37 results on '"Emerich, D. W."'
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2. Accumulation of the phytoalexin, glyceollin, in root nodules of soybean formed by effective and ineffective strains ofBradyrhizobium japonicum
- Author
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Karr, D. B., Emerich, D. W., and Karr, A. L.
- Published
- 1992
- Full Text
- View/download PDF
3. Alfalfa leaf senescence induced by drought stress: photosynthesis, hydrogen peroxide metabolism, lipid peroxidation and ethylene.
- Author
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Irigoyen, J. J., Emerich, D. W., and Sánchez-Díaz, M.
- Subjects
- *
ALFALFA , *DROUGHTS , *PHOTOSYNTHESIS , *HYDROGEN peroxide , *METABOLISM , *LIPIDS - Abstract
Exchange rates of CO2 and H2O and metabolism of hydrogen peroxide have been measured in leaves of alfalfa (Medicago sativa L. cv.. Aragón) under drought stress. The inhibitory effect of drought upon photosynthesis depended on the severity of the stress treatment. Leaf water potential (Ψleaf) down to -28 MPa reduced CO2 availability due to! stomata! closure and inhibited the rate of photosynthesis.. Leaf water potential lower than -2.8 MPa directly affected CO2 fixation, although CO2 was not limiting. Transpiration was more affected by stomata! closure than photosynthesis, which led to an apparent improvement in WUE (water use efficiency). Alfalfa leaves with Ψleaf lower than -.2.0 MPa had an increased quantum requirement, probably due to the severe stress. effect on photoenergetic reactions. Ethylene evolution from alfalfa leaves increased when they were subjected to Ψleaf of -1.6 MPa. Under more severe stress, the leaves showed low or almost no ethylene production. In parallel with the increase in ethylene production, alfalfa leaves exhibited an increased membrane lipid peroxidation index (malondialdehyd.e content) and an increased peroxide content. Superoxide .disniutase activity (SOD; EC 1.15.1.1) was not affected by drought stress. Catalase (EC 1.11.1.6) was inhibited at slight stress but significantly increased at a Ψleaf of -2.0 MPa. Peroxidase (EC 1.11.1.7) was progressively inhibited as drought stress developed. The possible implication of reactive O2 intermediates in drought stress-induced senescence of alfalfa leaves is discussed in the light of the pattern of enzymatic scavenging systems. [ABSTRACT FROM AUTHOR]
- Published
- 1992
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4. Phosphoenolpyruvate carboxylase, malate and alcohol dehydrogenase activities in alfalfa (Medicago sativa) nodules under water stress.
- Author
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Irigoyen, J. J., Emerich, D. W., and Sánchez‐Díaz, M.
- Subjects
- *
PYRUVATE kinase , *ALFALFA , *ALCOHOL dehydrogenase , *ALCOHOL , *MALATE dehydrogenase , *ZINC enzymes - Abstract
The effect of drought upon phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), malate dehydrogenase (MDH; EC 1.1.1.37), alcohol dehydrogenase (ADH; EC 1.1.1.1) and β-hydroxybutyrate dehydrogenase (β-OH-BDH; EC 1.1.130) enzyme activities as well as the leghemoglobin (Lb), malate and ethanol contents of alfalfa nodules (Medicago sativa L. cv Aragón) were examined. Both the leghemoglobin (Lb) content and the Lb/soluble protein ratio were significantly reduced at a nodule water potential (ψmod ) of -1.3 MPa. At lower ψmod,Lb content decreased further, but the ratio remained unchanged. Slight stress (-1.3 MPa) drastically affected acetylene reduction activity (ARA; 60% reduction)' whereas in vitro PEPC activity was maintained at relatively constant values. As stress progressed (-2.0 MPa), a simultaneous reduction in both activities was observed. Severe stress (ψmodlower than: -2.0 MPa) stimulated in vitro PEPC. Bacteroid β-OH-BDFI activity was stimulated by slight (-1.3 MPa) and moderate (-2.0 MPa) drought. MDH activity rose in slightly stressed nodules (ψmod -1.3 MPa). Greater water deficits sharply decreased. MDH activity to! values significant]y lower than those found in control nodules. Nodule malate content followed the same pattern as MDH. The plant fraction of the nodule showed constitutive ADH activity and contained ethanol, ADH was stimulated at slight (-1.3 MPa) and moderate drought levels (-2.0 MPa). Ethanol content showed similar behavior to ADH activity. Inhibition of ARA, reduction of Lb con tent and stimulation of the fermentative metabolism induced by water stress suggest some reduction in O2 availability within the nodule. [ABSTRACT FROM AUTHOR]
- Published
- 1992
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5. Functional Characterization of the Sinorhizobium meliloti Acetate Metabolism Genes aceA, SMc00767, and glcB.
