Bradyrhizobium Japonicum Bacteroids Research Articles

Gibberellin oxidase activities in Bradyrhizobium japonicum bacteroids

Bradyrhizobium japonicum bacteroids isolated from root nodules of soybean (Glycine max.) plants converted the gibberellin (GA) precursor [14C1]GA12 into several products identified by combined gas chromatography–mass spectrometry as [14C1]GA24, [14C1]GA9, [14C1]GA15, GA9 17-nor-16-one and unidentified products. The oxidation of GA12, catalyzed by the GA 20-oxidase, was present in symbiotic bacteroids from plants around flowering, but not in bacteroids from plants at either an early vegetative stage or at late growth stages. Expression of cps and ks genes, involved in ent-kaurene biosynthesis, was also demonstrated in bacteroids from soybean plants around flowering. Earlier precursors of the GA pathway, ent-[14C1]kaurenoic acid or [14C4]GA12-aldehyde, were efficiently utilized by B. japonicum bacteroids to give labelled GA9 plus intermediates partially oxidized at C-20, as well as GA9 17-nor-16-one and an unidentified product. No 3β or 13-hydroxylated [14C]GAs were detected in any of the incubations. Moreover the C19-GAs [14C1]GA4 or [14C1]GA20 were recovered unconverted upon incubation with the bacteroids which supports the absence of GA 3β-hydroxylase activity in B. japonicum. The bacterial 20-oxidase utilized the 13-hydroxylated substrates [14C1]GA53, [14C1]GA44 or [14C1]GA19, although with less efficiency than [14C1]GA12 to give [14C1]GA20 as final product, while the 3β-hydroxylated substrate [14C1]GA14 was converted to [14C1]GA4 to a very small extent. Endogenous GA9 and GA24 were identified by GC–MS in methanolic nodule extracts. These results suggest that B. japonicum bacteroids would synthesize GA9 under the symbiotic conditions present in soybean root nodules.

Bradyrhizobium japonicum bacteroid appendages expressed in senescing and argon-treated soybean nodules

Bradyrhizobium japonicum bacteroids in soybean nodules expressed fibrillar appendages during senescence. In both scanning and transmission electron microscopy (SEM and TEM), these structures were observed to connect adjacent bacteroids, and bacteroids to symbiotic membranes. They were 20–25 nm in diameter, 100–2,500 nm in length and were linear, branched, or part of a web-like matrix. Bacteroids expressing appendages were not uniformly distributed, but were abundant within localized regions in the senescing nodule. The root systems of nodulated greenhouse-grown plants flushed with argon induced the appendages at earlier plant ages, and they were more prolific and wide spread than those in untreated nodules. Bradyrhizobium japonicum symbiotic appendages appear to be a response to an environmental niche within senescing nodules.

Involvement of Bradyrhizobium japonicum denitrification in symbiotic nitrogen fixation by soybean plants subjected to flooding

Abstract Denitrification by Bradyrhizobium japonicum bacteroids contributes to nitric oxide (NO) production within soybean nodules in response to flooding conditions. However, the physiological relevance of NO production by denitrification in B. japonicum–Glycine max symbiosis is still unclear. In this work, soybean plants were inoculated with B. japonicum strains lacking the nirK or norC genes which encode the copper-containing nitrite reductase and the c-type nitric oxide reductase enzymes, respectively. 14 days flooding increased nodule number of plants inoculated with the WT and norC strains, but not of plants inoculated with the nirK mutant. However, nodule dry weight was not affected by 14 days flooding regardless of the strain used for inoculation. Supporting this observation, individual nodule growth was significantly higher in plants inoculated with nirK than those inoculated with WT or norC after 14 days flooding. Nodule functioning was strongly inhibited by flooding since leghemoglobin content of the nodules induced by any of the strains was significantly decreased after 7 or 14 days flooding compared to control plants. However, this effect was more relevant in nodules of plants inoculated with the WT or norC mutant than in those inoculated with the nirK mutant. Nitrogen fixation was also estimated by analyzing nitrogen content derived from biological nitrogen fixation in shoots, using the 15N isotope dilution technique. By using this approach, we observed that the negative effect of 14 days flooding on nitrogen fixation was more pronounced in plants inoculated with the norC mutant. However, nitrogen fixation of plants inoculated with nirK showed the highest tolerance to 14 days flooding. These findings allowed us to demonstrate the previously proposed hypothesis which suggests that NO formed by copper-containing nitrite reductase in soybean nodules, in response to flooding, has a negative effect on nitrogenase activity. We propose that inoculation of soybeans with a B. japonicum nirK mutant, which does not produce NO from nitrate, increases the tolerance of symbiotic nitrogen fixation to flooding.

