Soybean Nodule Bacteroids Research Articles

Integrated roles of BclA and DD-carboxypeptidase 1 in Bradyrhizobium differentiation within NCR-producing and NCR-lacking root nodules

Legumes harbor in their symbiotic nodule organs nitrogen fixing rhizobium bacteria called bacteroids. Some legumes produce Nodule-specific Cysteine-Rich (NCR) peptides in the nodule cells to control the intracellular bacterial population. NCR peptides have antimicrobial activity and drive bacteroids toward terminal differentiation. Other legumes do not produce NCR peptides and their bacteroids are not differentiated. Bradyrhizobia, infecting NCR-producing Aeschynomene plants, require the peptide uptake transporter BclA to cope with the NCR peptides as well as a specific peptidoglycan-modifying DD-carboxypeptidase, DD-CPase1. We show that Bradyrhizobium diazoefficiens strain USDA110 forms undifferentiated bacteroids in NCR-lacking soybean nodules. Unexpectedly, in Aeschynomene afraspera nodules the nitrogen fixing USDA110 bacteroids are hardly differentiated despite the fact that this host produces NCR peptides, suggesting that USDA110 is insensitive to the host peptide effectors and that nitrogen fixation can be uncoupled from differentiation. In agreement with the absence of bacteroid differentiation, USDA110 does not require its bclA gene for nitrogen fixing symbiosis with these two host plants. Furthermore, we show that the BclA and DD-CPase1 act independently in the NCR-induced morphological differentiation of bacteroids. Our results suggest that BclA is required to protect the rhizobia against the NCR stress but not to induce the terminal differentiation pathway.

Stable Fluorescent and Enzymatic Tagging of Bradyrhizobium diazoefficiens to Analyze Host-Plant Infection and Colonization.

Bradyrhizobium diazoefficiens USDA 110 (formerly named Bradyrhizobium japonicum) can fix dinitrogen when living as an endosymbiont in root nodules of soybean and some other legumes. Formation of a functional symbiosis relies on a defined developmental program mediated by controlled gene expression in both symbiotic partners. In contrast to other well-studied Rhizobium-legume model systems that have been thoroughly examined by means of genetically tagged strains, analysis of B. diazoefficiens host infection has been impaired due to the lack of suitable tagging systems. Here, we describe the construction of B. diazoefficiens strains constitutively expressing single-copy genes for fluorescent proteins (eBFP2, mTurquoise2, GFP+, sYFP2, mCherry, HcRed) and enzymes (GusA, LacZ). For stable inheritance, the constructs were recombined into the chromosome. Effectiveness and versatility of the tagged strains was demonstrated in plant infection assays. (i) The infection process was followed from root-hair attachment to colonization of nodule cells with epifluorescent microscopy. (ii) Monitoring mixed infections with two strains producing different fluorescent proteins allowed rapid analysis of nodule occupancy and revealed that the majority of nodules contained clonal populations. (iii) Microscopic analysis of nodules induced by fluorescent strains provided evidence for host-dependent control of B. diazoefficiens bacteroid morphology in nodules of Aeschynomene afraspera and Arachis hypogaea (peanut), as deduced from their altered morphology compared with bacteroids in soybean nodules.

Fraser John Bergersen 1929 - 2011

Fraser Bergersen rose from humble beginnings in New Zealand to become a leading microbiologist who specialised in the physiology and biochemistry of legume nitrogen fixation. He and his family emigrated to Australia in 1954.Virtually all of his careerwas spent in Canberra at CSIRO Plant Industry. In the 1970s, Bergersen and colleagues achieved world-wide prominence when they elucidated the role of leghaemoglobin in facilitating oxygen diffusion to the Bradyrhizobium bacteroids in soybean nodules and in the nitrogen fixation process itself. During the rest of his working life, Fraser Bergersen contributed greatly to understanding the role of oxygen, the mode of its delivery, and terminal oxidases in all forms of biological nitrogen fixation.

