Degradation of catechin by Bradyrhizobium japonicum

Rhizobia utilize phenolic substances as sole carbonsource. Bradyrhizobium japonicum utilizescatechin, a unit of condensed tannin as carbonsource. To establish the degradative pathway ofcatechin, the products of catechin degradation wereisolated by paper chromatography and TLC andidentified by HPLC, UV, IR and NMR spectra. B.japonicum cleaves catechin through catechinoxygenase. Phloroglucinolcarboxylic acid andprotocatechuic acid were identified as the initialproducts of degradation. Phloroglucinolcarboxylicacid is further decarboxylated to phloroglucinolwhich is dehydroxylated to resorcinol. Resorcinolis hydroxylated to hydroxyquinol. Protocatechuicacid and hydroxyquinol undergo intradiol cleavagethrough protocatechuate 3,4-dioxygenase andhydroxyquinol 1,2-dioxygenase to form-carboxy cis, cis-muconic acidand maleylacetate respectively. The enzymes ofcatechin degradative pathway are inducible. Estimation of all the enzymes involved in thecatabolism of catechin reveals the existence of acatechin degradative pathway in B. japonicum.     

Uptake and Metabolism of Carbohydrates by Bradyrhizobium japonicum Bacteroids

Bradyrhizobium japonicum bacteroids were isolated anaerobically and were supplied with ¹⁴C-labeled trehalose, sucrose, UDP-glucose, glucose, or fructose under low O₂ (2% in the gas phase). Uptake and conversion of ¹⁴C to CO₂ were measured at intervals up to 90 minutes. Of the five compounds studied, UDP-glucose was most rapidly absorbed but it was very slowly metabolized. Trehalose was the sugar most rapidly converted to CO₂, and fructose was respired at a rate at least double that of glucose. Sucrose and glucose were converted to CO₂ at a very low but measurable rate (<0.1 nanomoles per milligram protein per hour). Carbon Number 1 of glucose appeared in CO₂ at a rate 30 times greater than the conversion of carbon Number 6 to CO₂, indicating high activity of the pentose phosphate pathway. Enzymes of the Entner-Doudoroff pathway were not detected in bacteroids, but very low activities of sucrose synthase and phosphofructokinase were demonstrated. Although metabolism of sugars by B. japonicum bacteroids was clearly demonstrated, the rate of sugar uptake was only 1/30 to 1/50 the rate of succinate uptake. The overall results support the view that, although bacteroids metabolize sugars, the rates are very low and are inadequate to support nitrogenase.     

Anaerobic C1 Metabolism of the O-Methyl-14C-Labeled Substituent of Vanillate

The O-methyl substituents of aromatic compounds constitute a C₁ growth substrate for a number of taxonomically diverse anaerobic acetogens. In this study, strain TH-001, an O-demethylating obligate anaerobe, was chosen to represent this physiological group, and the carbon flow when cells were grown on O-methyl substituents as a C₁ substrate was determined by ¹⁴C radiotracer techniques. O-[methyl-¹⁴C]vanillate (4-hydroxy-3-methoxy-benzoate) was used as the labeled C₁ substrate. The data showed that for every O-methyl carbon converted to [¹⁴C]acetate, two were oxidized to ¹⁴CO₂. Quantitation of the carbon recovered in the two products, acetate and CO₂, indicated that acetate was formed in part by the fixation of unlabeled CO₂. The specific activity of ¹⁴C in acetate was 70% of that in the O-methyl substrate, suggesting that only one carbon of acetate was derived from the O-methyl group. Thus, it is postulated that the carboxyl carbon of the product acetate is derived from CO₂ and the methyl carbon is derived from the O-methyl substituent of vanillate. The metabolism of O-[methyl-¹⁴C]vanillate by strain TH-001 can be described as follows: 3¹⁴CH₃OC₇H₅O₃ + CO₂ + 4H₂O → ¹⁴CH₃COOH + 2¹⁴CO₂ + 10H⁺ + 10e^- + 3HOC₇H₅O₃.     