- Author
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Ramírez-Trujillo, J. A., Encarnación, S., Salazar, E., de los Santos, A. García, Dunn, M. F., Emerich, D. W., Calva, E., and Hernández-Lucas, I.
- Subjects
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RHIZOBIUM meliloti , *ACETATES , *BACTERIAL metabolism , *GENETIC mutation , *BACTERIAL genetics - Abstract
The genes encoding malate synthase (glcB) and isocitrate lyase (aceA) and a 240-bp open reading frame (SMc00767) located downstream of aceA were isolated and functionally characterized in Sinorhizobium meliloti. Independent and double interposon mutants of each gene were constructed, and the corresponding phenotypes were analyzed. aceA mutants failed to grow on acetate, and mutants deficient in SMc00767 were also affected in acetate utilization. In contrast, mutants deficient in glcB grew on acetate similar to wild-type strain Rm5000. Complementation experiments showed that aceA and SMc00767 gene constructs were able to restore the growth on acetate in the corresponding single mutants. aceA-glcB, aceA-SMc00767, and glcB-SMc00767 double knockouts were also unable to grow on acetate, but this ability was recovered when the wild-type aceA-glcB or aceA-SMc00767 loci were introduced into the double mutants. These data confirm the functional role of aceA and SMc00767 and show that glcB, in the absence of SMc00767, is required for acetate metabolism. Isocitrate lyase and malate synthase activities were measured in strain Rm5000, the mutant derivatives, and complemented strains. aceA and glcB were able to complement the enzymatic activity lacking in the corresponding single mutants. The enzymatic activities also showed that SMc00767 represses the activity of isocitrate lyase in cells grown on acetate. Gene fusions confirmed the repressor role of SMc00767, which regulates aceA expression at the transcriptional level. Comparison of the transcriptional profiles of the SMc00767 mutant and wild-type strain Rm5000 showed that SMc00767 represses the expression of a moderate number of open reading frames, including aceA; thus, we propose that SMc00767 is a novel repressor involved in acetate metabolism in S. meliloti. Genetic and functional analyses indicated that aceA and SMc00767 constitute a functional two-gene operon, which is conserved in other α-proteobacteria. Alfalfa plants infected with the aceA and glcB mutants were not impaired in nodulation or nitrogen fixation, and so the glyoxylate cycle is not required in the Rhizobium-legume symbiosis. [ABSTRACT FROM AUTHOR]
- Published
- 2007
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6. Altered exopolysaccharides of Bradyrhizobium japonicum mutants correlate with impaired soybean lectin binding, but not with effective nodule formation.
- Author
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Karr DB, Liang RT, Reuhs BL, and Emerich DW
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- Arabinose metabolism, Bradyrhizobium genetics, Conjugation, Genetic, Culture Media, Escherichia coli genetics, Gluconates metabolism, Malates metabolism, Mannitol metabolism, Mutation, Plant Lectins, Plasmids, Polysaccharides genetics, Bradyrhizobium physiology, Lectins metabolism, Polysaccharides physiology, Soybean Proteins, Glycine max microbiology, Symbiosis physiology
- Abstract
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.
- Published
- 2000
- Full Text
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7. Enhanced attachment of Bradyrhizobium japonicum to soybean through reduced root colonization of internally seedborne microorganisms.
- Author
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Oehrle NW, Karr DB, Kremer RJ, and Emerich DW
- Subjects
- Bacterial Adhesion, Penicillin G pharmacology, Plant Roots drug effects, Seeds drug effects, Seeds microbiology, Glycine max drug effects, Symbiosis, Bradyrhizobium growth & development, Plant Roots microbiology, Glycine max microbiology
- Abstract
Internally seedborne microorganisms are those surviving common surface sterilization procedures. Such microbes often colonize the radicle surface of a germinating soybean (Glycine max) seed, introducing an undefined parameter into studies on attachment and infection by Bradyrhizobium japonicum. Bacterial isolates from surface-sterilized soybean seed, cv. Williams 82 and cv. Maverick, used in our studies, were identified as Agrobacterium radiobacter, Aeromonas sp., Bacillus spp., Chryseomonas luteola, Flavimonas oryzihabitans, and Sphingomonas paucimobilis. Growth of these microbes during seed germination was reduced by treating germinating seeds with 500 micrograms/mL penicillin G. The effects of this antibiotic on seedling development and on B. japonicum 2143 attachment, nodulation, and nitrogen fixation are reported here. Penicillin G treatment of seeds did not reduce seed germination or root tip growth, or affect seedling development. No differences in nodulation kinetics, nitrogen fixation onset or rates were observed. However, the number of B. japonicum attached to treated intact seedlings was enhanced 200-325%, demonstrating that other root-colonizing bacteria can interfere with rhizobial attachment. Penicillin G treatment of soybean seedlings can be used to reduce the root colonizing microbes, which introduce an undefined parameter into studies of attachment of B. japonicum to the soybean root, without affecting plant development.
