Japonicum Cells Research Articles

Scarcity of fixed carbon transfer in a model microbial phototroph-heterotroph interaction.

Although the green alga Chlamydomonas reinhardtii has long served as a reference organism, few studies have interrogated its role as a primary producer in microbial interactions. Here, we quantitatively investigated C. reinhardtii's capacity to support a heterotrophic microbe using the established coculture system with Mesorhizobium japonicum, a vitamin B12-producing α-proteobacterium. Using stable isotope probing and nanoscale secondary ion mass spectrometry (nanoSIMS), we tracked the flow of photosynthetic fixed carbon and consequent bacterial biomass synthesis under continuous and diurnal light with single-cell resolution. We found that more 13C fixed by the alga was taken up by bacterial cells under continuous light, invalidating the hypothesis that the alga's fermentative degradation of starch reserves during the night would boost M. japonicum heterotrophy. 15NH4 assimilation rates and changes in cell size revealed that M. japonicum cells reduced new biomass synthesis in coculture with the alga but continued to divide-a hallmark of nutrient limitation often referred to as reductive division. Despite this sign of starvation, the bacterium still synthesized vitamin B12 and supported the growth of a B12-dependent C. reinhardtii mutant. Finally, we showed that bacterial proliferation could be supported solely by the algal lysis that occurred in coculture, highlighting the role of necromass in carbon cycling. Collectively, these results reveal the scarcity of fixed carbon in this microbial trophic relationship (particularly under environmentally relevant light regimes), demonstrate B12 exchange even during bacterial starvation, and underscore the importance of quantitative approaches for assessing metabolic coupling in algal-bacterial interactions.

Fluorescence lifetime microscopy reveals the biologically-related photophysical heterogeneity of oxyblepharismin in light-adapted (blue) Blepharisma japonicum cells.

The step-up photophobic response of the heterotrich ciliate Blepharisma japonicum is mediated by a hypericinic pigment, blepharismin, which is not present in any of the known six families of photoreceptors, namely rhodopsins, phytochromes, xanthopsins, cryptochromes, phototropins, and BLUF proteins. Upon irradiation, native cells become light-adapted (blue) by converting blepharismin into the photochemically stable oxyblepharismin (OxyBP). So far, OxyBP has been investigated mainly from a photophysical point of view in vitro, either alone or complexed with proteins. In this work, we exploit the vivid fluorescence of OxyBP to characterize its lifetime emission in blue B. Japonicum cells, on account of the recognized role of the fluorescence lifetime to provide physicochemical insights into the fluorophore environment at the nanoscale. In a biological context, OxyBP modifies its emission lifetime as compared to isotropic media. The phasor approach to fluorescence lifetime microscopy in confocal mode highlights that fluorescence originates from two excited states, whose relative balance changes throughout the cell body. Additionally, Cilia and kinetids, i.e., the organelles involved in photomovement, display lifetime asymmetry between the anterior and posterior part of the cell. From these data, some hypotheses on the phototransduction mechanism are proposed.

Optimization of culture medium for a Bradyrhizobium japonicum strain introduced from Russia

