Bacteroid Protein Research Articles

Nodule Physiology of a Supernodulating Soybean (Glyine max) Mutant

The nodule physiology of a supernodulating, nitrate tolerant symbiosis soybean (Glycine max (L.) Merr.) mutant (nts382) was compared to that of its wild-type parent, cv. Bragg. Nodule number and mass were greater in nts382 than cv. Bragg and individual nodule mass, bacteroid and haem content, and acetylene reduction activity per g nodule were less. Acetylene reduction activity expressed per mg bacteroid protein was the same in the two genotypes. In median sections, the ratio of infected to total nodule area was smaller in nts382, infected cell size was smaller and there were fewer bacteroids per peribacteroid envelope. When inoculum dose was decreased from 109 to 103 viable cells per pot, nodule number on nts382 decreased approximately to that on cv. Bragg; nodule size, bacteroid and haem contents increased as did nodule acetylene reduction activity. Application of moderate levels of nitrate, which did not significantly affect symbiotic parameters of cv. Bragg or high inoculum nts382, stimulated nodule growth and nitrogenase activity of low inoculum nts382. A combination of nitrate and low inoculum levels enhanced nodule parameters of nts382 to the level usually seen with cv. Bragg. When supernodulated, plant dry weight of nts382 was less than that of Bragg; decreasing inoculum dose had no significant effect but nitrate application increased plant growth; nitrate plus low inoculum induced similar plant growth to that of cv. Bragg. Nodule carbohydrate content was similar in both genotypes but nodule and xylem sap ureide contents were higher in nts382. In general, nodules of supernodulated nts382 resembled under-developed cv. Bragg nodules; when supernodulation was avoided by using low inoculum doses, ,nts382 nodules resembled those of cv. Bragg. Nitrogen metabolism in the mutant seems to be disturbed, resulting in ureide accumulation.

Organization and characterization of genes essential for symbiotic nitrogen fixation from Bradyrhizobium japonicum I110.

A total of 96 independent Tn5 insertions within a 39-kilobase-pair (kbp) segment of chromosomal DNA containing the three structural genes for nitrogenase (nifH, nifD, and nifK) from Bradyhizobium japonicum I110 were obtained in Escherichia coli and transferred to the wild-type strain by marker exchange. Individual transconjugants containing a Tn5 insertion were inoculated onto Glycine max cv. Wilkin (soybeans) and analyzed for their effect on symbiotic nitrogen fixation. In addition to the three structural genes, genes essential for nitrogen fixation (fix genes) were located in three separate regions: 9 kbp upstream of the nifDK operon; 1.5 kbp downstream of the nifDK operon; 4.5 kbp upstream of nifH. All of the fix::Tn5 insertion strains formed nodules which contained low or undetectable levels of nitrogenase activity. Bacteroids isolated from these nodules had approximately the same levels of the nifDK and nifH transcripts as those detectable from nodules formed by the wild-type strain. Western blot analysis of bacteroid proteins from nodules formed by the fix::Tn5 mutants or the wild-type strain showed the presence of similar levels of the nitrogenase protein subunits. The region upstream of nifH was characterized further by DNA sequence analysis and was shown to contain the nifB gene. The coding sequence of the nifB gene consisted of 1,494 nucleotides and was preceded by putative promoter (5' GTGG-10 base pairs [bp] TTGCA 3') and upstream activator (5' TGT-4 bp-T-5 bp-ACA 3') sequences.

Nitrate and nitrite reduction by alfalfa root nodules: Accumulation of nitrite in Rhizobium melioti bacteroids and senescence of nodules

Addition of NO−3 rapidly induced senescence of root nodules in alfalfa (Medicago sativa L. cv. Aragon). Loss of nodule dry matter began at the lowest NO−3 concentration (10 mM) but degradation of bacteroid proteins was only detected when nodules were supplied with NO−3 concentrations above 20 mM.Bacteroids from Rhizobium meliloti contained high specific activities of nitrate reductase (NR) and nitrite reductase (NiR). Both enzymes were presumably substrate‐induced although substantial enzyme activities were present in the absence of NO−3 Typical specific activities for soluble NR and NiR of bacteroids under NO−3 free conditions were 1.2 and 1.4 μmol (mg protein)−1h−1, respectively. In the presence of NO−3, the specific activity of NR was considerably greater than that of NiR, thus causing NO−2 accumulation in bacteroids. Nitrite levels in the bacteroids were linearly correlated with specific activities of NR and NiR, indicating that NO−2 is formed by bacteroid NR and that this NO−2 in turn, induces bacteroid NiR. Accumulation of NO−2 within bacteroids also indicates that NO−2 inhibits nodule activity after feeding plants with NO−3

