Bioavailability of mineral-associated trace metals as cofactors for nitrogen fixation by Azotobacter vinelandii

Life on Earth depends on N₂-fixing microbes to make ammonia from atmospheric N₂ gas by the nitrogenase enzyme. Most nitrogenases use Mo as a cofactor; however, V and Fe are also possible. N₂ fixation was once believed to have evolved during the Archean-Proterozoic times using Fe as a cofactor. However, δ¹⁵N values of paleo-ocean sediments suggest Mo and V cofactors despite their low concentrations in the paleo-oceans. This apparent paradox is based on an untested assumption that only soluble metals are bioavailable. In this study, laboratory experiments were performed to test the bioavailability of mineral-associated trace metals to a model N₂-fixing bacterium Azotobacter vinelandii. N₂ fixation was observed when Mo in molybdenite, V in cavansite, and Fe in ferrihydrite were used as the sole sources of cofactors, but the rate of N₂ fixation was greatly reduced. A physical separation between minerals and cells further reduced the rate of N₂ fixation. Biochemical assays detected five siderophores, including aminochelin, azotochelin, azotobactin, protochelin, and vibrioferrin, as possible chelators to extract metals from minerals. The results of this study demonstrate that mineral-associated trace metals are bioavailable as cofactors of nitrogenases to support N₂ fixation in those environments that lack soluble trace metals and may offer a partial answer to the paradox.     

Calorimetry of nitrogenase-mediated reductions in detached soybean nodules

Heat evolved by isolated soybean (Glycine max cv Clark) nodules was measured to estimate more directly the metabolic cost associated with the symbiotic N₂ fixation system. A calorimeter constructed by modifying standard laboratory equipment allowed measurement on 1 gram of detached nodules under a controlled gas stream. Simultaneous gas balance and heat output determinations were made.

There was major heat output by nodules for all of the nitrogenase substrates tested (H⁺, N₂, N₂O, and C₂H₂) further establishing the in vivo energy inefficiency of biological N₂ fixation. Exposure to a short burst of 100% O₂ partially inactivated nitrogenase to permit calculations of heat evolved per mole of substrate reduced. The specific rate of heat evolution for H⁺ reductions was 171 ± 6 kilocalories per mole H₂ evolved in an Ar-O₂ atmosphere, that for N₂ fixation was 784 ± 26 kilocalories per mole H₂ evolved and N₂ fixed, and that for C₂H₂ reduction was 250 ± 12 kilocalories/mole C₂H₄ formed. When the appropriate thermodynamic parameters are taken into account for the different substrates and products, a ΔH′ of −200 kilocalories per mole 2e⁻ is shown to be associated with active transfer of electrons by the nitrogenase system. These values lead to a calculated N₂ fixation cost of 9.5 grams glucose per gram N₂ fixed or 3.8 grams C per gram N₂, which is in close agreement with earlier calculations based on nodular CO₂ production.

     

Hydrogenase system in legume nodules: a mechanism of providing nitrogenase with energy and protection from oxygen damage.

Some strains of rhizobia possess a hydrogenase system which catalyzes the oxidation of the H₂ that is evolved from nitrogenase during N₂ fixation. Oxidation of H₂ by a hydrogen uptake positive strain of $\text{Rhizobium japonicum}$ provides energy for support of the N₂ fixation reactions and protects nitrogenase from O₂ damage     

Carbon substrate re-orders relative growth of a bacterium using Mo-, V-, or Fe-nitrogenase for nitrogen fixation

