2 fixation. This is followed by a brief account of the discovery of an alternative N2 fixation system that does not require molybdenum in the N2-fixing bacterum Azotobacter vinelandii. The next section cites observations from the early literature on N2 fixation suggesting may not always require molybdenum. Next, recent evidence for an alternative N2 fixation system in A. vinelandii is discussed. A brief description of our discovery of an alternative nitrogenase which is not a molybdenum or vanadium enzyme is presented, followed by a summary of recent papers describing an alternative vanadium-containing nitrogenase. Available information on the genetics and regulation of alternative N2 fixation systems is discussed. Finally, the possible/probable presence of alternative N2 fixation systems in bacteria other than Azotobacter species is covered.

Two nifA-like genes, designated anfA and vnfA, have been identified in Azotobacter vinelandii. The anfA gene is located upstream from the nitrogenase-3 structural gene cluster (anfHDGK) and is preceded by a sequence that is potentially part of a ntrA-dependent promoter. The product of anfA appears to be required for expression of nitrogenase-3, since cells of the anfA deletion strain CA66 were unable to synthesize this nitrogenase when derepressed in N-free, Mo-and V-deficient medium. The vnfA gene was identified after determination of the nucleotide sequence of DNA flanking the TnS insertion in mutant strain CA46. Two open reading frames (ORF1 and ORF2) were found located upstream from the vnfA gene, and a nifE-like ORF, preceded by a possible ntrA-dependent promoter, was found downstream from this gene. It is not known whether vnfA is expressed only under N2-fixing conditions. However, potential ntrA-dependent promoters were found immediately upstream from vnfA (within the 3' end of ORF2) and immediately downstream from ORF1. The region spanning ORF1 and ORF2 contained an A+T-rich sequence that was also found immediately upstream from the potential ntrA-dependent promoter of anfA. The product of vnfA appears to be required for the synthesis of nitrogenase-2, since cells of strain CA46 synthesized only nitrogenase-l and -3 but not nitrogenase-2 when grown in the presence of vanadium. The product of nifA, which is required for synthesis of nitrogenase-1, is not required for synthesis of either nitrogenase-2 or nitrogenase-3. However, growth data indicate that nifA is required for a factor (or factors) necessary for maximal diazotrophic growth under Moand V-deficient conditions.The diazotroph Azotobacter vinelandii contains three genetically distinct nitrogenases. Nitrogenase-1 is synthesized under Mo sufficiency, nitrogenase-2 is expressed under conditions in which Mo has been replaced by V, and nitrogenase-3 is present in cells grown under Mo-and V-deficient conditions (13, 19, and references therein). Transcription of the structural genes for these nitrogenases (and possibly of other nitrogen fixation genes) is regulated not only in response to fixed nitrogen but also in response to Mo and V (17; unpublished results). A number of genes whose products are involved in the regulation of N2 fixation genes in A. vinelandii have been primarily identified through their sequence similarities with well-characterized nif regulatory genes from Klebsiella pneumoniae. In this organism, a two-tier cascade control of nif regulation has been proposed whereby the products of the genes ntrA (rpoN) and ntrBC control the expression of the nif regulatory nifLA operon in response to nitrogen status. The products of nifLA and ntrA, in turn, regulate the expression of the other nif transcriptional units in response to nitrogen and oxygen status (for a review, see reference 27).

Multiple genomic regions homologous to nifJ were found in the diazotroph Azotobacter vinelandii. The nifHDK gene cluster, located on a 12.8-kilobase (kb) XhoI fragment and two additional XhoI fragments (7.4 and 8.4 kb) hybridized to a niJH-specific DNA template but the 7.4-and 8.4-kb fragments did not hybridize to nifD-or nifK-specific DNA probes. In vivo transcription of the nifHDK gene cluster was ammonia-repressible and required the presence of at least 50 nM molybdenum in the derepression medium. Three mRNA species were transcribed from the nifHDK gene cluster, a 4.2-kb transcript homologous to niff-, nifD-, and nifK-specific DNA templates, a 2.6-kb transcript homologous to nifH-and nijD-specific DNA templates, and a 1.2-kb transcript homologous only to the niff-specific DNA template. In strain CAll, a niJfDK deletion mutant, the nifHDK-specific transcripts were not produced and the strain was unable to grow in N-free medium in the presence of Na2MoO4 at concentrations of 50 nM or higher. However, at concentrations of 25 nM Mo or less, growth occurred in N-free medium. Under these conditions two niff-homologous (but not nijD-or nifK-homologous) transcripts were observed (1.2 and 1.8 kb). Presumably these were transcribed from the additional nW1H-homologous sequences present in the genome. These results are consistent with the existence of two N2 fixation systems in A. vinelandii which are regulated by molybdenum at the level of transcription.