Search

The exact mechanism(s) of infection and symbiotic development between rhizobia and legumes is not yet known, but changes in rhizobial exopolysaccharides (EPSs) affect both infection and nodule development of the legume host. Early events in the symbiotic process between Bradyrhizobium japonicum and soybean (Glycine max [L.] Merr.) were studied using two mutants, defective in soybean lectin (SBL) binding, which had been generated from B. japonicum 2143 (USDA 3I-1b-143 derivative) by Tn5 mutagenesis. In addition to their SBL-binding deficiency, these mutants produced less EPS than the parental strain. The composition of EPS varied with the genotype and with the carbon source used for growth. When grown on arabinose, gluconate, or mannitol, the wild-type parental strain, B. japonicum 2143, produced EPS typical of DNA homology group I Bradyrhizobium, designated EPS I. When grown on malate, strain 2143 produced a different EPS composed only of galactose and its acetylated derivative and designated EPS II. Mutant 1252 produced EPS II when grown on arabinose or malate, but when grown on gluconate or mannitol, mutant 1252 produced a different EPS comprised of glucose, galactose, xylose and glucuronic acid (1:5:1:1) and designated EPS III. Mutant 1251, grown on any of these carbon sources, produced EPS III. The EPS of strain 2143 and mutant 1252 contained SBL-binding polysaccharide. The amount of the SBL-binding polysaccharide produced by mutant 1252 varied with the carbon source used for growth. The capsular polysaccharide (CPS) produced by strain 2143 during growth on arabinose, gluconate or mannitol, showed a high level of SBL binding, whereas CPS produced during growth of strain 2143 on malate showed a low level of SBL binding. However, the change in EPS composition and SBL binding of strain 2143 grown on malate did not affect the wild-type nodulation and nitrogen fixation phenotype of 2143. Mutant 1251, which produced EPS III, nodulated 2 d later than parental strain 2143, but formed effective, nitrogen-fixing tap root nodules. Mutant 1252, which produced either EPS II or III, however nodulated 5-6 d later and formed few and ineffective tap root nodules. Restoration of EPS I production in mutant 1252 correlated with restored SBL binding, but not with wild-type nodulation and nitrogen fixation.

A complete tricarboxylic acid (TCA) cycle is generally considered necessary for energy production from the dicarboxylic acid substrates malate, succinate, and fumarate. However, a Bradyrhizobium japonicum sucA mutant that is missing alpha-ketoglutarate dehydrogenase is able to grow on malate as its sole source of carbon. This mutant also fixes nitrogen in symbiosis with soybean, where dicarboxylic acids are its principal carbon substrate. Using a flow chamber system to make direct measurements of oxygen consumption and ammonium excretion, we confirmed that bacteroids formed by the sucA mutant displayed wild-type rates of respiration and nitrogen fixation. Despite the absence of alpha-ketoglutarate dehydrogenase activity, whole cells of the mutant were able to decarboxylate alpha-[U-(14)C]ketoglutarate and [U-(14)C]glutamate at rates similar to those of wild-type B. japonicum, indicating that there was an alternative route for alpha-ketoglutarate catabolism. Because cell extracts from B. japonicum decarboxylated [U-(14)C]glutamate very slowly, the gamma-aminobutyrate shunt is unlikely to be the pathway responsible for alpha-ketoglutarate catabolism in the mutant. In contrast, cell extracts from both the wild type and mutant showed a coenzyme A (CoA)-independent alpha-ketoglutarate decarboxylation activity. This activity was independent of pyridine nucleotides and was stimulated by thiamine PP(i). Thin-layer chromatography showed that the product of alpha-ketoglutarate decarboxylation was succinic semialdehyde. The CoA-independent alpha-ketoglutarate decarboxylase, along with succinate semialdehyde dehydrogenase, may form an alternative pathway for alpha-ketoglutarate catabolism, and this pathway may enhance TCA cycle function during symbiotic nitrogen fixation.

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 15N2 excreted alanine highly enriched in 15N. The ammonium in these assays neither accumulated significantly nor was enriched in 15N. 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.

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.