Biological nitrogen fixation is catalyzed by the molybdenum (Mo), vanadium (V) and iron (Fe)-only nitrogenase metalloenzymes. Studies with purified enzymes have found that the ‘alternative’ V- and Fe-nitrogenases generally reduce N₂ more slowly and produce more byproduct H₂ than the Mo-nitrogenase, leading to an assumption that their usage results in slower growth. Here we show that, in the metabolically versatile photoheterotroph Rhodopseudomonas palustris, the type of carbon substrate influences the relative rates of diazotrophic growth based on different nitrogenase isoforms. The V-nitrogenase supports growth as fast as the Mo-nitrogenase on acetate but not on the more oxidized substrate succinate. Our data suggest that this is due to insufficient electron flux to the V-nitrogenase isoform on succinate compared with acetate. Despite slightly faster growth based on the V-nitrogenase on acetate, the wild-type strain uses exclusively the Mo-nitrogenase on both carbon substrates. Notably, the differences in H₂:N₂ stoichiometry by alternative nitrogenases (~1.5 for V-nitrogenase, ~4–7 for Fe-nitrogenase) and Mo-nitrogenase (~1) measured here are lower than prior in vitro estimates. These results indicate that the metabolic costs of V-based nitrogen fixation could be less significant for growth than previously assumed, helping explain why alternative nitrogenase genes persist in diverse diazotroph lineages and are broadly distributed in the environment.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Metal limitation of cyanobacterial N2 fixation and implications for the Precambrian nitrogen cycle

Nitrogen fixation is a critical part of the global nitrogen cycle, replacing biologically available reduced nitrogen lost by denitrification. The redox‐sensitive trace metals Fe and Mo are key components of the primary nitrogenase enzyme used by cyanobacteria (and other prokaryotes) to fix atmospheric N₂ into bioessential compounds. Progressive oxygenation of the Earth's atmosphere has forced changes in the redox state of the oceans through geologic time, from anoxic Fe‐enriched waters in the Archean to partially sulfidic deep waters by the mid‐Proterozoic. This development of ocean redox chemistry during the Precambrian led to fluctuations in Fe and Mo availability that could have significantly impacted the ability of prokaryotes to fix nitrogen. It has been suggested that metal limitation of nitrogen fixation and nitrate assimilation, along with increased rates of denitrification, could have resulted in globally reduced rates of primary production and nitrogen‐starved oceans through much of the Proterozoic. To test the first part of this hypothesis, we grew N₂‐fixing cyanobacteria in cultures with metal concentrations reflecting an anoxic Archean ocean (high Fe, low Mo), a sulfidic Proterozoic ocean (low Fe, moderate Mo), and an oxic Phanerozoic ocean (low Fe, high Mo). We measured low rates of cellular N₂ fixation under [Fe] and [Mo] estimated for the Archean ocean. With decreased [Fe] and higher [Mo] representing sulfidic Proterozoic conditions, N₂ fixation, growth, and biomass C:N were similar to those observed with metal concentrations of the fully oxygenated oceans that likely developed in the Phanerozoic. Our results raise the possibility that an initial rise in atmospheric oxygen could actually have enhanced nitrogen fixation rates to near modern marine levels, providing that phosphate was available and rising O₂ levels did not markedly inhibit nitrogenase activity.     

Respiratory and Nitrogenase Activities of Soybean Nodules Formed by Hydrogen Uptake Negative (Hup) Mutant and Revertant Strains of Rhizobium japonicum Characterized by Protein Patterns

Rates of respiratory CO₂ loss and nitrogenase activities of H₂ uptake-negative mutant strains and H₂ uptake-positive revertant strains of Rhizobium japonicum have been investigated. Two-dimensional gel protein patterns of bacteroids formed by inoculation of soybeans (Glycine max L.) with these two strains show that they are closely related and revealed only one obvious difference between them. On the basis of molecular weight standards, it was concluded that the missing protein spot in the H₂ uptake-negative mutant strain could be caused by a failure of the mutant to synthesize hydrogenase. Nodules formed by the H₂ uptake-negative mutant strain evolved respiratory CO₂ at a rate of about 10% higher than that of nodules formed by the H₂ uptake-positive revertant strain. During short-term experiments employed, rates of both C₂H₂ reduction and ¹⁵N₂ fixation varied considerably among replicate samples and no statistically significant differences between mutant and revertant strains were observed. It was observed that increasing the partial pressure of O₂ over nodules significantly decreased the proportion of nitrogenase electrons allocated to H⁺.     

