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PubMed1990

Nitrogen Fixation in Continuous Culture with NH(4)Cl-Containing Media.

N balance and N dilution were determined from growth of Azospirillum brasilense Sp7 and two unidentified gram-negative nitrogen-fixing microorganisms in continuous culture supplied with NH(4)Cl. At the 1.1 and 2.2 mM NH(4)Cl steady states (N-to-C ratios of 1:68 and 1:34, respectively), the organisms grew with NH(4)Cl and N(2) as N sources simultaneously under carbon limitation. No ammonium could be detected in the supernatant of these cultures.

Journal Article↗
PubMed1998

Salt stress sensitivity of nitrogen fixation in Enterobacter agglomerans strains.

Two strains 333 to 339 of Enterobacter agglomerans were selected in the present study to evaluate the response of increasing concentrations of NaCl on growth, N(2)-fixation, and nitrogenase activity/synthesis. E. agglomerans strains 333 and 339 showed optimum growth and acetylene-reducing activity with 0.5 to 1.0% NaCl in a nitrogen-free minimal medium (NFDM) with glucose, respectively, in 28 h incubation, although both strains displayed better growth and acetylene-reducing activity with 3.0% and 2.0% NaCl after 52 h and 100 h incubation periods than the 28 h culture did. Our experiments with shiftings of salt concentrations in NFDM medium indicated that a synthesis of nitrogenase enzyme was generally more sensitive to higher concentrations of NaCl than nitrogenase activity was.

Journal Article↗
PubMed1985

Cellulose Decomposition and Associated Nitrogen Fixation by Mixed Cultures of Cellulomonas gelida and Azospirillum Species or Bacillus macerans.

Mixed cultures of Cellulomonas gelida plus Azospirillum lipoferum or Azospirillum brasilense and C. gelida plus Bacillus macerans were shown to degrade cellulose and straw and to utilize the energy-yielding products to fix atmospheric nitrogen. This cooperative process was followed over 30 days in sand-based cultures in which the breakdown of 20% of the cellulose and 28 to 30% of the straw resulted in the fixation of 12 to 14.6 mg of N per g of cellulose and 17 to 19 mg of N per g of g straw consumed. Cellulomonas species have certain advantages over aerobic cellulose-degrading fungi in being able to degrade cellulose at oxygen concentrations as low as 1% O(2) (vol/vol) which would allow a close association between cellulose-degrading and microaerobic diazotrophic microorganisms. Cultures inoculated with initially different proportions of A. brasilense and C. gelida all reached a stable ratio of approximately 1 Azospirillum/3 Cellulomonas cells.

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PubMed2002

Nitrogen fixation by photosynthetic bacteria.

In 1949, Howard Gest and Martin Kamen published two brief papers in Science that changed our perceptions about the metabolic capabilities of photosynthetic bacteria. Their discovery of photoproduction of hydrogen and the ability of Rhodospirillum rubrum to fix nitrogen led to a greater understanding of both processes.

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PubMed1980

Nitrogen fixation by photosynthetic bacteria in lowland rice culture.

Propanil (3',4'-dichloropropionanilide) was a potent inhibitor of the nitrogenase activity of blue-green algae (cyanobacteria) in flooded soil, but the herbicide at comparable concentrations was not toxic to rice, protozoa, and nitrogen-fixing bacteria. Ethanol-amended flooded soils treated with propanil exhibited higher rates of nitrogenase activity than those not treated with the herbicide. The enhanced nitrogenase activity in propanil-treated soils was associated with a rise in the population of purple sulfur bacteria, especially of cells resembling Chromatium and Thiospirillum. By employing propanil and a means of excluding light from the floodwater to prevent the development of phototrophs during rice growth under lowland conditions, the relative activities of blue-green algae, photosynthetic bacteria, and the rhizosphere microflora were determined. The results suggest that the potential contribution of photosynthetic bacteria may be quite high.

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PubMed1979

Nitrogen fixation associated with the rice plant grown in water culture.

Acetylene reduction activity of intact rice plants was measured in closed assay chambers with plants grown in water culture. Acetylene was added to the liquid medium, and the ethylene formed was measured from both gas and liquid phases. After cutoff of mineral nitrogen supply and inoculation of fresh soil, rice plants grown from the seedling stage in water culture exhibited acetylene reduction activity after a lag period. However, rice plants grown in a paddy field and transferred to water culture were more suitable for N(2) fixation studies because of their higher, less variable acetylene reduction activity. The time course of acetylene reduction was monitored by continuous circulation of gas between the gas phase and the liquid phase, and the result showed an initial 2- or 3-h period of lower activity, followed by increased and almost constant activity up to 24 h. The effects on acetylene reduction activity of aeration, ammonium, chloramphenicol, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea addition are reported. Ammonium was inhibitive at 0.33 mM, and its depressive effect was alleviated by ammonium uptake by the plants.

