Binding characteristics of n(2)-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.     

Plant growth stimulation of wheat (Triticum aestivum L.) by inoculation of salinity tolerant Azotobacter strains

Five salinity tolerant Azotobacter strains i.e., ST3, ST6, ST9, ST17 and ST24 were obtained from saline soils. These Azotobacter strains were used as inoculant for wheat variety WH157 in earthen pots containing saline soil under pot house conditions, using three fertilizer treatment doses i.e., control (no fertilizer, no inoculation), 90 Kg N ha⁻¹ and 120 Kg N ha⁻¹. Inoculation with salinity tolerant Azotobacter strains caused significant increase in total nitrogen, biomass and grain yield of wheat. Maximum increase in plant growth parameters were obtained after inoculation with Azotobacter strain ST24 at fertilization dose of 120 kg N ha⁻¹ and its inoculation resulted in attaining 89.9 cms plant height, 6.1 g seed yield, 12.0 g shoot dry weight and 0.7 % total nitrogen. The survival of Azotobacter strain ST24 in the soil was also highest in all the treatments at 30, 60 and 90 days after sowing (DAS). However, the population of Azotobacter decreased on 90 DAS as compared to counts observed at 60 DAS at all the fertilization treatments.     

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.     

Purification and properties of ribulosebisphosphate carboxylase large subunit binding protein

The large subunit binding protein, an abundant plastid protein implicated in the assembly of ribulose-1,5-bisphosphate carboxylase-oxygenase (RubisCO), has been highly purified from leaves of Pisum sativum. The 720 kilodaltons purified binding protein is composed of two types of subunits of 60 and 61 kilodaltons. Highly specific polyclonal antibodies have been raised against the binding protein. The antibodies do not cross-react with the large subunit nor do anti-RubisCO antibodies cross-react with the binding protein. A higher molecular weight form of the binding protein is immunoprecipitated from products of P. sativum polysomes translated in a wheat-germ system, indicating that the binding protein is synthesized by cytoplasmic ribosomes. Immunoblotting reveals the presence of binding protein in extracts of tobacco, wheat and barley leaves and castor bean endosperm.

The previously reported dissociation of the binding protein-large subunit complex upon addition of ATP in vitro has been confirmed and the fates of the dissociated subunits further investigated. The dissociated binding protein subunits are not phosphorylated or adenylated in vitro by added ATP.

     

The dissociation of insulin from human insulin antibodies

The dissociation of insulin from human insulin antibodies has been investigated using a technique that is rapid and does not require addition of excess unlabelled insulin. A slow (k₁ = 2·1^?3 min^?1 and a fast (k₂ = 4·10^?2 min^?1) dissociating antibody component were identified in all studies. These have been shown to correspond, respectively, to the high and low affinity antibody components of equilibrium binding studies. The range of k₁ and k₂ values and their response to temperature change is small. Insulin resistance and stability of diabetes are not related to properties of antibody dissociation. Dissociation is faster in the presence of high (6–850 nM) insulin concentration due to increased binding to the fast dissociating component without change in the dissociation rate constants. When incubation time is increased beyond achivement of maximal binding there is a time-dependent rise in binding to the slow dissociating component, with a concomitant fall in k₁. The traditional concept that equilibrium is established at maximum binding requires further examination.     

Binding of lipophorin to the fat body of the migratory locust

The interaction of locust high density lipophorin (HDLp) with pieces of fat body tissue was studied at 33°C using a radiolabelled ligand binding assay. Under the assay conditions, binding of tritium-labelled HDLp ([³H]HDLp) was demonstrated to correlate linearly with tissue concentration up to ∼ 7 mg of fat body protein per ml of incubation medium. The [³H]HDLp binding that was displaceable by a 20-fold excess of unlabelled HDLp (which is an approximation of the specific binding) reached equilibrium after ∼ 2 h, whereas low levels of non-displaceable binding increased linearly during this time interval. Analysis of the concentration dependent total binding of [³H]HDLp revealed the presence of a specific binding site with an equilibrium dissociation constant of K_d = 3.1 (±0.5) × 10⁻⁷ M and a maximal binding capacity of 9.8 (±0.5) ng μg⁻¹ tissue protein. Competition experiments demonstrated that the affinity of unlabelled HDLp for the binding site is similar to the affinity of [³H]HDLp. Unlabelled low density lipophorin (LDLp), however, was shown to have an approx. 20-fold lower affinity for the binding site.     

