The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

Waugh, Sheila; Paulsen, D.; Mylona, Photini V.; Maynard, R H; Premakumar, R.; Bishop, Paul E.

doi:10.1128/jb.177.6.1505-1510.1995

Cited by 49 publications

(28 citation statements)

References 41 publications

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“…2C, bar 13). This observation concurs with previous results in A. vinelandii, which demonstrated that anfG is crucial for reduction of dinitrogen by FeFe nitrogenase but is not essential for acetylene reduction (8). In contrast, only minor effects on nitrogenase activity were observed with the anfOR deletion mutant ( Fig.…”

Section: Resultssupporting

confidence: 79%

“…The subunits of dinitrogenase reductase are encoded by anfH, whereas the α-and β-subunits of dinitrogenase are encoded by anfD and anfK, respectively. An additional δ-subunit encoded by anfG, which is specific for alternative nitrogenases, forms an α 2 β 2 δ 2 hexameric structure in FeFe nitrogenase (8). The predicted protein products of anfO and anfR genes do not show overall similarity to any known nif gene products, and the exact role of these two genes in the function of FeFe nitrogenase is unknown.…”

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confidence: 99%

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Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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All diazotrophic organisms sequenced to date encode a molybdenum-dependent nitrogenase, but some also have alternative nitrogenases that are dependent on either vanadium (VFe) or iron only (FeFe) for activity. In Azotobacter vinelandii, expression of the three different types of nitrogenase is regulated in response to metal availability. The majority of genes required for nitrogen fixation in this organism are encoded in the nitrogen fixation (nif) gene clusters, whereas genes specific for vanadium-or irondependent diazotophy are encoded by the vanadium nitrogen fixation (vnf) and alternative nitrogen fixation (anf) genes, respectively. Due to the complexities of metal-dependent regulation and gene redundancy in A. vinelandii, it has been difficult to determine the precise genetic requirements for alternative nitrogen fixation. In this study, we have used Escherichia coli as a chassis to build an artificial iron-only (Anf) nitrogenase system composed of defined anf and nif genes. Using this system, we demonstrate that the pathway for biosynthesis of the iron-only cofactor (FeFe-co) is likely to be simpler than the pathway for biosynthesis of the molybdenum-dependent cofactor (FeMo-co) equivalent. A number of genes considered to be essential for nitrogen fixation by FeFe nitrogenase, including nifM, vnfEN, and anfOR, are not required for the artificial Anf system in E. coli. This finding has enabled us to engineer a minimal FeFe nitrogenase system comprising the structural anfHDGK genes and the nifBUSV genes required for metallocluster biosynthesis, with nifF and nifJ providing electron transport to the alternative nitrogenase. This minimal Anf system has potential implications for engineering diazotrophy in eukaryotes, particularly in compartments (e.g., organelles) where molybdenum may be limiting.

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Section: Resultssupporting

confidence: 79%

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confidence: 99%

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Yang

Xie

Wang

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The ␣-and ␤-subunits are encoded by the anfD and anfK genes, respectively. The ␦-subunit, a small protein, characteristic of the alternative nitrogenases (33,39,41), is encoded by the anfG gene.In addition to the structural genes for nitrogenase 3, the anfHDGK operon contains two additional open reading frames (ORFs) (anfO and anfR) located immediately downstream of the anfK gene (4, 11). Recent studies show that anfO and anfR are cotranscribed with the anfHDGK genes in one polycistronic message that apparently undergoes posttranscriptional processing (31).…”

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confidence: 99%

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

et al. 1996

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In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo-and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15 N 2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo-and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative -independent transcription termination site has been identified 250 bp from the 3 end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo-and V-deficient conditions.Nitrogen fixation, the reduction of dinitrogen to ammonia, is an enzymatic process that occurs in a wide range of bacterial species, including the soil bacterium Azotobacter vinelandii. This process is catalyzed by the enzyme complex nitrogenase. Nitrogenase is a metalloenzyme composed of two protein components called dinitrogenase reductase and dinitrogenase. A. vinelandii harbors three genetically distinct nitrogenases (3, 4). The first nitrogenase is the molybdenum (Mo)-containing enzyme (nitrogenase 1) that is made in the absence of a fixed nitrogen source when Mo is available (9). The second enzyme, a vanadium (V)-containing nitrogenase (nitrogenase 2), is synthesized under Mo-deficient conditions in the presence of V (4, 9, 12). The third nitrogenase (nitrogenase 3) is expressed only in the absence of Mo and V (3,6,17,30).The protein subunits for the Mo nitrogenase are encoded by the nifHDK operon (10). The structural genes for V nitrogenase comprise two operons, vnfHorfFd and vnfDGK (12). The subunits for the third nitrogenase are encoded by the anfHDGK operon (11).Recently, three other diazotrophs, Clostridium pasteurianum (41), Rhodobacter capsulatus (16, 33), and Rhodospirillum rubrum (4), were found to have alternative nitrogenases that are similar to nitrogenase 3 of A. vinelandii. These nitrogenases are expressed only in the absence of Mo. Nitrogenase 3 is composed of dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase reductase 3 is a homodimer (␥ 2 ) encoded by the anfH gene, while dinitrogenase 3 is a hexamer (␣ 2 ␤ 2 ␦ 2 ). The ␣-and ␤-subunits are encoded by the anfD and anfK genes, respectively. The ␦-subunit, a small protein, characteristic of the alternative nitrogenases (33,39,41), is encoded by the anfG gene.In addition to the struct...

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“…The process is carried out by bacteria. In both symbiotic and free-living bacteria, BNF is genetically controlled by a group of nif, anf or vnf genes (Joerger et al, 1991;Waugh et al, 1995;Coelho et al, 2007a;Sugitha & Kumar, 2009). The nifH gene codes for dinitrogenase reductase and is part of the nif cluster that produces the nitrogenase catalytic enzyme complex and its regulatory proteins that are responsible for reducing atmospheric N 2 to ammonia (Sugitha & Kumar, 2009;Rosado et al, 1998;Poly et al, 2001).…”

Section: Introductionmentioning

confidence: 99%

The Impact of Mineral Nitrogen Fertilization on the Occurrence of Native Diazotrophic Bacteria in Kohlrabi (Brassica oleracea) Shoots and Roots

Ekandjo¹,

Ruppel²

2015

JAS

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Biological Nitrogen Fixation (BNF) is a process of great importance in crop production systems, as it provides additional natural sources of mineral nitrogen. BNF is catalyzed by diazotrophs that are identified by the nif operon presence comprising the nifH gene that encodes for enzyme nitrogenase synthesis. Thoroughly understanding of factors that influence diazotrophic abundance is crucial for their utilization to enhance sustainability and prevent land degradation in modern agriculture. In this study the impacts of nitrogen fertilization on diazotrophic abundance in Brassica oleracea roots and leaves was investigated in greenhouse experiments by real-time qPCR. One way ANOVA was used to compare means and bivariate Pearson correlation tested for relationships between variables. Increased nitrogen fertilization significantly increased the nitrogen content in leaves but not in roots. No significant changes in nifH gene copy numbers nor in proportion of nifH gene copy numbers were detectable. This indicates no effect of mineral N fertilization on the abundance of total native diazotrophic bacterial numbers in Brassica oleracea plants.

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The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii

Cited by 49 publications

References 41 publications

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Reconstruction and minimal gene requirements for the alternative iron-only nitrogenase in Escherichia coli

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii

The Impact of Mineral Nitrogen Fertilization on the Occurrence of Native Diazotrophic Bacteria in Kohlrabi (Brassica oleracea) Shoots and Roots

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