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PubMed1984

Rhizobium free-living nitrogen fixation occurs in specialized nongrowing cells.

A model for free-living N(2) fixation by Rhizobium sp. RC3200 is presented that asserts that this process occurs in nongrowing cells. Cultures containing mixed populations of cell types, N(2)-fixing and vegetative, grow cooperatively. In nitrogen-limited liquid suspension cultures, cooperative growth occurs by means of ammonium that is produced and exported by nongrowing, N(2)-fixing cells and transported to vegetative cells. This model implies prokaryotic differentiation: the creation of metabolically specialized cells, terminally nonviable, that functionally cooperate in a higher cell order. Here, the switch to a Rhizobium N(2)-fixing cell state is regulated by both O(2) and utilizable nitrogen.

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PubMed1982

Relative Efficacy of Different Alfalfa Cultivar-Rhizobium meliloti Strain Combinations for Symbiotic Nitrogen Fixation.

Five Rhizobium meliloti isolates known to have different capabilities for expression of nitrogenase activity under symbiotic conditions were used to inoculate four representative Medicago sativa cultivars under aseptic conditions. Nitrogenase activities and respiratory activity were measured for whole plants and excised nodules. Dry weights and nodule numbers were also recorded after 4 weeks of growth in plastic pouches on a nitrogen-free nutrient medium. Hydrogen evolution and acetylene reduction rates were used to calculate the fraction of reducing power allocated to dinitrogen reduction. Although the efficiency of the system defined in this way was poorly correlated with plant yield, a very high linear correlation was obtained between yield and the algebraic product of nitrogenase activity and efficiency. High correlation (r > 0.78) was obtained between respiration and nitrogenase activity for whole plants as well as for excised nodules. Nodular respiration accounted for between 10 and 20% of the total plant dark respiration. The four test cultivars exhibited significantly different symbiotic responses to the inocula, although trends in potential for expression of the nitrogenase system by the five R. meliloti strains were evident. There was significant interaction between the host plant and symbiont in determining nitrogenase activity and yield. This screening method allows quantitative discrimination between effective and ineffective host-inoculum combinations.

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PubMed1988

Nodulation and Nitrogen Fixation Efficacy of Rhizobium fredii with Phaseolus vulgaris Genotypes.

Phaseolus plant introduction (PI) genotypes (consisting of 684 P. vulgaris, 26 P. acutifolius, 39 P. lunatus, and 5 P. coccineus accessions) were evaluated for their ability to form effective symbioses with strains of six slow-growing (Bradyrhizobium) and four fast-growing (Rhizobium fredii) soybean rhizobia. Of the 684 P. vulgaris genotypes examined, three PIs were found to form effective nitrogen-fixing symbioses with the R. fredii strains. While none of the Bradyrhizobium strains nodulated any of the genotypes tested, some produced large numbers of undifferentiated root proliferations (hypertrophies). A symbiotic plasmid-cured R. fredii strain failed to nodulate the P. vulgaris PIs and cultivars, suggesting that P. vulgaris host range genes are Sym plasmid borne in the fast-growing soybean rhizobia.

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PubMed1991

Genealogy of regulation of human sex and adrenal function, prostaglandin action, snapdragon and petunia flower colors, antibiotics, and nitrogen fixation: functional diversity from two ancestral dehydrogenases.

Metabolism of steroid hormones by dehydrogenases is an important mechanism for regulating steroid hormone action. Analysis of recently reported amino acid sequences of 11 beta-hydroxysteroid dehydrogenase, 17 beta-hydroxysteroid dehydrogenase, and 3 alpha, 20 beta-hydroxysteroid dehydrogenase reveals that they are descended from a common ancestor. Unexpectedly, this superfamily of dehydrogenases has other interesting relatives: 15-hydroxyprostaglandin dehydrogenase, proteins found in nitrogen-fixing bacteria, and enzymes important in the synthesis of antibiotics. The novel lineage of these proteins and the actions of flavonoids in regulating gene transcription in nitrogen-fixing bacteria and mammals provide new insights into the evolution of regulation of gene transcription by intercellular signals in multicellular animals.

Amino Acid Sequence↗
PubMed1982

Relative abundance of ureides and nitrate in plant tissues of soybean as a quantitative assay of nitrogen fixation.