Elucidating the interactions and phytotoxicity of zinc oxide nanoparticles with agriculturally beneficial bacteria and selected crop plants

There is a growing interest in the use of bioinoculants to assist mineral fertilizers in improving crop production and yield. Azotobacter and Pseudomonas are two agriculturally relevant strains of bacteria which have been established as efficient bioinoculants. An experiment involving addition of graded concentrations of zinc oxide (ZnO) nanoparticles was undertaken using log phase cultures of Azotobacter and Pseudomonas. Growth kinetics revealed a clear trend of gradual decrease with Pseudomonas; however, Azotobacter exhibited a twofold enhancement in growth with increase in the concentration of ZnO concentration. Scanning electron microscopy (SEM), supported by energy-dispersive X-ray (EDX) analyses, illustrated the significant effect of ZnO nanoparticles on Azotobacter by the enhancement in the abundance of globular biofilm-like structures and the intracellular presence of ZnO, with the increase in its concentration. It can be surmised that extracellular mucilage production in Azotobacter may be providing a barrier to the nanoparticles. Further experiments with Azotobacter by inoculation of wheat and tomato seeds with ZnO nanoparticles alone or bacteria grown on ZnO-infused growth medium revealed interesting results. Vigour index of wheat seeds reduced by 40–50% in the presence of different concentrations of ZnO nanoparticles alone, which was alleviated by 15–20%, when ZnO and Azotobacter were present together. However, a drastic 50–60% decrease in vigour indices of tomato seeds was recorded, irrespective of Azotobacter inoculation.     

Cyanide-linked dimer-monomer equilibrium of Chromatium vinosum ferric cytochrome c′

Cyanide binding to Chromatium vinosum ferricytochrome c′ has been studied to further investigate possible allosteric interactions between the subunits of this dimeric protein. Cyanide binding to C. vinosum cytochrome c′ appears to be cooperative. However, the cyanide binding reaction is unusual in that the overall affinity of cyanide increases as the concentration of cytochrome c′ decreases and that cyanide binding causes the ligated dimer to dissociate to monomers as shown by gel-filtration chromatography. Therefore, the cyanide binding properties of C. vinosum ferricytochrome c′ are complicated by a cyanide-linked dimer to monomer dissociation equilibrium of the complexed protein. The dimer to monomer dissociation constant is 20-fold smaller than that for CO linked dissociation constant of ferrocytochrome c′. Furthermore, the pH dependence of both the intrinsic equilibrium binding constant and the dimer to monomer equilibrium dissociation constant was investigated over the pH range of 7.0 to 9.2 to examine the effect of any ionizable groups. The equilibrium constants did not exhibit a significant pH dependence over this pH range.     

Maize Root Lectins Mediate the Interaction with Herbaspirillum seropedicae via N-Acetyl Glucosamine Residues of Lipopolysaccharides

Herbaspirillum seropedicae is a plant growth-promoting diazotrophic betaproteobacterium which associates with important crops, such as maize, wheat, rice and sugar-cane. We have previously reported that intact lipopolysaccharide (LPS) is required for H. seropedicae attachment and endophytic colonization of maize roots. In this study, we present evidence that the LPS biosynthesis gene waaL (codes for the O-antigen ligase) is induced during rhizosphere colonization by H. seropedicae. Furthermore a waaL mutant strain lacking the O-antigen portion of the LPS is severely impaired in colonization. Since N-acetyl glucosamine inhibits H. seropedicae attachment to maize roots, lectin-like proteins from maize roots (MRLs) were isolated and mass spectrometry (MS) analysis showed that MRL-1 and MRL-2 correspond to maize proteins with a jacalin-like lectin domain, while MRL-3 contains a B-chain lectin domain. These proteins showed agglutination activity against wild type H. seropedicae, but failed to agglutinate the waaL mutant strain. The agglutination reaction was severely diminished in the presence of N-acetyl glucosamine. Moreover addition of the MRL proteins as competitors in H. seropedicae attachment assays decreased 80-fold the adhesion of the wild type to maize roots. The results suggest that N-acetyl glucosamine residues of the LPS O-antigen bind to maize root lectins, an essential step for efficient bacterial attachment and colonization.     

