Acetylene reduction,H2 evolution and 15N2 fixation in the Alnus incana-Frankia symbiosis

Acetylene reduction, ¹⁵N₂ reduction and H₂ evolution were measured in root systems of intact plants of grey alder (Alnus incana (L.) Moench) in symbiosis with Frankia. The ratios of C₂H₂: ¹⁵N₂ were compared with C₂H₂:N₂ ratios calculated from C₂H₂ reduction and H₂ evolution, and with C₂H₂:N₂ ratios calculated from accumulated C₂H₄ production and nitrogen content. It was possible to calculate C₂H₂:N₂ ratios from C₂H₂ reduction and H₂ evolution because this source of Frankia did not show any hydrogenase activity. The ratios obtained using the different methods ranged from 2.72 to 4.42, but these values were not significantly different. It was also shown that enriched ¹⁵N could be detected in the shoot after a 1-h incubation of the root-system. It is concluded that the measurement of H₂ evolution in combination with C₂H₂ reduction represents a nondestructive assay for nitrogen fixation in a Frankia symbiosis which shows no detectable hydrogenase activity.     

Respiratory capacity,nitrogenase activity and structural changes ofFrankia,in symbiosis withAlnus incana,in response to prolonged darkness

Plants ofAlnus incana (L.) Moench in symbiosis with a local source ofFrankia were exposed to prolonged darkness under controlled climate conditions.Frankia vesicle clusters were prepared from the root nodules, and the condition ofFrankia was measured as respiratory capacity by supplying the preparation with saturating amounts of four different substrates. During darkness, nitrogenase (EC 1.7.99.2) activity decreased in intact plants and in the vesicle-cluster preparations. The respiratory capacity ofFrankia also decreased. After 4 d in darkness most respiration was lost, though all nitrogenase activity was already lost after 3 d. When the dark treatment was ended after 2 d and normal light/dark conditions restored, nitrogenase activity immediately started to recover. The respiratory capacity continued to decrease and no recovery was observed until the third day after the end of the dark treatment. Whole-plant nitrogenase activity slowly increased at a rate similar to the rate of increase observed in untreated plants. Transmission electron micrographs of the root nodules showed that the cytoplasm of infected host cells and the cells ofFrankia were structurally degraded in response to dark treatment, while young vesicles were frequent during recovery. Growth and differentiation ofFrankia cells were apparently important for recovery of the enzyme activities studied.     

Diurnal Variation in Acetylene Reduction and Net Hydrogen Evolution in Five Tropical and Subtropical Nitrogen-Fixing Tree Symbioses

Diurnal variations in acetylene reduction and net hydrogen evolutionwere shown in five tropical and subtropical nitrogen-fixingtree symbioses. The symbioses studied in a growth chamber were:Acacia albida x TAL 1457, Leucaena leucocephala x TAL 1145,Prosopis chilensis x TAL 600, Casuarina glauca x HFP Cc13 andC. obesa x HFP Cc13. Acetylene reduction was highest at the end of the light periodin all symbioses studied. In the A. albida x TAL 1457 symbiosis,the diurnal variations in acetylene reduction and net hydrogenevolution showed a minor synchrony, while in the other symbiosesthe diurnal pattern of acetylene reduction and net hydrogenevolution clearly differed. Also, a diurnal variation in relativeefficiency of nitrogenase was shown in the A. albida x TAL 1457symbiosis. A hydrogen uptake enzyme was detected at a low substrate concentration(24.5 mmol m^–3 H₂) for L. leucocephala x TAL 1145, C.obesa x HFP Cc13 and has earlier been found for C. glauca xHFP CcI3. A hydrogen uptake system was also found for P. chilensisx TAL 600 and A. albida x TAL 1457 at a 17-fold higher substrateconcentration. The results show that a diurnal variation in C₂H₂ reductionand H₂ evolution occurs, and that diurnal variation in the conversionfactor between C₂H₂ reduction and N₂ fixation could occur. Thisfact raises criticisms regarding the use of a point estimateof this factor. Key words: Acetylene reduction, hydrogen evolution, uptake hydrogenase, nitrogen-fixing tree symbioses     

