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

Mylona, Photini V.; Premakumar, R.; Pau, Richard N.; Bishop, Paul E.

doi:10.1128/jb.178.1.204-208.1996

Cited by 19 publications

(34 citation statements)

References 42 publications

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“…2 B and C, bar 14). This finding is inconsistent with the phenotype of anfO and anfR mutants in A. vinelandii, which are essential for reduction of dinitrogen but not for acetylene (7). Finally, deletion mutations in nifF and nifJ, which provide electron transport to MoFe nitrogenase in K. oxytoca, reduced both the level of acetylene and nitrogen reduction by the reconstituted FeFe nitrogenase in E. coli ( Fig.…”

Section: Resultsmentioning

confidence: 84%

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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: Resultsmentioning

confidence: 84%

“…The structural genes encoding FeFe nitrogenase are organized as a single operon anfHDGKOR in Azotobacter vinelandii (6,7). The subunits of dinitrogenase reductase are encoded by anfH, whereas the α-and β-subunits of dinitrogenase are encoded by anfD and anfK, respectively.…”

mentioning

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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“…Mylona et al (26) found that mutants in two genes of the anf system of A. vinelandii, which encodes the non-Mo, non-V nitrogenase, have high acetylene reduction activities but (36). While a distinct non-Mo, non-V nitrogenase could account for the acetylene reduction observed in cells grown without metal, that seems unlikely given the very poor growth of cells in metal-deficient medium.…”

Section: Discussionmentioning

confidence: 99%

Isolation and characterization of the VnfEN genes of the cyanobacterium Anabaena variabilis

Thiel

1996

J Bacteriol

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The filamentous cyanobacterium Anabaena variabilis fixes nitrogen in the presence of vanadium (V) and in the absence of molybdenum (Mo), using a V-dependent nitrogenase (V-nitrogenase) encoded by the vnfDGK genes. Downstream from these genes are two genes that are similar to the vnfEN genes of Azotobacter vinelandii. Like the vnfDGK genes, the vnfEN genes were transcribed in the absence of Mo, whether or not V was present. A mutant with an insertion in the vnfN gene lacked V-nitrogenase activity; thus, the vnfEN genes were essential for the V-nitrogenase system in A. variabilis. Growth and acetylene reduction assays with wild-type and mutant strains suggested that the V-nitrogenase reduced dinitrogen better than acetylene. The similarity of the vnfEN genes of A. variabilis and A. vinelandii was not strong. The vnfEN genes of A. variabilis showed greater similarity to the vnfDK genes just upstream than to the A. vinelandii vnfEN genes. Sequence comparisons provide support for the idea that if the vnf genes were transferred laterally among bacterial strains, the vnf cluster was not transferred intact. It appears likely that the structural genes were transferred before a duplication event led to the evolution of the vnfEN genes independently in the two strains. The divergence of the vnfEN genes from the vnfDK genes suggests that this duplication, and hence the transfer of vnf genes, was an ancient event.Anabaena variabilis ATCC 29413 is a filamentous cyanobacterium that fixes dinitrogen under a variety of environmental conditions. Under aerobic conditions, nitrogen fixation occurs exclusively in cells called heterocysts (18,27). Heterocysts are terminally differentiated cells that develop in a pattern from certain vegetative cells in the filament in response to limitation in fixed nitrogen (reviewed in reference 44). The primary Modependent nitrogenase (Mo-nitrogenase) in this organism is encoded by a contiguous cluster of nif1 genes, including nifB1, fdxN, nifS1, nifU1, nifD1, nifK1, nifE1, and nifN1, that are transcribed only in heterocysts (8,22,39). These genes are very similar to the nif cluster of Anabaena sp. strain PCC 7120 (reviewed in reference 21), which is also transcribed exclusively in heterocysts (16). We have recently characterized an alternative Mo-nitrogenase gene cluster in this strain that is absent in Anabaena sp. strain PCC 7120; this second cluster contains genes nifB2, nifS2, nifU2, nifH2, nifD2, nifK2, nifEN2, nifX2, and nifW2, which are transcribed in vegetative cells and in heterocysts only under strictly anaerobic conditions (39). These nif2 genes are homologs of the nif1 genes; however, this cluster differs from the nif1 cluster: there is no fdxN gene, nor is there the 11-kb excision element that is present in nif1 and in the nif cluster of Anabaena sp. strain PCC 7120. In addition, the nifE1 and nifN1 homologs are fused into a single open reading frame, nifEN2. Certain fdx genes in this organism that are expressed only under anaerobic conditions may also be part of this second system (...

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“…1). Like R. capsulatus anf1, anf2, and anf3 mutants, A. vinelandii anfO and anfR mutants cannot reduce N 2 to ammonia but are still able to reduce acetylene via the iron-only nitrogenase (25). To date, no A. vinelandii mutant defective in the anf3-like gene has been constructed.…”

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confidence: 99%

“…vinelandii AnfO exhibits some similarity to the N-terminal part of dinitrogenase reductases (including a cysteine residue involved in coordination of the [4Fe-4S] cluster) and to the heme-binding domain of P-450 cytochromes, suggesting that AnfO might be an iron-containing protein (14,25). In contrast to AnfO, no putative conserved domain has been detected for AnfR.…”

mentioning

confidence: 99%

Identification of Two New Genes Involved in Diazotrophic Growth via the Alternative Fe-Only Nitrogenase in the Phototrophic Purple Bacterium Rhodobacter capsulatus

et al. 2005

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Growth of Rhodobacter capsulatus with molecular dinitrogen as the sole N source via the alternative Fe-only nitrogenase requires all seven gene products of the anfHDGK-1-2-3 operon. In contrast to mutant strains carrying lesions in the structural genes of nitrogenase (anfH, anfD, anfG, and anfK), strains defective for either anf1, anf2, or anf3 are still able to reduce the artificial substrate acetylene, although with diminished activity. To obtain further information on the role of Anf1, we screened an R. capsulatus genomic library designed for use in yeast two-hybrid studies with Anf1 as bait. Two genes, which we propose to call ranR and ranT (for genes related to alternative nitrogenase), coding for products that interact with Anf1 were identified. A ranR mutant exhibited a phenotype similar to that of an anf1 mutant strain (no growth with N 2 in the absence of molybdenum, but significant reduction of acetylene via the Fe-only nitrogenase), whereas a ranT mutant retained the ability to grow diazotrophically, but growth was clearly delayed compared to the parental strain. In contrast to the situation for anf1, expression of neither ranR nor ranT was regulated by ammonium or molybdenum. A putative role for Anf1, RanR, and RanT in the acquisition and/or processing of iron in connection with the Fe-only nitrogenase system is discussed.

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

Cited by 19 publications

References 42 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

Isolation and characterization of the VnfEN genes of the cyanobacterium Anabaena variabilis

Identification of Two New Genes Involved in Diazotrophic Growth via the Alternative Fe-Only Nitrogenase in the Phototrophic Purple Bacterium Rhodobacter capsulatus

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