Interactions of bacteriophage T4-coded gene 32 protein with nucleic acids: III. Binding properties of two specific proteolytic digestion products of the protein (G32P1I and G32P1III)

Brief treatment of gene 32 protein with proteolytic enzymes produces two specific digestion products in good yield (Moise & Hosoda, 1976). One, representing the native protein with ~60 amino acid residues removed from the C-terminus, is G32P¹I. The other, for which ~20 amino acid residues have been removed from the N-terminus in addition to the 60 residues from the C-terminus, is G32P¹III. Both of these specific “core” fragments of gene 32 protein have been isolated and purified, and their binding properties to single-stranded oligo- and polynucleotides have been studied. We find that the binding properties of G32P¹I are relatively little changed from those characteristic of the native gene 32 protein: (1) the apparent binding constants to short (l = 2 to 8) oligonucleotides are independent of lattice length and essentially independent of base and sugar composition, but do show an increased salt dependence of binding relative to that of the native protein; (2) the intrinsic association constants (K) for polynucleotides binding in the co-operative mode show the same binding specificities as seen with the native protein, but with absolute values increased two to fourfold; (3) the polynucleotide binding co-operativity parameter (ω^?2 × 10³) and the binding site size (n ～-7 nucleotide residues) are the same as for the native protein; (4) essentially the entire salt dependence of the net affinity (Kω) remains in K. However, unlike native gene 32 protein, G32P¹I can melt native DNA to equilibrium (Hosoda et al., 1974; Greve et al., 1978); this suggests that the kinetic pathways for DNA melting by these two species must differ, since the changes in equilibrium binding parameters measured here are far too small to account for the differences in melting behavior. In contrast to G32P¹I, for G32P¹III we find that: (1) binding is non-cooperative (ω ～-1); (2) the binding site size (n) for the protein has decreased by one to two nucleotide residues relative to that characteristic of the native protein and G32P¹I; (3) binding to short (l = 2 to 8) oligonucleotides is length and salt concentration dependent; (4) while binding to polynucleotides continues to show approximately the same base composition dependence as the native protein, the absolute values of K are somewhat different and the salt concentration dependencies of K are less. Polynucleotide ultraviolet light and circular dichroism spectra obtained in the presence of G32P¹I and G32P¹III are indistinguishable from those measured with the native protein at similar binding densities, indicating that all three protein species distort the polynucleotide lattice to comparable extents.These results are combined with the equilibrium binding data for native gene 32 protein (Kowalczykowski et al., 1980a: Newport et al., 1980) to obtain further insight into the molecular details of the interactions of this protein with its nucleic acid binding substrates.     

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.     

Root-to-Root Travel of the Beneficial Bacterium Azospirillum brasilense

The root-to-root travel of the beneficial bacterium Azospirillum brasilense on wheat and soybean roots in agar, sand, and light-textured soil was monitored. We used a motile wild-type (Mot⁺) strain and a motility-deficient (Mot^-) strain which was derived from the wild-type strain. The colonization levels of inoculated roots were similar for the two strains. Mot⁺ cells moved from inoculated roots (either natural or artificial roots in agar, sand, or light-textured soil) to noninoculated roots, where they formed a band-type colonization composed of bacterial aggregates encircling a limited part of the root, regardless of the plant species. The Mot^- strain did not move toward noninoculated roots of either plant species and usually stayed at the inoculation site and root tips. The effect of attractants and repellents was the primary factor governing the motility of Mot⁺ cells in the presence of adequate water. We propose that interroot travel of A. brasilense is an essential preliminary step in the root-bacterium recognition mechanism. Bacterial motility might have a general role in getting Azospirillum cells to the site where firmer attachment favors colonization of the root system. Azospirillum travel toward plants is a nonspecific active process which is not directly dependent on nutrient deficiency but is a consequence of a nonspecific bacterial chemotaxis, influenced by the balance between attractants and possibly repellents leaked by the root.     

