Nickel accumulation and storage in Bradyrhizobium japonicum

Hydrogenase-derepressed (chemolithotrophic growth conditions) and heterotrophically grown cultures of Bradyrhizobium japonicum accumulated nickel about equally over a 3-h period. Both types of cultures accumulated nickel primarily in a form that was not exchangeable with NiCl2, and they accumulated much more Ni than would be needed for the Ni-containing hydrogenase. The nickel accumulated by heterotrophically incubated cultures could later be mobilized to allow active hydrogenase synthesis during derepression in the absence of nickel, while cells both grown and derepressed without nickel had low hydrogenase activities. The level of activity in cells grown with Ni and then derepressed without nickel was about the same as that in cultures derepressed in the presence of nickel. The Ni accumulated by heterotrophically grown cultures was associated principally with soluble proteins rather than particulate material, and this Ni was not lost upon dialyzing an extract containing the soluble proteins against either Ni-containing or EDTA-containing buffer. However, this Ni was lost upon pronase or low pH treatments. The soluble Ni-binding proteins were partially purified by gel filtration and DEAE chromatography. They were not antigenically related to hydrogenase peptides. Much of the 63Ni eluted as a single peak of 48 kilodaltons. Experiments involving immunoprecipitation of 63Ni-containing hydrogenase suggested that the stored source of Ni in heterotrophic cultures that could later be mobilized into hydrogenase resided in the nonexchangeable Ni-containing fraction rather than in loosely bound or ionic forms.     

Nickel uptake in Bradyrhizobium japonicum.

Free-living Bradyrhizobium japonicum grown heterotrophically with 1 microM 63Ni2+ accumulated label. Strain SR470, a Hupc mutant, accumulated almost 10-fold more 63Ni2+ on a per-cell basis than did strain SR, the wild type. Nongrowing cells were also able to accumulate nickel over a 2-h period, with the Hupc mutant strain SR470 again accumulating significantly more 63Ni2+ than strain SR. These results suggest that this mutant is constitutive for nickel uptake as well as for hydrogenase expression. The apparent Kms for nickel uptake in strain SR and strain SR470 were found to be similar, approximately 26 and 50 microM, respectively. The Vmax values, however, were significantly different, 0.29 nmol of Ni/min per 10(8) cells for SR and 1.40 nmol of Ni/min per 10(8) cells for SR470. The uptake process was relatively specific for nickel; only Cu2+ and Zn2+ (10 microM) were found to appreciably inhibit the uptake of 1 microM Ni, while a 10-fold excess of Mg2+, Co2+, Fe3+, or Mn2+ did not affect Ni2+ uptake. The lack of inhibition by Mg2+ indicates that nickel is not transported by a magnesium uptake system. Nickel uptake was also inhibited by cold (53% inhibition at 4 degrees C) and slightly by the ionophores nigericin and carbonyl cyanide m-chlorophenylhydrazone. Other ionophores did not appreciably affect nickel uptake, even though they significantly stimulated O2 uptake. The cytochrome c oxidase inhibitors azide, cyanide, and hydroxylamine did not inhibit Ni2+ uptake, even at concentrations (of cyanide and hydroxylamine) that inhibited O2 uptake. The addition of oxidizable substrates such as succinate or gluconate did not increase nickel uptake, even though they increased respiratory activity. Nickel update showed a pH dependence with an optimum at 6.0. Most (approximately 85%) of the 63Ni2+ taken up in 1 min by strain SR470 was not exchangeable with cold nickel.     

Production and survival during storage of spray-dried Bradyrhizobium japonicum cell concentrates

Production of Bradyrhizobium japonicum cell concentrates by spray-drying in skim milk plus sucrose medium and the feasability of storing dried inocula over long periods were investigated. Storage of spray-dried cells under mild vacuum was equivalent to storage under nitrogen. Oxygen and ambient temperature were found detrimental for survival of dried cells. High initial cell concentration and storage under low relative humidities (< 23% RH) at 4°C increased the longevity of the inocula (> 10⁹ cfu g^-1 during at least a 25 week storage period) without altering the symbiotic properties of B. japonicum.     

Protein phosphorylation in Bradyrhizobium japonicum bacteroids and cultures.

Protein phosphorylation was demonstrated in Bradyrhizobium japonicum bacteroids in vivo and in cultures in vivo and in vitro. Comparison of in vivo-labeled phosphoproteins of bacteroids and of cultured cells showed differences in both the pattern and intensity of labeling. In cultured cells, comparison of the labeling patterns and intensities of in vivo- and in vitro-labeled phosphoproteins showed a number of similarities; however, several phosphoproteins were found only after one of the two labeling conditions. The labeling intensity was time dependent in both in vivo and in vitro assays and was dependent on the presence of magnesium in in vitro assays. Differences in the rates of phosphorylation and dephosphorylation were noted for a number of proteins. The level of incorporation of 32P into protein was only 2% or less of the total phosphate accumulated during the in vivo labeling period. Several isolation and sample preparation procedures resulted in differences in labeling patterns. Phosphatase inhibitors and several potential metabolic effectors had negligible effects on the phosphorylation pattern. There were no significant changes in the phosphorylation patterns of cells cultured on mannitol, acetate, and succinate, although the intensity of the labeling did vary with the carbon source.     