Levels of Ammonia, Nitrite, and Nitrate in Alfalfa Root Nodules Supplied With Nitrate

Protein content, protease activity, and levels of NH4+, NO2- and NO3- were determined in the plant and bacteroid fractions of alfalfa (Medicago sativa L. cv. Aragon) nodules supplied with NO3- (0—200mM). Protein of the host cell cytosol represented approximately 70 % of the total nodule protein. The effect of NO3- on plant cytosolic protein closely paralleled that on bacteroid protein. Protein of both nodule fractions remained constant upon exposure to NO3- concentrations below 20 mM and was reduced by 30% above 20 mM NO3-. Patterns of proteolytic activity were inversely correlated to those of the corresponding protein for both bacteroids and cytosol. The nodule level of NH4+ was basically unaffected by increasing added NO3- whereas NO2- accumulated in both bacteroids and plant cytosol, although to a greater extent in the latter. Likewise, NO3- accumulated in the cytosol with much higher relative values than those of NO2-. Only trace or null amounts of NO3- were found in the corresponding bacteroids. Linear correlations either between bacteroidal and cytosolic NO2-, or between NO2- in both fractions and cytosolic NO3- levels were highly significant. The overall results indicate that the whole nodule capacity of NO2- reduction is not sufficient to avoid accumulation of NO2-, and support the view that NO2- plays a more important role than NH4+ or NO3- itself as inhibitor of nodule activity.

Effects of Short-Term N2 Deficiency on N Metabolism in Legume Nodules

The study aimed to test the hypothesis that ammonia production by Rhizobium bacteroids provides not only a source of nitrogen for growth but has a central regulatory role in maintaining the metabolic activity and functional integrity of the legume nodule. Production of ammonia in intact, attached nodules was interrupted by short-term (up to 3 days) exposure of the nodulated root systems of cowpea (Vigna unguiculata L. Walp cv Vita 3: Rhizobium CB 756) and lupin (Lupinus albus L. cv Ultra: Rhizobium WU 425) to atmospheres of argon:oxygen (80:20; v/v). Treatment did not affect nodule growth, levels of plant cell and bacteroid protein, leghaemoglobin content, or nitrogenase (EC 1.7.99.2) activity (acetylene reduction) but severely reduced (by 90%) synthesis and export of the major nitrogenous solutes produced by the two symbioses (ureides in cowpea, amides in lupin). Glutamine synthetase (EC 6.3.1.2) and NAD:glutamate oxidoreductase (EC I.4.1.2) were more or less stable to Ar:O(2) treatment, but activities of the glutamine-utilizing enzymes, glutamate synthase (EC 2.6.1.53), asparagine synthetase (EC 6.3.5.4) (lupin only), and de novo purine synthesis (cowpea only), were all markedly reduced. Production and export of nitrogenous solutes by both symbioses resumed within 2 hours after transferring Ar:O(2)-treated plants back to air. In each case the major exported product of fixation after transfer was initially glutamine, reflecting the relative stability of glutamine synthetase activity. Subsequently, glutamine declined and products of its assimilation became predominant consistent with resurgence of enzymes for the synthesis of asparagine in lupin and ureides in cowpea. Enzymes not directly involved with either ammonia or glutamine assimilation (purine synthesis, purine oxidation, and carbon metabolism of both bacteroids and plant cells) also showed transient changes in activity following interruption of N(2) supply. These data have been interpreted to indicate a far-reaching effect of the production of ammonia by bacteroids on a wide range of enzymes, possibly through control of protein turnover, rather than a highly specific effect of ammonia, or some product of its assimilation, on a few enzyme species.

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.