Binding of [3H]batrachotoxinin A-20-α-benzoate and [3h]saxitoxin to receptor sites associated with sodium channels in trout brain synaptoneurosomes

1. [³H]Batrachotoxinin A-20-α-benzoate ([³H]BTX-b) and [³H]saxitoxin ([³H]STX), radioligands that bind to distinct sites on the voltage-sensitive sodium channel, were bound specifically to saturable sites in rainbow trout (Oncorhynchus mykiss) brain synaptoneurosomes.2. Specific [³H]BTX-B binding was temperature dependent with highest levels of specific [³H]BTX-B binding observed at 7°C. Specific binding was inversely correlated with assay temperature at temperatures above 7°C.3. Saturating concentrations of scorpion (Leiurus quinquestriatus) venom (ScV) stimulated specific [³H]BTX-B binding at 27°C, but not at 7°C. The dihydropyrazole insecticide RH 3421 inhibited specific [³H]BTX-B binding at 7°C but had no effect on specific binding at 27°C. The sodium channel activators veratridine and aconitine and the local anesthetic dibucaine inhibited specific [³H]BTX-B binding at both 7°C and 27°C.4. Displacement experiments in the presence of ScV at 27°C gave an equilibrium dissociation constant (K_d) for [³H]BTX-B of 710 nM and a maximal binding capacity (B_max) of 11.3 pmol/mg protein. Kinetic experiments established the rates of association (1.17 × 10⁵min⁻¹ nM⁻¹) and dissociation (0.0514min⁻¹) of the ligand-receptor complex.5. The binding of [³H]STX reached apparent saturation at 7.5 nM. Scatchard analysis of the saturation data indicated a K_d of 3.8nM and a B_max of 1.9 pmol/mg protein.6. These studies provide evidence for high affinity, saturable binding sites for [³H]BTX-B and [³H]STX in trout brain preparations. Whereas certain neurotoxins modified the specific binding of [³H]BTX-B in trout brain synaptoneurosomes in a predictable fashion, other compounds known to affect specific [³H]BTX-B binding in mammalian brain preparations had no effect on specific [³H]BTX-B binding in the trout.     

Legume-Rhizobium Interactions: Cowpea Root Exudate Elicits Faster Nodulation Response by Rhizobium Species

Preinfection events in legume-Rhizobium symbiosis were analyzed by studying the different nodulation behaviors of two rhizobial strains in cowpeas (Vigna sinensis). Log-phase cultures of Rhizobium sp. strain 1001, an isolate from the plant nodule, initiated host responses leading to infection within 2 h after inoculation, whereas log-phase cultures of Rhizobium sp. strain 32H1 took at least 7 h to trigger a discernible response. The delay observed with strain 32H1 could be eliminated by incubating the rhizobial suspension, before inoculation, for 4.5 h either in the cowpea rhizosphere/rhizoplane condition or in the root exudate of cowpea plants, grown without NH₄⁺ in the rooting medium. The delay could not be eliminated by incubating the rhizobial suspension in the rooting medium of plants grown in the presence of 5 mM NH₄⁺, indicating that there is a regulatory role of combined nitrogen in triggering preinfection events by the legume. The substance(s) in the root exudate which elicited the faster nodulation response by Rhizobium sp. strain 32H1 could be separated into a high-molecular-weight fraction by Sephadex G-100 gel filtration. The data support the notion that legume roots release substances that favor the development of rhizobial features essential for infection and nodulation.     

In vitro characterization of skeletal muscle β-adrenergic receptors coupled to adenylate cyclase