Acetylene degradation by new isolates of aerobic bacteria and comparison of acetylene hydratase enzymes

Aerobic acetylene-degrading bacteria were isolated from soil samples. Two isolates were assigned to the species Rhodococcus opacus, two others to Rhodococcus ruber and Gordona sp. They were compared with known strains of aerobic acetylene-, cyanide-, or nitrile-utilizing bacteria. The acetylene hydratases of R. opacus could be measured in cell-free extracts only in the presence of a strong reductant like titanium(III) citrate. Expression of these enzymes was molybdenum-dependent. Acetylene hydratases in cell-free extracts of R. ruber and Gordona spp. did not require addition of reductants. No cross-reactivity could be found between cell-free extracts of any of these aerobic isolates and antibodies raised against the acetylene hydratase of the strictly anaerobic fermenting bacterium Pelobacter acetylenicus. These results show that acetylene hydratases are a biochemically heterogeneous group of enzymes.     

Acetylene reduction by rumen microflora

Summary Acetylene reduction to ethylene by filtrates of rumen contents has been studied. The Km values for acetylene are comparable to those reported for nitrogenase enzymes from N₂ fixing bacteria. The enhancement of ethylene production from acetylene by phosphate and pyruvate suggests that the reduction was carried out by anaerobic microorganisms. Acetylene reduction occurred in the rumen only when a high nitrogen diet was fed to the sheep. Some microorganisms isolated from the rumen contents were grown anaerobically under N₂ gas on agar not supplemented with combined nitrogen. Methane production by filtrates of rumen contents was found to be inhibited by acetylene.     

Effect of supra-ambient oxygen on nitrogenase activity (c(2)h(2)) and root respiration of soybeans and isolated soybean bacteroids

Isolated soybean (Glycine max [L.] Merr. cv Wilkin) bacteroids have O₂-dependent nitrogenase activity which is strongly inhibited by supraoptimal O₂ concentrations. Oxygen-inhibited nitrogenase activity is recovered by addition of 10 millimolar sodium succinate or by lowering the O₂ concentration.

Brief treatment of roots of intact soybean plants with 1.0 atmosphere O₂ reduces nitrogenase activity (C₂H₂). There is a rapid partial recovery of activity within 2 to 3 hours, and a slower return to near normal levels by 36 hours. The drop and recovery of nitrogenase activity is accompanied by a parallel drop and increase in root respiration. There is a direct relationship between the change in respiration and the change in acetylene reduction following O₂ treatment. The O₂-mediated changes in nitrogenase activity and root respiration are not affected by the planting medium. The ratio of the change in respiration to the change in nitrogenase activity was the same in 13 soybean cultivars.

     

Effect of acetylene on root respiration and acetylene reducing activity in nodulated soya bean

Acetylene decreased root and nodule respiration, as measured by CO₂ evolution of nodulated or non-nodulated Glycine max. An inhibition of 25 to 35% in 15 to 30 minutes occurred when 13% C₂H₂ was introduced in the gas flux which aerated the root nutrient solution. When the light intensity was doubled to 800 microeinsteins per square meter per second, the inhibition increased to 50% and nodule acetylene reduction activity was inhibited 50%.     

Dinitrogen Fixation by Cultures of Frankia sp CpIl Demonstrated by N(2) Incorporation

The filamentous bacterium Frankia of the Actinomycetales, isolated from the nitrogen-fixing root nodules of certain woody plants, has shown nitrogenase activity in culture, using the acetylene reduction method. In the present work, nitrogenase activity in pure cultures of Frankia sp. CpIl is confirmed using mass spectrometric measurements of ¹⁵N₂ incorporation. After addition of carrier NH₄⁺ to digested cultures, those exposed to ¹⁵N₂ (25 atom%) had a ¹⁵N content of 3.16 atom% compared to 0.354 atom% ¹⁵N in the controls.     

