Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids

Oxyleghemoglobin was used to supply low concentrations of O₂ to H₂-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H₂ oxidation system of these bacteroids was capable of effectively utilizing O₂ at the low concentrations of O₂ expected to be found in soybean nodules. Apparent K_m values of approximately 10 nanomolar O₂ have been calculated for the oxyhydrogen reaction. These values include the K_m values for both H₂ oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H₂ additions stimulated C₂H₂ reduction, reduced the rate of endogenous respiration and maintained the ATP contents of bacteroids. In our reconstituted oxyleghemoglobin and bacteriod system, we estimate that the H₂ oxidation system is capable of recycling all of the H₂ evolved during the N₂ fixation process.     

Role of uptake hydrogenase in providing reductant for nitrogenase in Rhizobium leguminosarum bacteroids

The role of uptake hydrogenase in providing reducing power to nitrogenase was investigated in Rhizobium leguminosarum bacteroids from nodules of Pisum sativum L. (cv. Homesteader). H₂ increased the rate of C₂H₂ reduction in the absence of added substrates. Malate also increased nitrogenase (C₂H₂) activity while decreasing the effect of H₂. At exogenous malate concentrations above 0.05 mM no effect of H₂ was seen. Malate appeared to be more important as a source of reductant than of ATP. When iodoacetate was used to minimize the contribution of endogenous substrates to nitrogenase activity in an isolate in which H₂ uptake was not coupled to ATP formation, H₂ increased the rate of C₂H₂ reduction by 77%. In the presence of iodoacetate, an ATP-generating system did not enhance C₂H₂ reduction, but when H₂ was also included, the rate of C₂H₂ reduction was increased by 280% over that with the ATP-generating system alone. The data suggest that, under conditions of substrate starvation, the uptake hydrogenase in R. leguminosarum could provide reductant as well as ATP in an isolate in which the H₂ uptake is coupled to ATP formation, to the nitrogenase complex.     

Enumeration of Free-Living Aerobic N2-Fixing H2-Oxidizing Bacteria by Using a Heterotrophic Semisolid Medium and Most-Probable-Number Technique

A heterotrophic semisolid medium was used with two sensitive assay methods, C₂H₂ reduction and O₂-dependent tritium uptake, to determine nitrogenase and hydrogenase activities, respectively. Organisms known to be positive for both activities showed hydrogenase activity in both the presence and absence of 1% C₂H₂, and thus, it was possible to test a single culture for both activities. Hydrogen uptake activity was detected for the first time in N₂-fixing strains of Pseudomonas stutzeri. The method was then applied to the most-probable-number method of counting N₂-fixing and H₂-oxidizing bacteria in some natural systems. The numbers of H₂-oxidizing diazotrophs were considerably higher in soil surrounding nodules of white beans than they were in the other systems tested. This observation is consistent with reports that the rhizosphere may be an important ecological niche for H₂ transformation.     

Hydrogen inhibition of nitrogen reduction by nitrogenase in isolated soybean nodule bacteroids

Dihydrogen, a by-product of biological nitrogen fixation, is a competitive inhibitor of N₂ reduction by nitrogenase. To evaluate the significance of H₂ inhibition in vivo, we have measured the apparent inhibition constant for H₂ inhibition of N₂ reduction in Bradyrhizobium japonicum bacteroids isolated from soybean nodules. The rate of N₂ reduction was measured as ammonia production by bacteroids incubated in a buffer containing 200 micromolar leghemoglobin and 10 millimolar succinate under 0.02 atmosphere O₂ and various concentrations of N₂ and H₂. The apparent inhibition constant for H₂ under these conditions was determined to be approximately 0.03 atmosphere. This relatively low value strengthens the proposal that H₂ inhibition of N₂ reduction may be a significant factor in lowering the efficiency of nitrogen fixation in legume nodules.     