The aim and tasks of the study. The study was aimed at determination of ingredients and their concentrations to culture medium YMA, which would be the most suitable for cultivation of soybean nodule bacteria Bradyrhizobium japonicum. This will provide a scientific rationale for the production of nodule bacteria on an industrial scale.Materials and methods. The starting material was soybean nodule bacteria B. japonicum, strain GF, introduced from the All-Russian Research Institute of Soybean of the Russian Agricultural Academy. Culture media were YMA, SM, TY, PA, BSE.Concentrated solution of bacteria was cultured for two days at 28 ° C, then was spread in liquid nutrient medium YMA and cultured for four days. Thereafter, B. japonicum was devided among four media in 250 flasks of 100 ml. Optimal medium was spectrophotometrically determined for different periods of bacterium growth by growth curves. Thereafter, nutrient medium composition and concentration of its ingredients were optimized. In particular, mannitol in nutrient medium YMA was replaced with the same amount of glycerol, lactose, dextrose, sucrose, and maltose, while the other ingredients remained unchanged.Results and discussion. The results showed that B. japonicum cells grew the fastest in nutrient medium YMA as compared with the others - SM, TY, PA, BSE. Thus, culture medium YMA was chosen to optimize the composition.The growth rate of B. japonicum enhanced, when carbon from different sources was added to nutrient medium. Among several sources (glucose, mannitol, sucrose, glycerytan), glucose was the best. In terms of strain GF growth it was found that the optimal ratio of ingredients and concentrations was 10.0 g / L of glucose, 0.8 g / L of yeast powder, 0.2 g / L of MgSO4 • 7H2O, 0.8 g / L of K2HPO4, 0.1 g / L of NaCl, 0.01 g / L of CaCl2, and 4 ml / L of Rh solution.Optimized culture medium was compared with original medium YMA and medium 3. Results of culturing bacteria 12, 24, 36 and 48 hours after inoculation were assessed. It was proved that optimized nutrient medium by these parameters was better than original one and number 3.Conclusions. The study revealed that of the five culture media YMA was the best to inoculate soybean nodule bacteria B. japonicum, strain GF. Its composition was optimized, and it was demonstrated that the best ingredients and their concentrations were 10.0 g / L of glucose, 0.8 g / L of yeast powder, 0.2 g / L of MgSO4 • 7H2O, 0.8 g / L of K2HPO4, 0.1 g / L of NaCl, 0.01 g / L of CaCl2, and 4 ml / L of Rh solution. Optimized culture medium YMA will enable the production of nodule bacteria on an industrial scale in a short time.

Peribacteroid solution of soybean root nodules partly induces genomic loci for differentiation into bacteroids of free-living Bradyrhizobium japonicum cells

In leguminous root nodules, rhizobia differentiate into morphology specific to symbiosis, called bacteroids. As bacteroids are surrounded with peribacteroid membranes filled with peribacteroid solution (PBS), it is considered that PBS contains substances inducing differentiation of rhizobia into bacteroids. In this study, genome-wide expression profiles of Bradyrhizobium japonicum cells cultured in PBS purified from root nodule of soybean (Glycine max L.) were compared with those of bacteroids using macroarray. PBS treatment preferentially induced regions in a large symbiosis island including various symbiosis relevant genes such as nod, fix, nol and noe, in which 75% of regions were commonly induced in bacteroids, while general repressions outside of the symbiosis island seen in bacteroids were not observed in PBS treated cells. The present results suggest that PBS contained some, but not all, substances inducing expression of the genes which are involved in differentiation into bacteroids.

An overview of the metabolic differences betweenBradyrhizobium japonicum110 bacteria and differentiated bacteroids from soybean (Glycine max) root nodules: anin vitro13C- and31P-nuclear magnetic resonance spectroscopy study

Bradyrhizobium japonicum is a symbiotic nitrogen-fixing soil bacteria that induce root nodules formation in legume soybean (Glycine max.). Using (13)C- and (31)P-nuclear magnetic resonance (NMR) spectroscopy, we have analysed the metabolite profiles of cultivated B. japonicum cells and bacteroids isolated from soybean nodules. Our results revealed some quantitative and qualitative differences between the metabolite profiles of bacteroids and their vegetative state. This includes in bacteroids a huge accumulation of soluble carbohydrates such as trehalose, glutamate, myo-inositol and homospermidine as well as Pi, nucleotide pools and intermediates of the primary carbon metabolism. Using this novel approach, these data show that most of the compounds detected in bacteroids reflect the metabolic adaptation of rhizobia to the surrounding microenvironment with its host plant cells.

