Symbiotic Bradyrhizobium japonicum reduces N2O surrounding the soybean root system via nitrous oxide reductase

N(2)O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N(2)O uptake and conversion of (15)N-N(2)O into (15)N-N(2). Free-living cells of USDA110 showed N(2)O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N(2)O reductase activity. When detached soybean nodules formed with USDA110 were fed with (15)N-N(2)O, they rapidly emitted (15)N-N(2) outside the nodules at a ratio of 98.5% of (15)N-N(2)O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N(2)O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N(2)O (0.34 ppm). These results indicate that the conversion of N(2)O to N(2) depends exclusively on the respiratory N(2)O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N(2)O.     

Improved Soybean Root Association of N-Starved Bradyrhizobium japonicum

In this study, we addressed the effects of N limitation in Bradyrhizobium japonicum for its association with soybean roots. The wild-type strain LP 3001 grew for six generations with a growth rate of 1.2 day(-1) in a minimal medium with 28 mM mannitol as the carbon source and with the N source [(NH(4))(2)SO(4)] limited to only 20 microM. Under these conditions, the glutamine synthetase (GS) activity was five to six times higher than in similar cultures grown with 1 or 0.1 mM (NH(4))(2)SO(4). The NtrBC-inducible GSII form of this enzyme accounted for 60% of the specific activity in N-starved rhizobia, being negligible in the other two cultures. The exopolysaccharide (EPS) and capsular polysaccharide (CPS) contents relative to cell protein were significantly higher in the N-starved cultures, but on the other hand, the poly-3-hydroxybutyrate level did not rise in comparison with N-sufficient cultures. In agreement with the accumulation of CPS in N-starved cultures, soybean lectin (SBL) binding as well as stimulation of rhizobial adsorption to soybean roots by SBL pretreatment were higher. The last effect was evident only in cultures that had not entered stationary phase. We also studied nodC gene induction in relation to N starvation. In the chromosomal nodC::lacZ fusion Bj110-573, nodC gene expression was induced by genistein 2.7-fold more in N-starved young cultures than in nonstarved ones. In stationary-phase cultures, nodC gene expression was similarly induced in N-limited cultures, but induction was negligible in cultures limited by another nutrient. Nodulation profiles obtained with strain LP 3001 grown under N starvation indicated that these cultures nodulated faster. In addition, as culture age increased, the nodulation efficiency decreased for two reasons: fewer nodules were formed, and nodulation was delayed. However, their relative importance was different according to the nutrient condition: in older cultures the overall decrease in the number of nodules was the main effect in N-starved cultures, whereas a delay in nodulation was more responsible for a loss in efficiency of N-sufficient cultures. Competition for nodulation was studied with young cultures of two wild-type strains differing only in their antibiotic resistance, the N-starved cultures being the most competitive.     

The Type III Secretion System of Bradyrhizobium japonicum USDA122 Mediates Symbiotic Incompatibility with Rj2 Soybean Plants

The rhcJ and ttsI mutants of Bradyrhizobium japonicum USDA122 for the type III protein secretion system (T3SS) failed to secrete typical effector proteins and gained the ability to nodulate Rj2 soybean plants (Hardee), which are symbiotically incompatible with wild-type USDA122. This suggests that effectors secreted via the T3SS trigger incompatibility between these two partners.     

Bradyrhizobium japonicum Inoculant Mobility, Nodule Occupancy, and Acetylene Reduction in the Soybean Root System

In the American Midwest, superior inoculant rhizobia applied to soybeans usually occupy only 5 to 20% of nodules, and response to inoculation is the exception rather than the rule. Attempts to overcome this problem have met with limited success. We evaluated the ability of Bradyrhizobium japonicum, supplied as a seed coat inoculant, to stay abreast of the infectible region of the developing soybean root system. The rhizoplane population of the inoculant strain declined with distance from site of placement, the decrease being more pronounced on lateral than on taproots. This decline was paralleled by a decrease in inoculant-strain nodule occupancy. Inoculant bradyrhizobia contributed little to nodulation of lateral roots, which at pod-fill accounted for more than 50% of nodule number and mass, and were major contributors to acetylene reduction activity. From these data, it appears that inoculant bradyrhizobia are competitive with indigenous soil strains at the point of placement in the soil but have limited mobility and so are incapable of sustaining high populations throughout the developing root system. The result is low nodule occupancy by the inoculant strain in the tapand lateral roots. Future studies should address aspects of inoculant placement and establishment.     

