Genetics of alternative nitrogen fixation systems in Azotobacter vinelandii

Bishop, Paul E.; MacDougal, Sheela I.; Wolfinger, E D; Shermer, Casendra L.

doi:10.1007/978-1-4684-6432-0_67

Cited by 10 publications

(8 citation statements)

References 20 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This nitrogenase, which is synthesized when A. vinelandii is grown in the absence of both Mo and V, has recently been isolated from A. vinelandii strain CA11.6 grown under these conditions (6), and the third cluster of nifHDK-like genes has been isolated and sequenced (3). The present study provides definitive genetic evidence for the presence of a third nitrogenase which can be clearly distinguished from the Mo-and V-nitrogenases.…”

Section: Discussionsupporting

confidence: 51%

See 1 more Smart Citation

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium

1989

View full text Add to dashboard Cite

We have constructed a strain of Azotobacter vinelandii which has deletions in the genes for both the molybdenum (Mo) and vanadium (V) nitrogenases. This strain fixed nitrogen in medium that did not contain Mo or V. Growth and nitrogenase activity were inhibited by Mo and V. In highly purified medium, growth was limited by iron. Addition of other metals (Co, Cr, Cu, Mn, Ni, Re, Ti, W, and Zn) did not stimulate growth. Like the V-nitrogenase, the nitrogenase synthesized by the double deletion strain reduced acetylene to both ethylene and ethane (C2H6JC2H4 ratio, 0.046). There was an approximately 10-fold increase in ethane production when Mo was added to the deletion strain grown in medium lacking Mo and V. This change in reactivity may be due to the incorporation of an Mo-containing cofactor into the nitrogenase synthesized by the double-deletion strain. A strain synthesizing the V-nitrogenase did not show a similar increase in ethane production. The growth characteristics of the double-deletion strain, together with the metal composition reported for a nitrogenase isolated from a tungstate-tolerant strain lacking genes for the molydenum enzyme grown in the absence of Mo and V (J. R. Chisnell, R. Premakumar, and P. E. Bishop, J. Bacteriol. 170:27-33, 1988) show that A. vinelandii can synthesize a nitrogenase which lacks both Mo and V. Reduction of dinitrogen by nitrogenase can therefore occur at a center lacking both these metals.Until recently, nitrogenases isolated from all nitrogenfixing organisms consisted of an iron protein and a molybdenum (Mo)-iron protein. They contain an FeMo cofactor at the probable active site (21). The existence of Mo-independent nitrogenases was first proposed by Bishop et al. because NiW mutants of Azotobacter vinelandii were able to grow and reduce nitrogen in the absence of Mo (1). An alternative nitrogenase, with a vanadium (V)-iron protein and an iron protein, was first isolated from the related species Azotobacter chroococcum (18). In this organism three contiguous structural genes (nifHDK) encode the polypeptides of the molybdenum-containing nitrogenase (4). Polypeptides of the V-containing nitrogenase are encoded by a distinct cluster of nifHDK-like genes (19). Mo-and Vnitrogenases have also been isolated from A. vinelandii (5, 11). However, A. vinelandii apIpears to differ from A. chroococcum in having three instead of two niffl-like genes (12).One is in the cluster of genes (nifHDK) which encodes the Mo-nitrogenase, and one is presumably associated with genes which encode the V-nitrogenase. The third niffl-like gene is in another niffHDK-like cluster which encodes a third nitrogenase (3). We show here that A. vinelandii has genes which are homologous to the genes for the V-nitrogenase of A. chroococcum.A strain of A. vinelandii has been constructed with deletions in the genes encoding both the Mo-and V-nitrogenases. Characterization of nitrogen fixation by this doubledeletion strain indicates that the third nitrogenase synthesized by A. vinelandii has novel properties.(

show abstract

Section: Discussionsupporting

confidence: 51%

“…The third niffl-like gene is in another niffHDK-like cluster which encodes a third nitrogenase (3). We show here that A. vinelandii has genes which are homologous to the genes for the V-nitrogenase of A. chroococcum.…”

mentioning

confidence: 68%

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium

1989

View full text Add to dashboard Cite

show abstract

“…The technique would be more useful for assaying alternative nitrogenase systems which evolve H E at high rates [88].…”

Section: Determinative Testsmentioning

confidence: 99%

N2-fixing pseudomonads and related soil bacteria

Chan

1994

FEMS Microbiology Reviews

View full text Add to dashboard Cite

Pseudomonas-like organisms form a highly heterogeneous and ubiquitous group of bacteria. Recent identification of an authentic P~eudomonas genus should help to decrease difficulties which arose from taxonomic uncertainties. Further difficulties in recognising N2-fixing Pseudomonas species lie in the confirmation of diazotrophy. Optimised conditions are often needed for the detection of nitrogenase activity since it is controlled by specific environmental factors and physiological requirements. However, genetically constructed N2-fixing strains from authentic Pseudomonas species have demonstrated that at least some members of the genus possess mechanisms to accommodate and express nil (N z fixation) genes from a well-studied diazotroph, Klebsiella pneumoniae. Renowned for their catabolic versatility, pseudomonads can use a wide range of carbon and energy substrates. Hence, potential N2-fixing pseudomonads are conceivably less limited by carbon and energy sources available in the environment compared to other N2-fixing organisms. Pseudomonas species dominate in the rhizosphere of some plants from which isolates have been shown to be diazotrophic. Several strains are also chemolithotrophs, capable of using H 2 as energy and electron source and CO2 as carbon source. Besides assays for N2-fixing activity, DNA hybridisation to the well conserved molybdo-nitrogenase structural gene probe is an indicator of diazotrophy. However, absence of hybridisation to this probe, despite N 2 fixation activity, has been reported. Although the genetics of N 2 fixation in pseudomonads have hardly been studied, some nif genes have been shown to be plasmid-borne. Pseudomonas species are also predominant soil denitrifiers, reducing nitrate and nitrite to gaseous forms of nitrogen during anaerobic respiration. Hence, they play an important role in the global biological nitrogen cycle. Several diazotrophic species including a few pseudomonads can also denitrify. The potential contribution by Ne-fixing pseudomonads to the sinks and sources of soil nitrogen is considered small in the short term but essentially remains unclear in the absence of experimental data. Reliable rapid methods for their specific enumeration are indispensable for assessing their population dynamics and ascertaining their ecological significance.

show abstract

“…(Some preliminary results from this study were presented at the 7th International Congress on Nitrogen Fixation [7]. )…”

mentioning

confidence: 99%

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii

et al. 1989

View full text Add to dashboard Cite

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.

show abstract

Genetics of alternative nitrogen fixation systems in Azotobacter vinelandii

Cited by 10 publications

References 20 publications

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium

Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium

N2-fixing pseudomonads and related soil bacteria

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii

Contact Info

Product

Resources

About