Vanillate Metabolism in Corynebacterium glutamicum

Corynebacterium glutamicum, a Gram-positive soil bacterium belonging to the mycolic acids-containing actinomycetes, is able to use the lignin degradation products ferulate, vanillate, and protocatechuate as sole carbon sources. The gene cluster responsible for vanillate catabolism was identified and characterized. The vanAB genes encoding vanillate demethylase are organized in an operon together with the vanK gene, coding for a transport system most likely responsible for protocatechuate uptake. While gene disruption mutagenesis revealed that vanillate demethylase is indispensable for ferulate and vanillate utilization, a vanK mutation does not lead to a complete growth arrest but to a decreased growth rate on protocatechuate, indicating that one or more additional protocatechuate transporter(s) are present in C. glutamicum.     

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.     

Functional Role of Bradyrhizobium japonicum Trehalose Biosynthesis and Metabolism Genes during Physiological Stress and Nodulation

Trehalose, a disaccharide accumulated by many microorganisms, acts as a protectant during periods of physiological stress, such as salinity and desiccation. Previous studies reported that the trehalose biosynthetic genes (otsA, treS, and treY) in Bradyrhizobium japonicum were induced by salinity and desiccation stresses. Functional mutational analyses indicated that disruption of otsA decreased trehalose accumulation in cells and that an otsA treY double mutant accumulated an extremely low level of trehalose. In contrast, trehalose accumulated to a greater extent in a treS mutant, and maltose levels decreased relative to that seen with the wild-type strain. Mutant strains lacking the OtsA pathway, including the single, double, and triple ΔotsA, ΔotsA ΔtreS and ΔotsA ΔtreY, and ΔotsA ΔtreS ΔtreY mutants, were inhibited for growth on 60 mM NaCl. While mutants lacking functional OtsAB and TreYZ pathways failed to grow on complex medium containing 60 mM NaCl, there was no difference in the viability of the double mutant strain when cells were grown under conditions of desiccation stress. In contrast, mutants lacking a functional TreS pathway were less tolerant of desiccation stress than the wild-type strain. Soybean plants inoculated with mutants lacking the OtsAB and TreYZ pathways produced fewer mature nodules and a greater number of immature nodules relative to those produced by the wild-type strain. Taken together, results of these studies indicate that stress-induced trehalose biosynthesis in B. japonicum is due mainly to the OtsAB pathway and that the TreS pathway is likely involved in the degradation of trehalose to maltose. Trehalose accumulation in B. japonicum enhances survival under conditions of salinity stress and plays a role in the development of symbiotic nitrogen-fixing root nodules on soybean plants.Rhizobia induce the formation of nodules on the roots of legume plants, in which atmospheric nitrogen is fixed and supplied to the host plant, thereby enhancing growth under nitrogen-limiting conditions. The symbiotic interaction between rhizobia and their cognate leguminous plants is important for agricultural productivity, especially in less developed countries. However, physiological stresses, such as desiccation and salinity, negatively affect these symbiotic interactions by limiting nitrogen fixation (44). The osmotic environment within the rhizosphere may affect root colonization, infection thread development, nodule development, and the formation of effective N₂-fixing nodules (21). Moreover, when legume seeds are inoculated with appropriate rhizobial strains prior to planting in the field, the vast majority of nodules produced are often not formed by the inoculant bacteria but rather by indigenous strains in the soil (36). This is in part due to the death of inoculant strains from rapid seed coat-mediated desiccation. Therefore, improvement of the survival of rhizobia under conditions of physiological stresses may promote biological nitrogen fixation and enhance plant growth.Rhizobia synthesize and accumulate compatible solutes, including trehalose, in response to desiccation and solute-mediated physiological stresses (5, 21, 42). Trehalose, a nonreducing disaccharide with an α,α-1,1 linkage between the two glucose molecules, has been shown to protect cell membranes and proteins from stress-induced inactivation and denaturation (8, 23, 24). The relationship between trehalose accumulation and symbiotic phenotype is dependent on rhizobial species and host genotype. Suarez et al. (39) reported an increase in root nodule number and nitrogen fixation by Phaseolus vulgaris inoculated with a trehalose-6-phosphate synthase-overexpressing strain of Rhizobium etli. In contrast, trehalose accumulation in Rhizobium leguminosarum and Sinorhizobium meliloti cells did not result in an increase in nitrogen-fixing nodules but led to enhancement of competitiveness on clover and on certain alfalfa genotypes, respectively (1, 16, 20).Four trehalose biosynthetic pathways, mediated by OtsAB, TreS, TreYZ, and TreT, have been reported thus far for prokaryotes (8, 25). The OtsAB pathway results in the condensation of glucose-6-phosphate with UDP-glucose by trehalose-6-phosphate synthase (OtsA) to form trehalose-6-phosphate. Trehalose is subsequently formed from trehalose-6-phosphate by the action of trehalose-6-phosphate phosphatase (OtsB). The TreS pathway involves a reversible transglycosylation reaction in which trehalose synthase (TreS) converts maltose, a disaccharide with α,α-1,4 linkage between the two glucose molecules, to trehalose. The third pathway, mediated by TreYZ, involves the conversion of maltodextrins into trehalose. The terminal α-1,1-glycosylic bond at the end of the maltodextrin polymer is hydrolyzed by maltooligosyltrehalose synthase (TreY), and trehalose is subsequently released from the end of the polymer via hydrolysis by maltooligosyltrehalose trehalohydrolase (TreZ). More recently, a trehalose glycosyltransferring synthase (TreT) was shown to catalyze the reversible formation of trehalose from ADP-glucose and glucose (25).In addition to biosynthesis, Gram-negative bacteria have also been reported to have trehalose degradation systems. Typically, trehalose is hydrolyzed into two glucose moieties by periplasmic and cytoplasmic trehalase enzymes, TreA and TreF, respectively (13, 15). However, Sinorhizobium meliloti also uses ThuA and ThuB for trehalose utilization (16).Bradyrhizobium japonicum, the root nodule symbiont of soybeans, accumulates trehalose in cultured cells and bacteroids (34, 35). Biochemical studies indicated that B. japonicum has three independent trehalose biosynthetic pathways involving trehalose synthase (TreS), maltooligosyltrehalose synthase (TreYZ), and trehalose-6-phosphate synthetase (OtsAB) (38). Sequence analysis of the B. japonicum USDA 110 genome identified the genes that encode these biosynthetic pathways: otsAB (bll0322 to bll0323), two homologs of treS (blr6767 and bll0902), and treYZ (blr6770 to blr6771), but not treT (17). Orthologous gene sequences to the trehalose degradation genes treA, treF, and thuAB have not been found in the genome of B. japonicum USDA 110. Cytryn et al. (6) reported that expression of otsA, treS (blr6767), and treY genes were highly induced by desiccation stress. Moreover, the concentrations of these three enzymes increased when B. japonicum was cultured in the presence of salt (38). Trehalose concentration in B. japonicum has been reported to increase due to desiccation stress (6), and this sugar is purported to act as an osmoprotectant. The addition of exogenously supplied trehalose has been reported to enhance the survival of B. japonicum in response to desiccation and salinity stresses (9, 37). Despite this information, little is known about how the various trehalose biosynthetic pathways modulate stress tolerance and symbiotic performance in B. japonicum.The purpose of this study was to examine the functional role(s) of the B. japonicum trehalose biosynthetic pathways on stress survival by constructing single, double, and triple mutants and by producing strains that overexpress the trehalose biosynthesis enzymes. Here we report on the relationship between trehalose accumulation and physiological responses to salinity and desiccation stresses in mutant and overexpression strains and that mutations in the trehalose biosynthesis pathways altered the symbiotic performance of B. japonicum USDA 110 on soybeans. Results of these studies indicate that trehalose accumulation in B. japonicum plays a prominent role in the saprophytic and symbiotic competence of this agriculturally important soil bacterium.     

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.     

Aerobic growth and respiration of a delta-aminolevulinic acid synthase (hemA) mutant of Bradyrhizobium japonicum.