Loss of Nitrous Oxide Reductase in Pseudomonas aeruginosa Cultured under N2O as Determined by Rocket Immunoelectrophoresis

The mass ratio of nitrous oxide reductase to total protein in the soluble protein fraction of Pseudomonas aeruginosa P2 was highest in cells grown on nitrate, decreased in cells grown on N₂O following the exhaustion of the initial charge of nitrate, and was nearly zero in cells exposed solely to N₂O.     

Generation of Bradyrhizobium japonicum Mutants with Increased N2O Reductase Activity by Selection after Introduction of a Mutated dnaQ Gene

We obtained two beneficial mutants of Bradyrhizobium japonicum USDA110 with increased nitrous oxide (N₂O) reductase (N₂OR) activity by introducing a plasmid containing a mutated B. japonicum dnaQ gene (pKQ2) and performing enrichment culture under selection pressure for N₂O respiration. Mutation of dnaQ, which encodes the epsilon subunit of DNA polymerase III, gives a strong mutator phenotype in Escherichia coli. pKQ2 introduction into B. japonicum USDA110 increased the frequency of occurrence of colonies spontaneously resistant to kanamycin. A series of repeated cultivations of USDA110 with and without pKQ2 was conducted in anaerobic conditions under 5% (vol/vol) or 20% (vol/vol) N₂O atmosphere. At the 10th cultivation cycle, cell populations of USDA110(pKQ2) showed higher N₂OR activity than the wild-type strains. Four bacterial mutants lacking pKQ2 obtained by plant passage showed 7 to 12 times the N₂OR activity of the wild-type USDA110. Although two mutants had a weak or null fix phenotype for symbiotic nitrogen fixation, the remaining two (5M09 and 5M14) had the same symbiotic nitrogen fixation ability and heterotrophic growth in culture as wild-type USDA110.     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N2 Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO₃-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a “conventional” Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Nitric Oxide and Nitrous Oxide Production by Soybean and Winged Bean during the in Vivo Nitrate Reductase Assay

This study was conducted to determine by gas chromatography (GC) and mass spectrometry (MS) the identity and the quantity of volatile N products produced during the helium-purged in vivo NR assay of soybean (Glycine max [L.] Merr. cv Williams) and winged bean (Psophocarpus tetragonolobus [L.] DC. cv Lunita) leaflets. Gaseous material for identification and quantitation was collected by cryogenic trapping of volatile compounds carried in the He-purge gas stream. As opposed to an earlier report, acetaldehyde oxime production was not detected by our GC method, and acetaldehyde oxime was shown to be much more soluble in water than the compound(s) evolved from soybean leaflets. Nitric oxide (NO) and nitrous oxide (N₂O) were identified by GC and GC/MS as the main N products formed. NO and N₂O produced from soybean leaflets were both labeled with ¹⁵N when ¹⁵N-nitrate was used in the assay medium, demonstrating that both were produced from nitrate during nitrate reduction. Other compounds co-trapped with NO and N₂O were identified as air (N₂, O₂), CO₂, methanol, acetaldehyde, and ethanol. Leaves of winged bean, subjected to the purged in vivo NR assay, evolved greater quantities of NO and N₂O (13.9 and 0.37 micromole per gram fresh weight per 30 minutes, respectively) than did the soybean cv Williams (1.67 and 0.09 micromole per gram fresh weight per 30 minutes, respectively). In both species NO production was dominant. In contrast, with similar assays, NO and N₂O were not evolved from leaves of the nr₁ soybean mutant which lacks the constitutive NR enzymes. In addition to soybean cv Williams, six other Glycine sp. examined evolved significant quantities of NO_(x) (NO and NO₂). Other species including Neonotonia wightii (Arn.) Lackey comb. nov., Pueraria montana (Lour.) Merr., and Pueraria thunbergiana Benth. evolved lower levels of NO_(x).     

Mutant Strain of Bradyrhizobium japonicum with Increased Symbiotic N(2) Fixation Rates and Altered Mo Metabolism Properties