- Published
- 2000
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8. Catabolism of alpha-ketoglutarate by a sucA mutant of Bradyrhizobium japonicum: evidence for an alternative tricarboxylic acid cycle.
- Author
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Green LS, Li Y, Emerich DW, Bergersen FJ, and Day DA
- Subjects
- Bradyrhizobium genetics, Decarboxylation, Gene Deletion, Glutamate Decarboxylase metabolism, Glutamic Acid metabolism, Ketoglutarate Dehydrogenase Complex genetics, Mutagenesis, Nitrogen Fixation, Succinate-Semialdehyde Dehydrogenase, Aldehyde Oxidoreductases metabolism, Bradyrhizobium enzymology, Carboxy-Lyases metabolism, Citric Acid Cycle, Ketoglutarate Dehydrogenase Complex metabolism, Ketoglutaric Acids metabolism
- Abstract
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.
- Published
- 2000
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9. Bradyrhizobium japonicum isocitrate dehydrogenase exhibits calcium-dependent hysteresis.
- Author
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Karr DB and Emerich DW
- Subjects
- Amino Acid Sequence, Bradyrhizobium genetics, Calcium metabolism, Hydrogen-Ion Concentration, Isocitrate Dehydrogenase genetics, Isocitrate Dehydrogenase isolation & purification, Kinetics, Magnesium metabolism, Molecular Sequence Data, Molecular Weight, Sequence Homology, Amino Acid, Bradyrhizobium enzymology, Isocitrate Dehydrogenase metabolism
- Abstract
Bradyrhizobium japonicum NADP(+)-dependent isocitrate dehydrogenase was purified both from cultured cells and from the symbiotic form of the bacteria and was found to be identical in terms of N-terminal amino acid sequence, kinetics, and physicochemical properties. Magnesium and glycerol were absolute requirements for maintaining enzyme activity. The N-terminal amino acid sequence of the enzyme was more similar to the sequences from soybean and yeast than to other bacterial sequences. There was no immunological cross-reaction of antibodies from B. japonicum isocitrate dehydrogenase to extracts of soybean, pea, or Escherichia coli, but there was detectable, although weak, cross-reaction of antibodies from E. coli with the B. japonicum enzyme. B. japonicum isocitrate dehydrogenase displayed strong inhibition by NADH, indicating that during symbiotic nitrogen fixation the enzyme activity would be markedly reduced in planta. The enzyme displayed a calcium-dependent hysteresis, with a pronounced lag lasting as long as 2 min. Hysteresis was evident at concentrations of magnesium less than 0.5 mM and calcium greater than 1 microM. The hysteresis could be alleviated by excess magnesium or by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. The results suggest two roles for magnesium during catalysis; one magnesium may be needed to convert the enzyme into the steady-state form and the second needed for chelation of isocitrate for catalysis. The calcium-dependent hysteretic behavior of B. japonicum NADP(+)-isocitrate dehydrogenase suggested that this metal could serve as an intracellular regulator during symbiosis., (Copyright 2000 Academic Press.)
- Published
- 2000
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10. Alanine, not ammonia, is excreted from N2-fixing soybean nodule bacteroids.
- Author
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Waters JK, Hughes BL 2nd, Purcell LC, Gerhardt KO, Mawhinney TP, and Emerich DW
- Abstract
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.
- Published
- 1998
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11. Isocitrate dehydrogenase and glyoxylate cycle enzyme activities in Bradyrhizobium japonicum under various growth conditions.
- Author
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Green LS, Karr DB, and Emerich DW
- Subjects
- 3-Hydroxybutyric Acid, Acetates metabolism, Enzyme Induction, Enzyme Stability, Hydroxybutyrates metabolism, Isocitrate Lyase biosynthesis, Kinetics, Malate Synthase metabolism, Plant Roots microbiology, Glycine max microbiology, Symbiosis, Glyoxylates metabolism, Isocitrate Dehydrogenase metabolism, Isocitrate Lyase metabolism, Rhizobiaceae enzymology
- Abstract
Bradyrhizobium japonicum, the nitrogen-fixing symbiotic partner of soybean, was grown on various carbon substrates and assayed for the presence of the glyoxylate cycle enzymes, isocitrate lyase and malate synthase. The highest levels of isocitrate lyase [165-170 nmol min-1 (mg protein)-1] were found in cells grown on acetate or beta-hydroxybutyrate, intermediate activity was found after growth on pyruvate or galactose, and very little activity was found in cells grown on arabinose, malate, or glycerol. Malate synthase activity was present in arabinose- and malate-grown cultures and increased by only 50-80% when cells were grown on acetate. B. japonicum bacteroids, harvested at four different nodule ages, showed very little isocitrate lyase activity, implying that a complete glyoxylate cycle is not functional during symbiosis. The apparent Km of isocitrate lyase for D,L-isocitrate was fourfold higher than that of isocitrate dehydrogenase (61.5 and 15.5 microM, respectively) in desalted crude extracts from acetate-grown B. japonicum. When isocitrate lyase was induced, neither the Vmax nor the D,L-isocitrate Km of isocitrate dehydrogenase changed, implying that isocitrate dehydrogenase is not inhibited by covalent modification to facilitate operation of the glyoxylate cycle in B. japonicum.