Influence of Elevated Atmospheric Carbon Dioxide on Transcriptional Responses of <i>Bradyrhizobium japonicum</i> in the Soybean Rhizoplane

Elevated atmospheric CO2 can influence the structure and function of rhizoplane and rhizosphere microorganisms by altering root growth and the quality and quantity of compounds released into the rhizoplane and rhizosphere via root exudation. In these studies we investigated the transcriptional responses of Bradyrhizobium japonicum cells growing in the rhizoplane of soybean plants exposed to elevated atmospheric CO2. The results of microarray analyses indicated that elevated atmospheric CO2 concentration indirectly influenced the expression of a large number of genes in Bradyrhizobium attached to soybean roots. In addition, relative to plants and bacteria grown under ambient CO2 growth conditions, genes involved in C1 metabolism, denitrification and FixK2-associated genes, including those involved in nitrogen fixation, microaerobic respiration, respiratory nitrite reductase, and heme biosynthesis, were significantly up-regulated under conditions of elevated CO2 in the rhizosphere. The expression profile of genes involved in lipochitooligosaccharide Nod factor biosynthesis and negative transcriptional regulators of nodulation genes, nolA and nodD2, were also influenced by plant growth under conditions of elevated CO2. Taken together, the results of these studies indicate that the growth of soybeans under conditions of elevated atmospheric CO2 influences gene expressions in B. japonicum in the soybean rhizoplane, resulting in changes to carbon/nitrogen metabolism, respiration, and nodulation efficiency.

NifA is required for maximal expression of denitrification genes in Bradyrhizobium japonicum

In Bradyrhizobium japonicum the napEDABC, nirK, norCBQD and nosRZDYFLX genes, which encode reductases for nitrate, nitrite, nitric oxide and nitrous oxide, respectively, are required for denitrification. Microaerobic induction of these genes depends on fixLJ and fixK2, whose products form the FixLJ-FixK2 regulatory cascade. In B. japonicum, a second oxygen-responsive regulatory cascade mediated by the nitrogen fixation regulatory protein, NifA, has been described. In this study, we show that disruption of nifA caused a growth defect in B. japonicum cells, when grown under denitrifying conditions, and decreased activity of periplasmic nitrate and nitrite reductase enzymes was also observed. Furthermore, expression of napE-lacZ, nirK-lacZ or norC-lacZ transcriptional fusions, as well as levels of nirK transcripts were significantly reduced in the nifA mutant after incubation under nitrate-respiring conditions. Haem c staining analyses revealed that NifA is required for full synthesis of the NapC and NorC proteins, which are required for denitrification. A B. japonicum rpoN1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor sigma54, was able to grow anaerobically with nitrate as terminal electron acceptor and showed wild-type levels of nitrate and nitrite reductase activities. We propose that the nitrogen fixation regulatory protein, NifA, is involved in the maximal expression of the denitrification genes in B. japonicum. This influence is independent of sigma54.

Genome-Wide Transcript Analysis of Bradyrhizobium japonicum Bacteroids in Soybean Root Nodules

The transcriptome of endosymbiotic Bradyrhizobium japonicum bacteroids was assessed, using RNA extracted from determinate soybean root nodules. Results were compared with the transcript profiles of B. japonicum cells grown in either aerobic or microaerobic culture. Microoxia is a known trigger for the induction of symbiotically relevant genes. In fact, one third of the genes induced in bacteroids at day 21 after inoculation are congruent with those up-regulated in culture by a decreased oxygen concentration. The other induced genes, however, may be regulated by cues other than oxygen limitation. Both groups of genes provide a rich source for the possible discovery of novel functions related to symbiosis. Samples taken at different timepoints in nodule development have led to the distinction of genes expressed early and late in bacteroids. The experimental approach applied here is also useful for B. japonicum mutant analyses. As an example, we compared the transcriptome of wild-type bacteroids with that of bacteroids formed by a mutant defective in the RNA polymerase transcription factor sigma54. This led to a collection of hitherto unrecognized B. japonicum genes potentially transcribed in planta in a sigma54-dependent manner.

Pre‐incubation of Bradyrhizobium japonicum with jasmonates accelerates nodulation and nitrogen fixation in soybean (Glycine max) at optimal and suboptimal root zone temperatures