Bradyrhizobium japonicum bacteroid appendages expressed in senescing and argon-treated soybean nodules

Bradyrhizobium japonicum bacteroids in soybean nodules expressed fibrillar appendages during senescence. In both scanning and transmission electron microscopy (SEM and TEM), these structures were observed to connect adjacent bacteroids, and bacteroids to symbiotic membranes. They were 20–25 nm in diameter, 100–2,500 nm in length and were linear, branched, or part of a web-like matrix. Bacteroids expressing appendages were not uniformly distributed, but were abundant within localized regions in the senescing nodule. The root systems of nodulated greenhouse-grown plants flushed with argon induced the appendages at earlier plant ages, and they were more prolific and wide spread than those in untreated nodules. Bradyrhizobium japonicum symbiotic appendages appear to be a response to an environmental niche within senescing nodules.

Differential protein profiles of Bradyrhizobium japonicum USDA110 bacteroid during soybean nodule development

During nodule development of legumes, together with the morphological alteration of plant cells, the rhizobia undergo marked biochemical and physiological changes and differentiate to bacteroids. Bacteroids invading a nodule are no longer free-growing cells, instead depending on the plant cell for all resources. To elucidate the molecular mechanism of the bacteroid differentiation of Bradyrhizobium japonicum, a time-course analysis of bacteroid protein profiles was studied by 2-D gel electrophoresis in soybean nodules. Using proteomic analysis, protein expressions in soybean nodule bacteroids 7, 10, 14, 28, and 49 days after inoculation (DAI) were monitored. The time points coincide with the early stage of nodule formation (7–10 DAI), the onset of nitrogen fixation (14–28 DAI), and nodule senescence (49 DAI). In this study, 275 annotated protein spots were successfully identified, and a cluster analysis of their expression was performed. A large portion of putative, upregulated proteins were observed in bacteroids at 7 or 10 DAI, and these upregulated proteins were mainly related to transcription, translation, protein folding, and degradation. In the later period (14–28 DAI) of bacteroid differentiation, a number of Nif and Fix proteins were upregulated. In addition, proteins related to the chaperonin and a synthetic enzyme of the poly-beta-hydroxybutyrate were expressed at high abundance. A number of proteins related to solute transporter were upregulated in the bacteroids and dominantly detected throughout nodule development. Changes of relative abundance of these proteins are discussed in relation to symbiosis.

Recovery of nitrogenase from aerobically isolated soybean nodule bacteroids

Bacteroids, the symbiotic forms of rhizobia, express nitrogenase, which is the enzyme that catalyzes the reduction of atmospheric dinitrogen to ammonium. The extreme oxygen lability of nitrogenase requires that bacteroids be isolated under anaerobic conditions to preserve their ability to reduce atmospheric dinitrogen. Aerobically isolated bacteroids were found to exhibit nitrogenase activity as measured via the acetylene reduction method but only after incubation of the bacteroids for about 1 h under low partial pressures of oxygen. The recovery of nitrogenase activity was dependent upon the partial pressure of oxygen to which the bacteroids were exposed during the one-hour incubation. The recovery of acetylene reduction activity was prevented by addition of inhibitors of protein synthesis, but not by inhibitors of transcription. Acetylene reduction could be expressed from aerobically isolated bacteroids stored for up to three days after isolation. The recovery of nitrogenase demonstrates that both the plant and the bacteroids possess mechanisms to protect the enzyme against oxygen. The recovery of nitrogen fixation activity from aerobically isolated bacteroids may provide a facile way to obtain bacteroids for studies on symbiotic functioning.

Formation of pyridine nucleotides under symbiotic and non-symbiotic conditions between soybean nodules and free-living rhizobia

Enzymatic regulation of pyricline nucleotide formation, under symbiotic and non-symbiotic conditions, was analyzed using soybeans ( Glycine max L. cv. ‘Akisengoku’) and rhizobia ( Bradyrhizobia japonicum strain A1017), respectively. It was found that levels of pyridine nucleotides in bacteroids in root nodules were different from those in free-living cells of rhizobia. This difference was associated with differences in activities of enzymes involved in the pathway from l-tryptophan to NAD and NADP. That is, these activities were lower in bacteroids than in free-living bacteria and lower in the nodule cytosol than in root extracts. The optimum pH for NAD synthetase in bacteroids, was 9.0. Additionally, the optimum pH for ATP-nicotinamide mononucleotide (NMN) adenyltransferase, final step enzyme in NAD formation, was estimated to be 7.6. In the bacteroid fraction, the K m of NAD synthetase (22 μM) was ∼1/22 of that of ATP-NMN adenyltransferase (482 μM). Vmax values were estimated to be almost in the same order for both NAD synthetase and ATP-NMN adenyltransferase. This is the first report on the formation of pyridine nucleotides originating from l-tryptophan in bacteroids in soybean nodules and free-living bacteria.