- Published
- 1998
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12. The Formation of Nitrogen-Fixing Bacteroids Is Delayed but Not Abolished in Soybean Infected by an [alpha]-Ketoglutarate Dehydrogenase-Deficient Mutant of Bradyrhizobium japonicum.
- Author
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Green LS and Emerich DW
- Abstract
A mutant strain of Bradyrhizobium japonicum USDA 110 devoid of [alpha]-ketoglutarate dehydrogenase activity (LSG184) was used to test whether this tricarboxylic acid cycle enzyme is necessary to support nitrogen fixation during symbiosis with soybean (Glycine max). LSG184 formed nodules about 5 d later than the wild-type strain, and the nodules, although otherwise normal in structure, contained many fewer infected host cells than is typical. At 19 d after inoculation cells infected with the mutant strain were only partially filled with bacteroids and showed large accumulations of starch, but by 32 d after inoculation the host cells infected with the mutant appeared normal. The onset of nitrogen fixation was delayed about 15 d for plants inoculated with LSG184, and the rate, on a per nodule fresh weight basis, reached only about 20% of normal. However, because nodules formed by LSG184 contained only about 20% of the normal number of bacteroids, it could be inferred that the mutant, on an individual bacteroid basis, was fixing nitrogen at near wild-type rates. Therefore, the loss of [alpha]-ketoglutarate dehydrogenase in B. japonicum does not prevent the formation or the functioning of nitrogen-fixing bacteroids in soybean.
- Published
- 1997
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13. Bradyrhizobium japonicum does not require alpha-ketoglutarate dehydrogenase for growth on succinate or malate.
- Author
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Green LS and Emerich DW
- Subjects
- Amino Acid Sequence, Carbohydrate Metabolism, Cloning, Molecular, Codon genetics, Genes, Bacterial genetics, Glutamic Acid metabolism, Isocitrate Lyase metabolism, Ketoglutarate Dehydrogenase Complex genetics, Molecular Sequence Data, Mutation, Quaternary Ammonium Compounds metabolism, Restriction Mapping, Rhizobiaceae enzymology, Rhizobiaceae genetics, Rhizobiaceae metabolism, Sequence Analysis, DNA, Succinic Acid, Ketoglutarate Dehydrogenase Complex physiology, Malates metabolism, Rhizobiaceae growth & development, Succinates metabolism
- Abstract
The sucA gene, encoding the E1 component of alpha-ketoglutarate dehydrogenase, was cloned from Bradyrhizobium japonicum USDA110, and its nucleotide sequence was determined. The gene shows a codon usage bias typical of non-nif and non-fix genes from this bacterium, with 89.1% of the codons being G or C in the third position. A mutant strain of B. japonicum, LSG184, was constructed with the sucA gene interrupted by a kanamycin resistance marker. LSG184 is devoid of alpha-ketoglutarate dehydrogenase activity, indicating that there is only one copy of sucA in B. japonicum and that it is completely inactivated in the mutant. Batch culture experiments on minimal medium revealed that LSG184 grows well on a variety of carbon substrates, including arabinose, malate, succinate, beta-hydroxybutyrate, glycerol, formate, and galactose. The sucA mutant is not a succinate auxotroph but has a reduced ability to use glutamate as a carbon or nitrogen source and an increased sensitivity to growth inhibition by acetate, relative to the parental strain. Because LSG184 grows well on malate or succinate as its sole carbon source, we conclude that B. japonicum, unlike most other bacteria, does not require an intact tricarboxylic acid (TCA) cycle to meet its energy needs when growing on the four-carbon TCA cycle intermediates. Our data support the idea that B. japonicum has alternate energy-yielding pathways that could potentially compensate for inhibition of alpha-ketoglutarate dehydrogenase during symbiotic nitrogen fixation under oxygen-limiting conditions.
- Published
- 1997
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14. Protein Synthesis by Bradyrhizobium japonicum Bacteroids Declines as a Function of Nodule Age.