Citrate as a siderophore in Bradyrhizobium japonicum.

Under iron-limiting conditions, many bacteria secrete ferric iron-specific ligands, generically termed siderophores, to aid in the sequestering and transport of iron. One strain of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum, 61A152, was shown to produce a siderophore when 20 B. japonicum strains were screened with all six chemical assays commonly used to detect such production. Production by strain 61A152 was detected via the chrome azurol S assay, a general test for siderophores which is independent of siderophore structure. The iron-chelating compound was neither a catechol nor a hydroxamate and was ninhydrin negative. It was determined to be citric acid via a combination of thin-layer chromatography and high-voltage paper electrophoresis; this identification was verified by a specific enzymatic assay for citric acid. The inverse correlation which was observed between citric acid release and the iron content of the medium suggested that ferric citrate could serve as an iron source. This was confirmed via growth and transport assays. Exogenously added ferric citrate could be used to overcome iron starvation, and iron-deficient cells actively transported radiolabeled ferric citrate. These results, taken together, indicate a role for ferric citrate in the iron nutrition of this strain, which has been shown to be an efficient nitrogen-fixing strain on a variety of soybean cultivars.     

Molybdate transport by Bradyrhizobium japonicum bacteroids.

Bacteroid suspensions of Bradyrhizobium japonicum USDA 136 isolated from soybeans grown in Mo-deficient conditions were able to transport molybdate at a nearly constant rate for up to 1 min. The apparent Km for molybdate was 0.1 microM, and the Vmax was about 5 pmol/min per mg (dry weight) of bacteroid. Supplementation of bacteroid suspensions with oxidizable carbon sources did not markedly increase molybdate uptake rates. Anaerobically isolated bacteroids accumulated twice as much Mo in 1 h as aerobically isolated cells did, but the first 5 min of molybdate uptake was not dependent on the isolation condition with respect to O2. Respiratory inhibitors such as cyanide, azide, and hydroxylamine did not appreciably affect molybdate uptake, even at concentrations that inhibited O2 uptake. The uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) and the ionophores nigericin and monensin significantly inhibited molybdate uptake. The electrogenic ionophores valinomycin and gramicidin stimulated molybdate uptake. Rapid pH shift experiments indicated that molybdate transport depends on a transmembrane proton gradient (delta pH), and it is probably transported electroneutrally as H2MoO4. Most of the 99MoO4(2-) taken up was not exchangeable with a 100-fold excess of unlabeled MoO4(2-). Tungstate was a competitive inhibitor of molybdate uptake, with a Ki of 0.034 microM, and vanadate inhibited molybdate uptake slightly.     

Dual roles of Bradyrhizobium japonicum nickelin protein in nickel storage and GTP-dependent Ni mobilization

The hydrogenase accessory protein HypB, or nickelin, has two functions in the N(2)-fixing, H(2)-oxidizing bacterium Bradyrhizobium japonicum. One function of HypB involves the mobilization of nickel into hydrogenase. HypB also carries out a nickel storage/sequestering function in B. japonicum, binding nine nickel ions per monomer. Here we report that the two roles (nickel mobilization and storage) of HypB can be separated in vitro and in vivo using molecular and biochemical approaches. The role of HypB in hydrogenase maturation is completely dependent on its intrinsic GTPase activity; strains which produce a HypB protein that is severely deficient in GTPase activity but that fully retains nickel-sequestering ability cannot produce active hydrogenase even upon prolonged nickel supplementation. A HypB protein that lacks the nickel-binding polyhistidine region near the N terminus lacks only the nickel storage capacity function; it is still able to bind a single nickel ion and also retains complete GTPase activity.     

Conservation of symbiotic nitrogen fixation gene sequences in Rhizobium japonicum and Bradyrhizobium japonicum.