Oxygen-dependent growth of the Bradyrhizobium japonicum hemA mutant MLG1 (M.L. Guerinot and B.K. Chelm, Proc. Natl. Acad. Sci. USA 83:1837-1841, 1986) was demonstrated in cultured cells in the absence of exogenous delta-aminolevulinic acid (ALA), but growth of analogous mutants of Rhizobium meliloti or of Escherichia coli was not observed unless ALA was added to the yeast extract-containing media. No heme could be detected in extracts of strain MLG1 cells as measured by the absorption or by the peroxidase activity of the heme moiety, but the rates of growth and endogenous respiration of the mutant were essentially identical to those found in the parent strain. A role for ALA in the viability of strain MLG1 could not be ruled out since the ALA analog levulinic acid inhibited growth, but neither ALA synthase nor glutamate-dependent ALA synthesis activity was found in the mutant. The data show that the cytochromes normally discerned in wild-type B. japonicum cultured cells by absorption spectroscopy are not essential for aerobic growth or respiration.     

Codon usage and G+C content in Bradyrhizobium japonicum genes are not uniform

To date, the sequences of 45 Bradyrhizobium japonicum genes are known. This provides sufficient information to determine their codon usage and G+C content. Surprisingly, B. japonicum nodulation and NifA-regulated genes were found to have a less biased codon usage and a lower G+C content than genes not belonging to these two groups. Thus, the coding regions of nodulation genes and NifA-regulated genes could hardly be identified in codon preference plots whereas this was not difficult with other genes. The codon frequency table of the highly biased genes was used in a codon preference plot to analyze the RSRj9 sequence which is an insertion sequence (IS)-like element. The plot helped identify a new open reading frame (ORF355) that escaped previous detection because of two sequencing errors. These were now corrected. The deduced gene product of ORF355 in RSRj9 showed extensive similarity to a putative protein encoded by an ORF in the T-DNA of Agrobacterium rhizogenes. The DNA sequences bordering both ORFs showed inverted repeats and potential target site duplications which supported the assumption that they were IS-like elements.     

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.     

A dominant-negative fur mutation in Bradyrhizobium japonicum

In many bacteria, the ferric uptake regulator (Fur) protein plays a central role in the regulation of iron uptake genes. Because iron figures prominently in the agriculturally important symbiosis between soybean and its nitrogen-fixing endosymbiont Bradyrhizobium japonicum, we wanted to assess the role of Fur in the interaction. We identified a fur mutant by selecting for manganese resistance. Manganese interacts with the Fur protein and represses iron uptake genes. In the presence of high levels of manganese, bacteria with a wild-type copy of the fur gene repress iron uptake systems and starve for iron, whereas fur mutants fail to repress iron uptake systems and survive. The B. japonicum fur mutant, as expected, fails to repress iron-regulated outer membrane proteins in the presence of iron. Unexpectedly, a wild-type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild-type cells leads to a fur phenotype. Unlike a B. japonicum fur-null mutant, the strain carrying the dominant-negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, mung bean, or cowpea, suggesting a role for a Fur-regulated protein or proteins in the symbiosis.     

Autecology of Bradyrhizobium japonicum in soybean-rice rotations

The effect of rice culture on changes in the number of a strain of soybean root-nodule bacteria, (Bradyrhizobium japonicum CB1809), already established in the soil by growing inoculated soybean crops, was investigated in transitional red-brown earth soils at two sites in south-western New South Wales. At the first site, 5.5 years elapsed between the harvest of the last of four successive crops of soybean and the sowing of the next. In this period three crops of rice and one crop of triticale were sown and in the intervals between these crops, and after the crop of triticale, the land was fallowed. Before sowing the first rice crop, the number of Bradyrhizobium japonicum was 1.32×10⁵ g^–1 soil. The respective numbers of bradyrhizobia after the first, second and third rice crops were 4.52 ×10⁴, 1.26×10⁴ and 6.40×10² g^–1 soil. In the following two years the population remained constant. Thus sufficient bradyrhizobia survived in soil to nodulate and allow N₂-fixation by the succeeding soybean crop. At the second site, numbers of bradyrhizobia declined during a rice crop, but the decline was less than when the soil was fallowed (400-fold cf. 2200-fold). Multiplication of bradyrhizobia was rapid in the rhizosphere of soybean seedlings sown without inoculation in the rice bays. At 16 days after sowing, their numbers were not significantly different (p<0.05) from those in plots where rice had not been sown. Nodulation of soybeans was greatest in plots where rice had not been grown, but yield and grain nitrogen were not significantly different (p<0.05). Our results indicate that flooding soil has a deleterious effect on the survival of bradyrhizobia but, under the conditions of the experiments, sufficient B. japonicum strain CB 1809 survived to provide good nodulation after three crops of rice covering a total period of 5.5 years between crops of soybean.     