Diazotrophy and Nitrogenase Activity in the Archaebacterium Methanosarcina barkeri 227

Nitrogen fixation (diazotrophy) has recently been demonstrated in several methanogenic archaebacteria. To compare the process in an archaebacterium with that in eubacteria, we examined the properties of diazotrophic growth and nitrogenase activity in Methanosarcina barkeri 227. Growth yields with methanol or acetate as a growth substrate were significantly lower in N₂-grown cultures than in NH₄⁺-grown cultures, and the culture doubling times were increased, indicating that diazotrophy was energetically costly, as it is in eubacteria. Growth of nitrogen-fixing cells was inhibited when molybdenum was omitted from the medium; addition of 10 nM molybdate stimulated growth, while 1 μM molybdate restored maximum diazotrophic growth. Omission of molybdenum did not inhibit growth of ammonia-grown cells. Tungstate (100 μM) strongly inhibited growth of molybdenum-deficient diazotrophic cells, while ammonia-grown cells were unaffected. The addition of 100 nM vanadate or chromate did not stimulate diazotrophic growth of molybdenum-starved cells. These results are consistent with the presence of a molybdenum-containing nitrogenase in M. barkeri. Acetylene, the usual substrate for assaying nitrogenase activity, inhibited methanogenesis by M. barkeri and consequently needed to be used at a low partial pressure (0.3% of the headspace) when acetylene reduction by whole cells was assayed. Whole cells reduced 0.3% acetylene to ethylene at a very low rate (1 to 2 nmol h⁻¹ mg of protein⁻¹), and they “switched off” acetylene reduction in response to added ammonia or glutamine. Crude extracts from diazotrophic cells reduced 10% acetylene at a rate of 4 to 5 nmol of C₂H₄ formed h⁻¹ mg of protein⁻¹ when supplied with ATP and reducing power, while extracts of Klebsiella pneumoniae prepared by the same procedures had rates 100-fold higher. Acetylene reduction by extracts required ATP and was completely inhibited by 1 mM ADP in the presence of 5 mM ATP. The low rates of C₂H₂ reduction could be due to improper assay conditions, to switched-off enzyme, or to the nitrogenase's having lower activity towards acetylene than towards dinitrogen.     

Quantification and Removal of Some Contaminating Gases from Acetylene Used to Study Gas-Utilizing Enzymes and Microorganisms

Acetylene generated from various grades of calcium carbide and obtained from commercial- and purified-grade acetylene cylinders was shown to contain high concentrations of various contaminants. Dependent on the source of acetylene, these included, at maximal values, H₂ (0.023%), O₂ (0.779%), N₂ (3.78%), PH₃ (0.06%), CH₄ (0.073%), and acetone (1 to 10%). The concentration of the contaminants in cylinder acetylene was highly dependent on the extent of cylinder discharge. Several conventional methods used to partially purify cylinder acetylene were compared. A small-scale method for extensively purifying acetylene is described. An effect of acetylene quality on acetylene reduction assays conducted with purified nitrogenase from Azotobacter vinelandii was demonstrated.     

Further Errors in the Acetylene Reduction Assay: Effects of Plant Disturbance

A flow-through gas system was used to study the effects of disturbanceon nitrogenase (acetylene reduction) activity of nodulated rootsystems of soyabean (Glycine max) and white clover (Trifoliumrepens). Detopping plus removal of the rooting medium (by shaking)produced a substantial decrease in maximum nitrogenase activity.This response is due to a reduction in oxygen flux to the bacteroidscaused by an increase in the oxygen diffusion resistance ofthe nodule. The decrease in maximum nitrogenase activity wasmuch smaller for roots subjected to detopping only. Thus, theeffect of root shaking is more important than that of shootremoval. The effect of detopping plus root shaking on nitrogenase activityoccurred whether the plants were equilibrated and assayed at25°C or 15°C. However, the effect of disturbance onthe oxygen diffusion resistance of the nodules, and thus onnitrogenase activity, was greater at the higher temperature.At the lower temperature the oxygen diffusion resistance ofthe nodules had already been increased in response to the reducedrequirement for oxygen. These nodules were less susceptibleto the effects of disturbance. Thus, comparisons of the effectsof equilibration temperature on nitrogenase activity produceddifferent results depending on whether intact or disturbed systemswere used. With intact systems activity was lower at the lowertemperature but with detopped/shaken roots the lowest activityoccurred at the higher temperature. It is concluded that the use of detopped/shaken roots can producesubstantial errors in the acetylene reduction assay, which makesthe assay invalid even when used for comparative purposes. However,comparisons with rates of ¹⁵N₂ fixation and H₂ production showthat accurate measurements of nitrogenase activity can be obtainedfrom maximum rates of acetylene reduction by intact plants ina flow-through gas system. The continued use of assay proceduresin which cumulated ethylene production from disturbed systemsis measured in closed vessels must be questioned. Key words: Nodules, acetylene, nitrogenase activity     

