para-Hydroxybenzoate supported nitrogen fixation in Azotobacter vinelandii strain OP (13705)

1988 ◽  
Vol 34 (11) ◽  
pp. 1271-1275 ◽  
Author(s):  
Jay B. Peterson ◽  
Lynn S. Peterson
Keyword(s):  
Nitrogen Fixation ◽  
Cytoplasmic Membrane ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Molecular Nitrogen ◽  
Carbon Sources ◽  
Gel Filtration ◽  
Cell Extracts ◽  
Reduction Assay ◽  
Membrane Bound

Azotobacter vinelandii cells grew with molecular nitrogen and p-hydroxybenzoate as the sole added nitrogen and carbon sources. Nitrogenase activity in p-hydroxybenzoate grown cells was demonstrated with the acetylene reduction assay. Cell extracts contained the enzymes p-hydroxybenzoate hydroxylase (EC 1.14.13.2) and protocatechuate 3,4-dioxygenase (EC 1.13.1.3); oxygenases associated with p-hydroxybenzoate metabolism. These enzymes separated from respiration particles with gel filtration chromatography, indicating that they are soluble and not membrane bound. This evidence indicates that oxygen enters to the inner face of the cytoplasmic membrane during nitrogen fixation.

Molybdenum enhancement of nitrogen fixation in a Mo-starved Azotobacter vinelandii Nif− mutant

1982 ◽  
Vol 28 (10) ◽  
pp. 1173-1180 ◽  
Author(s):  
William J. Page ◽  
S. Karen Collinson
Keyword(s):  
Molecular Weight ◽  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Nitrogen Fixing ◽  
Wild Type ◽  
Cell Extracts ◽  
In The Wild ◽  
Dinitrogenase Reductase

Molybdenum (Mo)-starved wild-type and Nif− strains of Azotobacter vinelandii reduced acetylene (fixed nitrogen) in Mo-limited nitrogen-free medium. Vanadate enhanced this activity in all of the strains. Molybdate caused repression of nitrogenase activity in the Nif− mutants and enhanced the nitrogenase activity in the wild type. The nitrogenase activity in the Nif− mutant UW3, however, was enhanced by Mo, became maximal after 3 h, and then declined to zero after 10 h of incubation. The activation of nitrogenase by Mo followed a 5- to 10-min lag and was inhibited when streptomycin or rifampin was added with Mo. Examination of Mo-starved nitrogen-fixing UW3 cell extracts by two-dimensional polyacrylamide gel electrophoresis revealed molecular weight 57 000, 50 000, and 30 000 proteins that were Mo and NH4+ repressive. The molecular weight 30 000 protein appeared in the same position on the gel as the wild-type dinitrogenase reductase, although UW3 did not produce this protein under Mo-sufficient nitrogen-fixing conditions. Cell extracts prepared 3 h after Mo addition lacked the molecular weight 57 000 and 50 000 proteins but contained a new protein corresponding to the β subunit of dinitrogenase. When UW3 nitrogenase activity was lost, the dinitrogenase reductase-like protein also was absent. The results suggest that a complex active in nitrogen fixation may form between components of the traditional Mo-sufficient and alternative Mo-starved cell nitrogen fixation systems.

Seasonal variation in nitrogen fixation, associated microbial populations, and carbohydrates in roots and rhizomes of Typha latifolia (Typhaceae)

1984 ◽  
Vol 62 (9) ◽  
pp. 1965-1967 ◽  
Author(s):  
David D. Biesboer
Keyword(s):  
Nitrogen Fixation ◽  
Nitrogenase Activity ◽  
Anaerobic Bacteria ◽  
Typha Latifolia ◽  
Root Surface ◽  
Microbial Populations ◽  
Aerobic Bacteria ◽  
Free Sugar ◽  
Fixation Rate ◽  
Reduction Assay

Seasonal changes in nitrogen fixation, numbers of nitrogen-fixing bacteria associated with the roots, and rhizome–root carbohydrates were studied for the broad-leaved cattail, Typha latifolia L. Populations of anaerobic and aerobic diazotrophic bacteria were present on the root surface. Anaerobic bacteria predominated in the diazotrophic association, were more active in the acetylene reduction assay, and generally outnumbered aerobic bacteria by 2 to 1 during maximum rates of seasonal nitrogen fixation. The observed maximum nitrogen fixation rate coincided closely with reproductive development in Typha and peak microbial populations. Starch levels in rhizomes were nearly depleted during the middle of the growing season, whereas free sugar concentrations remained stable. Sugar concentrations in the roots increased rapidly during rhizome–root growth and decreased rapidly prior to peak nitrogenase activity.