Jasmonic acid (JA) and methyl jasmonate, collectively known as jasmonates, are naturally occurring in plants; they are important signal molecules involved in induced disease resistance and mediate many physiological activities in plants. We studied the effect of JA and its methyl ester, methyl jasmonate (MeJA), on the induction of nod genes in Bradyrhizobium japonicum GG4 (USDA3) carrying a plasmid with a translational fusion between B. japonicum nodY and lacZ of Escherichia coli, and the expression activity was measured by β‐galactosidase activity. Both JA and MeJA strongly induced the expression of nod genes. They have little or no deleterious effects on the growth of B. japonicum cells, while genistein (Gen) showed inhibitory effects. We further studied the effect of JA‐ and MeJA‐induced B. japonicum on soybean nodulation and nitrogen fixation under optimal (25°C) and suboptimal (17°C) root zone temperature (RZT) conditions. B. japonicum cells were grown in liquid yeast extract mannitol media and induced with a range of Gen, JA, and MeJA concentrations, including a treatment control with no inducer added. Soybean seedlings were grown at 25 or 17°C RZT with a constant air temperature (25°C) and inoculated, at the vegetative cotyledonary stage, with various B. japonicum induction treatments. Addition of Gen or jasmonates to B. japonicum, prior to inoculation, enhanced nodulation, nitrogen fixation, and plant growth at suboptimal RZT conditions. A higher concentration of Gen was inhibitory at 25°C, while this same concentration was stimulatory at 17°C. Interestingly, pre‐incubation of B. japonicum with JA and MeJA enhanced soybean nodulation and nitrogen fixation under both optimal and suboptimal RZTs. We show that jasmonates are thus a new class of signaling molecules in the B. japonicum‐soybean symbiosis and that pre‐induction of B. japonicum with jasmonates can be used to enhance soybean nodulation, nitrogen fixation, and early plant growth.

Alterations of ciliate phosducin phosphorylation in Blepharisma japonicum cells

We have previously reported that motile photophobic response in ciliate Blepharisma japonicum correlates with dephosphorylation of a cytosolic 28 kDa phosphoprotein (PP28) exhibiting properties similar to those of phosducin. Here we demonstrate in in vivo phosphorylation assay that the light-elicited dephosphorylation of the PP28 is significantly modified by cell incubation with substances known to modulate protein phosphatase and kinase activities. Immunoblot analyses showed that incubation of ciliates with okadaic acid and calyculin A, potent inhibitors of type 1 or 2A protein phosphatases, distinctly increased phosphorylation of PP28 in dark-adapted cells and markedly weakened dephosphorylation of the ciliate phosducin following cell illumination. An enhancement of PP28 phosphorylation was also observed in dark-adapted ciliates exposed to 8-Br-cAMP and 8-Br-cGMP, slowly hydrolysable cyclic nucleotide analogs and 3-isobutyryl-1-methylxanthine (IBMX), a non-specific cyclic nucleotide phosphodiesterase (PDEs) inhibitor. Only slight changes in light-evoked dephosphorylation levels of PP28 were observed in cells treated with the cyclic nucleotide analogs and IBMX. Incubation of ciliates with H 89 or KT 5823, highly selective inhibitor of cAMP-dependent protein kinase (PKA) and cGMP-dependent protein kinase (PKG), respectively, decreased PP28 phosphorylation levels in dark-adapted cells, whereas the extent of light-evoked dephosphorylation of the phosphoprotein was only slightly influenced. Cell treatment with higher Ca 2+ concentration together with ionophore A23187 in culture medium resulted in marked increase in PP28 phosphorylation levels, while quite an opposite effect was observed in cells exposed to Ca 2+ chelators, EGTA or BAPTA/AM as well as calmodulin antagonists, such as trifluoperazine (TFP), W-7 or calmidazolium. Light-dependent dephosphorylation was not considerably affected by these treatments. The experimental findings presented here suggest that an endogenous light-dependent protein kinase-phosphatase system may be engaged in the alteration of phosducin phosphorylation in ciliate B. japonicum thereby to modulate the cell motile photophobic behavior.

KatG is the primary detoxifier of hydrogen peroxide produced by aerobic metabolism in Bradyrhizobium japonicum.