Molecular Cloning, Functional Characterization, and Subcellular Localization of Soybean Nodule Dihydrolipoamide Reductase

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed K mand k cat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

Molecular Cloning, Functional Characterization, and Subcellular Localization of Soybean Nodule Dihydrolipoamide Reductase,

Nodule ferric leghemoglobin reductase (FLbR) and leaf dihydrolipoamide reductase (DLDH) belong to the same family of pyridine nucleotide-disulfide oxidoreductases. We report here the cloning, expression, and characterization of a second protein with FLbR activity, FLbR-2, from soybean (Glycine max) nodules. The cDNA is 1,779 bp in length and codes for a precursor protein comprising a 30-residue mitochondrial transit peptide and a 470-residue mature protein of 50 kD. The derived protein has considerable homology with soybean nodule FLbR-1 (93% identity) and pea (Pisum sativum) leaf mitochondria DLDH (89% identity). The cDNA encoding the mature protein was overexpressed in Escherichia coli. The recombinant enzyme showed Km and kcat values for ferric leghemoglobin that were very similar to those of DLDH. The transcripts of FLbR-2 were more abundant in stems and roots than in nodules and leaves. Immunoblots of nodule fractions revealed that an antibody raised against pea leaf DLDH cross-reacted with recombinant FLbR-2, native FLbR-2 of soybean nodule mitochondria, DLDH from bacteroids, and an unknown protein of approximately 70 kD localized in the nodule cytosol. Immunogold labeling was also observed in the mitochondria, cytosol, and bacteroids of soybean nodules. The similar biochemical, kinetic, and immunological properties, as well as the high amino acid sequence identity and mitochondrial localization, draw us to conclude that FLbR-2 is soybean DLDH.

Identification of a gene involved in the malonate-malonamate shuttle from the leguminous symbiotic bacteria, Bradyrhizobium japonicum

Summary The role of malonate was suggested for the nitrogen transport between bacteroids and legume plants. The malonate-malonamate shuttle was proposed for the nitrogen transport. The key enzyme of the malonate-malonamate shuttle, malonamidase MAE2, was purified and the gene encoding the MAE2 from Bradyrhizobium japonicum was isolated and sequenced. The amidation site was identified from the primary structure of malonamidase, MAE2. The conservative regions of the MAE2 have the myristoylation sites, which might be a clue for transmembrane function between cytosol and bacteroids in soybean nodules.

Alanine, not ammonia, is excreted from N 2 -fixing soybean nodule bacteroids

Symbiotic nitrogen fixation, the process whereby nitrogen-fixing bacteria enter into associations with plants, provides the major source of nitrogen for the biosphere. Nitrogenase, a bacterial enzyme, catalyzes the reduction of atmospheric dinitrogen to ammonium. In rhizobia-leguminous plant symbioses, the current model of nitrogen transfer from the symbiotic form of the bacteria, called a bacteroid, to the plant is that nitrogenase-generated ammonia diffuses across the bacteroid membrane and is assimilated into amino acids outside of the bacteroid. We purified soybean nodule bacteroids by a procedure that removed contaminating plant proteins and found that alanine was the major nitrogen-containing compound excreted. Bacteroids incubated in the presence of 15 N 2 excreted alanine highly enriched in 15 N. The ammonium in these assays neither accumulated significantly nor was enriched in 15 N. The results demonstrate that a transport mechanism rather than diffusion functions at this critical step of nitrogen transfer from the bacteroids to the plant host. Alanine may serve only as a transport species, but this would permit physiological separation of the transport of fixed nitrogen from other nitrogen metabolic functions commonly mediated through glutamate.

