Binding Characteristics of N2-Fixing Bacteria to Cereal Roots

The attachment of Rhizobium japonicum 61A89 and Rhizobium spp. 32H1 to the roots of wheat and rice seedlings is analyzed in terms of an equilibrium model. A Langmuir adsorption isotherm describes the binding. Strain 61A89 binds to a greater extent than does strain 32H1, and the equilibrium constants for each strain binding to wheat are strongly temperature dependent. Both time-dependent dissociation and association, predicted by an equilibrium model, have been found. The dissociation rate constant for 32H1 is approximately twice that of 61A89, and each is weakly temperature dependent. The rate equation for the binding of exponentially growing 61A89 to wheat roots has been solved as a function of time. Theory and experiment both indicate that the binding at very short times is much less than the equilibrium values. The binding of Azotobacter vinelandii 12837 to wheat roots has also been measured. Root-associated Azotobacter fixes nitrogen, whereas under aerobic growth conditions, root-associated 61A89 and 32H1 do not. The effect of metabolic inhibitors and antibiotics on the binding of Rhizobia and Azotobacter was examined.     

Associative effect of inoculation of different strains of Azotobacter and homologous rhizobium on the yield of moong (Vigna radiata), soybean (Glycine max) and pea (Pisum sativum)

Summary It has been observed that in the case ofVigna radiata andGlycine max incorporation of suitable strain of Azotobacter gave higher yield than obtained by the use of Rhizobium as inoculant. In the case ofVigna radiata even a strain of Azotobacter isolated from the rhizosphere of berseem gave similar yields as Rhizobium. In the case ofPisum sativum association of Rhizobium with a strain ofAzotobacter chroococcum isolated from the rhizosphere of pea gave numerically higher yield than Rhizobium alone. It may be possible that statistically higher yield may be obtained when a suitable strain of Azotobacter is used after screening a large number of strains of Azotobacter from the rhizosphere of pea.     

Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.     

Transposon mediation allows a symbiotic plasmid of Rhizobium leguminosarum bv. trifolii to become a symbiosis island in Agrobacterium and Rhizobium

The symbiotic plasmid (pSym) of Rhizobium leguminosarum bv. trifolii 4S5, which carries Tn5-mob, was successfully transferred into Agrobacterium tumefaciens A136 by using a conjugation method. The resulting transconjugants induced the development of ineffective nitrogen-fixing nodules on the roots of white clover seedlings. Depending on the manner in which the pSym was retained, the transconjugants were divided into two groups of strains, Afp and Afcs. pSym was retained as a plasmid in the Afp strains but was integrated into the int gene encoding a phage-related integrase on the linear chromosome of A. tumefaciens A136 in strain Afcs1 (one of the Afcs strains) to form a symbiosis island. Conjugation was performed between strain Afcs1 and R. leguminosarum bv. trifolii H1 (a pSym-cured derivative of wild-type strain 4S), and the Rhizobium H1tr strains were screened as transconjugants. Eighteen of the H1tr strains induced effective nitrogen-fixing nodules on the roots of the host plants. pSym was transferred into all of the transconjugants, except for strain H1tr1, at the same size as pSym of strain 4S5. In strain H1tr1, pSym was integrated into the chromosome as a symbiosis island. These data suggest that pSym can exist among Rhizobium and Agrobacterium strains both as a plasmid and as a symbiosis island with transposon mediation.     

Root Hair Deformation, Bacterial Attachment, and Plant Growth in Wheat-Azospirillum Associations

Seven Azospirillum strains induced more deformation of root hairs of wheat than did strains of Rhizobium leguminosarum, Azotobacter chroococcum, or Escherichia coli. Azospirillum sp. strain Sp245 caused the most deformation. Strain Sp245 (isolated from surface sterile roots of wheat) and strain Sp7 (isolated from the rhizosphere of a forage grass) were compared with regard to their effects on root hair deformation, their attachment to roots, and their effects on the growth of four wheat cultivars. The amount of deformation caused by the two strains in the four cultivars increased in the following order: cv. Tobari, cv. Tonari, cv. BH1146, cv. Lagoa. Strain Sp245 attached to the roots of all cultivars in low numbers, and attachment did not increase with time (up to 48 h). Strain Sp7 attached in higher numbers, and attachment increased with time. Inoculation of the four cultivars of wheat had pronounced effects on root mass measured at maturity. The magnitude of the effects in the four cultivars increased in the following order: Tobari, Tonari, BH1146, Lagoa; these effects were progressively more positive for strain Sp245 and progressively more negative for strain Sp7. Concentrations of N in wheat did not vary substantially between cultivars or strains. Concentrations of K and P did not vary substantially between cultivars but did vary between strains, Sp245 effecting increases and Sp7 effecting decreases.     