Biological nitrogen fixation by alternative nitrogenases in terrestrial ecosystems: a review

Biological nitrogen fixation (BNF), a key reaction of the nitrogen cycle, is catalyzed by the enzyme nitrogenase. The best studied isoform of this metalloenzyme requires molybdenum (Mo) at its active center to reduce atmospheric dinitrogen (N₂) into bioavailable ammonium. The Mo-dependent nitrogenase is found in all diazotrophs and is the only nitrogenase reported in diazotrophs that form N₂-fixing symbioses with higher plants. In addition to the canonical Mo nitrogenase, two alternative nitrogenases, which use either vanadium (V) or iron (Fe) instead of Mo are known to fix nitrogen. They have been identified in ecologically important groups including free-living bacteria in soils and freshwaters and as symbionts of certain cryptogamic covers. Despite the discovery of these alternative isoforms more than 40 years ago, BNF is still believed to primarily rely on Mo. Here, we review existing studies on alternative nitrogenases in terrestrial settings, spanning inland forests to coastal ecosystems. These studies show frequent Mo limitation of BNF, ubiquitous distribution of alternative nitrogenase genes and significant contributions of alternative nitrogenases to N₂ fixation in ecosystems ranging from the tropics to the subarctic. The effect of temperature on nitrogenase isoform activity and regulation is also discussed. We present recently developed methods for measuring alternative nitrogenase activity in the field and discuss the associated analytical challenges. Finally, we discuss how the enzymatic diversity of nitrogenase forces a re-examination of existing knowledge gaps and our understanding of BNF in nature.

     

Coordinated regulation of nitrogen fixation and molybdate transport by molybdenum

Biological nitrogen fixation, the reduction of chemically inert dinitrogen to bioavailable ammonia, is a central process in the global nitrogen cycle highly relevant for life on earth. N₂ reduction to NH₃ is catalyzed by nitrogenases exclusively synthesized by diazotrophic prokaryotes. All diazotrophs have a molybdenum nitrogenase containing the unique iron‐molybdenum cofactor FeMoco. In addition, some diazotrophs encode one or two alternative Mo‐free nitrogenases that are less efficient at reducing N₂ than Mo‐nitrogenase. To permit biogenesis of Mo‐nitrogenase and other molybdoenzymes when Mo is scarce, bacteria synthesize the high‐affinity molybdate transporter ModABC. Generally, Mo supports expression of Mo‐nitrogenase genes, while it represses production of Mo‐free nitrogenases and ModABC. Since all three nitrogenases and ModABC can reach very high levels at suitable Mo concentrations, tight Mo‐mediated control saves considerable resources and energy. This review outlines the similarities and differences in Mo‐responsive regulation of nitrogen fixation and molybdate transport in diverse diazotrophs.     

N(2)-Fixation by Freshly Isolated Nostoc from Coralloid Roots of the Cycad Macrozamia riedlei (Fisch. ex Gaud.) Gardn