Nitrogenase activity of pea bacteroids as affected by carbohydrates and ammonium chloride

Summary Regulation and efficiency of the nitrogen-fixing system of the rhizobium-pea symbiosis were investigated. Acetylene reduction of detached root nodules was measured with various substrates added. Succinate, fumarate and malate were most effective in stimulating nitrogenase activity; glucose, pyruvate and citrate were also active. Acetylene reducing activity of detached nodules was inhibited by the addition of NH₄Cl, irrespective of the substrate present. Nitrogenase activity of isolated bacteroids was not influenced by NH₄Cl.Respiration of detached nodules was not significantly stimulated by the addition of substrates. Ammonium chloride did not influence respiration. With detached nodules and isolated bacteroids a consumption of about 16 g of carbohydrate per g of nitrogen fixed could be calculated. Detached nodules produced more hydrogen relative to the acetylene reduced than did isolated bacteroids and intact plants.Results obtained indicate that the regulation of nitrogenase activity and the efficiency of substrate consumption depend on environmental conditions.     

The Relationship between H(2) Evolution and Acetylene Reduction in Pisum sativum-Rhizobium leguminosarum Symbioses Differing in Uptake Hydrogenase Activity

Peas (Pisum sativum L.) were inoculated with strains of Rhizobium leguminosarum having different levels of uptake hydrogenase (Hup) activity and were grown in sterile Leonard jars under controlled conditions. Rates of H₂ evolution and acetylene reduction were determined for intact nodulated roots at intervals after the onset of darkness or after removal of the shoots. Hup activity was estimated using treatment plants or equivalent plants from the growth chamber, by measuring the uptake of H₂ or ³H₂ in the presence of acetylene. In all cases, the rate of H₂ evolution was a continuous function of the rate of acetylene reduction. In symbioses with no demonstrable Hup activity, H₂ evolution increased in direct proportion to acetylene reduction and the slopes were similar with the Hup⁻ strains NA502 and 128C79. Hup activity was similar in strains 128C30 and 128C52 but significantly lower in strain 128C54. With these strains, the slopes of the H₂ evolution versus acetylene reduction curves initially increased with acetylene reduction, but became constant and similar to those for the Hup⁻ strains at high rates of acetylene reduction. On these parallel portions of the curves, the decreases in H₂ evolution by Hup⁺ strains were similar in magnitude to their H₂-saturated rates of Hup activity. The curvilinear relationship between H₂ evolution and acetylene reduction for a representative Hup⁺ strain (128C52) was the same, regardless of the experimental conditions used to vary the nitrogenase activity.     

Interaction between hydrogenase, nitrogenase, and respiratory activities in a Frankia isolate from Alnus rubra

H2 uptake and H2-supported O2 uptake were measured in N2-fixing cultures of Frankia strain ArI3 isolated from root nodules of Alnus rubra. H2 uptake by intact cells was O2 dependent and maximum rates were observed at ambient O2 concentrations. No hydrogenase activity could be detected in NH4+-grown, undifferentiated filaments cultured aerobically indicating that uptake hydrogenase activity was associated with the vesicles, the cellular site of nitrogen fixation in Frankia. Hydrogenase activity was inhibited by acetylene but inhibition could be alleviated by pretreatment with H2. H2 stimulated acetylene reduction at supraoptimal but not suboptimal O2 concentrations. These results suggest that uptake hydrogenase activity in ArI3 may play a role in O2 protection of nitrogenase, especially under conditions of carbon limitation.     