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.

The contribution of bacteroidal nitrate and nitrite reduction to the formation of nitrosylleghaemoglobin complexes in soybean root nodules

It is becoming recognized that leghaemoglobin constitutes an important buffer for the cytotoxic nitric oxide radical (NO(*)) in root nodules, although the sources of this NO(*) within nodules are unclear. In Bradyrhizobium japonicum bacteroids, NO(*) can be produced through the denitrification process, during which nitrate is reduced to nitrite by the periplasmic nitrate reductase Nap, and nitrite is reduced to NO(*) by the respiratory nitrite reductase NirK. To assess the contribution of bacteroidal denitrification to the NO(*) within nitrate-treated soybean nodules, electron paramagnetic resonance and UV-visible spectroscopy were employed to study the presence of nitrosylleghaemoglobin (LbNO) within nodules from plants inoculated with wild-type, napA or nirK B. japonicum strains. Since it has been found that hypoxia induces NO(*) production in plant root tissue, and that plant roots can be subjected to hypoxic stress during drought and flooding, the effect of hypoxic stress on the formation of LbNO complexes within nodules was also investigated. Maximal levels of LbNO were observed in nodules from plants treated with nitrate and subjected to hypoxic conditions. It is shown that, in the presence of nitrate, all of the LbNO within normoxic nodules arises from nitrate reduction by the bacteroidal periplasmic nitrate reductase, whereas Nap activity is only responsible for half of the LbNO within hypoxic nodules. In contrast to Nap, NirK is not essential for LbNO formation under any condition tested.

The role of Bradyrhizobium japonicum nitric oxide reductase in nitric oxide detoxification in soya bean root nodules

The identification of nitric oxide-bound leghaemoglobin within soya bean nodules has led to the question of how Bradyrhizobium japonicum bacteroids overcome the toxicity of this nitric oxide. It has previously been shown that one candidate for nitric oxide detoxification, the respiratory nitric oxide reductase, is expressed in soya bean nodules from plants supplied with nitrate. In this paper, the role of this enzyme in nitric oxide detoxification is assessed and discussion is provided on other possible B. japonicum nitric oxide detoxification systems.

Cloning and characterization of a NADP+-malic enzyme gene from Bradyrhizobium japonicum USDA110

Malic enzymes have been considered to play a key role in energy metabolism for nitrogenase reaction in bacteroids. To elucidate the physiological role of the malic enzymes in Bradyrhizobium japonicum bacteroids, a putative malic enzyme gene Bjtme1 was cloned by polymerase chain reaction (PCR) using degenerated primers from conserved regions of the protein sequences of bacterial malic enzymes and draft sequence data of the Bradyrhizobium japonicum USDA110 genome sequence project. To confirm the characteristics of the Bjtme1 gene, the protein encoded by this gene was over-expressed using a pET32a(+) system and it exhibited a NADP+-malic enzyme (EC 1.1.1.40) activity, indicating that Bjtme1 was the gene of the NADP+-malic enzyme. This is the first report on the cloning and characterization of the NADP+-malic enzyme gene from B. japonicum, and the gene structure was compared with that of NADP+-malic enzyme genes of other rhizobia.

Partial purification and characterization of pyruvate kinase from the plant fraction of soybean root nodules.