Nitrogenase (EC 1.7.99.2) activity (acetylene reduction) and nitrogen fixation (¹⁵N₂ fixation) were measured in cyanobacteria freshly isolated from the coralloid roots of Macrozamia riedlei (Fisch. ex Gaud.) Gardn. Light and gas phase oxygen concentration had marked interactive effects on activity, with higher (up to 100-fold) rates of acetylene reduction and ¹⁵N₂ fixation in light. The relationship between ethylene formation and N₂-fixation varied in the freshly isolated cyanobacteria from 4 to 7 nanomoles of C₂H₄ per nanomole ¹⁵N₂. Intact coralloid roots, incubated in darkness and ambient air, showed a value of 4.3. Maximum rates of nitrogenase activity occurred at about 0.6% O₂ in light, while in darkness there was a broad optimum around 5 to 8% O₂. Inhibition of nitrogenase, in light, by pO₂ above 0.6% was irreversible. Measurements of light-dependent O₂ evolution and ¹⁴CO₂ fixation indicated negligible photosynthetic electron transport involving photosystem II and, on the basis of inhibitor studies, the stimulatory effect of light was attributed to cyclic photophos-phorylation. Nitrogenase activity of free-living culture of an isolate from Macrozamia (Nostoc PCC 73102) was only slightly inhibited by O₂ levels above 6% O₂ and the inhibition was reversible. These cells showed rates of light-dependent O₂ evolution and ¹⁴CO₂ fixation which were 100- to 200-fold higher than those by the freshly isolated symbiont. Furthermore, nitrogenase activity was dependent on both photosynthetic electron transport and photophosphorylation. These data indicate that cyanobacteria within cycad coralloid roots are differentiated specifically for symbiotic functioning in a microaerobic environment. Specializations include a high heterocyst frequency, enhanced permeability to O₂, and a direct dependence on the cycad for substrates to support nitrogenase activity.     

Hydrogen Recycling by Rhizobium leguminosarum Isolates and Growth and Nitrogen Contents of Pea Plants (Pisum sativum L.)

The ability to recycle H₂ evolved by nitrogenase is thought to be of importance in increasing the efficiency of N₂ fixation and to be a factor in increasing plant yield in symbiotic systems. To determine whether this ability is a significant factor in the Rhizobium leguminosarum-Pisum sativum L. system, plants were inoculated with R. leguminosarum isolates which differed in their ability to oxidize H₂ and in their relative efficiency of N₂ fixation. These plants were grown at three levels of irradiance and harvested after 3, 4, and 5 weeks of growth for determination of C₂H₂ reduction, H₂ evolution and uptake, plant dry weight, and N content. Plants inoculated with uptake hydrogenase-positive (Hup⁺) isolates did not exhibit higher dry weight or N content than those inoculated with Hup⁻ isolates under any of the growth conditions studied. The efficiency of the nitrogenase system of Hup⁻ isolates increased at a low irradiance, a factor which may allow them to compete successfully with Hup⁺ isolates. In some Hup⁺R. leguminosarum isolates, H₂ oxidation is coupled to ATP formation, whereas in others, it is not. There were no differences in plant dry weight and N content in plants inoculated with the two types and grown for 5 weeks at three irradiance levels. The addition of H₂ to Hup⁺ nodules whose supply of photosynthate had been removed by stem excision did not increase C₂H₂ reduction in either coupled or uncoupled types.     

TheParasponia parviflora — Rhizobium symbiosis. Isotopic nitrogen fixation,hydrogen evolution and nitrogen-fixation efficiency,and oxygen relations