Root nodule bacteria to improve yield of wheat

Summary Beneficial effects on yield and N-assimilation in wheat cv. HD2329 due to inoculation with nine strains belonging to six species and cowpea miscellany of genus rhizobium and Azotobacter were observed. Stimulation of soil Azotobacter and Azospirillum in the root region of wheat occurred due to seed inoculation with Rhizobium.     

Evidence for a single forskolin-binding site in rat adipocyte membrane. Studies of [14,15-3H]dihydroforskolin binding and adenylate cyclase activation

[14,15-3H] Dihydroforskolin has been used as a tracer in the study of forskolin binding to adipocyte plasma membrane and the subsequent activation of adenylate cyclase (EC 4.6.1.1) of this membrane. The specific binding of radioactivity to the membrane was rapid, temperature-dependent, saturable, and readily reversible. The equilibrium dissociation reaction constant (KD) for the binding was 13 microM, with a maximum binding (Bmax) of 61 pmol of forskolin per mg of membrane protein. The Hill coefficient was 1.0. The bound [14,15-3H] dihydroforskolin was displaced by forskolin with rate constants of 0.07 X 10(6) M-1 min-1 and 1.2 min-1 for the association and dissociation reactions, respectively (30 degrees C). The equilibrium dissociation constant (KD) was approximately the same as the concentration that produced half-maximum activation (EC50) of the adenylate cyclase of rat adipocyte plasma membrane. There was a linear correlation between forskolin binding and adenylate cyclase activation. The results are consistent with the concept of a single class of binding site which binds forskolin. [14,15-3H] Dihydroforskolin appears to be a potentially useful tracer in the study of the mechanism of activation of the catalytic unit of adipocyte adenylate cyclase.     

Selective modulation of protein C affinity for EPCR and phospholipids by Gla domain mutation

Uniquely amongst vitamin K-dependent coagulation proteins, protein C interacts via its Gla domain both with a receptor, the endothelial cell protein C receptor (EPCR), and with phospholipids. We have studied naturally occurring and recombinant protein C Gla domain variants for soluble (s)EPCR binding, cell surface activation to activated protein C (APC) by the thrombin-thrombomodulin complex, and phospholipid dependent factor Va (FVa) inactivation by APC, to establish if these functions are concordant. Wild-type protein C binding to sEPCR was characterized with surface plasmon resonance to have an association rate constant of 5.23 x 10(5) m(-1).s(-1), a dissociation rate constant of 7.61 x 10(-2) s(-1) and equilibrium binding constant (K(D)) of 147 nm. It was activated by thrombin over endothelial cells with a K(m) of 213 nm and once activated to APC, rapidly inactivated FVa. Each of these interactions was dramatically reduced for variants causing gross Gla domain misfolding (R-1L, R-1C, E16D and E26K). Recombinant variants Q32A, V34A and D35A had essentially normal functions. However, R9H and H10Q/S11G/S12N/D23S/Q32E/N33D/H44Y (QGNSEDY) variants had slightly reduced (< twofold) binding to sEPCR, arising from an increased rate of dissociation, and increased K(m) (358 nm for QGNSEDY) for endothelial cell surface activation by thrombin. Interestingly, these variants had greatly reduced (R9H) or greatly enhanced (QGNSEDY) ability to inactivate FVa. Therefore, protein C binding to sEPCR and phospholipids is broadly dependent on correct Gla domain folding, but can be selectively influenced by judicious mutation.     

Adsorption of rhizobia to cereal roots

The attachment of $\text{Rhizobium}\text{japonicum}$ 61A89 to the roots of wheat and rice seedlings is an equilibrium process that follows a Langmuir adsorption isotherm. This model predicts a maximum of 8 × 10⁹ viable bound bacteria per g of root. Different strains of bound rhizobia have both characteristic appearances and surface densities on the root surface. The bound rhizobia did not fix nitrogen.     

Molecular analysis of an essential gene upstream of rpoN in Rhizobium NGR234

Rhizobium sp. NGR234 is a broad-host range strain. The rpoN gene of this organism encodes a sigma factor which is a primary co-regulator of endosymbiosis. We characterized the locus upstream of rpoN, and identified a contiguous open reading frame, here termed ORF1. DNA sequence analysis of this ORF showed that it encoded a polypeptide highly conserved with a corresponding ORF of Rhizobium meliloti. The gene product contained two ATP/GTP binding pockets. Codon usage in the ORF and the nitrogenase operon nifKDH of NGR234 was similar. Although we used a non-transposable cassette flanked by appropriate sized DNA fragments, we were unable to isolate site-directed mutants in the ORF, whose ATP/GTP binding protein product is thus probably of essential biological function. ORF1 and rpoN exhibited conserved linkage among diverse rhizobia, and in Azotobacter vinelandii. Intragenomic and interspecific homology studies confirmed directly that ORF1 (NGR234) belonged to a large family of ATP-binding protein genes.     