- Author
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Karr DB and Emerich DW
- Abstract
Isolated bacteroids of Bradyrhizobium japonicum accumulated exogenously supplied [(sup35)S]methionine or [(sup3)H]leucine and incorporated them into cytosolic proteins. The accumulation of these labeled amino acids was inhibited by azide. Only 3 to 6% of these accumulated amino acids were incorporated into protein. Protein synthesis was not stimulated by incubation of bacteroids in the presence of potassium salts, malate, or amino acids, but azide, chloramphenicol, and acridine did inhibit the process. No prominent differences were observed in autoradiograms after sodium dodecyl sulfate-polyacrylamide gel electrophoresis of (sup35)S-labeled bacteroid proteins as a function of nodule age. The rates of protein synthesis and protein turnover declined during nodule development. Protein synthesis declined about 60% between 14 and 20 days after planting, which is the period of a rapid increase in acetylene reduction activity. This correlation suggests a metabolic mechanism by which significant amounts of cellular energy are diverted to the nitrogen fixation process.
- Published
- 1996
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15. Alanine dehydrogenase from soybean nodule bacteroids: purification and properties.
- Author
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Smith MT and Emerich DW
- Subjects
- Alanine Dehydrogenase, Amino Acid Oxidoreductases drug effects, Amino Acid Oxidoreductases isolation & purification, Ammonia metabolism, Cell Compartmentation, Cytosol enzymology, NAD metabolism, Pyruvates metabolism, Sodium Chloride pharmacology, Glycine max microbiology, Amino Acid Oxidoreductases metabolism, Rhizobiaceae enzymology, Symbiosis
- Abstract
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, NH4+, pyruvate, L-alanine, and NAD were 86 microM, 8.9 mM, 0.49 mM, 1 mM and 200 microM, respectively. High concentrations of pyruvate, L-alanine, or NH4+ caused inhibition of activity with Ki'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.
- Published
- 1993
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16. Alanine dehydrogenase from soybean nodule bacteroids. Kinetic mechanism and pH studies.
- Author
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Smith MT and Emerich DW
- Subjects
- Alanine Dehydrogenase, Ammonia metabolism, Hydrogen-Ion Concentration, Kinetics, Microcomputers, Models, Theoretical, NAD metabolism, Oxidation-Reduction, Pyruvates metabolism, Amino Acid Oxidoreductases metabolism, Rhizobiaceae enzymology, Glycine max microbiology
- Abstract
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.
- Published
- 1993
17. Fluorescence studies with malate dehydrogenase from Bradyrhizobium japonicum 3I1B-143 bacteroids: a two-tryptophan containing protein.
- Author
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Ghiron CA, Eftink MR, Waters JK, and Emerich DW
- Subjects
- Kinetics, Lasers, NAD metabolism, Oxidation-Reduction, Protein Binding, Spectrometry, Fluorescence methods, Malate Dehydrogenase metabolism, Rhizobiaceae enzymology, Tryptophan
- Abstract
A number of fluorescence studies, both of trp residues and bound NADH, have been reported for porcine malate dehydrogenase (MDH). The large number of trp residues (six) complicates the interpretation of some studies. To circumvent this we have performed studies with a two-tryptophan (per subunit) MDH from Bradyrhizobium japonicum 3I1B-143 bacteroids. We have performed phase/modulation fluorescence lifetime measurements, as a function of temperature and added quencher KI, in order to resolved the 1.2-ns (blue) and 6.5-ns (red) contributions from the two classes of trp residues. Anisotropy decay studies have also been performed. The binding of NADH dynamically quenches the fluorescence of both trp residues, but, unlike mammalian cytoplasmic and mitochondrial MDH, there is not a large enhancement in fluorescence of bound NADH upon forming a ternary complex with either tartronic acid or D-malate.
- Published
- 1990
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18. Purification and properties of acetyl-CoA synthetase from Bradyrhizobium japonicum bacteroids.
- Author
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Preston GG, Wall JD, and Emerich DW
- Subjects
- Acetate-CoA Ligase metabolism, Acetates metabolism, Acetic Acid, Adenosine Triphosphate metabolism, Cations, Divalent, Chromatography, Coenzyme A metabolism, Electrophoresis, Polyacrylamide Gel, Hydrogen-Ion Concentration, Kinetics, Magnesium Chloride pharmacology, Molecular Weight, Nucleotides metabolism, Substrate Specificity, Acetate-CoA Ligase isolation & purification, Coenzyme A Ligases isolation & purification, Rhizobiaceae enzymology
- Abstract
Acetyl-CoA synthetase was purified 800-fold from Bradyrhizobium japonicum bacteroids. A specific activity of 16 mumol/min per mg of protein was achieved, with a 30-40% yield. The purification scheme consisted of only three consecutive chromatography steps. The enzyme has a native Mr of 150,000, estimated by gel-permeation chromatography, and a subunit Mr of 72,000, determined by SDS/polyacrylamide-gel electrophoresis. The optimum pH and temperature are 8.5 and 50 degrees C respectively. The Km values for acetate, CoA and ATP were 146, 202 and 275 microM respectively. The reaction was specific for acetate, as propionate and oleate were used very poorly. Likewise, the enzyme used only ATP, ADP or dATP; AMP, GTP, XTP and UTP could not replace ATP. Acetyl-CoA synthetase showed a broad specificity for metals; MnCl2 could replace MgCl2. In addition, CaCl2 and CoCl2 were approx. 50% as effective as MgCl2, but FeCl3, NiCl2 or ZnCl2 could not effectively substitute for MgCl2. The enzyme may be regulated by NADP+ and pyruvate; no effect was seen of amino acids, glucose catabolites, reduced nicotinamide nucleotides or acetyl-CoA. Inhibition was seen with AMP, PPi, FMN and pyridoxal phosphate, with Ki values of 720, 222, 397 and 1050 microM respectively.