Pyruvate kinase (PK, EC 2.7.1.40) was partially purified from the plant cytosolic fraction of N2-fixing soybean (Glycine max [L.] Merr.) root nodules. The partially purified PK preparation was completely free of contamination by phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), the other major phosphoenolpyruvate (PEP)-utilizing enzyme in legume root nodules. Latency experiments with sonicated nodule extracts showed that Bradyrhizobium japonicum bacteroids do not express either PK or PEPC activity in symbiosis. In contrast, free-living B. japonicum bacteria expressed PK activity, but not PEPC activity. Antibodies specific for the cytosolic isoform of PK from castor bean endosperm cross-reacted with a 52-kDa polypeptide in the partially purified PK preparation. At the optimal assay pH (pH 8.0 for PEPC and pH 6.9 for PK) and in the absence of malate, PEPC activity in crude nodule extracts was 2.6 times the corresponding PK activity. This would tend to favour PEP metabolism by PEPC over PEP metabolism by PK. However, at pH 7.0 in the presence of 5 mM malate, PEPC activity was strongly inhibited, but PK activity was unaffected. Thus, we propose that PK and PEPC activity in legume root nodules may be coordinately regulated by fluctuations in malate concentration in the plant cytosolic fraction of the bacteroid-containing cells. Reduced uptake of malate by the bacteroids, as a result of reduced rates of N2 fixation, may favour PEP metabolism by PK over PEP metabolism by PEPC.

Effectiveness of ascorbate and ascorbate peroxidase in promoting nitrogen fixation in model systems

Ascorbate and ascorbate peroxidase are important antioxidants that are abundant in N 2-fixing legume root nodules. Antioxidants are especially critical in root nodules because leghemoglobin, which is present at high concentrations in nodules, is prone to autoxidation and production of activated oxygen species such as O 2 − and H 2O 2. The merits of ascorbate and ascorbate peroxidase for maintaining conditions favorable for N 2 fixation were examined in two model systems containing oxygen-binding proteins (purified myoglobin or leghemoglobin) and N 2-fixing microorganisms (free-living Azorhizobium or bacteroids of Bradyrhizobium japonicum) in sealed vials. The inclusion of ascorbate alone to these systems led to enhanced oxygenation of hemeproteins, as well as to increases in nitrogenase (acetylene reduction) activity. The inclusion of both ascorbate and ascorbate peroxidase resulted in even greater positive responses, including increases of up to 4.5-fold in nitrogenase activity. In contrast, superoxide dismutase did not provide beneficial antioxidant action and catalase alone provided only very marginal benefit. Optimal concentrations were 2 mM for ascorbate and 200 μg/ml for ascorbate peroxidase. These concentrations are similar to those found in intact soybean nodules. These results support the conclusion that ascorbate and ascorbate peroxidase are beneficial for maintaining conditions favorable for N 2 fixation in nodules.

Protein Synthesis by Bradyrhizobium japonicum Bacteroids Declines as a Function of Nodule Age

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.

Identification of Bradyrhizobium Japonicum malonamidase E2 in the periplasmic space of soybean nodule bacteroids

Summary MalonamidaseE2 (MAE2) was found both in the plant cytosolic fraction and in the Bradyrhizobium japonicum bacteroids of soybean nodules, and was proposed to play an important role in the transfer of fixed nitrogen (NH 3 ) to plant cells. The enzyme in the cytosol was identical in its molecular size and was immunologically homologous with MAE2 from free-living B. japonicum . The enzyme was not found in soybean root that is not infected by symbiotic bacteria. B. japonicum MAE2 presence in the periplasmic space was confirmed by the determination of the enzyme activity using intact bacteria in the presence of Triton X-100 (0.02%). Under microaerobic conditions, B. japonicum secreted MAE2 into the medium. However, the presence of malonate did not affect the secretion, indicating that malonate is not directly involved in the secretion of MAE2. The evidence described above strongly suggests that B. japonicum MAE2 located in the periplasmic space may be secreted into the peribacteroidal space of nodules during symbiosis.

Purification and Properties of NAD- and NADP-dependent Malic Enzymes from Bradyrhizobium japonicum Bacteroids

Summary Bacteroids were isolated from 6 weeks-old soybean nodules and the NAD- and NADP-dependent malic enzymes purified by ammonium sulfate precipitation, DEAE-cellulose ion exchange chromatography, Bio-Gel chromatography and fast flow Q-Sepharose ion-exchange chromatography. NADP-ME (E.C. 1.1.1.40) was purified 40-fold and NAD-ME (E.C. 1.1.1.38) 14-fold. Native PAGE demonstrated that NAD-ME and NADP-ME from bacteroids are two distinct enzymes. The molecular weight of the native enzymes was estimated by means of gel filtration and SDS-PAGE. It was shown that NADP-ME is a 60 kD monomer and NAD-ME is a heterodimer with subunits of 60 and 80 kD. NADP-ME (K M : 0.18 mM) exhibited much higher affinity for malate than NAD-ME (K M : 5 mM). The role of both enzymes in bacteroid metabolism is discussed.