Identification of Bradyrhizobium Japonicum malonamidase E2 in the periplasmic space of soybean nodule bacteroids

Summary MalonamidaseE2 (MAE2) was found both in the plant cytosolic fraction and in the Bradyrhizobium japonicum bacteroids of soybean nodules, and was proposed to play an important role in the transfer of fixed nitrogen (NH 3 ) to plant cells. The enzyme in the cytosol was identical in its molecular size and was immunologically homologous with MAE2 from free-living B. japonicum . The enzyme was not found in soybean root that is not infected by symbiotic bacteria. B. japonicum MAE2 presence in the periplasmic space was confirmed by the determination of the enzyme activity using intact bacteria in the presence of Triton X-100 (0.02%). Under microaerobic conditions, B. japonicum secreted MAE2 into the medium. However, the presence of malonate did not affect the secretion, indicating that malonate is not directly involved in the secretion of MAE2. The evidence described above strongly suggests that B. japonicum MAE2 located in the periplasmic space may be secreted into the peribacteroidal space of nodules during symbiosis.

Factors Influencing the Synthesis of Polysaccharide by Bradyrhizobium japonicum Bacteroids in Field-Grown Soybean Nodules

Certain strains of Bradyrhizobium japonicum produce large quantities of polysaccharide in soybean (Glycine max (L.) Merr.) nodules, and nodule polysaccharide (NPS) is different from that produced in culture. A previous survey of field-grown plants showed highly variable levels of NPS among field sites. To obtain clues about the possible function of NPS, we conducted two additional surveys of field-grown plants. The amount of polysaccharide in bulk samples of nodules was not associated with soil type, texture, slope, drainage, or any of the measured soil chemical properties except pH and [Ca]. Correlations with pH and [Ca] were positive and highly significant for two independent surveys involving a total of 77 sites in two years. In a preliminary comparison of high and low levels of Ca supplied to soybean plants grown in silica sand in a greenhouse, a high level of Ca (200 mg of Ca liter) increased the NPS level and increased the Ca content of the polysaccharide fraction. B. japonicum isolates from 450 nodules collected at 10 field sites in 1993 were used to form nodules on soybean plants grown in sand culture in a greenhouse in order to examine bacterial phenotype under controlled conditions. Results showed that the NPS level in the bulk nodule sample from any given site was a function of the proportion of nodule occupants that were capable of NPS synthesis. Thus, a higher soil pH and/or [Ca] may positively influence the survival of B. japonicum capable of synthesis of the nodule-specific polysaccharide.

Composition and Distribution of Adenylates in Soybean (Glycine max L.) Nodule Tissue.

Adenylates (ATP, ADP, and AMP) may play a central role in the regulation of the O2-limited C and N metabolism of soybean nodules. To be able to interpret measurements of adenylate levels in whole nodules and to appreciate the significance of observed changes in adenylates associated with changes in O2-limited metabolism, methods were developed for measuring in vivo levels of adenylate pools in the cortex, plant central zone, and bacteroid fractions of soybean (Glycine max L. Merr cv Maple Arrow x Bradyrhizobium japonicum strain USDA 16) nodules. Intact nodulated roots were either frozen in situ by flushing with prechilled Freon-113(-156[deg]C) or by rapidly (<1 s) uprooting plants and plunging them into liquid N2. The adenylate energy charge (AEC = [ATP + 0.5 x ADP]/[ATP + ADP + AMP]) of whole-nodule tissue (0.65 [plus or minus] 0.01, n = 4) was low compared to that of subtending roots (0.80 [plus or minus] 0.03, n = 4), a finding indicative of hypoxic metabolism in nodules. The cortex and central zone tissues were dissected apart in lyophilized nodules, and AEC values were 0.84 [plus or minus] 0.04 and 0.61 [plus or minus] 0.03, respectively. Although the total adenylate pool in the lyophilized nodules was only 41% of that measured in hydrated tissues, the AEC values were similar, and the lyophilized nodules were assumed to provide useful material for assessing adenylate distribution. The nodule cortex contained 4.4% of whole-nodule adenylates, with 95.6% being located in the central zone. Aqueous fractionation of bacteroids from the plant fraction of whole nodules and the use of marker enzymes or compounds to correct for recovery of bacteroids and cross-contamination of the bacteroid and plant fractions resulted in estimates that 36.2% of the total adenylate pool was in bacteroids, and 59.4% was in the plant fraction of the central zone. These are the first quantitative assessments of adenylate distribution in the plant and bacteroid fractions of legume nodules. These estimates were combined with theoretical calculations of rates of ATP consumption in the cortex (9.5 nmol g-1 fresh weight of nodule s-1), plant central zone (38 nmol g-1 fresh weight of nodule s-1), and bacteroids (62 nmol g-1 fresh weight of nodule s-1) of soybean nodules to estimate the time constants for turnover of the total adenylate pool and the ATP pool within each nodule fraction. The low values for time constant (1.6-5.8 s for total adenylate, 0.9-2.5 s for ATP only) in each fraction reflect the high metabolic activity of soybean nodules and provide a background for further studies of the role of adenylates in O2-limited nodule metabolism.

