Nitrogenase Activity Of Bacteroids Research Articles

NAD-Malic Enzyme Affects Nitrogen Fixing Activity of Bradyrhizobium japonicum USDA 110 Bacteroids in Soybean Nodules

The NAD(+)-dependent malic enzyme (DME) has been reported to play a key role supporting nitrogenase activity in bacteroids of Sinorhizobium meliloti. Genetic evidence for a similar role in Bradyrhizobium japonicum USDA110 was obtained by constructing a dme mutant. Soybean plants inoculated with a dme mutant did not show delayed nodulation, but formed small root nodules and exhibited significant nitrogen-deficiency symptoms. Nodule numbers and the acetylene reducting activity per nodule as a dry weight value 14 and 28 days after inoculation with the dme mutant were comparable to those of plants inoculated with wild-type B. japonicum. However, shoot dry weight and acetylene reducting activity per nodule decreased to ca. 30% of the values in plants with wild-type B. japonicum. The sucrose and organic acid (malate, succinate, acetate, α-ketoglutarate and lactate) contents of the nodules were investigated. Amounts of sucrose, malate and a-ketoglutarate increased on inoculation with the dme mutant, suggesting that the decreased DME and nitrogenase activities in the bacteroids resulted in a reduction in the consumption of these respiratory metabolites by the nodules. The data suggest that the DME activity of B. japonicum bacteroids plays a role in nodule metabolism and supports nitrogen fixation.

Impact of glyphosate on the Bradyrhizobium japonicum symbiosis with glyphosate-resistant transgenic soybean: a minireview.

Glyphosate-resistant (GR) soybean [Glycine max (L.) Merr.] expressing an insensitive 5-enolpyruvylshikimic acid-3-phosphate synthase (EPSPS) gene has revolutionized weed control in soybean production. The soybean nitrogen fixing symbiont, Bradyrhizobium japonicum, possesses a glyphosate-sensitive enzyme and upon exposure to glyphosate accumulates shikimic acid and hydroxybenzoic acids such as protocatechuic acid (PCA), accompanied with B. japonicum growth inhibition and death at high concentrations. In a series of greenhouse and field experiments, glyphosate inhibited nodulation and nodule leghemoglobin content of GR soybean. Glyphosate accumulated in nodules of field-grown GR soybean, but its effect on nitrogenase activity of GR soybean was inconsistent in field studies. In greenhouse studies, nitrogenase activity of GR soybean following glyphosate application was transiently inhibited especially in early growth stages, with the greatest inhibition occurring under moisture stress. Studies using bacteroid preparations showed that the level of glyphosate inhibition of bacteroid nitrogenase activity was related to in vitro glyphosate sensitivity of the B. japonicum strains. These studies indicate the potential for reduced nitrogen fixation in the GR soybean system; however, yield reductions due to this reduced N2 fixation in early stages of growth have not been demonstrated.

Adenylate gradients and Ar:O(2) effects on legume nodules: I. Mathematical models.

Mathematical models were developed to test the likelihood that large cytosolic adenylate concentration gradients exist across the bacteria-infected cells of legume nodules. Previous studies hypothesized that this may be the case to account for the unusually low adenylate energy charge (AEC; 0.65) measured in the plant fraction of metabolically active nodules (M.M. Kuzma, H. Winter, P. Storer, I. Oresnik, C.A. Atkins, D.B. Layzell [1999] Plant Physiol 119: 399-407). Simulations coupled leghemoglobin-facilitated O(2) diffusion into the infected cell, through bacteroid nitrogenase activity, with the ATP demand for transport and ammonia assimilation in the plant fraction of ureide- and amide-producing nodules. Although large cytosolic adenylate gradients were predicted to exist in both nodule types, amide nodules were predicted to have steeper AEC gradients (0.82-0.52) than ureide nodules (0.82-0.61). The differences were attributed to an additional ATP demand for Asn synthesis in the amide nodule. Simulations for nodules transferred to an Ar:O(2) atmosphere predicted a major reduction in the magnitude of adenylate gradients and an increase in the AEC of the plant fraction. Results were consistent with a number of experimental studies and were used to propose an experimental test of the models.

Glyphosate effects on phenolic metabolism of nodulated soybean (Glycine max L. merr.).

Glyphosate is a herbicide that blocks the shikimic acid pathway. Three Bradyrhizobium japonicum strains with different sensitivities to glyphosate were used to test the effect of this herbicide on the phenolic metabolism of nodulated soybeans and on the bacteroid nitrogenase activity. Glyphosate caused an inhibition in the bacteroid nitrogenase activity that was related with the sensitivity of the nodule-forming strains. Both leaves and nodules accumulated huge amounts of shikimate and phenolic acids (mainly protocatechuic acid), indicating that the herbicide was translocated to the nodule and disturbed phenolic metabolism. However, this accumulation was not clearly related to the sensitivity of the different strains. Bacteroids from control plants were incubated with the same concentration of shikimate, and phenolic acid accumulated in glyphosate-treated plants. Despite the high levels found in nodules, they were not responsible for the decrease of the nitrogenase activity. Glyphosate by itself caused a small inhibition of the bacteroid nitrogenase activity.