Summary Isotopic¹⁵N₂ experiments confirmed nitrogen fixation inParasponia parviflora. The conversion ratio C₂H₄/N₂ was 6.7 under the experimental conditions employed. Measurements of the δ¹⁵N in leaves of Parasponia and Trema showed on the basis of these determinations thatParasponia parviflora possesses N₂-fixing capacity and can be distinguished in this respect from the non-nitrogen-fixingTrema cannabina tested by the same method. Therefore, δ¹⁵N can be used to monitor N₂ fixation in natural ecosystems. Hydrogen evolution and the relative efficiency of N₂ fixation in this relation have been determined. DetachedParasponia parviflora root nodules grown in soil and tested in an argon/oxygen atmosphere produced appr. 4 μmol H₂.h⁻¹.g⁻¹ fresh weight root nodules. The relative efficiency of hydrogen utilization as measured in argon, air, and in the presence of C₂H₂ 10% (v/v) was for both equations used for to express this efficiency 0.96 and 0.97, respectively. This indicates that Parasponia like the root nodules of some actinorhizal symbioses (Alnus, Myrica, Elaeagnus) and some tropical legumes (Vigna sinensis) has evolved mechanisms of minimizing net hydrogen production in air, thus increasing the efficiency of electron transfer to nitrogen. The oxygen relation of nitrogen fixation (C₂H₂) inParasponia parviflora root nodules was determined. The nitrogenase activity of Parasponia root nodules increased at increasing oxygen concentrations up till c. 40% O₂. At oxygen levels above 40% O₂, the nitrogenase activity of the root nodules was nil or very erratic suggesting that at these oxygen levels the nitrogenase is not longer protected against the harmful effect of oxygen. In this respect Parasponia root nodules differ from actinorhizal root nodules in other nonlegumes, where optimal nitrogenase activity was observed in the range of 12–25% oxygen. Respiration experiments with Parasponia root nodules showed that in the range 10, 20, and 40% oxygen, the respiration rate (CO₂ evolution) increased concomitantly with an increase of the acetylene reduction rate. The CO₂/C₂H₄ values obtained varied between 8.1 and 19.2, being therefore 2–3 times higher than similar estimations in some actinorhizal and legume root nodules. The respiratory quotient (RQ) of detachedParasponia parviflora root nodules was in air initially approximately 2.0, but this value dropped to about 1.0 in a 3-hours period.     

Gas Exchange in the Filamentous Cyanobacterium Nostoc punctiforme Strain ATCC 29133 and Its Hydrogenase-Deficient Mutant Strain NHM5

Nostoc punctiforme ATCC 29133 is a nitrogen-fixing, heterocystous cyanobacterium of symbiotic origin. During nitrogen fixation, it produces molecular hydrogen (H₂), which is recaptured by an uptake hydrogenase. Gas exchange in cultures of N. punctiforme ATCC 29133 and its hydrogenase-free mutant strain NHM5 was studied. Exchange of O₂, CO₂, N₂, and H₂ was followed simultaneously with a mass spectrometer in cultures grown under nitrogen-fixing conditions. Isotopic tracing was used to separate evolution and uptake of CO₂ and O₂. The amount of H₂ produced per molecule of N₂ fixed was found to vary with light conditions, high light giving a greater increase in H₂ production than N₂ fixation. The ratio under low light and high light was approximately 1.4 and 6.1 molecules of H₂ produced per molecule of N₂ fixed, respectively. Incubation under high light for a longer time, until the culture was depleted of CO₂, caused a decrease in the nitrogen fixation rate. At the same time, hydrogen production in the hydrogenase-deficient strain was increased from an initial rate of approximately 6 μmol (mg of chlorophyll a)⁻¹ h⁻¹ to 9 μmol (mg of chlorophyll a)⁻¹ h⁻¹ after about 50 min. A light-stimulated hydrogen-deuterium exchange activity stemming from the nitrogenase was observed in the two strains. The present findings are important for understanding this nitrogenase-based system, aiming at photobiological hydrogen production, as we have identified the conditions under which the energy flow through the nitrogenase can be directed towards hydrogen production rather than nitrogen fixation.     

Evidence for the involvement of a genetic determinant controlling functional specificity of group VI B elements in the metabolism of N2 and NO-3 in the blue-green alga Nostoc muscorum.

The parent $\text{N. muscorum}$ is Mo-requiring for growth on N₂ or NO₃^? as nitrogen source. W and Cr both are observed to competitively inhibit the function of Mo in growth on N₂ and NO₃^? media in parent strain. Spontaneous mutants growing in the presence of W or Cr were isolated and when examined, found to be W- or Cr-requiring for growth with both N₂ and NO₃^? as nitrogen source. The results of the characterization of the three strains with respect to mutation frequency, interaction among Mo, W and Cr in growth on N₂ as nitrogen source, and requirement of W or Cr for NO₃^? inhibition of heterocyst formation in W- or Cr-requiring strain growing in NO₃^? medium, all suggest the operation of a single genetic determinant in specifying whether Mo, W or Cr (group VI B elements) is required for growth with both N₂ and NO₃^? as nitrogen sources. They also further suggest that this single genetic determinant is common to nitrogenase and nitrate reductase.     