Alanine Dehydrogenase from Soybean Nodule Bacteroids: Purification and Properties

Alanine dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH + 4, pyruvate, L-alanine, and NAD were 86 μM, 8.9 mM, 0.49 mM, 1 mM and 200 μM, respectively. High concentrations of pyruvate, L-alanine, or NH + 4 caused inhibition of activity with K i ′s of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible. s. Inc.

Alanine dehydrogenase from soybean nodule bacteroids. Kinetic mechanism and pH studies

The kinetic mechanism of alanine dehydrogenase from soybean nodule bacteroids was studied by initial velocity experiments with or without product inhibitors, dead-end inhibitors, or alternate substrates. Without inhibitors, double-reciprocal plots of initial velocity experiments showed intersecting lines, indicating a sequential mechanism. These initial velocity experiments also revealed rapid-equilibrium ordered binding of NH4+ prior to pyruvate. When NAD was varied at changing-fixed concentrations of L-alanine, a nonlinear, concave down double-reciprocal plot was obtained. Substrate inhibition by pyruvate or L-alanine with cosubstrates varied was uncompetitive giving further support to an ordered mechanism. Product inhibition studies showed that both NAD and NADH and pyruvate and L-alanine were competitive. This suggested a Theorell-Chance mechanism. When product inhibition by L-alanine was studied with NH4+ varied in a series of experiments at increasing concentrations of pyruvate, the inhibition was eliminated, as expected for a Theorell-Chance mechanism. Furthermore, when NADH, NH4+, and pyruvate were varied simultaneously, maintaining their concentrations at a constant ratio to each other, an infinite Vmax was obtained. pH studies of the kinetic parameters indicated that NH4+, rather than NH3, was the true substrate that binds to a residue on the enzyme with a pK of 8.1. In conclusion, the kinetic mechanism at pH 8.5 was determined to be a Ter-Bi Theorell-Chance. In the amination direction, the substrates add in the order: NADH, NH4+, pyruvate, with NH4+ binding in rapid-equilibrium. In the reverse direction, NAD adds first, followed by L-alanine.

The consequences of the isotope effect on proline dehydrogenation rates estimated by the tritium loss method

Loss of tritium from a substrate is often used to estimate the rate of dehydrogenation. However, loss of 3H may be much slower than loss of H because of the tritium isotope effect. In order to assess the impact of the tritium isotope effect, loss of 3H from the C-5 position of proline during dehydrogenation by rat liver mitochondria and bacteroids from soybean ( Glycine max [L.] Merrill) nodules was compared with appearance of 14C in products of [ 14C]proline dehydrogenation. Incubations were carried out in the presence of o-aminobenzaldehyde (added to trap the initial product, Δ 1-pyroline-5-carboxylate). The fraction of total 14C products trapped by o-aminobenzaldehyde varied from 0.07 to 0.75 depending upon experimental conditions. With rat liver mitochondria, dehydrogenation of [ 14C]proline was between 3.27 and 9.25 times faster than dehydrogenation of 3H proline, depending upon assay conditions. Soybean nodule bacteroids dehydrogenated [ 14C]proline about 5 times faster than [ 3H]proline. We conclude the following: (i) the rate of proline dehydrogenation may be greatly underestimated by the tritium assay because of the tritium isotope effect, and (ii) the 14C assay may underestimate the rate of proline dehydrogenation if it is assumed that o-aminobenzaldehyde quantitatively traps Δ 1-pyrroline-5-carboxylate under all conditions. The simplicity of the tritium assay makes it attractive for routine use. However, its use requires determination of the tritium isotope effect, under the specific conditions of the assay, in order to correct the results. The considerations discussed here have broad applicability to any dehydrogenase assay employing tritium loss.