Fate of Nodule-Specific Polysaccharide Produced by Bradyrhizobium japonicum Bacteroids.

A polysaccharide produced by Bradyrhizobium japonicum bacteroids in nodules (NPS) on soybean (Glycine max [L.] Merr.) roots is different in composition and structure from the extracellular polysaccharide produced in culture by this organism. Isogenic strains either capable or incapable of NPS synthesis supported similar rates of plant growth and nitrogenase activity, indicating that polysaccharide deposition was not detrimental. The possibility that NPS may have some protective or nutritional role for bacteroids was considered. Analysis of disintegrating nodules over periods of 1 to 3 months indicated greater recovery of viable bacteria from NPS+ nodules prior to the breakdown of NPS. During and after the breakdown of NPS, the decline in viable bacteria was similar for NPS+ and NPS- strains. Bacteroid destruction in senescing nodules may be accelerated by exposure to proteolytic enzymes in host cytoplasm; however, highly purified NPS had no significant effect on the in vitro activity of partially purified proteases, so protection of bacteroids via this mechanism is unlikely. B. japonicum USDA 438 did not utilize NPS as a carbon source for growth in liquid culture. In vitro assays of NPS depolymerase activity in cultured bacteria and bacteroids were negative using a variety of strains, all of which contained extracellular polysaccharide depolymerase. It seems highly unlikely that B. japonicum can utilize the polysaccharide it synthesizes in nodules, and NPS breakdown in senescing nodules is probably caused by saprophytic fungi.

Cloning and Mutagenesis of a Cytochrome P-450 Locus from Bradyrhizobium japonicum That Is Expressed Anaerobically and Symbiotically

Cytochromes P-450, which in many organisms participate in the metabolism of a variety of endobiotic and xenobiotic substances, are synthesized by symbiotic bacteroids of Bradyrhizobium japonicum. Polyclonal antibodies were raised against two cytochromes P-450 (CYP112 and CYP114) purified from bacteroids. A lambda gt11 expression clone of B. japonicum USDA 110 DNA that reacted with the anti-CYP112 antibody was obtained and was used to screen a library of USDA 110 genomic DNA in pLAFR1 for a clone of the P-450 locus. Forced expression of subclones of the P-450 locus in Escherichia coli produced polypeptides that reacted with either the anti-CYP112 antibody or the anti-CYP114 antibody; no cross-reactivity was evident. A Western blot (immunoblot) analysis showed that neither protein was present in free-living aerobically grown B. japonicum cells, but that both proteins were present in cells grown anaerobically, as well as in bacteroids. A mutant strain disrupted in the CYP112 locus produced neither CYP112 nor CYP114, indicating that the mutation was polar for CYP114. The mutant produced effective nodules on soybeans, even though the bacteroids contained no detectable P-450. This suggests that the cytochromes P-450 which we examined are not involved in an essential symbiotic function.

Distribution of the two types of polysaccharide formed by Bradyrhizobium japonicum bacteroids in nodules on field-grown soybean plants ( Glycine max (L.) Merr.)

In a comparison of pure strains of Bradyrhizobium japonicum with greenhouse-grown nodules, it was found that polysaccharide synthesis in soybean nodules ( Glycine max (L.) Merr.) is common in serogroups 31, 46 and 123. Among strains in these three serogroups plus serogroup 76, two-thirds of the 54 strains tested were polysaccharide formers, and these four serogroups are among the most commonly found in surveys of field-grown plants. In a survey of 66 samples of soybean nodules from field-grown plants, B. japonicum polysaccharide was found in all samples, although there were as much as 20-fold differences in polysaccharide concentration among samples. Some of this variation among field sites is probably explained by the fact that, on any given soybean plant, some of the nodules may contain B. japonicum polysaccharide while others may not. Different types of nodule polysaccharide are formed by the two DNA homology groups of B. japonicum, and the distribution of polysaccharide types in field-grown nodules from different regions of the U.S. corresponded roughly to the distribution of the two DNA homology groups of B. japonicum in different regions of the U.S.