Proteolytic Activity in Soybean Root Nodules

Root nodules were harvested from chamber-grown soybean (Glycine max L. Merrill cv Woodworth) plants throughout development. Apparent nitrogenase activity (acetylene reduction) peaked before seeds began to develop, but a significant amount of activity remained as the seeds matured. Nodule senescence was defined as the period in which residual nitrogenase activity was lost. During this time, soluble protein and leghemoglobin levels in the host cell cytosol decreased, and proteolytic activity against azocasein increased. Degradative changes were not detected in bacteroids during nodule senescence. Total soluble bacteroid protein per gram of nodule remained constant, and an increase in proteolytic activity in bacteroid extracts was not observed. These results are consistent with the view that soybean nodule bacteroids are capable of redifferentiation into free-living bacteria upon deterioration of the legume-rhizobia symbiosis.

Nodule Protein Synthesis and Nitrogenase Activity of Soybeans Exposed to Fixed Nitrogen

Nitrate or ammonium was added to soybean (Glycine max L. Merrill cv Corsoy) plants grown in plastic pouches 10 days after nodules first appeared. By the third day of treatment with 10 millimolar nitrate, nitrogenase specific activity (per unit nodule weight) had decreased to 15% to 25% of that of untreated plants. Longer incubations and higher concentrations of nitrate had no greater effect. In addition, exogenous nitrate or ammonium resulted in slower nodule growth and decreased total protein synthesis in both the bacterial and the plant portion of the nodule (as measured by incorporation of (35)S). Two-dimensional gel electrophoresis revealed that the nitrogenase components were not repressed or degraded relative to other bacteroid proteins. In the presence of an optimal carbon source, the nitrogenase specific activity of nodules detached from nitrate-treated plants was equivalent to that of nodules from untreated plants. These results are consistent with models that propose decreased availability or utilization of photosynthate in root nodules when legumes are exposed to fixed nitrogen.

Rhizobium japonicum mutants defective in symbiotic nitrogen fixation.

Rhizobium japonicum strains 3I1b110 and 61A76 were mutagenized to obtain 25 independently derived mutants that produced soybean nodules defective in nitrogen fixation, as assayed by acetylene reduction. The proteins of both the bacterial and the plant portions of the nodules were analyzed by two-dimensional polyacrylamide gel electrophoresis. All of the mutants had lower-than-normal levels of the nitrogenase components, and all but four contained a prominent bacteroid protein not observed in wild-type bacteroids. Experiments with bacteria grown ex planta suggested that this protein was derepressed by the absence of ammonia. Nitrogenase component II of one mutant was altered in isoelectric point. The soluble plant fraction of the nodules of seven mutants had very low levels of heme, yet the nodules of five of these seven mutants contained the polypeptide of leghemoglobin. Thus, the synthesis of the globin may not be coupled to the content of available heme in soybean nodules. The nodules of the other two of these seven mutants lacked not only leghemoglobin but most of the other normal plant and bacteroid proteins. Ultrastructural examination of nodules formed by these two mutants indicated normal ramification of infection threads but suggested a problem in subsequent survival of the bacteria and their release from the infection threads.

Turnover of nitrogenase and leghemoglobin in root nodules of Pisum sativum

Turnover rates of the two nitrogenase components and leghemoglobin in root nodules of pea plants nodulated with Rhizobium leguminosarum were determined with three different methods: 1, Kinetics of 35S incorporation into protein; 2, pulse-chase experiments; 3, chloramphenicol inhibition of bacteroid protein synthesis. Methods 1 and 3 revealed that the turnover rates of the two nitrogenase components and leghemoglobin are identical to the average rate of bacteroid and plant nodule protein turnover. The t 1 2 times of component I and II and leghemoglobin were about 2 days. Pulse-chase experiments with 35SO 2− 4 appeared to be rather unsuitable for determination of turnover rates in pea root nodules.