Activities, occurrence, and localization of hydrogenase in free-living and symbiotic frankia

Symbiotic and free-living Frankia were investigated for correlation between hydrogenase activities (in vivo/in vitro assays) and for occurrence and localization of hydrogenase protein by Western blots and immuno-gold localization, respectively. Freshly prepared nodule homogenates from the symbiosis between Alnus incana and a local source of Frankia did not show any detectable in vivo or in vitro hydrogenase uptake activity, as also has been shown earlier. However, a free-living Frankia strain originally isolated from these nodules clearly showed both in vivo and in vitro hydrogenase activity, with the latter being approximately four times higher. Frankia strain Cpl1 showed hydrogen uptake activity both in symbiosis with Alnus incana and in a free-living state. Western blots on the different combinations of host plants and Frankia strains used in the present study revealed that all the Frankia sources contained a hydrogenase protein, even the local source where no in vivo or in vitro activity could be measured. The 72 kilodalton protein found in the symbiotic Frankia as well as in the free-living Frankia strains were immunologically related to the large subunit of a dimeric hydrogenase purified from Alcaligenes latus. Recognitions to polypeptides with molecular masses of about 41 and 19.5 kilodaltons were also observed in Frankia strain UGL011101 and in the local source of Frankia, respectively. Immunogold localization of the protein demonstrated that in both the symbiotic state and the free-living nitrogen-fixing Frankia, the protein is located in vesicles and in hyphae. The inability to measure any uptake hydrogenase activity is therefore not due to the absence of hydrogenase enzyme. However, the possibility of an inactive hydrogenase enzyme cannot be ruled out.     

Biological Dinitrogen Fixation (Acetylene Reduction) Associated with Florida Mangroves

Biological dinitrogen fixation in mangrove communities of the Tampa Bay region of South Florida was investigated using the acetylene reduction technique. Low rates of acetylene reduction (0.01 to 1.84 nmol of C₂H₄/g [wet weight] per h) were associated with plant-free sediments, while plant-associated sediments gave rise to slightly higher rates. Activity in sediments increased greatly upon the addition of various carbon sources, indicating an energy limitation for nitrogenase (C₂H₂) activity. In situ determinations of dinitrogen fixation in sediments also indicated low rates and exhibited a similar response to glucose amendment. Litter from the green macroalga, Ulva spp., mangrove leaves, and sea grass also gave rise to significant rates of acetylene reduction.     

Respiration and nitrogenase activity in nodules ofCasuarina cunninghamiana and cultures ofFrankia sp. HFP020203: Effects of temperature and partial pressure of O2

The effects of time after exposure to acetylene and of nodule excision were examined using a flow-through system. After a transient depression in the rate of acetylene reduction that began about 1.5 min after exposure to acetylene, the rate recovered to 98% of the initial maximum value after 40 min. After nodule excision the rate stabilized to 90% of the initial maximum value observed in the intact plant.Excised nodules, measured at 6-min intervals in a closed system, with frequent changes of the gas mixture, were used for the remaining experiments. Acetylene reduction by the nodules increased rapidly as temperature was increased between 6 and 26°C. Between 26 and 36°C there was relatively little effect of temperature on acetylene reduction.Nodules and cultures ofFrankia were compared with respect to the effect of temperature and pO₂ (partial pressure of oxygen) on oxygen uptake. Cultures ofFrankia were grown on a nitrogen-free medium at either 0.3 kPa O₂ (vesicles absent) or 20 kPa O₂ (vesicles present). Oxygen uptake by nodules (vesicles absent) and by vesicle-containing cultures was strongly dependent on pO₂ at values below 20 kPa. This suggests the presence of a barrier to oxygen diffusion. Oxygen uptake was dependent on temperature as well as on pO₂, but the Q₁₀ was much larger for the cultures than for the nodules. This suggests that vesicles or related structures are not the source of the diffusion barrier in Casuarina nodules. Respiration by cultures ofFrankia lacking vesicles became O₂-saturated at low pO₂ values. Thus these cultures did not have a significant diffusion barrier. From these results it is concluded that nodules ofCasuarina cunninghamiana have a barrier to oxygen diffusion supplied by the host tissue and not byFrankia.     