scholarly journals Rhizobium etli Genetically Engineered for the Heterologous Expression of Vitreoscilla sp. Hemoglobin: Effects on Free-Living and Symbiosis

1999 ◽  
Vol 12 (11) ◽  
pp. 1008-1015 ◽  
Author(s):  
Mario Ramírez ◽  
Brenda Valderrama ◽  
Raúl Arredondo-Peter ◽  
Mario Soberón ◽  
Jaime Mora ◽  
...  
Keyword(s):  
Nitrogen Fixation ◽  
Heterologous Expression ◽  
Energy Content ◽  
Nitrogenase Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Rhizobium Etli ◽  
Free Living ◽  
Cell Extracts ◽  
Bean Plants

Oxygen concentration is an environmental signal that regulates nitrogen fixation in the Rhizobium-legume symbiosis. We investigated the effect of the heterologous expression of Vitreoscilla sp. hemoglobin (VHb), which is an oxygen-binding protein, in Rhizobium etli. The vhb gene and its native promoter were subcloned in the plasmid pMR4 and transformed into the R. etli strain CE3. Free-living cultures of engineered R. etli CE3 expressed the vhb gene, as shown by the CO-difference spectral and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of cell extracts. The expression of vhb in free-living R. etli grown under most limiting oxygen concentrations resulted in an increase in respiratory activity, chemical energy content, and expression of the nitrogen-fixation gene nifHc. Bacteroids isolated from nodules of bean plants inoculated with the engineered R. etli CE3 expressed the vhb gene, as shown by RNA slot-blot analysis. Bean plants inoculated with the engineered strain exhibited higher nitrogenase activity and total nitrogen content (68% and 14 to 53%, respectively) than bean plants inoculated with the R. etli wild type. These results suggest that the synthesis of VHb in engineered R. etli stimulated the respiratory efficiency of free-living rhizobia, and also probably of symbiotic bacteroids, thus leading to higher levels of symbiotic nitrogen fixation.

scholarly journals Characterization of Diazotrophic Rhizobacteria under Various Conditions

2013 ◽  
Vol 1 (3) ◽  
pp. 110-117
Author(s):  
Umesh Prasad Shrivastava
Keyword(s):  
Nitrogenase Activity ◽  
Nifh Gene ◽  
Carbon Sources ◽  
Nitrogen Sources ◽  
Accession Number ◽  
Carbon And Nitrogen Sources ◽  
Carbon And Nitrogen ◽  
Reduction Assay ◽  
Assay Methods

Nitrogenase activity was analysed after supplementation of various carbon and nitrogen sources in the growth medium by Acetylene Reduction Assay methods in selected 9 isolates from 74 diazotrophic isolates.  Enhancement in nitrogenase activity was recorded many fold by the addition of different organic carbon sources in which maltose and pyruvate showed better result than others. In case of supplementation of nitrogen sources, reduction of nitrogenase activity was observed.  Nitrogenase activity increased from 22.7 to 72.7% in various strains when they are tested in anaerobic condition, Amplification of fragment of 390 bp showed that nitrogenase activity due to presence of nifH gene.  Sequences were submitted to NCBI GeneBank and the accession number of nifH sequence of ECI-10A (FJ032023), AF-4B (FJ032024), AF-4C (FJ032022) and BN-2A (FJ032021) has been obtained. Phylogenetic analysis based on showed that these 4 isolates belong to the member of γ-proteobacteria, but show appreciable genetic diversity.DOI: http://dx.doi.org/10.3126/ijasbt.v1i3.8607 Int J Appl Sci Biotechnol, Vol. 1(3) 2013 : 110-117

Internal Membranes in the Nitrogen Fixing Organisms: Free-Living Azotobacter vinelandii and Rhizobium japonicum Bacteriods