Bacteria are exposed to reactive oxygen species from the environment and from those generated by aerobic metabolism. Catalases are heme proteins that detoxify H(2)O(2), and many bacteria contain more than one catalase enzyme. Also, the nonheme peroxidase alkyl hydroperoxide reductase (Ahp) is the major scavenger of endogenous H(2)O(2) in Escherichia coli. Here, we show that aerobically grown Bradyrhizobium japonicum cells express a single catalase activity. Four genes encoding putative catalases in the B. japonicum genome were identified, including a katG homolog encoding a catalase-peroxidase. Deletion of the katG gene resulted in loss of catalase activity in cell extracts and of exogenous H(2)O(2) consumption by whole cells. The katG strain had a severe aerobic growth phenotype but showed improved growth in the absence of O(2). By contrast, a B. japonicum ahpCD mutant grew well aerobically and consumed H(2)O(2) at wild-type rates. A heme-deficient hemA mutant expressed about one-third of the KatG activity as the wild type but grew well aerobically and scavenged low concentrations of exogenous H(2)O(2). However, cells of the hemA strain were deficient in consumption of high concentrations of H(2)O(2) and were very sensitive to killing by short exposure to H(2)O(2). In addition, KatG activity did not decrease as a result of mutation of the gene encoding the transcriptional activator OxyR. We conclude that aerobic metabolism produces toxic levels of H(2)O(2) in B. japonicum, which is detoxified primarily by KatG. Furthermore, the katG level sufficient for detoxification does not require OxyR.

The Chemotactic Properties of Bradyrhizobium japonicum in the Presence of Natural Fine-Grained Minerals

The natural argillaceous minerals montmorillonite and palygorskite were found to enhance the motility of Bradyrhizobium japonicum cells and to slow down their chemotactic motion to glucose. The latter effect of the minerals is probably due to the adsorption of mineral particles on the cell surface and the blockade of the receptors that are responsible for the chemotactic behavior of the bacterium.

Effects of Interactions Between Chemical Seed Dressings And Bradyrhizobium Japonicum on Soybean Seeds

Soybean seeds, both untreated and dusted with selected pesticides, were inoculated with Bradyrhizobium japonicum and stored in the laboratory at room temperature. Approximately 1 hour, 24 hours and 48 hours following inoculation samples of the seeds were taken to count the surviving rhizobia and to grow soybean plants in the greenhouse experiment. Numbers of B. japonicum cells surviving on soybean seeds (two cultivars) treated with the tested pesticidal dressings and on the untreated seeds did not differ significantly when analysed within the first hour following seed inoculation with the rhizobia. However, analyses performed 24 hours and 48 hours later showed no B. japonicum cells surviving on the seeds dressed with mancozeb (fungicide), while the numbers of these bacteria on the untreated seeds and on the seeds treated with a mixture of carbendazim + thiram (fungicides) or with carbosulfan (insecticide) were similar and remained on relatively high levels. Only soybean plants that developed from mancozeb treated seeds had significantly less nodules, both in terms of numbers and mass, as well as lower fresh and dry weight of shoots as compared to the plants grown from the control seeds and the seeds treated with the other pesticides tested. The toxicity of mancozeb to the symbiosis was particularly severe when soybean seeds inoculated with B. japonicum were stored for 24-48 hours before planting

Bradyrhizobium japonicum mutants with enhanced sensitivity to genistein resulting in altered nod gene regulation.

Bradyrhizobium japonicum mutants with altered nod gene induction characteristics were isolated by screening mutants for genistein-independent nod gene expression. Plasmid pZB32, carrying a nodY::lacZ transcriptional gene fusion, was introduced into B. japonicum cells that had been subjected to UV mutagenesis. Ten independent transformants producing a blue color on plates containing 5bromo-4chloro-3indolyl-beta-D-galactopyranoside but lacking genistein, indicative of constitutive expression of the nodY::lacZ reporter gene, were isolated. Beta-galactosidase activity assays revealed that while all of the 10 strains were sensitive to low concentrations of genistein, none exhibited truly constitutive nodY::lacZ expression in liquid culture. Soybean plants inoculated with three of the mutants were chlorotic and stunted, with shoot dry weights close to those of the uninoculated plants, indicating the absence of nitrogen fixation. Differences in the kinetics of nodY::lacZ expression and lipochitin oligosaccharide Nod signal production suggested that the strains carried different mutations. Some of these strains may be useful in mitigating the low root zone temperature-associated delay in soybean nodulation at the northern extent of soybean cultivation.