Southern hybridization with nif (nitrogen fixation) and nod (nodulation) DNA probes from Rhizobium meliloti against intact plasmid DNA of Rhizobium japonicum and Bradyrhizobium japonicum strains indicated that both nif and nod sequences are on plasmid DNA in most R. japonicum strains. An exception is found with R. japonicum strain USDA194 and all B. japonicum strains where nif and nod sequences are on the chromosome. In R. japonicum strains, with the exception of strain USDA205, both nif and nod sequences are on the same plasmid. In strain USDA205, the nif genes are on a 112-megadalton plasmid, and nod genes are on a 195-megadalton plasmid. Hybridization to EcoRI digests of total DNA to nif and nod probes from R. meliloti show that the nif and nod sequences are conserved in both R. japonicum and B. japonicum strains regardless of the plasmid or chromosomal location of these genes. In addition, nif DNA hybridization patterns were identical among all R. japonicum strains and with most of the B. japonicum strains examined. Similarly, many of the bands that hybridize to the nodulation probe isolated from R. meliloti were found to be common among R. japonicum strains. Under reduced hybridization stringency conditions, strong conservation of nodulation sequences was observed in strains of B. japonicum. We have also found that the plasmid pRjaUSDA193, which possess nif and nod sequences, does not possess sequence homology with any plasmid of USDA194, but is homologous to parts of the chromosome of USDA194. Strain USDA194 is unique, since nif and nod sequences are present on the chromosome instead of on a plasmid as observed with all other strains examined.     

Carbon metabolism in Bradyrhizobium japonicum bacteroids

Abstract Carbon metabolism in Bradyrhizobium japonicum bacteroids is reviewed. Additionally, the bacteroid tricarboxylic acid (TCA) cycle and its regulation under oxygen-limited conditions is considered, with emphasis on possible sites of TCA cycle rate-limiting reactions. Furthermore, we consider other adaptive pathways that may be employed by these organisms while in symbiosis. These pathways include: (1) a poly-β-hydroxy-butyrate shunt, (2) a malate-aspartate shuttle, (3) an α-ketoglutarate-glutamate shunt, (4) a modified dicarboxylic acid cycle, and (5) fermentation pathways leading to lactate, acetaldehyde and ethanol. The effects of oxygen limitation on host carbon metabolism are also considered briefly.     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

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 °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. Received: 20 September 1999 / Received revision: 3 March 2000 / Accepted: 6 March 2000     

Chemotaxis of Bradyrhizobium japonicum to soybean exudates.

The chemotactic response of Bradyrhizobium japonicum toward soybean seed and root exudates was examined. Assays using various isoflavones and fractionated exudate indicated that isoflavones are not the principal attractants in exudates. Likewise, induction of nod genes with isoflavones or seed exudate before assay did not enhance chemotaxis. Screening of numerous compounds revealed that only dicarboxylic acids and the amino acids glutamate and aspartate were strong attractants. The presence of glutamate, aspartate, and dicarboxylic acids in appreciable concentrations in soybean seed and root exudates indicates that these compounds likely represent natural chemoattractants for B. japonicum.     

Viability of Bradyrhizobium japonicum bacteriods

Homogenates from soybean nodules, formed by 12 strains of Bradyrhizobium japonicum, were plated into yeast-extract mannitol agar containing 3 or 37 g mannitol 1^-1. Viable counts ranged from 8.298 to 11.265 log₁₀ cells-gram nodule^-1. When monitored over the life cycle of the symbiosis, the viability of strains USDA 110 and USDA 123 increased with days after planting (DAP), and at 70 DAP was 95% and 81%, respectively. By contrast, the viability of USDA 38 bacteroids decreased with time, and at 70 DAP was only 1.9%. At 49 DAP, nodules induced by USDA 38 had significantly fewer bacteroids per peribacteroid membrane than those formed by USDA 110 or USDA 123, and at 70 DAP, 27% of the USDA 38 bacteroids showed some degree of degeneration. Viable counts of USDA 123 and USDA 110 bacteroids, isolated from the nodules of 12 different cultivars, ranged from 10.963 to 11.463 and from 10.683 to 11.117 log₁₀ viable cells-gram nodule^-1, respectively. Varying the osmolarity of the medium had no predictable effect on bacteroid viability. When surface-sterilized nodules of IPAGO 587 (high bacteroid viability) and USDA 38 (low bacteroid viability) were inoculated into a nonsterile silt loam soil, at rates equivalent to 5.0×10⁸ and 5.0×10⁶ viable bacteroids g^-1 soil, respectively, and then incubated at 28° C for 60 days, 4.3×10⁴ and 1.5×10⁴ surviving cells g^-1 soil, respectively, were recovered. Thus, despite differences due to host and strain variation, bacteroid viability appears to be unrelated to persistence of individual strains following an annual legume crop cycle.Journal paper No. 14930, Agricultural Experiment Station University of Minnesota, St. Paul, MN 55108, USA     

Siderophore Utilization by Bradyrhizobium japonicum

Bradyrhizobium japonicum USDA 110 and 61A152 can utilize the hydroxamate-type siderophores ferrichrome and rhodotorulate, in addition to ferric citrate, to overcome iron starvation. These strains can also utilize the pyoverdin-type siderophore pseudobactin St3. The ability to utilize another organism's siderophores may confer a selective advantage in the rhizosphere.     