Nitrogen fixing activity of rhizobial strain 32H1 in peanut and cowpea nodules

Rhizobial bacteroids undergo unique modifications in peanut root nodules. To determine the likely effect of such modifications on nitrogen fixation, peanuts and cowpeas were nodulated by the same strain of Rhizobium and nitrogenase activities (C 2H 2-reduction) were compared over 4 weeks. Peanuts were found to have one and a half to three times higher C 2H 2-reduction activity/unit nodule mass than cowpeas at different nodule ages. Nitrogen accumulated in the plant tops/unit weight of nodules on the roots was three times greater in peanuts. The bacteroid protein content/unit of nodule mass was very similar for the two species.

The effect of waterlogging on the synthesis of the nitrogenase components in bacteroids of Rhizobium leguminosarum in root nodules of Pisum sativum

The effect of waterlogging of root nodules on nitrogenase activity and synthesis was studied in Pisum sativum inoculated with Rhizobium leguminosarum (strain PRE). It was shown that: 1. nitrogenase activity of intact pea plants was decreased by waterlogging, 2. this decrease was paralleled by a decline of the amount of active nitrogenase determined in toluene EDTA treated bacteroids, 3. SDS-polyacrylamide gel electrophoresis revealed that the amount of nitrogenase component II (CII) decreased by waterlogging while the amount of component I (CI) was not markedly affected, and 4. analysis of bacteroid proteins after 35SO 4 labeling of pea plants showed that CII synthesis was repressed while CI synthesis continued indicating that the synthesis of CI and CII is regulated by independent mechanisms.

Effects of Protein Synthesis Inhibitors on Acetylene Reduction Activity of Lupin Root Nodules

Rifampicin and D-threo-chloramphenicol inhibited the incorporation of [35S]methionine into purified bacteroid suspensions, and into the bacteroid fraction but not the plant cytoplasmic fraction of cultured nodules. Cycloheximide and anisomycin inhibited [35S]methionine incorporation into the plant cytoplasmic fraction of cultured nodules; at early times they inhibited incorporation into the bacteroid fraction, but at later times this effect was reversed. Chloramphenicol, rifampicin, spectinomycin, cycloheximide and anisomycin all prevented the induction of acetylene reduction activity in immature nodules; spectinomycin did not prevent induction in nodules containing a spectinomycin-resistant Rhizobium. Neither rifampicin nor chloramphenicol inhibited the acetylene reduction activity of mature nodules, but cycloheximide and anisomycin caused rapid loss of activity. Cycloheximide did not inhibit the acetylene reduction activity of Rhizobium strain 32H1 in pure cultures. The results suggest that both plant cytoplasmic protein synthesis and bacteroid protein synthesis are needed for the induction of nitrogenase activity in developing lupin nodules, and that plant cytoplasmic protein synthesis but not bacteroid protein synthesis is needed for the maintenance of nitrogenase activity at high levels.

Development of the nitrogen-fixing and protein-synthesizing apparatus of bacteroids in pea root nodules

Some aspects of root nodule development of Pisum sativum inoculated with Rhizobium leguminosarum were examined. 1. 1. Nitrogenase activity (measured as acetylene reduction) appears to be preceded by leghemoglobin synthesis (measured immunologically). 2. 2. Syntheses of component I and component II of nitrogenase are not strictly coordinated. Synthesis of component I starts before component II. 3. 3. Plant and bacteroid protein synthesis (measured by [ 35S]sulfate labeling) in root nodules declines rapidly during nodule development. Corresponding with this decline is a decrease in quantity and quality of rRNA.

Analysis of DNA Content, Nitrogenase Activity and In Vivo Protein Synthesis of Rhizobium leguminosarum Bacteroids on Sucrose Gradients

Bacteroids of Rhizobium leguminosarum (strain PRE) isolated from pea root nodules (Pisum sativum) were separated by sucrose density gradient centrifugation, according to their stage of development from bacteria to mature bacteroids. The DNA content per cell, as measured by cytofluorometry, increased with development. Nitrogenase components in soluble bacteroid proteins were present in highest concentrations in mature bacteroids. The ratio of the amounts of Mo–Fe protein to Fe protein was not constant in different stages of development. Incorporation of 35SO4 2– into soluble bacteroid proteins in the nodule was maximal in the youngest stages. In mature bacteroids nitrogenase was synthesized preferentially although less 35SO4 2– was incorporated into total soluble protein. Nitrogenase activity, measured as ATP- and S2O4 2–-dependent acetylene reduction by EDTA/toluene treated bacteroids, was high in mature bacteroids and low in the youngest stages.