Experimental determination of the respiration associated with soybean/rhizobium nitrogenase function, nodule maintenance, and total nodule nitrogen fixation

The total metabolic cost of soybean (Glycine max L. Mer Clark) nodule nitrogen fixation was empirically separated into respiration associated with electron flow through nitrogenase and respiration associated with maintenance of nodule function.

Rates of CO₂ evolution and H₂ evolution from intact, nodulated root systems under Ar:O₂ atmospheres decreased in parallel when plants were maintained in an extended dark period. While H₂ evolution approached zero after 36 hours of darkness at 22°C, CO₂ evolution rate remained at 38° of the rate measured in light. Of the remaining CO₂ evolution, 62% was estimated to originate from the nodules and represents a measure of nodule maintenance respiration. The nodule maintenance requirement was temperature dependent and was estimated at 79 and 137 micromoles CO₂ (per gram dry weight nodule) per hour at 22°C and 30°C, respectively.

The cost of N₂ fixation in terms of CO₂ evolved per electron pair utilized by nitrogenase was estimated from the slope of H₂ evolution rate versus CO₂ evolution rate. The cost was 2 moles CO₂ evolved per mole H₂ evolved and was independent of temperature.

In this symbiosis, nodule maintenance consumed 22% of total respiratory energy while the functioning of nitrogenase consumed a further 52%. The remaining respiratory energy was calculated to be associated with ammonia assimilation, transport of reduced N, and H₂ evolution.

     

Effect of light and atmospheric carbon dioxide concentration on nitrogen fixation by herbage legumes

Summary Lucerne, red clover and white clover were grown at two atmospheric concentrations of CO₂ (300 and 1000 μl l⁻¹) and the effects on N₂ fixation, nodule mass/number and root/shoot dry matter production determined. Pea plants were similarly evaluated as a comparison with grain legumes. CO₂ enrichment increased N₂ fixation activity in all cases but activity/unit nodule mass was significantly increased only in the pea. The enhancement of N₂ fixation in herbage legumes by CO₂ enrichment reflected an increase in nodule mass which in turn was attributed to increased nodule number, and results show that under the experimental conditions obtaining here photosynthate supply did not limit nodule N₂ fixation in these plants though it was limiting in the case of peas. White clover growing in a 6 and 14 hour photoperiod was studied for response of the N₂ fixing system to light. Long photoperiod (14 hour) plants assayed at constant temperature (20°C) did not show a significant response to light at the end of the dark period either in terms of fixation per plant or per unit nodule mass, in contrast with short photoperiod (6 hour) plants which showed significant responses. Short photoperiod plants compensated for reduced photosynthates by maintaining only half the root nodule mass and fixation activity of 14 hour photoperiod plants though plants in both systems supported similar rates of N₂ fixation per unit mass of nodule during the photoperiod. Comparison of N₂ fixation activities in whole and decapitated plant systems indicates the importance of shoot reserves for sustaining nitrogenase activity in white clover during short-term interruption of photosynthesis. These results support the conclusion of the CO₂ enrichment studies, that herbage legumes have the potential for supplying their nodule photosynthate requirements for sustaining optimum rates of N₂ fixation and excess carbon supply is used solely to promote further nodulation. Nodules of short photoperiod white clover plants were less efficient in N₂ fixation in that they evolved more H₂ relative to N₂ (C₂H₂) reduced than did long photoperiod plants.     