Effect of proline on nitrogenase activity in symbiosomes from root nodules of soybean (Glycine maxL.) subjected to drought stress

Experiments were carried out to investigate if drought stress affects the ability of bacteroids from soybean (Glycine max L.) root nodules to utilize proline and malate to support nitrogenase activity. The bacteroids were isolated in sub-ambient oxygen and nitrogenase activity was measured by acetylene reduction. Nitrogenase activity supported by proline was 8-fold higher in bacteroids from drought-stressed nodules than in bacteroids from control nodules. In contrast to the results with proline there was no significant response to drought stress in the rate of bacteroid nitrogenase activity supported by malate. The effect of drought stress on transport of proline and malate across the symbiosome membrane was investigated by incubation of symbiosomes isolated in sub-ambient oxygen with radioactive tracers. Drought stress tended to increase the rate of proline uptake relative to a minor decrease in malate uptake into symbiosomes in response to drought. There was no indication of a saturable carrier in the symbiosome membrane for either substrate at concentrations in the range 0.1-2 mM. The rate of malate uptake into symbiosomes was twice as high as the rate of proline uptake at all substrate levels tested. The protein composition of the symbiosome membrane was altered in response to drought stress and these changes may relate to the permeability of the symbiosome membrane.

Uptake of glycine betaine and its analogues by bacteroids of Rhizobium meliloti

Bacteroids isolated from alfalfa nodules induced by Rhizobium meliloti 102F34 transported glycine betaine at a constant rate for up to 30 min. Addition of sodium salts greatly increased the uptake activity, whereas other salts or non-electrolytes had less effect. The apparent Km for glycine betaine uptake was 8.3 microM and V was about 0.84 nmol min-1 (mg protein)-1 in the presence of 200 mM-NaCl which gave maximum stimulation of the transport. Supplementing bacteroid suspensions with various energy-yielding substrates, or ATP, did not increase glycine betaine uptake rates. The uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and the respiratory inhibitor potassium cyanide strongly inhibited glycine betaine uptake, but arsenate was totally inactive. Glycine betaine transport showed considerable structural specificity: choline, proline betaine, gamma-butyrobetaine and trigonelline did not competitively inhibit the system, although choline and proline betaine were transported by bacteroids. Both a high-affinity activity and a low-affinity activity were found for choline uptake. These osmoprotective compounds might have a significant role in the maintenance of nitrogenase activity in bacteroids subjected to salt stress.

Effects of O2 Concentrations and Various Haemoglobins on Respiration and Nitrogenase Activity of Bacteroids from Stem and Root Nodules of Sesbania rostrata and of the Same Bacteria from Continuous Cultures

Summary: Bacteroids from both stem and root nodules of Sesbania rostrata inoculated with Rhizobium sp. strain ORS571 were studied in the presence of the respective leghaemoglobins or soybean root nodule leghaemoglobin. Concentrations of free dissolved O2 and rates of O2 consumption and nitrogenase activity were measured during steady states in a special reaction chamber. The two types of bacteroids had high-affinity terminal oxidase systems. They were capable of respiration-coupled nitrogenase activity at concentrations below 10 nM free dissolved O2; at such concentrations the homologous leghaemoglobins were more effective than soybean leghaemoglobin because of their higher affinity for O2. S. rostrata bacteroids were less efficient in nitrogenase activity (consuming 4 to 11 mol O2 per mol of C2H2 reduced) than soybean bacteroids (02/C2H4 = 3.6) in comparable experiments, but efficiency was not affected by the source of leghaemoglobin. Similar experiments with ORS571 grown in N2-fixing continuous cultures showed that these bacteria also had high-affinity terminal oxidase systems coupled to nitrogenase, but those grown at <1 μM dissolved O2 were less efficient (O2/C2H4 >21) than those grown at 7 to 11 μM-O2 (O2/C2H4 <8). Nitrogenase activity of bacteria grown at the higher O2 concentration increased when the O2 concentration in the chamber was raised from 0.1 to 3 μM in experiments in which mammalian myoglobin replaced leghaemoglobin in the reaction solution. With bacteria grown at < 1 μM-O2, nitrogenase activity was inhibited (reversibly) by 50% after an increase from 0.1 to 1.4 μM-O2 in the reaction chamber. After changes in rates of supply of dissolved O2 there were oscillations in rates of O2 consumption before establishment of new steady states. These effects were greater in bacteria grown at 7 to 11 μM-O2 than in those grown at < 1 μM-O2, but with the latter, a sixfold increase in O2 flux produced only very small increases in concentration of dissolved O2.

Flux of O 2 affects apparent optimal concentrations of O 2 in suspensions of bacteria respiring microaerobically

Calculations of the flux of O 2 at very low concentrations in aqueous suspensions of bacteria show that different substrate-induced O 2 demands may induce apparently different optima for O 2 -related activities. This arises from different concentration gradients near to the bacterial surfaces. These effects are illustrated from published experimental data showing apparently different optimal concentrations of O 2 for nitrogenase activity of bacteroids isolated from root nodules of haricot bean, according to whether glucose or succinate were supplied as exogenous substrate. Profiles of O 2 concentration within the spherical domain occupied by each bacterium were calculated for each of the experimental conditions discussed. Special attention was given to the effects of the presence of the macromolecular O 2 -carrying protein leghaemoglobin, which diminishes the O 2 concentration gradients within the sphere of solution occupied by each bacterium.