Survival and change in physiological state of Bradyrhizobium japonicum in soybean (Glycine max L. Merril) liquid inoculants after long-term storage

Commercial liquid inoculants for soybean, stored at 20 °C for 1–8 years in 400 ml bottles or in 5000 ml containers, were assessed for their efficacy and changes in the physiological activity of Bradyrhizobium japonicum. A decrease in viable counts and in bacterial survival on seeds was observed in inoculants stored for several years. The number of nodules produced per plant in a growth chamber decreased and was correlated to the number of bacteria surviving on the seeds. Changes in physiological properties were assessed using biochemical, physiological and microscopic methods. The cell total sugars content decreased with increased storage of the inoculants. High calculated ratios of suspended solid dry matter/carbon/nitrogen/proteins weight per c.f.u. strongly suggested the presence of dead or viable but non-culturable (VBNC) cells in the inoculants. This was confirmed in a study of bacterial respiratory activity, using p-iodonitrotetrazolium reduction. The time of colony appearance on plates increased in the old inoculants stored for a long time, especially on yeast-free culture medium. The heterogeneity in colony size also increased with storage length. Inoculants stored for more than 2 years could be differentiated from the others by using nalidixic acid against cellular division. Nucleic acid staining of cells showed that the percentage of membrane-compromised bacteria in all the inoculants increased with increased storage length, whatever the type of packaging used for the inoculants. These results demonstrated that the physiological activity of B. japonicum cells in commercial liquid inoculants changes after storage. To complete c.f.u. determination, three methods were proposed to assess the fitness of stored bradyrhizobia, but they remain to be checked for reliability on a variety of commercial inoculants.

Response of Soybean [Glycine max (L.) Merr.] Cultivars to Genistein‐Preincubated Bradyrhizobium japonicum: Nodulation and Dry Matter Accumulation under Canadian Short‐Season Conditions

The addition of genistein, a plant‐to‐bacteria signal molecule, to Bradyrhizobium japonicum cells prior to use as inocula has been shown to increase nodule number and promote soybean N2 fixation at low root zone temperatures. Previous greenhouse and field experiments involving only two cultivars have indicated that soybean [Glycine max (L.) Merr.] cultivars can vary in their response to genistein application. The objective of this study was to evaluate a range of soybean cultivars for response to genistein application under short‐season cool‐spring Canadian conditions. A 2‐year field study was conducted in 1997 and 1998 with a range of soybean cultivars recommended for Quebec. The 11 cultivars tested represented a range of yield potentials and maturity groups. They were inoculated with genistein‐preincubated B. japonicum inocula or regular inocula, applied into the furrow at the time of planting. The results of these experiments indicated that neither maturity nor yield was correlated with increases in nodulation, biomass, and plant total nitrogen content resulting from genistein treatment and that all maturity groups responded to genistein application in essentially the same way. Thus, response of soybean cultivars to genistein addition is regulated by genotype characteristics other than maturity or yield level.

Changes in the physiological and agricultural characteristics of peat-based Bradyrhizobium japonicum inoculants after long-term storage.

Commercial soybean inoculants processed with sterilised peat and stored at 20 degrees C for 1-8 years were used as experimental materials to assess the changes in the physiological activity of Bradyrhizobium japonicum after storage. Viable counts decreased and physiological characteristics of the bacterium changed during storage, with an increase in the time taken for colony appearance on a medium without yeast extract, an increase in the lag time for nodule appearance on soybean grown in glass tubes and a decrease in survival on seeds. All the inoculants produced a significant increase in grain yield in a field experiment. The percentage of efficient cells in the field (relative to the plate counts) decreased as the length of storage increased. These results suggest that the physiological activity of B. japonicum cells changes after storage. Practical implications for inoculant quality control are discussed.