Microbial inoculation of plants

Summary Seed of maize, tomato, and wheat was inoculated with cultures of Azotobacter, Clostridium, and a nitrogen-fixing facultative Bacillus and grown in a nutrient-deficient sand and a highly fertile silt loam.In sand, wheat showed a significant positive response to inoculation with Azotobacter and Clostridium but maize and tomato were unaffected by inoculation.When inoculated seed was planted in Lima silt loam there were significant increases in the growth of maize, tomato, and wheat to treatment with Clostridium, inoculated maize and wheat responded to Azotobacter inoculation while only wheat responded to inoculation with the facultative Bacillus.In pure-culture studies of the ability of these cultures to establish upon plant roots it was shown that Azotobacter did not colonize the roots of lucerne, maize, tomato, or wheat to any great extent. Bacillus and Clostridium were moderate colonizers of plant roots reaching from 1 to 20 per cent the levels reached byPseudomonas fluorescens on the same plants.The author held a Fulbright Travel Grant for the 1961–1962 academic year.Agronomy Paper No. 595. Supported in part by funds provided by Regional Research Project NE 39.     

Binding of platelet activating factor by isolated membranes from U937 cells

Platelet activating factor (PAF), 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC) has been known to have biological effect on cells. The mechanisms of the effect of the potent phospholipid on cells has not been established. We have used 1-O-[3H]alkyl-2-acetyl-GPC [( 3H]PAF) to study the interaction on the isolated membranes of U937 cells. The binding process was time, protein concentration, temperature dependent and reversible. The binding of [3H]PAF to the U937 cell membranes was slightly inhibited by the addition of PAF analogue, 3-O-Hexadecyl-2-acetyl-sn-glycerol-1-phosphorylcholine. U937 cell membranes showed high affinity binding sites for PAF with equilibrium dissociation constant (Kd) of 5 x 10(-9) M. The displacement of bound [3H]PAF with 500-fold excess of nonlabeled PAF was not altered suggesting that the bound [3H]PAF was not degraded during the binding. Binding of [3H]PAF on U937 cell membranes was inhibited by PAF antagonist, 59227RP. The kinetic of the inhibition by PAF antagonist is competitive suggesting that PAF and PAF antagonist bind at the same site.     

Effect of protein additives on acetylene reduction (nitrogen fixation) by Rhizobium in the presence and absence of soybean cells

The effect of protein additives on acetylene reduction (N(2) fixation) by Rhizobium associated with soybean cells (Glycine max [L.] Merr.) in vitro was studied. Acetylene reduction was promoted on the basal medium supplemented with 1.4 mg of N/ml supplied as aqueous extracts of hexane-extracted soybean, red kidney beans (Phaseolus vulgaris L.), or peas (Pisum sativum L.). Commercial samples of alpha-casein, or bovine serum albumin also promoted acetylene reduction at a concentration of 1.4 mg of N/ml of basal medium, but egg albumin supplying an equal amount of nitrogen to the basal medium completely suppressed acetylene reduction. Autoclaving the aqueous extract of hexane-extracted soybean meal had no effect on its ability to promote acetylene reduction. The presence of 40 mm succinate decreased acetylene reduction with leguminous proteins supplying 1.4 mg of N/ml but promoted acetylene reduction by Rhizobium 32H1-soybean cell associations on media containing alpha-casein, bovine serum albumin, or egg albumin suppling 1.4 mg of N/ml. Similar results were obtained with both cowpea Rhizobium 32H1 and Rhizobium japonicum 61A96. Pure cultures of Rhizobium 32H1 developed acetylene-reducing activity in the presence of soybean extract on basal agar medium and in vermiculite supplied with N-free mineral salts plus crude soybean meal. The results suggest that in certain situations, free living Rhizobium may reduce N(2) under field conditions.     

Modified Fluorescent Technique, Using Rhodamine, for Studies of Rhizobium japonicum-Soybean Symbiosis

Rhodamine-conjugated capsular polysaccharides isolated from Rhizobium japonicum 61A76NS were used to examine binding between the labeled polysaccharides and soybean roots. Fluorescein-labeled polysaccharides were not satisfactory because soybean root hairs autofluoresce in the fluorescein region.     