Electron allocation to H+ and N2 by nitrogenase in Rhizobium leguminosarum bacteroids.

Electron allocation to H+ and N2 by nitrogenase in intact Rhizobium leguminosarum bacteroids has been studied. Nitrogenase activity was measured in intact cells with succinate and oxygen substrates. When whole cell nitrogenase activity was inhibited by oxygen-limitation or by the addition of the H+-conducting ionophore carbonylcyanide m-chlorophenylhydrazone, both inducing a low intracellular ATP/ADP ratio, the electron allocation to H+ was favoured over that to N2. When whole cell nitrogenase activity was inhibited by excess oxygen or by the addition of the K+-conducting ionophore valinomycin, both inhibiting electron transport to nitrogenase without affecting the intracellular ATP/ADP ratio, no effect upon the electron allocation to H+ and N2 was observed. The whole cell experiments could be confirmed by experiments with bacteroids treated with hexadecyltrimethylammonium bromide. Nitrogenase is highly active in these preparations with Na2S2O4 and MgATP as substrates. No effect was observed upon electron allocation to H+ and N2 when nitrogenase was inhibited by limitation of reductant (Na2S2O4) or MgATP. Only when nitrogenase was inhibited by MgADP, electron allocation to H+ was favoured. The amount of nitrogenase component 1 and 2 in bacteroids was estimated with protein blotting, followed by an immunological detection. It was found that 17% +/- 3% of total bacteroid protein is component 1 and 12% +/- 2% is component 2. The specific nitrogenase activity of bacteroids treated with hexadecyltrimethylammonium bromide is 178 +/- 62 nmol C2H4 formed X min-1 X mg total protein-1. Despite the high protein concentrations, nitrogenase is not inhibited. With cell-free extracts or with purified nitrogenase components isolated from R. leguminosarum bacteroids, electron allocation to H+ was favoured over that to N2, independently of the mechanism of inhibition. The discrepancies between the whole cell studies and those with isolated enzyme will be discussed with respect to the present mechanism of action of nitrogenase.

Regulation of the expression of leghaemoglobin genes in effective and ineffective root nodules of soybean

The expression of leghaemoglobin genes in effective and ineffective (unable to fix nitrogen) root nodules of soybean developed by Rhizobium japonicum strains 61A76, 61A24 and SM5 was measured by using a cDNA probe or a cloned leghaemoglobin sequence and in vitro translation of Lb-mRNA. Hybridization of the poly(A)-containing nodule polysomal RNA from 3-week-old nodules with a kinetically purified Lb-cDNA or with plasmid (pLbl) containing a leghaemoglobin sequence showed that Lb-mRNA is present in ineffective nodules formed by strains SM5 and 61A24 at reduced levels. Of the two major classes of electrophoretically distinguishable leghaemoglobins in soybean, LbS was not synthesized in 3-week-old strain 61A24-induced nodules while both sorts of leghaemoglobin were synthesized and accumulated in ineffective nodules formed by strain SM5 of Rhizobium. Ineffective nodules formed by strain 61A24 are green inside and do not appear to accumulate leghaemoglobin as measured by the haemochromogen assay, although low levels of apoleghaemoglobin were detected using leghaemoglobin antibodies. SM5-induced nodules were found to have about half as much as leghaemoglobin of that of effective (61A76-induced) nodules. This study demonstrates that while the appearance of leghaemoglobin is independent of nitrogenase activity in bacteroids, its synthesis is influenced to different degrees both by a mutation (SM5) and incompatibility (61A24) of Rhizobium. The primary regulation appears to be at the level of transcription or processing of mRNA since ineffective nodules contain Lb-mRNA approximately in proportion to the amount of apoleghaemoglobin present in these nodules.

Influence of ammonium chloride on the nitrogenase activity of nodulated pea plants (Pisum sativum).

A study was made on the short-term effect of ammonium ions on the nitrogenase activity of pea root nodules. Nodulated pea plants (Pisum sativum), having reached maximum acetylene-reducing activity, were supplied with NH4Cl (20 mM). Nitrogenase activity of intact plants, detached nodules, and isolated bacteroids was measured at differed time intervals. A significant drop (20 to 40%) in the acetylene-reducing activity of treated intact plants and their detached nodules was observed after 1 day. No drop in the nitrogenase activity of bacteroids (assayed aerobically, or anaerobically after treatment with ethylenediaminetetraacetic acid-toluene) occurred for 2 to 4 days after the addition of NH4+ to the plants, depending on cultural conditions. From these results it is concluded that the adverse effect of NH4+ on acetylene reduction by intact plants and detached nodules during the first 2 days is not due to a decrease in the amount of nitrogenase in the bacteroids. It is suggested that the effect has to be attributed to a reduced supply to the bacteroids of energy-delivery photosynthates.