Soybean (Glycine max) nodules formed by inoculation with either an effective strain or an ineffective (noninvasive, nodule-forming) strain of Bradyrhizobium japonicum were assayed for changes in developmental patterns of carbon metabolic enzymes of the plant nodule cells. Of the enzyme activities measured, only sucrose synthase, glutamine synthetase, and alcohol dehydrogenase were altered in the ineffective nodules relative to the effective nodules. Sucrose synthase and glutamine synthetase activities were greatly reduced, whereas alcohol dehydrogenase activity was elevated. Dark-induced senescence severely affected sucrose synthase but had little, if any, effect on the other enzymes measured. The developmental patterns of the anaerobically induced enzymes, aldolase and alcohol dehydrogenase, were different from those expected, implying that their development is not regulated solely by oxygen deprivation. However, anaerobic treatment of nodules resulted in responses similar to those enzymes in maize. The developmental profiles of the carbon metabolic enzymes suggest that carbohydrates are metabolized via the sucrose synthase and pentose phosphate pathways. This route of carbon metabolism, compared to glycolysis, would reduce the requirement of ATP for carbohydrate catabolism, generate NADPH for biosynthetic reactions, and provide intermediates for plant secondary metabolism.

Oxyleghemoglobin was used to supply low concentrations of O(2) to H(2)-oxidizing bacteroids from Rhizobium japonicum USDA 122 DES. The H(2) oxidation system of these bacteroids was capable of effectively utilizing O(2) at the low concentrations of O(2) expected to be found in soybean nodules. Apparent K(m) values of approximately 10 nanomolar O(2) have been calculated for the oxyhydrogen reaction. These values include the K(m) values for both H(2) oxidation and endogenous substrate oxidation. Even in the presence of oxyleghemoglobin, H(2) additions stimulated C(2)H(2) 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 H(2) oxidation system is capable of recycling all of the H(2) evolved during the N(2) fixation process.

Ion-exclusion high-pressure liquid chromatography (HPLC) was used to measure poly-beta-hydroxybutyrate (PHB) in Rhizobium japonicum bacteroids. The products in the acid digest of PHB-containing material were fractionated by HPLC on Aminex HPX-87H ion-exclusion resin for organic acid analysis. Crotonic acid formed from PHB during acid digestion was detected by its intense absorbance at 210 nm. The Aminex-HPLC method provides a rapid and simple chromatographic technique for routine analysis of organic acids. Results of PHB analysis by Aminex-HPLC were confirmed by gas chromatography and spectrophotometric analysis.

Rhizobium japonicum 122 DES bacteroids from soybean nodules possess an active H(2)-oxidizing system that recycles all of the H(2) lost through nitrogenase-dependent H(2) evolution. The addition of 72 muM H(2) to suspensions of bacteroids increased O(2) uptake 300% and the rate of C(2)H(2) reduction 300 to 500%. The optimal partial pressure of O(2) was increased, and the partial pressure of O(2) range for C(2)H(2) reduction was extended by adding H(2). A supply of succinate to bacteroids resulted in effects similar to those obtained by adding H(2). Both H(2) and succinate provided respiratory protection for the N(2)-fixing system in bacteroids. The oxidation of H(2) by bacteroids increased the steady-state pool of ATP by 20 to 40%. In the presence of 50 mM iodoacetate, which caused much greater inhibition of endogenous respiration than of H(2) oxidation, the addition of H(2) increased the steady-state pool of ATP in bacteroids by 500%. Inhibitor evidence and an absolute requirement for O(2) indicated that the H(2)-stimulated ATP synthesis occurred through oxidative phosphorylation. In the presence of 50 mM iodoacetate, H(2)-dependent ATP synthesis occurred at a rate sufficient to support nitrogenase activity. The addition of H(2) to H(2) uptake-negative strains of R. japonicum had no effect on ATP formation or C(2)H(2) reduction. It is concluded that the H(2)-oxidizing system in H(2) uptake-positive bacteroids benefits the N(2)-fixing process by providing respiratory protection of the O(2)-labile nitrogenase proteins and generating ATP to support maximal rates of C(2)H(2) reduction by oxidation of the H(2) produced from the nitrogenase system.

Some Rhizobium strains synthesize a unidirectional hydrogenase system in legume nodule bacteroids; this system participates in the recycling of hydrogen that otherwise would be lost as a by-product of the nitrogen fixation process. Soybeans inoculated with Rhizobium japonicum strains that synthesized the hydrogenase system fixed significantly more nitrogen and produced greater yields than plants inoculated with strains lacking hydrogen-uptake capacity. Rhizobium strains used as inocula for legumes should have the capability to synthesize the hydrogenase system as one of their desirable characteristics.

