Role of Pili (Fimbriae) in Attachment of Bradyrhizobium japonicum to Soybean Roots

Pili (fimbriae) were observed on cells of each of the five strains of Bradyrhizobium japonicum and the one strain of Rhizobium trifolii examined. Pili on B. japonicum were about 4 nm in diameter and polarly expressed. Piliated cells were estimated by transmission electron microscopy and hydrophobic attachment to polystyrene to constitute only a small percentage of the total population. The proportion of piliated cells in these populations was dependent on culture age in some strains. Piliated B. japonicum cells were selectively and quantitatively removed from suspension when cultures were incubated with either soybean roots or hydrophobic plastic surfaces, indicating that pili were involved in the attachment of the bacteria to these surfaces. Pili from B. japonicum 110 ARS were purified and found to have a subunit molecular weight of approximately 21,000. Treatment of B. japonicum suspensions with antiserum against the isolated pili reduced attachment to soybean roots by about 90% and nodulation by about 80%. Pili appear to be important mediators of attachment of B. japonicum to soybean roots under the conditions examined.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H2

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO₂ and H₂ (Aut⁻) were isolated. Five Aut⁻ mutants lacked hydrogen uptake activity (Hup⁻). The other seven Aut⁻ mutants possessed wild-type levels of hydrogen uptake activity (Hup⁺), both in free-living culture and symbiotically. Three of the Hup⁻ mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup⁻ only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O₂. All five Hup⁻ mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut⁻ Hup⁺ mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut⁻ Hup⁺ mutant, had CO₂ fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut⁻ mutant. The Aut⁻ Hup⁻ mutants that were Hup⁻ both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup⁻ mutants.     

Transposon Tn5-Generated Bradyrhizobium japonicum Mutants Unable To Grow Chemoautotrophically with H(2)

Twelve Tn5-induced mutants of Bradyrhizobium japonicum unable to grow chemoautotrophically with CO(2) and H(2) (Aut) were isolated. Five Aut mutants lacked hydrogen uptake activity (Hup). The other seven Aut mutants possessed wild-type levels of hydrogen uptake activity (Hup), both in free-living culture and symbiotically. Three of the Hup mutants lacked hydrogenase activity both in free-living culture and as nodule bacteroids. The other two mutants were Hup only in free-living culture. The latter two mutants appeared to be hypersensitive to repression by oxygen, since Hup activity could be derepressed under 0.4% O(2). All five Hup mutants expressed both ex planta and symbiotic nitrogenase activities. Two of the seven Aut Hup mutants expressed no free-living nitrogenase activity, but they did express it symbiotically. These two strains, plus one other Aut Hup mutant, had CO(2) fixation activities 20 to 32% of the wild-type level. The cosmid pSH22, which was shown previously to contain hydrogenase-related genes of B. japonicum, was conjugated into each Aut mutant. The Aut Hup mutants that were Hup both in free-living culture and symbiotically were complemented by the cosmid. None of the other mutants was complemented by pSH22. Individual subcloned fragments of pSH22 were used to complement two of the Hup mutants.     

Altered cell surface hydrophobicity of lipopolysaccharide-deficient mutant of Bradyrhizobium japonicum

The authors previously isolated a lipopolysaccharide (LPS) deficient Tn5-mutant of Bradyrhizobium japonicum, and subsequently isolated the LPS gene region. In this study the LPS deficiency of B. japonicum was studied in terms of its cell surface characteristics. By monitoring the kinetics of the partition with hexadecane the LPS-mutant was found to be far more hydrophobic than the wild type strain; the partition coefficients were 3.19 min(-1) for the mutant, as compared with only 1.40 min(-1) for the wild type. When the mutant was transformed with the cloned LPS gene, the transformant regained the wild type phenotypes, including the cell surface hydrophobicity (CSH) and LPS profile. A polyacrylamide gel electrophoretic analysis of LPS demonstrated that the O-antigenic part of LPS was completely absent in the mutant. The LPS-mutant of B. japonicum was visually distinguishable from the wild type after a simple centrifugation of the cells.     