1974 ◽  
Vol 32 ◽  
pp. 172-173
Author(s):  
D. Raveed ◽  
D. W. Reed ◽  
R. E. Toia
Keyword(s):  
Cytoplasmic Membrane ◽  
Azotobacter Vinelandii ◽  
Molecular Nitrogen ◽  
Membrane System ◽  
Rhizobium Japonicum ◽  
Intracellular Membrane ◽  
Free Living ◽  
Cytoplasmic Material ◽  
Living Bacterium ◽  
Free Living Bacterium

The free-living bacterium Azotobacter vinelandii carries out the fixation of molecular nitrogen under aerobic conditions. During growth at high oxygen concentrations, these bacteria exhibit high rates of both respiration and nitrogen fixation. Internal membranes are not easily seen in sections of intact bacteria but an extensive intracellular membrane system is apparent in osmotically-lysed cells (Fig. 1). Equilibration of the cells with 0.3 M glycerol and rapid dilution into ten volumes of 0.01 M Tris-HCl buffer, pH 7.4, permits the escape of electron dense cytoplasmic material. These intracellular membranes which contain the respiratory components of the cell are spherical invaginations of the cytoplasmic membrane and are completely separable from the smaller vesicular azotophore membranes which contain the nitrogenase in A. vinelandii.

A comparison of carbon source utilization for growth and nitrogenase activity in two Frankia isolates

1986 ◽  
Vol 32 (4) ◽  
pp. 353-358 ◽  
Author(s):  
Mary F. Lopez ◽  
Patricia Young ◽  
John G. Torrey
Keyword(s):  
Nitrogen Fixation ◽  
Carbon Source ◽  
Oxygen Uptake ◽  
Acetylene Reduction ◽  
Nitrogenase Activity ◽  
Carbon Sources ◽  
Alnus Rubra ◽  
Nitrogen Fixing ◽  
Pure Cultures

The carbon source requirements for the growth and nitrogen fixation of two morphologically distinct Frankia isolates were examined. Isolate ArI3 (from Alnus rubra) grew well on propionate, malate, acetate, and trehalose, and isolate CcI2 (from Casuarina cunninghamiana) grew best on pyruvate, acetate, and propionate. In general, the same carbon sources that supported growth supported both the development of vesicles and nitrogenase activity in long-term induction experiments in both isolates. However, ArI3 cultures induced on proprionate had 7 to 26 times the activity of other carbon sources and ArI3 cultures induced on acetate did not develop any detectable acetylene reduction. In a parallel set of experiments, cultures of both isolates were induced for nitrogenase activity on propionate and the resulting nitrogen fixing cultures were washed free of the organic acid by centrifugation. The washed cultures were incubated in the presence of various carbon sources to determine the ability of a particular substrate to supply energy directly for nitrogen fixation when vesicles and nitrogenase were already present. As was observed in the long-term induction experiments, pyruvate, propionate, and acetate supported the greatest activity in CcI2. Succinate and malate supported the greatest activity in ArI3, and propionate had very little stimulation of acetylene reduction. The reason for the lack of stimulation by propionate for washed cells of ArI3 was unclear but may have been due to toxic concentrations of the organic acid. In an attempt to compare the carbon utilization of ArI3 in pure culture with that in the alder symbiosis, oxygen uptake in the presence of various carbon sources of vesicles clusters isolate from Alnus rubra nodules inoculated with ArI3 was compared with the oxygen uptake of nitrogen-fixing pure cultures of ArI3. The oxygen uptake of the isolated vesicle clusters was stimulated by sucrose, trehalose, and glucose, but not by a variety of organic acids. In comparison, nitrogen-fixing pure cultures of ArI3 readily oxidized sugars and organic acids.

Construction of recombinant Escherichia coli producing nitrogenase-related proteins from Azotobacter vinelandii

2021 ◽  
Author(s):  
Yuki Tatemichi ◽  
Takeharu Nakahara ◽  
Mitsuyoshi Ueda ◽  
Kouichi Kuroda
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Fixation ◽  
Biological Nitrogen Fixation ◽  
Fossil Fuels ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Gene Clusters ◽  
Fixation Method ◽  
Overlap Extension ◽  
Biological Nitrogen