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

The interaction between the ATP-dependent evolution of H₂ catalyzed by nitrogenase and the oxidation of H₂ via a hydrogenase has been postulated to influence the efficiency of the N₂-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 H₂ in air and the H₂ produced accounted for about 30% of the energy transferred to the nitrogenase system during the period of active N₂ fixation. In contrast the same soybean cultivar inoculated with USDA 110 produced nodules with an active hydrogenase and consequently did not evolve H₂ in air. A comparison of Anoka soybeans inoculated with the two different strains of R. japonicum showed that mean rates of C₂H₂ reduction and O₂ 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 N₂ 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 H₂-evolving strain 176A27 produced increases in plant dry weight and total N₂ fixed of 11 and 15%, respectively. This apparent increase in the efficiency of N₂ fixation for nodulated legumes capable of reutilizing the H₂ evolved from nitrogenase is considered and it is concluded that provision of conclusive evidence of the role of the H₂-recycling process in N₂-fixing efficiency of legumes will require comparison of Rhizobium strains that are genetically identical with the exception of the presence of hydrogenase.

     

Contemporaneous N2 Fixation and Oxygenic Photosynthesis in the Nonheterocystous Mat-Forming Cyanobacterium Lyngbya aestuarii

The nonheterocystous filamentous cyanobacterial genus Lyngbya is a widespread and frequently dominant component of marine microbial mats. It is suspected of contributing to relatively high rates of N₂ fixation associated with mats. The ability to contemporaneously conduct O₂-sensitive N₂ fixation and oxygenic photosynthesis was investigated in Lyngbya aestuarii isolates from a North Carolina intertidal mat. Short-term (<4-h) additions of the photosystem II (O₂ evolution) inhibitor 3(3,4-dichlorophenyl)-1,1-dimethylurea stimulated light-mediated N₂ fixation (nitrogenase activity), indicating potential inhibition of N₂ fixation by O₂ production. However, some degree of light-mediated N₂ fixation in the absence of 3(3,4-dichlorophenyl)-1,1-dimethylurea was observed. Electron microscopic immunocytochemical localization of nitrogenase, coupled to microautoradiographic studies of ¹⁴CO₂ fixation and cellular deposition of the tetrazolium salt 2,4,5-triphenyltetrazolium chloride, revealed that (i) nitrogenase was widely distributed throughout individual filaments during illuminated and dark periods, (ii) ¹⁴CO₂ fixation was most active in intercalary regions, and (iii) daylight 2,4,5-triphenyltetrazolium chloride reduction (formazan deposition) was most intense in terminal regions. Results suggest lateral partitioning of photosynthesis and N₂ fixation during illumination, with N₂ fixation being confined to terminal regions. During darkness, a larger share of the filament appears capable of N₂ fixation.     

The acetylene-ethylene assay for n(2) fixation: laboratory and field evaluation

The methodology, characteristics and application of the sensitive C₂H₂-C₂H₄ assay for N₂ fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N₂ase-catalyzed reduction of C₂H₂ to C₂H₄, gas chromatographic isolation of C₂H₂ and C₂H₄, and quantitative measurement with a H₂-flame analyzer. As little as 1 μμmole C₂H₄ can be detected, providing a sensitivity 10³-fold greater than is possible with ¹⁵N analysis.

A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C₂H₂-reducing activity in the field. Applications to field samples included an evaluation of N₂ fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N₂ fixation rate of 30 to 33 kg N₂ fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N₂ fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N₂ fixing activity of soybeans. The validity of measuring N₂ fixation in terms of C₂H₂ reduction was established through extensive comparisons of these activities using defined systems, including purified N₂ase preparations and pure cultures of N₂-fixing bacteria.

With this assay it now becomes possible and practicable to conduct comprehensive surveys of N₂ fixation, to make detailed comparisons among different N₂-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N₂ fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N₂ fixation reaction.

     

Azolla-Anabaena azollae Relationship: V. N(2) Fixation, Acetylene Reduction, and H(2) Production

In order to characterize the reactions catalyzed by nitrogenase in the Azolla-Anabaena association, ¹⁵N₂ fixation, C₂H₂ reduction, and ATP-dependent H₂ production were measured in both the Azolla-Anabaena complex and in the alga isolated from the complex.