Cytokinin Production by Bradyrhizobium japonicum

Although there is considerable circumstantial evidence for the involvement of cytokinins in legume nodulation, the cytokinins produced by rhizobia have not been well characterized. Bradyrhizobium japonicum 61A68, a bacterium which nodulates soybean (Glycine max [L.] Merr.), was grown in defined medium. Cytokinins were purified from the culture medium by Amberlite XAD-2 chromatography and fractionated by column chromatography on Sephadex LH-20 in 35% ethanol. Pooled fractions from the Sephadex column were analyzed for cytokinin activity with the tobacco callus bioassay. Cytokinin activity was observed in fractions corresponding to the elution volumes of zeatin, ribosylzeatin, and methylthiozeatin. No activity corresponding to the elution volumes of isopentenyladenine or its riboside was found. Total cytokinin activity in the B. japonicum culture filtrate was equivalent to approximately 1 microgram of kinetin per liter. Transfer RNA was isolated from B. japonicum cells by phenol extraction, followed by potassium acetate extraction, cetyltrimethylammonium bromide precipitation, and DEAE cellulose chromatography. Transfer RNA was enzymically hydrolyzed to nucleosides. High performance liquid chromatographic analysis of cytokinin nucleosides showed peaks corresponding to the retention times of trans-ribosylzeatin, methylthioribosylzeatin, isopentenyladenosine, and methylthioisopentenyladenosine. Analysis of the tRNA hydrolysate by Sephadex LH-20 chromatography and tobacco bioassay showed cytokinin activity in fractions corresponding to ribosylzeatin, methylthioribosylzeatin, and isopentenyladenosine. The presence of the trans isomer of ribosylzeatin was also determined by enzyme immunoassay.     

Variability in molybdenum uptake activity in Bradyrhizobium japonicum strains.

Twenty naturally occurring strains of Bradyrhizobium japonicum in 11 serogroups were screened for the ability to take up Mo as bacteroids from soybean root nodules. The strains varied greatly in their ability to take up Mo in a 1-min period. The best strain was USDA 136, which had an Mo uptake activity of almost 3.0 pmol/min per mg of bacteroid (dry weight). In contrast, the poorest strain, USDA 62, had an Mo uptake activity of 0.35 pmol of Mo per min per mg of bacteroid. There were similarities in Mo uptake ability among most of the same serogroup members. The variability in Mo uptake rates between the best (USDA 136 and USDA 122) and poorest (USDA 62 and USDA 140) strains was attributed to their differing affinities for Mo. Double-reciprocal plots of velocity versus substrate indicated a Km for USDA 136 and USDA 122 of 0.045 and 0.054 microM, respectively, whereas strains USDA 62 and USDA 140 both exhibited an apparent Km for MoO42- of about 0.36 microM. The two strains with the higher-affinity Mo binding also accumulated four to five times as much Mo over a 30-min period as the other strains. Soybeans were grown in Mo-deficient and Mo-supplemented conditions after inoculation with the three top-ranking Mo uptake strains and the three poorest Mo uptake strains. Two separate greenhouse studies indicated that Mo supplementation significantly increased the N2 fixation activity of USDA 140 nodules; up to a 35% increase in specific nitrogen fixation activity of nodules due to Mo supplementation was observed. Strain USDA 62 nodule N2 fixation responded positively to Mo supplementation in one of the two experiments. The results indicate that MoO42- transport and, specifically, affinity for Mo by the bacteroid may ultimately affect symbiotic N2 fixation activity. Attempts to reactivate nitrogenase by adding molybdate to bacteroids from plants grown in Mo-deficient conditions were unsuccessful.     

Adenylate cyclase and cyclic AMP phosphodiesterase in Bradyrhizobium japonicum bacteroids.

Adenylate cyclase and cyclic AMP (cAMP) phosphodiesterase have been identified and partially characterized in bacteroids of Bradyrhizobium japonicum 3I1b-143. Adenylate cyclase activity was found in the bacteroid membrane fraction, whereas cAMP phosphodiesterase activity was located in both the membrane and the cytosol. In contrast to other microorganisms, B. japonicum adenylate cyclase remained firmly bound to the membrane during treatment with detergents. Adenylate cyclase was activated four- to fivefold by 0.01% sodium dodecyl sulfate (SDS), whereas other detergents gave only slight activation. SDS had no effect on the membrane-bound cAMP phosphodiesterase but strongly inhibited the soluble enzyme, indicating that the two enzymes are different. All three enzymes were characterized by their kinetic constants, pH optima, and divalent metal ion requirements. With increasing nodule age, adenylate cyclase activity increased, the membrane-bound cAMP phosphodiesterase decreased, and the soluble cAMP phosphodiesterase remained largely unchanged. These results suggest that cAMP plays a role in symbiosis.