Metabolism of glutamate and aspartate in bacteroids isolated from soybean root nodules

SUMMARY: Bacteroids were isolated anaerobically from root nodules of soybean (Glycine max L. Merr.) and supplied with 14C- or 15N-labelled glutamate or aspartate under microaerobic conditions where bacteroids retained a high activity of N2 fixation. Glutamate and aspartate significantly stimulated N2 fixation (C2H2 reduction) and respiration (CO2 evolution) of isolated bacteroids. They were both utilized as substrates for bacteroid respiration and their nitrogen was rapidly released from the bacteroids as NH3. Amino-oxyacetate (AOA) strongly inhibited glutamate utilization, but aspartate utilization was only slightly affected by AOA. Substantial activity of aspartate ammonia-lyase (aspartase) was detected in the cell-free extract of bacteroids. These results suggest that the major pathway of glutamate utilization in bacteroids is transamination to form aspartate followed by direct deamination of aspartate by aspartase. It is also suggested that the 4-aminobutyrate pathway (GABA-shunt) is partly responsible for the catabolism of glutamate by soybean nodule bacteroids. The results are discussed in terms of the possible relationships between C4-dicarboxylates and glutamate in carbon and nitrogen metabolism in soybean nodule bacteroids.

Activities of the Pentose Phosphate Pathway and Enzymes of Proline Metabolism in Legume Root Nodules

Based on localization and high activities of pyrroline-5-carboxylate reductase and proline dehydrogenase activities in soybean nodules, we previously suggested two major roles for pyrroline-5-carboxylate reductase in addition to the production of the considerable quantity of proline needed for biosynthesis; namely, transfer of energy to the location of biological N(2) fixation, and production of NADP(+) to drive the pentose phosphate pathway. The latter produces ribose-5-phosphate which can be used in de novo purine synthesis required for synthesis of ureides, the major form in which biologically fixed N(2) is transported from soybean root nodules to the plant shoot. In this paper, we report rapid induction (in soybean nodules) and exceptionally high activities (in nodules of eight species of N(2)-fixing plants) of pentose phosphate pathway and pyrroline-5-carboxylate reductase. There was a marked increase in proline dehydrogenase activity during soybean (Glycine max) ontogeny. The magnitude of proline dehydrogenase activity in bacteroids of soybean nodules was sufficiently high during most of the time course to supply a significant fraction of the energy requirement for N(2) fixation. Proline dehydrogenase activity in bacteroids from nodules of other species was also high. These observations support the above hypothesis. However, comparison of pentose phosphate pathway and pyrroline-5-carboxylate reductase activities of ureide versus amide-exporting nodules offers no support. The hypothesis predicts that pyrroline-5-carboxylate and pentose phosphate pathway activities should be higher in ureide-exporting nodules than in amide-exporting nodules. This predicted distinction was not observed in the results of in vitro assays of these activities.

Succinate degradation through the citric acid cycle in Bradyrhizobium japonicum J501 bacteroids under low oxygen concentrations.

The succinate metabolizing pathway under low oxygen concentrations in soybean nodule bacteroids was surveyed by pulse-chase experiments using [2,3-14C] succinate and by radiorespirometry. Oxidation of exogenous succinate through the complete citric acid cycle was strongly suggested. Amino acid and sugar formations from exogenous succinate were also observed in the bacteroids. Addition of succinate to the bacteroids increased both the NAD+/NADH ratio from 1.8 to 3.0 and the adenylate energy value from 0.25 to 0.53.