[³H]Dihydroalprenolol, a potent ß-adrenergic antagonist, was used to identify the adenylate cyclase-coupled ß-adrenoceptors in isolated membranes of rat skeletal muscle. The receptor sites, as revealed [³H]dihydroalprenolol binding, were predominantly localized in plasmalemmal fraction. That skeletal muscle fraction may also contain the plasmalemma of other intramuscular cells, especially that of blood vessels. Hence, the [³H]dihydroalprenolol binding observed in that fraction may be due partly to its binding to the plasmalemma of blood vessels. Small but consistent binding was also observed in sarcoplasmic reticulum and mitochondria. The level of [³H]dihydroalprenolol binding in different subcellular fractions closely correlated with the level of adenylate cyclase present in those fractions.The binding of [³H]dihydroalprenolol to plasmalemma exhibited saturation kinetics. The binding was rapid, reaching equilibrium within 5 min, and it was readily dissociable. From the kinetics of binding, association (K₁) and dissociation (K₂) rate constants of 2.21 · M^? · min^?1 and 3.21 · 10^?1, respectively, were obtained. The dissociation constant (K_d) of 15 nM for [³H]dihydroalprenolol obtained from saturation binding data closely agreed with the (K_d) derived from the ratio of dissociation and association rate constants (K₂/K₁).Several β-adrenergic agents known to be active on intact skeletal muscle also competed for [³H]dihydroalprenolol binding sites in isolated plasmalemma with essentially similar selectivity and stereospecificity. Catecholamines competed for [³H]dihydroalprenolol binding sites with a potency of isoproterenol > epinephrine > norepinephrine. A similar order of potency was noted for catecholamines in the activation of adenylate cyclase. Effects of catecholamines were stereospecific, (?)-isomers being more than potent than (+)-isomers. Phenylephrine, an α-adrenergic agonist, showed no effect either on [³H]dihydroalprenolol binding or on adenylate cyclase. Known ß-adrenergic antagonists, propranolol and alprenolol, stereospecifically inhibited the [³H]dihydroalprenolol binding and the isoproterenol-stimulated adenylate cyclase. The (K_i) values for the antagonists determined from inhibition of [³H]dihydroalprenolol binding agreed closely with the (K_i) values obtained from the inhibition of adenylate cyclase. The data suggest that the binding of [³H]dihydroalprenolol in skeletal muscle membranes possess the characteristics of a substance binding to the ß-adrenergic receptor.     

Pesticide tolerant Azotobacter isolates from paddy growing areas of northern Karnataka, India

A total of 14 Azotobacter strains were isolated from different paddy cultivating soils with pH ranging from 6.5 to 9.5 by using serial dilution agar plate method. The strains were Gram negative, rod shaped, cyst forming and developed brown to black colored colonies, which were glistening, smooth, slimy on Ashby’s agar plates. Biochemically they were positive for biochemical tests namely, indole production, citrate, catalase, carbohydrate fermentation and Voges–Proskauer test. Further, sequence analysis of PCR amplicons obtained from these cultures revealed the presence of five different Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris, Azotobacter sp., Azotobacter nigricans subsp. nigricans and Azotobacter tropicalis. Phylogenetically these strains were grouped into two distinct clusters. These strains were tested for their ability to grow on a media containing four different pesticides such as pendimethalin, glyphosate, chloropyrifos and phorate, which are commonly used for the paddy. Out of 14 strains tested, 13 strains were able to grow on a media containing herbicides such as pendimethalin, glyphosate and insecticides like chloropyrifos and phorate. However, five Azotobacter strains were able to grow at higher concentration of 5 % pesticides, without affecting their growth rate. Further, the effect of pesticides on the indole acetic acid (IAA) production by Azotobacter strains was also estimated. Azotobacter-16 strain was found to produce 34.4 μg ml^?l of IAA in a media supplemented with 1,000 mg of tryptophan and 5 % of pendimethalin. Present study reveals that species of Azotobacter are able to grow and survive in the presence of pesticides and no significant effects were observed on the metabolic activities of Azotobacter species.     

Biofilm of <Emphasis Type="Italic">Aureobasidium pullulans</Emphasis> var. <Emphasis Type="Italic">pullulans</Emphasis> on winter wheat kernels and its effect on other microorganisms

Winter wheat, grown under greenhouse conditions, was protected four times with a cell suspension of Aureobasidium pullulans var. pullulans during the growing season. After harvest, the distribution and survival rates of the studied biocontrol agent were analyzed under a scanning electron microscope. The abundance of filamentous fungi, yeasts, pseudomonads and Azotobacter bacteria was determined by inoculation onto selective agar media. A. pullulans produced mostly unicellular chlamydospores on the surface and in the brush of kernels. Multicellular blastospore conglomerates secreted extracellular polymeric substances (EPS), and their biofilms were found in the brush and crease of kernels. The application of a cell suspension of A. pullulans with the density of 10⁴ CFU to winter wheat spikes, repeated four times, inhibited the growth of pseudomonads, Azotobacter bacteria and filamentous fungi.     