Transfer from Rhizobium japonicum to Azotobacter vinelandii of genes required for nodulation

A mutant strain of Azotobacter vinelandii that is unable to fix N2 (Nif-) was transformed to Nif+ with DNA from Rhizobium japonicum. Of 50 Nif+ transformants tested, 3 contained the O antigen-related polysaccharide that is present on the cell surface of a nodulating R. japonicum strain, but is absent from a non-nodulating mutant strain.     

Purification of the dihydropyridine receptor of the voltage-dependent Ca2+ channel from skeletal muscle transverse tubules using (+) [3H]PN 200-110

The rabbit skeletal muscle T-tubule membranes preparation is the richest source of organic Ca2+ blocker receptor associated with the voltage-dependent Ca2+ channel. Solubilization by 3-[(3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate (CHAPS) in the presence of glycerol leads to a 52% recovery of active receptors as determined by (+)[3H]PN 200-110 binding experiments. The dissociation constant of the (+) [3H]PN 200-110 solubilized-receptor complex was 0.4 +/- 0.2 nM by equilibrium binding and 0.13 nM from the rate constants of association (k1 = 0.116 nM-1 min-1) and dissociation (k-1 = 1.5 10(-2) min-1). The (+) [3H]PN 200-110 receptor has been substantially purified by a combination of filtration of Ultrogel A2 column and lectin affinity chromatography in the presence of trace amount of specifically bound (+) [3H]PN 200-110. The purified material contained polypeptides of apparent molecular weights of 142 000, 32 000 and 33 000. These three components copurified with (+)[3H]PN 200-110 binding activity.     

Identification of stereospecific [3H]spiroperidol binding sites in mammalian lymphocytes

Direct binding to intact rat lymphocytes has been shown for the potent dopaminergic antagonist [³H]spiroperidol. The specific binding is saturable with two components (K_D1 = 1.9 nM, K_D2 = 36.2 nM). Determination of the K_D by kinetic studies measuring rate constants for association and dissociation provided K_D values similar to those obtained in equilibrium experiments. The specific binding is proportional to cell concentration and temperature dependent with a maximum at 37°C. [³H]spiroperidol binding is stereospecific since (+)butaclamol was more effective than (?)butaclamol. The relative potencies of different antidopaminergic agents in competing for [³H]spiroperidol binding sites parallel their activity in the striatum. Dopaminergic receptors have also been demonstrated in other mammalian lymphocytes (rabbit, dog, human). Lymphocyte dopaminergic receptors could be implicated in lymphocytes mediated immune response.     

Somatostatin binding to dissociated cells from rat cerebral cortex

A method has been developed for the study of somatostatin (SS) binding to dissociated cells from rat cerebral cortex. Binding of [¹²⁵I][Tyr¹¹]SS to cells obtained by mechanical dissociation of rat cerebral cortex was dependent on time and temperature, saturable, reversible and highly specific. Under conditions of equilibrium, i.e., 60 min at 25°C, native SS inhibited tracer binding in a dose-dependent manner. The Scatchard analysis of binding data was linear and yielded a dissociation constant of 0.60±0.08 nM with a maximal binding capacity of 160±16 fmol/mg protein. The binding of [¹²⁵I][Tyr¹¹]SS was specific as shown in experiments on tracer displacement by the native peptide, SS analogues, and unrelated peptides.     

Identification of a prostacyclin receptor coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mouse mastocytoma P-815 cells.

A stable analogue of prostacyclin, iloprost, specifically bound to 30,000 x g pellet (the membrane fraction) prepared from mouse mastocytoma P-815 cells. The binding was dependent on time, temperature and pH, and absolutely required a divalent cation. The equilibrium dissociation constant and the maximal concentration of the binding site as determined by Scatchard plot analysis were 10.4 nM and 1.12 pmol/mg of protein, respectively. The Hill coefficient was 1.0, indicating a single entity of binding site and no cooperativity. The binding site was highly specific for iloprost among PGs tested (iloprost much greater than PGE1 greater than carbacyclin greater than PGE2). In contrast, the membrane fraction had the binding site specific for PGE2 and PGE1, which was distinct from the prostacyclin receptor. The dissociation of bound [3H]iloprost from the membrane fraction was specifically enhanced by guanine nucleotides. Furthermore, iloprost dose-dependently enhanced the activity of adenylate cyclase in a GTP-dependent manner. These results indicate that a specific prostacyclin receptor is coupled to the adenylate cyclase system via a stimulatory GTP-binding protein in mastocytoma cells.