- Published
- 1990
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19. Developmental regulation of enzymes of sucrose and hexose metabolism in effective and ineffective soybean nodules.
- Author
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Anthon GE and Emerich DW
- Abstract
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.
- Published
- 1990
- Full Text
- View/download PDF
20. Oxyleghemoglobin-mediated Hydrogen Oxidation by Rhizobium japonicum USDA 122 DES Bacteroids.
- Author
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Emerich DW, Albrecht SL, Russell SA, Ching T, and Evans HJ
- Abstract
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.
- Published
- 1980
- Full Text
- View/download PDF
21. Hydrogenase system in legume nodules: a mechanism of providing nitrogenase with energy and protection from oxygen damage.
- Author
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Ruiz-Argüeso T, Emerich DW, and Evans HJ
- Subjects
- Hydrogen pharmacology, Nitrogen Fixation, Partial Pressure, Succinates pharmacology, Nitrogenase metabolism, Oxidoreductases metabolism, Oxygen pharmacology, Rhizobium enzymology
- Published
- 1979
- Full Text
- View/download PDF
22. Interactions of dinitrogenase and dinitrogenase reductase.
- Author
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Emerich DW, Hageman RV, and Burris RH
- Subjects
- Adenosine Triphosphate, Bacteria enzymology, Electron Transport, Kinetics, Macromolecular Substances, Ferredoxins metabolism, Nitrogenase metabolism
- Published
- 1981
- Full Text
- View/download PDF
23. Adenylate cyclase and cyclic AMP phosphodiesterase in Bradyrhizobium japonicum bacteroids.
- Author
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Catanese CA, Emerich DW, and Zahler WL
- Subjects
- Detergents pharmacology, Enzyme Activation, Kinetics, Rhizobiaceae ultrastructure, Sodium Dodecyl Sulfate pharmacology, Subcellular Fractions enzymology, 3',5'-Cyclic-AMP Phosphodiesterases metabolism, Adenylyl Cyclases metabolism, Rhizobiaceae enzymology
- Abstract
Adenylate cyclase and cyclic AMP (cAMP) phosphodiesterase have been identified and partially characterized in bacteroids of Bradyrhizobium japonicum 3I1b-143. Adenylate cyclase activity was found in the bacteroid membrane fraction, whereas cAMP phosphodiesterase activity was located in both the membrane and the cytosol. In contrast to other microorganisms, B. japonicum adenylate cyclase remained firmly bound to the membrane during treatment with detergents. Adenylate cyclase was activated four- to fivefold by 0.01% sodium dodecyl sulfate (SDS), whereas other detergents gave only slight activation. SDS had no effect on the membrane-bound cAMP phosphodiesterase but strongly inhibited the soluble enzyme, indicating that the two enzymes are different. All three enzymes were characterized by their kinetic constants, pH optima, and divalent metal ion requirements. With increasing nodule age, adenylate cyclase activity increased, the membrane-bound cAMP phosphodiesterase decreased, and the soluble cAMP phosphodiesterase remained largely unchanged. These results suggest that cAMP plays a role in symbiosis.
- Published
- 1989
- Full Text
- View/download PDF
24. Analysis of Poly-beta-Hydroxybutyrate in Rhizobium japonicum Bacteroids by Ion-Exclusion High-Pressure Liquid Chromatography and UV Detection.
- Author
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Karr DB, Waters JK, and Emerich DW
- Abstract
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.
- Published
- 1983
- Full Text
- View/download PDF
25. Hydrogen-dependent nitrogenase activity and ATP formation in Rhizobium japonicum bacteroids.
- Author
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Emerich DW, Ruiz-Argüeso T, Ching TM, and Evans HJ
- Subjects
- Ethane metabolism, Hydrogen pharmacology, Iodoacetates pharmacology, Oxidative Phosphorylation, Oxygen Consumption drug effects, Succinates pharmacology, Adenosine Triphosphate biosynthesis, Hydrogen metabolism, Nitrogenase metabolism, Rhizobium metabolism
- Abstract
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.
- Published
- 1979
- Full Text
- View/download PDF
26. Hydrogenase in Rhizobium japonicum Increases Nitrogen Fixation by Nodulated Soybeans.
- Author
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Albrecht SL, Maier RJ, Hanus FJ, Russell SA, Emerich DW, and Evans HJ
- Abstract
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.