The Mechanism of VWF-Mediated Platelet GPIbα Binding

The binding of Von Willebrand Factor to platelets is dependent on the conformation of the A1 domain which binds to platelet GPIbα. This interaction initiates the adherence of platelets to the subendothelial vasculature under the high shear that occurs in pathological thrombosis. We have developed a thermodynamic strategy that defines the A1:GPIbα interaction in terms of the free energies (ΔG values) of A1 unfolding from the native to intermediate state and the binding of these conformational states to GPIbα. We have isolated the intermediate conformation of A1 under nondenaturing conditions by reduction and carboxyamidation of the disulfide bond. The circular dichroism spectrum of reduction and carboxyamidation A1 indicates that the intermediate has ∼10% less α-helical structure that the native conformation. The loss of α-helical secondary structure increases the GPIbα binding affinity of the A1 domain ∼20-fold relative to the native conformation. Knowledge of these ΔG values illustrates that the A1:GPIbα complex exists in equilibrium between these two thermodynamically distinct conformations. Using this thermodynamic foundation, we have developed a quantitative allosteric model of the force-dependent catch-to-slip bonding that occurs between Von Willebrand Factor and platelets under elevated shear stress. Forced dissociation of GPIbα from A1 shifts the equilibrium from the low affinity native conformation to the high affinity intermediate conformation. Our results demonstrate that A1 binding to GPIbα is thermodynamically coupled to A1 unfolding and catch-to-slip bonding is a manifestation of this coupling. Our analysis unites thermodynamics of protein unfolding and conformation-specific binding with the force dependence of biological catch bonds and it encompasses the effects of two subtypes of mutations that cause Von Willebrand Disease.     

Epibatidine binds to four sites on the Torpedo nicotinic acetylcholine receptor

The nicotinic acetylcholine receptor (nAChR) from Torpedo electric organ is a pentamer of homologous subunits. This receptor is generally thought to carry two high affinity sites for agonists under equilibrium conditions. Here we demonstrate directly that each Torpedo nAChR carries at least four binding sites for the potent neuronal nAChR agonist, epibatidine, i.e., twice as many sites as for α-bungarotoxin. Using radiolabeled ligand binding techniques, we show that the binding of [³H]-(±)-epibatidine is heterogeneous and is characterized by two classes of binding sites with equilibrium dissociation constants of about 15 nM and 1 μM. These classes of sites exist in approximately equal numbers and all [³H]-(±)-epibatidine binding is competitively displaced by acetylcholine, suberyldicholine and d-tubocurarine. These results provide further evidence for the complexity of agonist binding to the nAChR and underscore the difficulties in determining simple relationships between site occupancy and functional responses.     

Diversity and characterization of <Emphasis Type="Italic">Azotobacter</Emphasis> isolates obtained from rice rhizosphere soils in Taiwan

Azotobacter species, free-living nitrogen-fixing bacteria, have been used as biofertilizers to improve the productivity of non-leguminous crops, including rice, due to their various plant growth-promoting traits. The purposes of this study were to characterize Azotobacter species isolated from rice rhizospheres in Taiwan and to determine the relationship between the species diversity of Azotobacter and soil properties. A total of 98 Azotobacter isolates were isolated from 27 paddy fields, and 16S rRNA gene sequences were used to identify Azotobacter species. The characteristics of these Azotobacter strains were analyzed including carbon source utilization and plant growth-promoting traits such as nitrogen fixation activity, indole acetic acid production, phosphate-solubilizing ability, and siderophore secretion. Of the 98 strains isolated in this study, 12 were selected to evaluate their effects on rice growth. Four species of Azotobacter were identified within these 98 strains, including A. beijerinckii, A. chroococcum, A. tropicalis, and A. vinelandii. Of these four species, A. chroococcum was predominant (51.0%) but A. beijerinckii had the highest level of nucleotide diversity. Strains within individual Azotobacter species showed diverse profiles in carbon source utilization. In addition, the species diversity of Azotobacter was significantly related to soil pH, Mn, and Zn. Members of the same Azotobacter species showed diverse plant growth-promoting traits, suggesting that the 98 strains isolated in this study may not equally effective in promoting rice growth. Of the 12 strains evaluated, A. beijerinckii CHB 461, A. chroococcum CHB 846, and A. chroococcum CHB 869 may be used to develop biofertilizers for rice cultivation because they significantly promoted rice growth. This study contributes to the selection of suitable Azotobacter strains for developing biofertilizer formulations and soil management strategies of Azotobacter for paddy fields.