The H(2)-oxidizing complex in Rhizobium japonicum 122 DES bacteroids failed to catalyze, at a measurable rate, (2)H(1)H exchange from a mixture of (2)H(2) and (1)H(2) in presence of (2)H(2)O and (1)H(2)O, providing no evidence for reversibility of the hydrogenase reaction in vivo. In the H(2) oxidation reaction, there was no significant discrimination between (2)H(2) and (1)H(2), indicating that the initial H(2)-activation step in the over-all H(2) oxidation reaction is not rate-limiting. By use of improved methods, an apparent K(m) for H(2) of 0.05 micromolar was determined. The H(2) 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 H(2) oxidation and stimulated O(2) uptake. This and other evidence suggest the involvement of cytochromes and nonheme iron proteins in the pathway of electron transport from H(2) to O(2). Partial pressures of H(2) at 0.03 atmosphere and below had a pronounced inhibitory effect on endogenous respiration by bacteroid suspensions. The inhibition of CO(2) evolution by low partial pressures of H(2) suggests that H(2) 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 H(2) 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 CO(2) (initial concentration) in solution inhibited H(2) uptake by bacteroid suspensions by 18%. Further research is necessary to establish the significance of the inhibition of H(2) uptake by succinate, acetate, formate, and CO(2) in the metabolism of the H(2)-uptake-positive strains of Rhizobium.

The microsymbiont population in soybean root nodules (Glycine max L. cv Williams 82 inoculated with Bradyrhizobium japonicum 2143) was characterized during symbiotic development to determine the extent of heterogeneity in this population. The microsymbiont population was isolated by centrifugation through a continuous sucrose gradient (44 to 57% weight to weight ratio) and appeared homogeneous at each age examined up to 26 days after planting based on the symmetrical distribution of the population, enzyme activities, poly-beta-hydroxybutyrate contents, protein contents, and viabilities. Some differences in viability, protein content, and acetylene reduction activity were observed at later ages. The population migrated to progressively lighter buoyant densities with increasing age until a density equivalent to 48% sucrose was reached. The changing density correlated directly with the increasing poly-beta-hydroxybutyrate to protein ratio. The acetylene reduction activity, based on microsymbiont concentration, followed the same developmental pattern as whole nodules. On a protein basis, the decline of acetylene reduction activity was later and reflected the decrease in protein content per cell. These results suggested that the microsymbiont population, which resulted from inoculation of B. japonicum 2143 onto Williams 82 cultivar of soybeans, developed as a homogeneous population.

The activities of several enzymes of the citric acid and poly-beta-hydroxybutyrate cycles were measured in Rhizobium japonicum 3I1B-143 bacteroids which had been isolated from soybean nodules by sucrose gradient centrifugation. During the period of developing nitrogenase activity, the specific activity of fumarase, hydroxybutyrate dehydrogenase, beta-ketothiolase, and pyruvate dehydrogenase complex increased whereas acetoacetate-succinyl-CoA transferase and isocitrate dehydrogenase decreased. Malate dehydrogenase activity remained constant. The amount of available acetyl-CoA, based on pyruvate dehydrogenase activity, should be sufficient to support both metabolic cycles concurrently. The temporal relationship between nitrogenase activity and poly-beta-hydroxybutyrate accumulation has been reexamined.

Acetyl coenzyme A (acetyl-CoA) synthetase and acetate kinase were localized within the soluble portion of Bradyrhizobium japonicum bacteroids, and no appreciable activity was found elsewhere in the nodule. The presence of each acetate-activating enzyme was confirmed by separation of the two enzyme activities on a hydroxylapatite column, by substrate dependence of each enzyme in both the forward and reverse directions, by substrate specificity, by inhibition patterns, and also by identification of the reaction products by C(18) reverse-phase high-pressure liquid chromatography. Phosphotransacetylase activity, found in the soluble portion of the bacteroid, was dependent on the presence of potassium and was inhibited by added sodium. The greatest acetyl-CoA hydrolase activity was found in the root nodule cytosol, although appreciable activity also was found within the bacteroids. The combined specific activities of acetyl-CoA synthetase and acetate kinase-phosphotransacetylase were approximate to that of the pyruvate dehydrogenase complex, thus providing B. japonicum with sufficient capacity to generate acetyl-CoA.