- Published
- 1979
- Full Text
- View/download PDF
27. Investigation of the H(2) Oxidation System in Rhizobium japonicum 122 DES Nodule Bacteroids.
- Author
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Emerich DW, Ruiz-Argüeso T, Russell SA, and Evans HJ
- Abstract
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.
- Published
- 1980
- Full Text
- View/download PDF
28. Uniformity of the microsymbiont population from soybean nodules with respect to buoyant density.
- Author
-
Karr DB and Emerich DW
- Abstract
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.
- Published
- 1988
- Full Text
- View/download PDF
29. Nitrogenase from Bacillus polymyxa. Purification and properties of the component proteins.
- Author
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Emerich DW and Burris RH
- Subjects
- Adenosine Triphosphate, Electron Spin Resonance Spectroscopy, Iron analysis, Lipopolysaccharides isolation & purification, Molecular Weight, Molybdenum analysis, Protein Binding, Species Specificity, Sulfides analysis, Bacillus enzymology, Nitrogenase isolation & purification
- Abstract
A purification procedure is described for the components of Bacillus polymyxa nitrogenase. The procedure requires the removal of interfering mucopolysaccharides before the two nitrogenase proteins can be purified by the methods used with other nitrogenase components. The highest specific activities obtained were 2750 nmol C2H4 formed . min-1 . mg-1 MoFe protein and 2521 nmol C2H4 formed . min-1 . mg-1 Fe protein. The MoFe protein has a molecular weight of 215 000 and contains 2 molybdenum atoms, 33 iron atoms and 21 atoms of acid-labile sulfur per protein molecule. The Fe protein contains 3.2 iron atoms and 3.6 acid-labile sulfur atoms per molecule of 55 500 molecular weight. Each Fe protein binds two ATP molecules. The EPR spectra are similar to those of other nitrogenase proteins. MgATP changes the EPR of the Fe protein from a rhombic to an axial-type signal.
- Published
- 1978
- Full Text
- View/download PDF
30. Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids: purification and properties.
- Author
-
Suzuki F, Zahler WL, and Emerich DW
- Subjects
- Acetyl-CoA C-Acetyltransferase antagonists & inhibitors, Acetyl-CoA C-Acetyltransferase isolation & purification, Kinetics, Magnesium pharmacology, Molecular Weight, NAD pharmacology, Acetyl-CoA C-Acetyltransferase metabolism, Acetyltransferases metabolism, Rhizobiaceae enzymology
- Abstract
Acetoacetyl-CoA thiolase of Bradyrhizobium japonicum bacteroids has been purified greater than 130-fold. The enzyme has a molecular weight of 180,000 +/- 15,000 and consists of four identical subunits of 44,000 +/- 2,000. The enzyme was specific for acetoacetyl-CoA; ketodecanoyl-CoA did not serve as a substrate. Catalysis proceeds via a ping-pong mechanism. Iodoacetamide effectively inhibited the enzyme but acetoacetyl-CoA provided considerable protection against this compound. Magnesium was found to inhibit both the thiolysis reaction and the condensation reaction. Acetoacetyl-CoA thiolysis activity was not affected by potassium, ammonium, or several organic acids but was found to be inhibited by NADH. The inhibition by NADH may have an effect during the decline of the symbiosis.
- Published
- 1987
- Full Text
- View/download PDF
31. Analysis of lectin binding by Bradyrhizobium japonicum strains grown on nitrocellulose filters using peroxidase-labeled lectin.
- Author
-
Liang R and Emerich DW
- Subjects
- Binding Sites, Collodion, Fluorescein-5-isothiocyanate, Fluoresceins, Mutation, Peroxidases, Rhizobium genetics, Thiocyanates, Lectins metabolism, Plant Lectins, Rhizobium metabolism, Soybean Proteins
- Abstract
A procedure was developed to assess the ability of wild-type and mutant strains of Bradyrhizobium japonicum to bind soybean lectin. The lectin-binding ability of bacteria grown on nitrocellulose filters was determined using peroxidase-labeled soybean lectin. The assay produced clear differences between strains known to be unable to bind soybean lectin and those which can. The assay gave results identical to those of the fluorescein isothiocyanate-soybean lectin-binding assay of T. V. Bhuvaneswari, S. G. Pueppke, and W. D. Bauer (1977, Plant Physiol. 60, 486-491) with regard both to the ability of particular B. japonicum strains to bind lectin and to the inhibition caused by N-acetyl-D-galactosamine. The method was used to screen Tn5-induced mutants of B. japonicum 2143 for their inability to bind soybean lectin. The procedure provides a sensitive and convenient method to screen Bradyrhizobium strains for the ability to bind soybean lectin.
- Published
- 1987
- Full Text
- View/download PDF
32. Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.