The effect of application of genistein to Bradyrhizobium japonicum culture and its rooting medium on soyabean growth nodulation and nitrogen assimilation in the presence of nitrate

In the soyabean [Glycine max (L.) Merr.]–B. japonicum symbiosis, genistein has been identified as one of the major compounds in soyabean seed and root extracts responsible for inducing the expression of the B. japonicum nod genes. High combined nitrogen in the growth medium inhibits nodulation and nitrogen assimilation. Two experiments were conducted to test the possibility of overcoming this inhibition by adding genistein to the rooting medium and by incubation of B. japonicum cells with genistein. One soyabean cultivar was used in the first experiment, and two in the second experiment. The experiments were conducted in a glasshouse using a completely randomized design with three rooting medium nitrate concentrations (0, 5 and 10 mM) and four genistein treatments. The genistein treatments were 0 (control), incubation of B. japonicum cells with 5 μM genistein, and regular watering with 5 μM or 20 μM genistein. A two way interaction existed in the first experiment, and two and three way interactions existed in the second experiment. Root growth was inhibited by repeated watering with 20 μM genistein. Weight per nodule was greater at 5 mM than at 0 mM nitrate. At 10 mM nitrate watering with genistein resulted in significant increases in nodule dry weight per plant. Shoot nitrogen contents were significantly increased at 5 mM nitrate by genistein incubation and watering with 20 μM genistein. Watering with 5 μM genistein significantly increased nodule nitrogen concentrations at both 5 and 10 mM nitrate. The two soyabean cultivars responded differently to the genistein and nitrate treatments in terms of nodule number, nodule weight, leaf nitrogen concentration and nodule nitrogen content. Genistein could, at least partially, overcome the inhibition of soyabean nodulation and nitrogen assimilation by nitrate.

Fur-independent regulation of iron metabolism by Irr in Bradyrhizobium japonicum.

Bradyrhizobium japonicum expresses both Fur and Irr, proteins that mediate iron-dependent regulation of gene expression. Control of irr mRNA accumulation by iron was aberrant in a fur mutant strain, and Fur repressed an irr::lacZ promoter fusion in the presence of iron. Furthermore, metal-dependent binding of Fur to an irr gene promoter was demonstrated in a region with no significant similarity to the Fur-binding consensus DNA element. These data suggest that the modest control of irr transcription by iron is mediated by Fur. However, Irr protein levels were regulated normally by iron in the fur strain, indicating that Fur is not required for post-transcriptional control of the irr gene. Accordingly, regulation of hemB, a haem biosynthesis gene regulated by Irr, was controlled normally by iron in a fur strain. In addition, the hemA gene was shown to be controlled by Fur, but not by Irr. It was concluded that Fur cannot be the only protein by which B. japonicum cells sense and respond to iron, and that Irr may be involved in Fur-independent signal transduction. Furthermore, iron-dependent regulation of haem biosynthesis involves both Irr and Fur.

Preincubation of B. japonicum cells with genistein reduces the inhibitory effects of mineral nitrogen on soybean nodulation and nitrogen fixation under field conditions.

Genistein is the major root produced isoflavonoid inducer of nod genes in the symbiosis between B. japonicum and soybean plants. Reduction in the isoflavonoid content of the host plants has recently been suggested as a possible explanation for the inhibition of mineral nitrogen (N) on the establishment of the symbiosis. In order to determine whether genistein addition could overcome this inhibition, we incubated B. japonicum cells (strain 532C) with genistein. Mineral N (in the form of NH4NO3) was applied at 0, 20 and 100 kg ha-1. The experiments were conducted on both a sandy-loam soil and a clay-loam soil. Preincubation of B. japonicum cells with genistein increased soybean nodule number and nodule weight, especially in the low-N-containing sandy-loam soil and the low N fertilizer treatment. Plant growth and yield were less affected by genistein preincubation treatments than nitrogen assimilation. Total plant nitrogen content was increased by the two genistein preincubation treatments at the early flowering stage. At maturity, shoot and total plant nitrogen contents were increased by the 40 μM genistein preincubation treatment at the sandy-loam soil site. Total nitrogen contents were increased by the 20 μM genistein preincubation treatment only at the 0 and 20 kg ha-1 nitrate levels in clay-loam soil. Forty μM genistein preincubation treatment increased soybean yield on the sandy-loam soil. There was no difference among treatments for 100-seed weight. The results suggest that preincubation of B. japonicum cells with genistein could improve soybean nodulation and nitrogen fixation, and at least partially overcome the inhibition of mineral nitrogen on soybean nodulation and nitrogen fixation.