Characteristics of the H2 oxidizing system in soybean nodule bacteroids

A derivative of Rhizobium japonicum (strain 122 DES) has been isolated which forms nodules on soybeans that evolve little or no H₂ in air and efficiently fixes N₂. Bacteroids isolated from nodules formed by strain 122 DES took up H₂ with O₂ as the physiological acceptor and appeared to be typical of those R. japonicum strains that possess the H₂ uptake system. The hydrogenase system in soybean nodules is located within the bacteroids and activity in macerated bacteroids is concentrated in a particulate fraction. The pH optimum for the reaction is near 8.0 and apparent K _m values for H₂ and O₂ are 2 M and 1 M, respectively. The H₂ oxidizing activity of a suspension of 122 DES bacteroids was stable at 4°C for at least 4 weeks and was not particularly sensitive to O₂. Neither C₂H₂ nor CO inhibited O₂ dependent H₂ uptake activity.Non-physiological electron acceptors of positive oxidation reduction potential also supported H₂ uptake by bacteroids. The rate of H₂ uptake with phenazine methosulfate as the acceptor was greater than that with O₂. When methylene blue, triphenyltetrazolium, potassium ferricyanide or dichlorophenolindophenol were added to bacteriod suspensions, without preincubation, rates of H₂ uptake were supported that were lower than those in the presence of O₂. Preincubation of the bacteroids with acceptors increased the rates of H₂ uptake. No H₂ evolution was observed from reaction mixtures containing bacteroid suspensions and reduced methyl or benzyl viologens. Of a series of carbon substrates added to bacteroid suspensions only acetate, formate or succinate at concentrations of 50 mM resulted in 20% or greater inhibition of H₂ oxidation.The H₂ uptake capacity of isolated 122 DES bacteroids (expressed on a dry bacteroid basis) was at least 10-fold higher than the rate of the nitrogenase reaction in nodules expressed on a comparable basis. Since about 1 mol of H₂ is evolved for every mol of N₂ reduced during the N₂ fixation reaction, these observations explain why soybean nodules formed by strain 122 DES and other strains with high H₂ uptake activities have a capacity for recycling all the H₂ produced from the nitrogenase reaction.Abbreviations PMS PHenazine methosulfate - MB Methylene blue     

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.

     

Nitrogenase Activity Associated with Roots and Stems of Field-Grown Corn (Zea mays L.) Plants

Corn (Zea mays L.) plants were assayed for nitrogenase activity (C₂H₂ reduction) during early ear development. Hybrid corn and inbred lines were grown separately at two experimental fields in New Jersey. Acetylene-dependent ethylene production was observed a few hours after harvest, from the field, on intact plants, root-soil cores, lower stem segments, and excised roots, all assayed under air and not preincubated previously. Incubation of excised roots at 1% O₂ resulted in lower rates of C₂H₂ reduction. The time course of C₂H₂ reduction by excised roots, assayed in air, was similar for all genotypes studied (two hybrids, eight inbreds, and a cross of corn × teosinte) and indicated that a long preincubation at reduced O₂ is not absolutely required for early detection of nitrogenase activity. Isolation of N₂-fixing bacteria from within the roots and stems, together with the diurnal fluctuation of nitrogenase activity in response to day/night cycles, were indicative of a close association with plant function. Collectively, the results provided strong evidence for the occurrence of nitrogenase activity associated with corn plants growing in a temperate climate and dependent upon indigenous N₂-fixing bacteria.     

Effect of Oxygen and Malate on NO(3) Inhibition of Nitrogenase in Soybean Nodules

Soybean (Glycine max cv Hodgson) nitrogenase activity (C₂H₂ reduction) in the presence or absence of nitrate was studied at various external O₂ tensions. Nitrogenase activity increased with oxygen partial pressure up to 30 kilopascals, which appeared to be the optimum. A parallel increase in ATP/ADP ratios indicated a limitation of respiration rate by low O₂ tensions in the nodule, and the values found for adenine nucleotide ratios suggested that the nitrogenase activity was limited by the rate of ATP regeneration. In the presence of nitrate, the nitrogenase activity was low and less stimulated by increased pO₂, although the nitrite content per gram of nodules decreased from 0.05 to 0.02 micromole when pO₂ increased from 10 to 30 kilopascals. Therefore, the accumulation of nitrite inside the nodule was probably not the major cause of the inhibition. Instead, inhibition by nitrate could be due to competition for reducing power between nitrate reduction and bacteroid or mitochondrial respiration inside the nodule. This is supported by the observation of decrease in ATP/ADP ratios from 1.65, in absence of nitrate, to 0.93 in the presence of this anion at 30 kilopascals O₂. Furthermore, the inhibition was suppressed by the addition, to the plant nutrient solution, of 15 millimolar l-malate, a carbon substrate that is considered to be the major source of reductant for the bacteroids in the symbiosis.     

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⁺.     

Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids

The H₂-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, ²H¹H exchange from a mixture of ²H₂ and ¹H₂ in presence of ²H₂O and ¹H₂O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H₂ oxidation reaction, there was no significant discrimination between ²H₂ and ¹H₂, indicating that the initial H₂-activation step in the over-all H₂ oxidation reaction is not rate-limiting. By use of improved methods, an apparent K_m for H₂ of 0.05 micromolar was determined. The H₂ oxidation reaction in bacteroids was strongly inhibited by cyanide (88% at 0.05 millimolar), theonyltrifluoroacetone, and other metal-complexing agents. Carbonyl cyanide m-chlorophenylhydrazone at 0.005 millimolar and 2,4-dinitrophenol at 0.5 millimolar inhibited H₂ oxidation and stimulated O₂ uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H₂ to O₂. Partial pressures of H₂ at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO₂ evolution by low partial pressures of H₂ suggests that H₂ utilization may result in conservation of oxidizable substrates and benefits the symbiosis under physiological conditions. Succinate, acetate, and formate at concentrations of 50 millimolar inhibited rates of H₂ uptake by 8, 29, and 25%, respectively. The inhibition by succinate was noncompetitive and that by acetate and formate was uncompetitive. A concentration of 11.6 millimolar CO₂ (initial concentration) in solution inhibited H₂ uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H₂ uptake by succinate, acetate, formate, and CO₂ in the metabolism of the H₂-uptake-positive strains of Rhizobium.     

Occurrence of Two Pathways for Malate Oxidation in Bacteroids Isolated from Sesbania rostrata Stem Nodules during C(2)H(2) Reduction

Malate oxidation supported C₂H₂ reduction by bacteroids isolated from Sesbania rostrata stem nodules. Optimal activity reached 7.5 nanomoles per minute per milligram of dry weight and was in the same order of magnitude as that observed with succinate but always required a lower O₂ tension. Malate dehydrogenase (EC 1.1.1.37), purified 66-fold from bacteroids, actively oxidized malate (K_m = 0.19 millimolar). Malic enzyme (EC 1.1.1.39) from Sesbania bacteroids had a lower affinity for malate (K_m = 2.32 millimolar). Both enzymes exclusively required NAD⁺ as cofactor and required an alkaline pH for optimal activity. 2-Oxoglutarate and oxalate, inhibiting malate dehydrogenase and malic enzyme, respectively, were used to specifically block each malate oxidation pathway in bacteroids. The predominance of malate dehydrogenase activity to support bacteroid N₂ fixation was demonstrated. The inhibition of O₂ consumption by 2-oxoglutarate confirmed the importance of the malate dehydrogenase pathway in malate oxidation. It is proposed that the utilization of malate, with regard to O₂, is important in a general strategy of this legume to maintain N₂ fixation under O₂ limited conditions.     

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.

     

Effect of temperature on h(2) evolution and acetylene reduction in pea nodules and in isolated bacteroids

Nitrogenase (EC 1.7.99.2) activity in pea (Pisum savitum) nodules formed after infection with Rhizobium leguminosarum (lacking uptake hydrogenase) was measured as acetylene reduction, H₂ evolution in air and H₂ evolution in Ar:O₂. With detached roots the relative efficiency, calculated from acetylene reduction, showed a decrease (from 55 to below 0%) with increasing temperature. With excised nodules and isolated bacteroids similar results were obtained. However, the relative efficiency calculated from H₂ evolution in Ar:O₂ was unaffected by temperature. Measurements on both excised nodules and isolated bacteroids showed a marked difference between acetylene reduction and H₂ evolution in Ar:O₂ with increased temperature, indicating that either acetylene reduction or H₂ evolution in Ar:O₂ are inadequate measures of nitrogenase activity at higher temperature.     

Adaptation of Nitrogen Fixation by Intact Soybean Nodules to Altered Rhizosphere pO(2)

The N₂-fixing legume nodule requires O₂ for ATP production; however, the O₂ sensitivity of nitrogenase dictates a requirement for a low pO₂ inside the nodule. The effects of long term exposures to various pO₂s on N₂[C₂H₂] fixation were evaluated with intact soybean (Glycine max [L.] Merr., var. Wye) plants. Continuous exposure of their rhizosphere to a pO₂ of 0.06 atmospheres initially reduced nitrogenase activity by 37 to 45% with restoration of original activity in 4 to 24 hours and with no further change in tests up to 95 hours; continuous exposure to 0.02 atmosphere of O₂ initially reduced nitrogenase activity 72%, with only partial recovery by 95 hours. Similar exposures to a pO₂ of 0.32 atmospheres had little effect on N₂[C₂H₂] fixation; a pO₂ of 0.89 atmospheres initially reduced nitrogenase activity by 98% with restoration to only 14 to 24% of that of the ambient O₂ controls by 95 hours. Re-exposure to ambient pO₂ of plants adapted to nonambient pO₂s reduced N₂[C₂H₂] fixation to similar magnitudes as the reductions which occurred upon initial exposure to variant pO₂ conditions, and a time period was required to readapt to ambient O₂. It is concluded that the N₂[C₂H₂]-fixing system of intact soybean plants is able to adapt to a wide range of external pO₂s as probably occur in soil. We postulate that this occurs through an undefined mechanism which enables the nodule to maintain an internal pO₂ optimal for nitrogenase activity.     