The effect of ammonium nitrate on the synthesis of nitrogenase and the concentration of leghemoglobin in pea root nodules induced by Rhizobium leguminosarum

The effects of NH 4NO 3 on the development of root nodules of Pisum sativum after infection with Rhizobium leguminosarum (strain PRE) and on the nitrogenase activity of the bacteriods in the nodule tissue were studied. The addition of NH 4NO 3 decreased the nitrogenase activity measured on intact nodules. This reduction of nitrogen fixation did not result from a reduced number of bacteroids or a decreased amount of bacteroid proteins per gram of nodule. The synthesis of nitrogenase, measured as the relative amount of incorporation of [ 35S]sulfate into the components I and II of nitrogenase was similarly not affected. The addition of NH 4NO 3 decreased the amount of leghemoglobin in the nodules and there was a quantitative correlation between the leghemoglobin content and the nitrogen-fixing capacity of the nodules. The conclusion is that the decrease of nitrogen-fixing capacity is caused by a decrease of the leghemoglobin content of the root nodules and not by repression of the nitrogenase synthesis.

Synthesis and turnover of leghaemoglobin in lupin root nodules

1. The problem of whether leghaemoglobin is synthesized on plant or bacterial ribosomes in root nodules of yellow lupin has been examined. 2. Leghaemoglobin, soluble plant protein and soluble bacteroid protein were labelled with 14C administered by uptake of 14CO 2. 3. Exposure of roots to 1 mM d- threo-chloramphenical resulted in inhibition of soluble bacteroid protein synthesis, but leghaemoglobin synthesis and soluble plant protein synthesis were unaffected. This result is consistent with leghaemoglobin being synthesized on plant ribosomes. 4. After nitrogen-fixing plants had been supplied with a pulse of 14CO 2, the decay of specific radioactivity of nodule protein fractions was observed. Leghaemoglobin had an apparent half-life of 18 days and is a stable protein in nitrogen-fixing yellow lupin nodules.

Investigations into the pathway of electron transport to the nitrogenase from nodule bacteroids

A series of investigations were conducted with the objective of elucidating natural pathways of electron transport from respiratory processes to the site of N2 fixation in nodule bacteroids. A survey of dehydrogenase activities in a crude extract of soybean nodule bacteroids revealed relatively high activities of NAD-specific β-hydroxybutyrate and glyceraldehyde-3-phosphate dehydrogenases. Moderate activities of NADP-specific isocitrate and glucose-6-phosphate dehydrogenases were observed. By use of the ATP-dependent acetylene reduction reaction catalyzed by soybean bacteroid nitrogenase, and enzymes and cofactors from bacteroids and other sources, the following sequences of electron transport to bacteroid nitrogenase were demonstrated: (1) H2 to bacteroid nitrogenase in presence of a nitrogenase-free extract ofC. pasteurianum; (2) β-hydroxybutyrate to bacteroid nitrogenase in a reaction containing β-hydroxybutyrate dehydrogenase, NADH dehydrogenase, NAD and benzyl viologen; (3) β-hydroxybutyrate dehydrogenase, to nitrogenase in reaction containing NADH dehydrogenase, NAD and either FMN or FAD; (4) light-dependent transfer of electrons from ascorbate to bacteroid nitrogenase in a reaction containing photosystem I from spinach chloroplasts, 2,6-dichlorophenolindophenol, and either azotoflavin from Azotobacter or non-heme iron protein from bacteroids; (5) glucose-6-phosphate to bacteroid nitrogenase in a system that included glucose-6-phosphate dehydrogenase, NADP, NADP-ferredoxin reductase from spinach, azotoflavin from Azotobacter and bacteroid non-heme iron protein. The electron transport factors, azotoflavin and bacteroid non-heme iron protein, failed to function in the transfer of electrons from an NADH-generating system to bacteroid nitrogenase. When FMN or FAD were added to systems containing azotoflavin and bacteroid non-heme iron protein, electrons apparently were transferred to the flavin-nucleotides and then nitrogenase without involvement of azotoflavin and bacteroid non-heme iron protein.