Diffusion of Gases through Plant Tissues : Entry of Acetylene into Legume Nodules

I have measured acetylene diffusion through plant tissues including nodules from several species of legume—vetch, peas, soybeans, and Sesbania rostrata. The observed half-time for reequilibration of internal and external concentration is less than 1 minute for typical nodules. Inward diffusion of acetylene in air is rapid relative to the use of acetylene by nitrogenase so that diffusion of acetylene would not be a significant limiting factor for nitrogenase activity in air. However, under an atmosphere of Ar:O₂ where there is no N₂ reduction, the inward diffusion rate of acetylene into larger nodules could produce a measurable limitation of observed nitrogenase activity at low acetylene concentrations.     

Variation in nitrogenase and hydrogenase activity of alaska pea root nodules

Hydrogenase activity of root nodules in the symbiotic association between Pisum sativum L. and Rhizobium leguminosarum was determined by incubating unexcised nodules with tritiated H₂ and measuring tissue HTO. Hydrogenase activity saturated at 0.50 millimolar H₂ and was not inhibited by the presence of 0.10 atmosphere C₂H₂, which prevented H₂ evolution from nitrogenase. Total H₂ production from nitogenase was estimated as net H₂ evolution in air plus H₂ exchange in 0.10 atmosphere C₂H₂. Although such an estimate of nitrogenase function may not be quantitatively exact, due to uncertain relationships between H₂ exchange and H₂ uptake activity of hydrogenase, differences observed in H₂ exchange under various conditions represent an indication of changes in hydrogenase activity. Hydrogenase activity was lower in associations grown under higher photosynthetic photon flux densities and decreased relative to total H₂ production by nitrogenase. Total H₂ production and hydrogenase activity were maximum 28 days after planting. Thereafter, hydrogenase activity and H₂ production declined, but the potential proportion of nitrogenase-produced H₂ recovered by the uptake hydrogenase system increased. Of five R. leguminosarum strains tested two possessed hydrogenase activity. Strains which had the potential to reassimilate H₂ had significantly higher rates of N₂ reduction than those which did not exhibit hydrogenase activity.     

Nitrogenase Activity and Amounts of Nitrogenase Proteins in a Frankia-Alnus incana Symbiosis Subjected to Darkness

Effects of prolonged darkness on nitrogenase activity in vivo, nitrogenase activity in vitro, and the amounts of nitrogenase proteins were studied in symbiotic Frankia. Plants of Alnus incana (L.) Moench in symbiosis with a local source of Frankia were grown for 9 to 10 weeks in an 18/6 hour light/darkness cycle. After 12 hours of a light period, the plants were exposed to darkness for up to 40 hours. Nitrogenase activity (acetylene reduction activity) of intact plants was measured repeatedly. Frankia vesicle clusters were prepared from the nodules with an anaerobic homogenization and filtration technique and were used for measurements of in vitro nitrogenase activity and for measurements of the amounts of nitrogenase proteins on Western blots. Antisera made against dinitrogenase reductase (Fe-protein) of Rhodospirillum rubrum and against dinitrogenase (MoFe-protein) of Azotobacter vinelandii were used. Western blots were made transparent and nitrogenase proteins were quantified spectrophotometrically. Nitrogenase activity both in vivo and in vitro decreased after about 23 hours of darkness and continued to decrease to about 25% and 16% of initial activity, respectively, after 40 hours. The amount of Fe-protein and MoFe-protein in Frankia of the same plants decreased to 60% and 35%, respectively, after 40 hours of darkness. Loss of nitrogenase activity thus appeared to be largely explained by loss of MoFe-protein.