The relationship between the relative abundance of ureides ([ureide-N/ureide-N plus nitrate-N] x 100) in the shoot axis (stems plus petioles), nodulated roots and leaflets of "Bragg" soybean (Glycine max [L.] Merrill) and the symbiotic dependence of these plants was examined under glass-house conditions. Plants, inoculated with effective Rhizobium japonicum CB1809, were grown with their roots exposed continuously to a nutrient solution containing either 0, 1.5, 3.0, 6.0 or 12.0 millimolar NO(3)-N per liter. Nodulation and N(2)-acetylene fixation were correlated inversely with the level of nitrate. Seasonal acetylene reduction profiles for each of the nitrate treatments were integrated and the symbiotic dependence ([N(2) fixed per total plant N] x 100) determined using a conversion ratio of 1.5:1 (acetylene reduced:N(2) fixed), calculated from the zero NO(3) treatment. Examination of the nitrogenous solutes of the shoot axis and nodulated roots showed linear relationships between the relative abundance of ureides and the symbiotic dependence of the plants. Two standard curves, depicting these relationships during vegetative and reproductive growth, were drawn for each plant part. The overriding effect of plant age invalidated any attempt to develop a standard relationship for leaflets. Data from two diurnal studies suggested that relative ureides were insensitive to diurnal fluctuations, thus simplifying sampling procedures. Plant material could be stored at ambient temperatures (20-30 degrees C) for up to 24 h without affecting the relative concentration of ureides and nitrate. It is suggested that the shoot axis provides the most suitable target organ when using this technique as a quantitative assay for N(2) fixation because of ease of sampling of these tissues, especially with field-grown plants.

Journal Article↗
PubMed1978

Hydrogen Reactions of Nodulated Leguminous Plants: II. Effects on Dry Matter Accumulation and Nitrogen Fixation.

The interaction between the ATP-dependent evolution of H(2) catalyzed by nitrogenase and the oxidation of H(2) via a hydrogenase has been postulated to influence the efficiency of the N(2)-fixing process in nodulated legumes. A comparative study using soybean (Glycine max L. Merr.) cv. Anoka inoculated with either Rhizobium japonicum strain USDA 31 or USDA 110 and cowpea (Vigna unguiculata L. Walp.) cv. Whippoorwill inoculated with Rhizobium strain 176A27 or 176A28 cultured on a N-free medium was conducted to address this question. Nodules from the Anoka cultivar inoculated with USDA 31 evolved H(2) in air and the H(2) produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N(2) fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H(2) in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C(2)H(2) reduction and O(2) consumption and mean mass of nodules taken at four times during vegetative growth were not significantly different.When compared to Anoka inoculated with USDA 31, the same cultivar inoculated with USDA 110 showed increases in total dry matter, per cent nitrogen, and total N(2) fixed of 24, 7, and 31%, respectively. Cowpeas in symbiosis with the hydrogenase-producing strain 176A28 in comparison with the same cultivar inoculated with the H(2)-evolving strain 176A27 produced increases in plant dry weight and total N(2) fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N(2) fixation for nodulated legumes capable of reutilizing the H(2) evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H(2)-recycling process in N(2)-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase.

Journal Article↗
PubMed2005

Nitrogen fixation under mild ambient conditions: part I--the initial dissociation/association step at molybdenum triamidoamine complexes.

In several recent studies Schrock and collaborators demonstrated for the first time how molecular dinitrogen can be catalytically transformed under mild and ambient conditions to ammonia by a molybdenum triamidoamine complex. In this work, we investigate the geometrical and electronic structures involved in this process of dinitrogen activation with quantum chemical methods. Density functional theory (DFT) has been employed to calculate the coordination energies of ammonia and dinitrogen relevant for the dissociation/association step in which ammonia is substituted by dinitrogen. In the DFT calculations the triamidoamine chelate ligand has been modeled by a systematic hierarchy of increasingly complex substituents at the amide nitrogen atoms. The most complex ligand considered is an experimentally known ligand with an HMT = 3,5-(2,4,6-Me3C6H2)2C6H3 substituent. Several assumptions by Schrock and collaborators on key reaction steps are confirmed by our calculations. Additional information is provided on many species not yet observed experimentally. Particular attention is paid to the role of the charge of the complexes. The investigation demonstrates that dinitrogen coordination is enhanced for the negatively charged metal fragment, that is, coordination is more favorable for the anionic metal fragment than for the neutral species. Coordination of N2 is least favorable for the cationic metal fragment. Furthermore, ammonia abstraction from the cationic complex is energetically unfavorable, while NH3 abstraction is less difficult from the neutral and easily feasible from the anionic low-spin complex.

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