Carbon monoxide dehydrogenase activity in Bradyrhizobium japonicum

Bradyrhizobium japonicum strain 110spc4 was capable of chemolithoautotrophic growth with carbon monoxide (CO) as a sole energy and carbon source under aerobic conditions. The enzyme carbon monoxide dehydrogenase (CODH; EC 1.2.99.2) has been purified 21-fold, with a yield of 16% and a specific activity of 58 nmol of CO oxidized/min/mg of protein, by a procedure that involved differential ultracentrifugation, anion-exchange chromatography, hydrophobic interaction chromatography, and gel filtration. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide gel electrophoresis and had a molecular mass of 230,000 Da. The 230-kDa enzyme was composed of large (L; 75-kDa), medium (M; 28.4-kDa), and small (S; 17.2-kDa) subunits occurring in heterohexameric (LMS)(2) subunit composition. The 75-kDa polypeptide exhibited immunological cross-reactivity with the large subunit of the CODH of Oligotropha carboxidovorans. The B. japonicum enzyme contained, per mole, 2.29 atoms of Mo, 7.96 atoms of Fe, 7.60 atoms of labile S, and 1.99 mol of flavin. Treatment of the enzyme with iodoacetamide yielded di(carboxamidomethyl)molybdopterin cytosine dinucleotide, identifying molybdopterin cytosine dinucleotide as the organic portion of the B. japonicum CODH molybdenum cofactor. The absorption spectrum of the purified enzyme was characteristic of a molybdenum-containing iron-sulfur flavoprotein.     

Characterization of the self-cleaving effector protein NopE1 of Bradyrhizobium japonicum

NopE1 is a type III-secreted protein of the symbiont Bradyrhizobium japonicum which is expressed in nodules. In vitro it exhibits self-cleavage in a duplicated domain of unknown function (DUF1521) but only in the presence of calcium. Here we show that either domain is self-sufficient for cleavage. An exchange of the aspartic acid residue at the cleavage site with asparagine prevented cleavage; however, cleavage was still observed with glutamic acid at the same position, indicating that a negative charge at the cleavage site is sufficient. Close to each cleavage site, an EF-hand-like motif is present. A replacement of one of the conserved aspartic acid residues with alanine prevented cleavage at the neighboring site. Except for EDTA, none of several protease inhibitors blocked cleavage, suggesting that a known protease-like mechanism is not involved in the reaction. In line with this, the reaction takes place within a broad pH and temperature range. Interestingly, magnesium, manganese, and several other divalent cations did not induce cleavage, indicating a highly specific calcium-binding site. Based on results obtained by blue-native gel electrophoresis, it is likely that the uncleaved protein forms a dimer and that the fragments of the cleaved protein oligomerize. A database search reveals that the DUF1521 domain is present in proteins encoded by Burkholderia phytofirmans PsNJ (a plant growth-promoting betaproteobacterium) and Vibrio coralliilyticus ATCC BAA450 (a pathogenic gammaproteobacterium). Obviously, this domain is more widespread in proteobacteria, and it might contribute to the interaction with hosts.     

Isolation and characterization of the major nod factor of Bradyrhizobium japonicum strain 532C

Bradyrhizobium japonicum 532C nodulates soybean effectively under cool Canadian spring conditions and is used in Canadian commercial inoculants. The major lipo-chitooligosaccharide (LCO), bacteria-to-plant signal was characterized by HPLC, FAB-mass spectroscopy MALDI-TOF mass spectroscopy and revealed to be LCO Nod Bj-V (C18:1, MeFuc). This LCO is produced by type I strains of B. japonicum and is therefore unlikely to account for this strains superior ability to nodulate soybean under Canadian conditions. We also found that use of yeast extract mannitol medium gave similar results to that of Bergerson minimal medium.     

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.     

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.     

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.