Mutant strains of Bradyrhizobium japonicum that required higher levels of molybdate than the wild-type strain for growth on NO(3)-containing medium were obtained after transposon Tn5 mutagenesis of the wild-type strain. The mutant strains expressed more than fivefold-greater nitrate reductase activities in the range of 0.1 to 1.0 mM added molybdate compared with activities expressed upon incubation in non-Mo-supplemented medium, whereas the nitrate reductase activity of the wild-type strain (JH) was not markedly influenced by Mo supplementation. In free-living culture, mutant strains JH310 and JH359 expressed substantial nitrogenase activity, even in medium treated to remove molybdate, and nitrogenase activity was influenced little by Mo supplementation, whereas the wild-type strain required 100 nM added Mo for highest nitrogenase activity. Double-reciprocal plots of Mo uptake rates versus Mo concentration showed that both bacteroids and free-living cells of mutant strain JH359 had about the same affinity for Mo as did the parent strain. Bacteroids of both the mutants and the wild type also exhibited similar Mo accumulation rates over a 9-min period under very-low-Mo (4 nM) conditions. Nitrogenase activities for strain JH359 and for the wild-type strain in free-living culture were both strongly inhibited by tungsten; thus, the nitrogenase activities of both strains are probably the result of a "conventional" Mo-containing nitrogenase. Soybeans inoculated with strain JH359 and grown under either Mo-supplemented or Mo-deficient conditions had greater specific acetylene reduction rates and significantly greater plant fresh weight than those inoculated with the wild-type strain. Under Mo-deficient conditions, the acetylene reduction rates and plant fresh weights were up to 35 and 58% greater, respectively, for mutant-nodulated plants compared with wild-type-strain-nodulated plants.     

Influence of 5-Methyltryptophan-Resistant Bradyrhizobium japonicum on Soybean Root Nodule Indole-3-Acetic Acid Content

Bradyrhizobium japonicum mutants resistant to 5-methyltryptophan were isolated. Some of these mutants were found to accumulate indole-3-acetic acid (IAA) and tryptophan in culture. In greenhouse studies, nodules from control plants inoculated with wild-type bradyrhizobia contained 0.04, 0.10, and 0.58 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. Nodules from plants inoculated with 5-methyltryptophan-resistant bradyrhizobia contained 0.94, 1.30, and 10.6 μg of free, ester-linked, and peptidyl IAA g (fresh weight) of nodules⁻¹, respectively. This manyfold increase in nodule IAA content indicates that the Bradyrhizobium inoculum can have a considerable influence on the endogenous IAA level of the nodule. Further, these data imply that much of the IAA that accumulated in the high-IAA-containing nodules was of bacterial rather than plant origin. These high-IAA-producing 5-methyltryptophan-resistant bacteria were poor symbiotic nitrogen fixers. Plants inoculated with these bacteria had a lower nodule mass and fixed less nitrogen per gram of nodule than did plants inoculated with wild-type bacteria.     

Preincubation of Bradyrhizobium japonicum with Genistein Accelerates Nodule Development of Soybean at Suboptimal Root Zone Temperatures

In the soybean (Glycine max [L.] Merr.) N2-fixing symbiosis, suboptimal root zone temperatures (RZTs) slow nodule development, especially at temperatures below 17[deg]C. A step in the infection process that occurs within the first 24 h is particularly sensitive to suboptimal RZT. The first phase in the establishment of the soybean-Bradyrhizobium japonicum symbiosis is the exchange of recognition molecules. The most effective plant-to-bacterium signal is genistein. Binding of genistein to B. japonicum activates many of the B. japonicum nod genes. To our knowledge, the potential of sub-optimal RZT to disrupt this interorganismal signaling has not previously been investigated. Controlled environment experiments were conducted to determine whether the preincubation of B. japonicum with genistein increases soybean nodulation and N2 fixation at suboptimal RZT and whether the time between inoculation and root-hair curling is shortened by genistein application. The results of these experiments indicated that (a) genistein application increased soybean nodulation at suboptimal RZTs (17.5 and 15[deg]C) but not at the optimal RZT (25[deg]C); (b) the period between inoculation and root-hair curling was shortened by inoculation with bradyrhizobia preincubated with genistein; (c) at 17.5 and 15[deg]C RZT, the onset of N2 fixation occurred earlier in plants that received genistein-treated bradyrhizobia than in plants inoculated with untreated bradyrhizobia; (d) over the tested concentration range, genistein application at 15 to 20 [mu]M was the most effective in stimulating nodulation; and (e) between 25 and 15[deg]C, as RZT decreased, there was an increase in the nodulation-stimulating potential of genistein.     

Nitrous Oxide (N2O) Emissions from Subsurface Soils of Agricultural Ecosystems

Ecosystems - Nitrous oxide (N2O) is a major greenhouse gas and cultivated soils are the most important anthropogenic source. N2O production and consumption are known to occur at depths below the A...     

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 K₁ 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.     