Pyrroline-5-Carboxylate Reductase in Soybean Nodules : Comparison of the Enzymes in Host Cytosol, Bradyrhizobium japonicum Bacteroids, and Cultures

Characteristics of pyrroline-5-carboxylate reductase (P5CR) from Bradyrhizobium japonicum bacteroids and cultured rhizobia were compared with those of the enzyme in soybean nodule host cytosol. Reductase from host cytosol differed from that in bacteroids in: (a) the effect of pH on enzymic activity, (b) the capacity to catalyze both reduction of pyrroline-5-carboxylic acid and NAD(+)-dependent proline oxidation, (c) apparent affinities for pyrroline-5-carboxylic acid, and (d) sensitivities to inhibition by NADP(+) and proline. The K(1) for proline inhibition of P5CR in bacteroid cytosol was 1.8 millimolar. The properties of P5CR in B. japonicum and bacteroid cytosol were similar. The specific activities of P5CR in the cytosolic fractions of the nodule host and the bacteroid compartment were also comparable.

Analysis of DNA sequences transcribed at high levels in Bradyrhizobium japonicum bacteroids but not necessary for symbiotic effectiveness.

Five regions of the Bradyrhizobium japonicum genome, which are transcribed at high levels in nitrogen-fixing soybean (Glycine max) nodules, were identified. None of these regions contained previously identified genes (e.g., nif, nod, and fix genes) that are known to be essential for development of functional nitrogen-fixing nodules. To assess the role of these regions in the development of the B. japonicum-soybean symbiosis, we cloned and used them to construct B. japonicum strains, in which large DNA segments (2.0-6.8 kilobases) containing the highly transcribed regions were deleted. The deletion strains were examined for symbiotic effectiveness and were found to be indistinguishable from the wild-type strain. Transcription of the cloned regions under a variety of physiological conditions and in several defined mutant B. japonicum strains was also examined. The transcriptional start sites for one pair of divergent transcripts were determined; the promoters do not contain any of the conserved sequences found in B. japonicum genes involved in symbiosis or nitrogen metabolism.

Improved analysis of metabolite uptake by Bradyrhizobium japonicum bacteroids

Uptake of a variety of metabolites by Bradyrhizobium japonicum bacteroids under aerobic conditions was studied with the goal of identifying factors that would influence the accuracy of kinetic parameters. Saturable and nonsaturable components of absorption were identified, allowing separate analysis of only the active component. A significant proportion of labeled substrate was released as CO2. The CO2 release was linear with time and occurred without a noticeable lag period. The kinetic parameters of saturable, active uptake indicated that if the label converted to CO2 was ignored, this would result in underestimation of the total uptake of malate by more than 50%. The proportion of the labeled substrate converted to CO2 depended on the compound but was high in all cases. Glucose showed the smallest difference, with 13% of the label converted to CO2 in a 5-min incubation. The uptake of glutamate was inhibited by aspartate, whereas no effect by equimolar dicarboxylic acids was found. Succinate or fumarate inhibited malate uptake, whereas amino acids showed no effect. Thus, organic acids appeared to have a common carrier distinct from that for amino acids. Techniques for separation of active and passive uptake components and for collection and analysis of CO2 respired are described; these techniques should be used for accurate analysis of metabolite uptake. Key words: Bradyrhizobium japonicum, bacteroids, metabolite uptake, CO2.

Characterization of a soluble catalase-peroxidase hemoprotein b-590, previously identified as 'cytochrome alpha 1' from Bradyrhizobium japonicum bacteroids.

The cytochrome "a1" or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified. The water-soluble native hemoprotein has an Mr of 136,000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b-590 (Escherichia coli; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO. It has catalase and peroxidase activities, again resembling the E. coli b-590. Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish peroxidase reacts with oxygen to form such a complex. The hemoprotein, which we name hemoprotein b-590 (Bradyrhizobium japonicum), may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species. The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.

Characterization of a soluble catalase-peroxidase hemoproteinb-590, previously identified as ‘cytochromea1’ fromBradyrhizobium japonicumbacteroids

Abstract The cytochrome “ a 1 ” or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified. The water-soluble native hemoprotein has an M r of 136 000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b -590 ( Escherichia coli ; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO. It has catalase and peroxidase activities, again resembling the E. coli b -590. Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish per-oxidase reacts with oxygen to form such a complex. The hemoprotein, which we name hemoprotein, b -590 ( Bradyrhizobium japonicum , may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species. The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.