Abstract Biological nitrogen fixation by nitrogenase has attracted attention as an alternative method to chemical nitrogen fixation, which requires large amounts of fossil fuels. Azotobacter vinelandii, which produces an oxygen-sensitive nitrogenase, can fix nitrogen even under aerobic conditions; therefore, the heterologous expression of nif-related genes from A. vinelandii is a promising strategy for developing a biological nitrogen fixation method. We assembled 17 nif-related genes, which are scattered throughout the genome of A. vinelandii, into synthetic gene clusters by overlap-extension-PCR and seamless cloning and expressed them in Escherichia coli. The transcription and translation of the 17 nif-related genes were evaluated by RT-qPCR and LC-MS/MS, respectively. The constructed E. coli showed nitrogenase activity under anaerobic and microaerobic conditions. This strain would be a useful model for examining the effect of other genes from A. vinelandii on nitrogen fixation by expressing them in addition to the minimal set of nif-related genes.

The acetylene reduction assay for measuring nitrogen fixation in waterlogged soil

1971 ◽  
Vol 17 (8) ◽  
pp. 1049-1056 ◽  
Author(s):  
W. A. Rice ◽  
E. A. Paul
Keyword(s):  
Nitrogen Fixation ◽  
Aqueous Phase ◽  
Nitrogenase Activity ◽  
Total Sample ◽  
Gas Diffusion ◽  
N Fixation ◽  
Waterlogged Soil ◽  
Sample Depth ◽  
Reduction Assay ◽  
Glucose System

Nitrogen fixation in waterlogged, soil–straw, and sand–clay–straw mixtures was measured with the C2H2 reduction assay, the 15N-tracer technique, and the Kjeldahl method. The reduction of 6 to 15 moles C2H2 corresponded to the fixation of 1 mole N2. The theoretical ratio is 3 moles C2H2 to 1 mole N2. A ratio of 3 moles C2H2 reduced for each mole of N2 fixed was obtained when samples of sand–clay–straw were incubated under conditions that minimized effects that were due to gas diffusion through the aqueous phase. Calculations indicated that N2 at a partial pressure of 0.2 atm above the samples was not sufficient to saturate the nitrogenase enzyme of the organisms in lower layers of soil–straw samples. Thus the concentration of N2 dissolved in the aqueous phase limited nitrogen fixation. C2H2 is more soluble in water than N2; the C2H2 reduction was not as limited by the C2H2 concentration in the aqueous phase.N2 was experimentally shown to be limited at depth in a sand–clay–glucose system in that fixation decreased from 128 to 36 μg N/g of sand–clay incubated so that the total sample depth ranged from 0.2 to 3 cm.The C2H2 reduction assay provides a method for measuring the potential nitrogenase activity in the waterlogged soil amended with straw; however, this assay must be calibrated for specific conditions. The data also indicate that where N2 diffusion rates may limit N fixation, a normal atmosphere (80%) of N2 should be used in the experiment.

scholarly journals Crystal structure of the [2Fe–2S] protein I (Shethna protein I) from Azotobacter vinelandii

2021 ◽  
Vol 77 (11) ◽  
Author(s):  
Burak V. Kabasakal ◽  
Charles A. R. Cotton ◽  
James W. Murray
Keyword(s):  
Crystal Structure ◽  
Nitrogen Fixation ◽  
Azotobacter Vinelandii ◽  
Nitrogenase Activity ◽  
Redox Chemistry ◽  
Evolutionary Relationships ◽  
Cysteine Residues ◽  
Aquifex Aeolicus ◽  
Iron Sulfur ◽  
The 1960S

Azotobacter vinelandii is a model diazotroph and is the source of most nitrogenase material for structural and biochemical work. Azotobacter can grow in above-atmospheric levels of oxygen, despite the sensitivity of nitrogenase activity to oxygen. Azotobacter has many iron–sulfur proteins in its genome, which were identified as far back as the 1960s and probably play roles in the complex redox chemistry that Azotobacter must maintain when fixing nitrogen. Here, the 2.1 Å resolution crystal structure of the [2Fe–2S] protein I (Shethna protein I) from A. vinelandii is presented, revealing a homodimer with the [2Fe–2S] cluster coordinated by the surrounding conserved cysteine residues. It is similar to the structure of the thioredoxin-like [2Fe–2S] protein from Aquifex aeolicus, including the positions of the [2Fe–2S] clusters and conserved cysteine residues. The structure of Shethna protein I will provide information for understanding its function in relation to nitrogen fixation and its evolutionary relationships to other ferredoxins.

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