- Author
-
Karr DB and Emerich DW
- Subjects
- Culture Media, In Vitro Techniques, Kinetics, Mannitol metabolism, Molecular Weight, Phosphorylation, Glycine max microbiology, Succinates metabolism, Symbiosis, Bacterial Proteins metabolism, Phosphoproteins metabolism, Rhizobiaceae metabolism
- Abstract
Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.
- Published
- 1989
- Full Text
- View/download PDF
33. Complementary functioning of the component proteins of nitrogenase from several bacteria.
- Author
-
Emerich DW and Burris RH
- Subjects
- Acetylene metabolism, Ammonia metabolism, Cross Reactions, Nitrogen Fixation, Oxidation-Reduction, Protein Binding, Species Specificity, Bacteria enzymology, Bacterial Proteins metabolism, Nitrogenase metabolism
- Abstract
The nitrogenase proteins from eight organisms have been highly purified, and a survey of their cross-reactions shows that the nitrogenase proteins from a wide variety of organisms can interact with one another. An active cross-reaction is the complementary functioning of the MoFe protein and the Fe protein from different organisms. Of 64 possible combinations of component proteins, 8 yielded homologous nitrogenases (components from the same organism); 45 of the 56 possible heterologous crosses generated active hybrid nitrogenases; 4 heterologous crosses yielded no measurable nitrogenase activity but did form inactive tight-binding complexes; 6 crosses did not give measurable activity; and 1 cross was not made. All these crosses were assayed for acetylene reduction, and some also were assayed for ammonia formation, hydrogen evolution, and ATP hydrolysis activity. The activity generated by combining two complementary heterologous nitrogenase components depended on pH, component ratio, and protein concentration, the same factors that determine the activity of homologous nitrogenases. However, several crosses showed an unusual dependency on component ratio and protein concentration, and some cross-reactions showed interesting ATP hydrolysis activity.
- Published
- 1978
- Full Text
- View/download PDF
34. Enzymes of the Poly-beta-Hydroxybutyrate and Citric Acid Cycles of Rhizobium japonicum Bacteroids.
- Author
-
Karr DB, Waters JK, Suzuki F, and Emerich DW
- Abstract
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.
- Published
- 1984
- Full Text
- View/download PDF
35. Acetate-Activating Enzymes of Bradyrhizobium japonicum Bacteroids.
- Author
-
Preston GG, Zeiher C, Wall JD, and Emerich DW
- Abstract
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.
- Published
- 1989
- Full Text
- View/download PDF
36. Nitrogenase: properties of the catalytically inactive complex between the Azotobacter vinelandii MoFe protein and the Clostridium pasteurianum Fe protein.
- Author
-
Emerich DW, Ljones T, and Burris RH
- Subjects
- Adenosine Triphosphate metabolism, Azotobacter enzymology, Binding Sites, Chelating Agents, Clostridium enzymology, Macromolecular Substances, Metalloproteins pharmacology, Nitrogenase antagonists & inhibitors, Oxidation-Reduction, Protein Binding, Iron, Metalloproteins metabolism, Molybdenum, Nitrogenase metabolism
- Abstract
The catalytically inactive complex generated by the combination of the Azotobacter vinelandii MoFe protein (Av1) and the Clostridium pasteurianum Fe protein (Cp2) inhibits N2 reduction, C2H3 reduction, H+ reduction and ATP hydrolysis catalyzed by the homologous nitrogenases. Kinetic data indicate that the inactive complex consists of two molecules of Cp2 to one molecule of Av1, with values for the inhibitor constant in the range of 1--10 nM. Inhibition of C. pasteurianum nitrogenase by Av1 produces a lag phase in acetylene reduction that increases with increasing Av1. The lag phase is found only at levels of Av1 sufficient to keep the ratio of Cp2 : Cp1 lower than 2. Gel filtration of a mixture of Av1 and Cp2 provides evidence for complex formation and indicates that each Av1 molecule binds more than one Cp2 molecule. The Av1-Cp2 complex binds two molecules of MgATP per molecule of Cp2. MgATP is not required for complex formation, but complex formation lowers the MgATP-Cp2 dissociation constant approx. 3-fold. Av1 protects the iron-sulfur center in Cp2 completely against the MgATP-induced reaction with chelators. This provides additional evidence for formation of the Av1-Cp2 complex and together with the results of the MgATP-binding studies suggests that the two binding sites for MgATP are some distance away from the iron-sulfur site on Cp2.
- Published
- 1978
- Full Text
- View/download PDF
37. Preparation of nitrogenase.
- Author
-
Emerich DW and Burris RH
- Subjects
- Crystallization, Iron, Metalloproteins isolation & purification, Molybdenum, Nitrogenase metabolism, Species Specificity, Azotobacter enzymology, Bacillus enzymology, Nitrogenase isolation & purification, Rhodospirillum rubrum enzymology
- Published
- 1978
- Full Text
- View/download PDF
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