Factors Influencing the Synthesis of Polysaccharide by Bradyrhizobium japonicum Bacteroids in Field-Grown Soybean Nodules

Certain strains of Bradyrhizobium japonicum produce large quantities of polysaccharide in soybean (Glycine max (L.) Merr.) nodules, and nodule polysaccharide (NPS) is different from that produced in culture. A previous survey of field-grown plants showed highly variable levels of NPS among field sites. To obtain clues about the possible function of NPS, we conducted two additional surveys of field-grown plants. The amount of polysaccharide in bulk samples of nodules was not associated with soil type, texture, slope, drainage, or any of the measured soil chemical properties except pH and [Ca]. Correlations with pH and [Ca] were positive and highly significant for two independent surveys involving a total of 77 sites in two years. In a preliminary comparison of high and low levels of Ca supplied to soybean plants grown in silica sand in a greenhouse, a high level of Ca (200 mg of Ca liter^-1) increased the NPS level and increased the Ca content of the polysaccharide fraction. B. japonicum isolates from 450 nodules collected at 10 field sites in 1993 were used to form nodules on soybean plants grown in sand culture in a greenhouse in order to examine bacterial phenotype under controlled conditions. Results showed that the NPS level in the bulk nodule sample from any given site was a function of the proportion of nodule occupants that were capable of NPS synthesis. Thus, a higher soil pH and/or [Ca] may positively influence the survival of B. japonicum capable of synthesis of the nodule-specific polysaccharide.     

Structure-based mechanism of O2 sensing and ligand discrimination by the FixL heme domain of Bradyrhizobium japonicum

Structures of the Bradyrhizobium japonicum FixL heme domain have been determined in the absence and presence of specific ligands to elucidate the detailed features of its O2 sensing mechanism. The putative roles of spin-state and steric hindrance were evaluated by the structure determination of ferrous CO-bound BjFixLH and correlating its features with other ligand-bound structures. As found for NO-BjFixLH, no protein conformational change was observed in CO-BjFixLH, suggesting a more complicated mechanism than solely spin state or ligand sterics. To evaluate the role of oxidation state, the structure of the ferrous deoxy-BjFixLH was determined. The structure of deoxy-BjFixLH was found to be virtually identical to the structure of the ferric met-BjFixLH. The role of hydrogen bonding of substrates to a heme-pocket water was evaluated by determining the structure of BjFixLH bound to 1-methyl-imidazole that cannot form a hydrogen bond with this water. In this case, the heme-mediated conformational change was observed, limiting the potential importance of this interaction. Finally, the structure of cyanomet-BjFixLH was revisited to rule out concerns regarding the partial occupancy of the cyanide ligand in a previous structure. In the revised structure, Arg 220 was found to move into the heme pocket to form a hydrogen bond to the bound cyanide ligand. The implications of these results on FixL's sensing mechanism are discussed.     

Competition for electrons favours N2O reduction in denitrifying Bradyrhizobium isolates

Bradyrhizobia are common members of soil microbiomes and known as N₂-fixing symbionts of economically important legumes. Many are also denitrifiers, which can act as sinks or sources for N₂O. Inoculation with compatible rhizobia is often needed for optimal N₂-fixation, but the choice of inoculant may have consequences for N₂O emission. Here, we determined the phylogeny and denitrification capacity of Bradyrhizobium strains, most of them isolated from peanut-nodules. Analyses of genomes and denitrification end-points showed that all were denitrifiers, but only ~1/3 could reduce N₂O. The N₂O-reducing isolates had strong preference for N₂O- over NO₃⁻-reduction. Such preference was also observed in a study of other bradyrhizobia and tentatively ascribed to competition between the electron pathways to Nap (periplasmic NO₃⁻ reductase) and Nos (N₂O reductase). Another possible explanation is lower abundance of Nap than Nos. Here, proteomics revealed that Nap was instead more abundant than Nos, supporting the hypothesis that the electron pathway to Nos outcompetes that to Nap. In contrast, Paracoccus denitrificans, which has membrane-bond NO₃⁻ reductase (Nar), reduced N₂O and NO₃⁻ simultaneously. We propose that the control at the metabolic level, favouring N₂O reduction over NO₃⁻ reduction, applies also to other denitrifiers carrying Nos and Nap but lacking Nar.     

Cyclic [beta]-1,6 -1,3 Glucans Are Synthesized by Bradyrhizobium japonicum Bacteroids within Soybean (Glycine max) Root Nodules

We have previously reported that free-living cultures of Bradyrhizobium species produce novel oligosaccharides that are cyclic, contain between 10 and 13 glucose residues, and are linked by [beta]-1,6 and [beta]-1,3 glycosidic bonds (K.J. Miller, R.S. Gore, R. Johnson, A.J. Benesi, V.N. Reinhold [1990] J Bacteriol 172: 136-142). In the present study, we show that these glucans are also synthesized by bacteroids of Bradyrhizobium japonicum USDA 110 within Glycine max root nodules.