Uptake and Metabolism of Carbohydrates by Bradyrhizobium japonicum Bacteroids

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with ¹⁴C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O₂ (2% in the gas phase). Uptake and conversion of ¹⁴C to CO₂ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO₂, and fructose was respired at a rate at least double that of glucose. Sucrose and glucose were converted to CO₂ at a very low but measurable rate (<0.1 nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO₂ at a rate 30 times greater than the conversion of carbon Number 6 to CO₂, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.     

Nitrite and Nitric Oxide as Inhibitors of Nitrogenase from Soybean Bacteroids

Nitrite was able to strongly inhibit C₂H₂ reduction by nitrogenase from soybean bacteroids, whereas H₂ evolution was unaffected under the same conditions. NO inhibited both C₂H₂ reduction and H₂ evolution; during C₂H₂ reduction, sensitivity of nitrogenase to NO was higher than to NO₂⁻, and the K_i values were, respectively, 0.056 and 0.52 mM. Production of NO resulting from a reduction of NO₂⁻ by dithionite in nitrogenase incubations was observed. However, the characteristics of inhibitions and the low level of NO generated by nitrite reduction ruled out the suggestion concerning a direct role of NO to explain the inhibitory effect of NO₂⁻ on nitrogenase.     

Acetylene Reduction (Nitrogen Fixation) by Pulp and Paper Mill Effluents and by Klebsiella Isolated from Effluents and Environmental Situations

High rates of acetylene (C₂H₂) reduction (nitrogenase activity) were observed in woodroom effluent from a neutral sulfite semi-chemical mill under aerobic (up to 644 nmol of C₂H₄ produced per ml per h) and under anaerobic (up to 135 nmol of C₂H₄ produced per ml per h) conditions. Pasteurized effluent developed C₂H₂ reduction activity when incubated under anaerobic but not under aerobic conditions. Activities were increased by addition of 0.5 to 3.0% glucose or xylose. Enrichment and enumeration studies showed that N₂-fixing Azotobacter and Klebsiella were abundant, and N₂-fixing Bacillus was present. Of 129 isolates of Klebsiella from pulp mills, lakes, rivers, and drainage and sewage systems, 32% possessed nitrogen-fixing ability.     

Nitrogenase Activity (C2H2) of Isolated Peanut and Cowpea Bacteroids under Nitrogen, Argon, and Helium Atmospheres

Peanut nodules have been reported to have several times highernitrogenase activity (C₂H₂) than cowpea and siratro nodulesinduced by the same rhizobial strains. The unique morphologicalmodification of the peanut bacteroids has been considered tobe the cause for such enhanced activity. To investigate thispossibility, nitrogenase activities of isolated peanut and cowpeabacteroids were compared. Peanut bacteroids showed low initialrates of C₂H₂ reduction which increased with time, but for cowpeabacteroids higher initial rates decreased with time. Moreover,the gases used as diluent for O₂ (N₂, Ar, or He) were foundto influence O₂ tolerance and C₂H₂-reduction rates of bacteroids.     

Hydrogen Evolution from Alfalfa and Clover Nodules and Hydrogen Uptake by Free-Living Rhizobium meliloti

A series of Rhizobium meliloti and Rhizobium trifolii strains were used as inocula for alfalfa and clover, respectively, grown under bacteriologically controlled conditions. Replicate samples of nodules formed by each strain were assayed for rates of H₂ evolution in air, rates of H₂ evolution under Ar and O₂, and rates of C₂H₂ reduction. Nodules formed by all strains of R. meliloti and R. trifolii on their respective hosts lost at least 17% of the electron flow through nitrogenase as evolved H₂. The mean loss from alfalfa nodules formed by 19 R. meliloti strains was 25%, and the mean loss from clover nodules formed by seven R. trifolii strains was 35%. R. meliloti and R. trifolii strains also were cultured under conditions that were previously established for derepression of hydrogenase synthesis. Only strains 102F65 and 102F51 of R. meliloti showed measurable activity under free-living conditions. Bacteroids from nodules formed by the two strains showing hydrogenase activity under free-living conditions also oxidized H₂ at low rates. The specific activity of hydrogenase in bacteroids formed by either strain 102F65 or strain 102F51 of R. meliloti was less than 0.1% of the specific activity of the hydrogenase system in bacteroids formed by H₂ uptake-positive Rhizobium japonicum USDA 110, which has been investigated previously. R. meliloti and R. trifolii strains tested possessed insufficient hydrogenase to recycle a substantial proportion of the H₂ evolved from the nitrogenase reaction in nodules of their hosts. Additional research is needed, therefore, to develop strains of R. meliloti and R. trifolii that possess an adequate H₂-recycling system.