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.     

Chemotaxis of Bradyrhizobium japonicum to various organic compounds

The investigation of the chemotactic response of Bradyrhizobium japonicum to amino acids, carbohydrates, multiatomic alcohols, organic acids, and soybean extracts showed that the extracts of some soybean varieties (Chernoburaya and Beskluben'kovaya) contain repellents. This indicates that the soybeans of host plants contain effectors that may play a role at the early stages of their interaction with nodule bacteria.     

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     

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.     

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.     

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.     

Restriction of Nodulation by Bradyrhizobium japonicum Is Mediated by Factors Present in the Roots of Glycine max

Reciprocal grafting experiments done using soybean plant introduction genotypes indicated that restriction of nodulation by Bradyrhizobium japonicum is determined by the genotype of the root and is dependent on plant growth temperature. Microscopic analyses indicated that the soybean plant introduction genotypes restrict nodulation of B. japonicum at symbiotic stages which occur both before and after the formation of nodule primordia.     

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.     

Acetate-Activating Enzymes of Bradyrhizobium japonicum Bacteroids

Acetyl coenzyme A (acetyl-CoA) synthetase and acetate kinase were localized within the soluble portion of Bradyrhizobium japonicum bacteroids, and no appreciable activity was found elsewhere in the nodule. The presence of each acetate-activating enzyme was confirmed by separation of the two enzyme activities on a hydroxylapatite column, by substrate dependence of each enzyme in both the forward and reverse directions, by substrate specificity, by inhibition patterns, and also by identification of the reaction products by C₁₈ reverse-phase high-pressure liquid chromatography. Phosphotransacetylase activity, found in the soluble portion of the bacteroid, was dependent on the presence of potassium and was inhibited by added sodium. The greatest acetyl-CoA hydrolase activity was found in the root nodule cytosol, although appreciable activity also was found within the bacteroids. The combined specific activities of acetyl-CoA synthetase and acetate kinase-phosphotransacetylase were approximate to that of the pyruvate dehydrogenase complex, thus providing B. japonicum with sufficient capacity to generate acetyl-CoA.     

Hydrogen Oxidation by the Host-Controlled Uptake Hydrogenase Phenotype of Bradyrhizobium japonicum in Symbiosis with Soybean Host Plants

Symbioses between uptake hydrogenase host-regulated (Hup-hr) phenotypes of Bradyrhizobium japonicum and exotic, agronomically unadapted soybean germ plasm were examined for expression of uptake hydrogenase activity. Determinations for hydrogen evolution and uptake hydrogenase activity identified five plant introduction (PI) lines which formed hydrogen-oxidizing symbioses with strains USDA 61 and PA3 6c. Hup-hr strains belonging to serogroup 94 expressed uptake hydrogenase activity in symbioses with PI 181696 and PI 219655 at rates sufficient to prevent hydrogen from escaping the nodules. The identification of soybean germ plasm forming hydrogen-oxidizing symbioses with Hup-hr bradyrhizobia potentially has implications for enhancing nitrogen fixation efficiency in soybean production.     

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.     

Host Plant Effects on Nodulation and Competitiveness of the Bradyrhizobium japonicum Serotype Strains Constituting Serocluster 123

Strains in Bradyrhizobium japonicum serocluster 123 are the major indigenous competitors for nodulation in a large portion of the soybean production area of the United States. Serocluster 123 is defined by the serotype strains USDA 123, USDA 127, and USDA 129. The objective of the work reported here was to evaluate the ability of two soybean genotypes, PI 377578 and PI 417566, to restrict the nodulation and reduce the competitiveness of serotype strains USDA 123, USDA 127, and USDA 129 in favor of the highly effective strain CB1809 and to determine how these soybean genotypes alter the competitive relationships among the three serotype strains in the serocluster. The soybean genotypes PI 377578 and PI 417566 along with the commonly grown cultivar Williams were planted in soil essentially free of soybean rhizobia and inoculated with single-strain treatments of USDA 123, USDA 127, USDA 129, or CB1809 and six dual-strain competition treatments of USDA 123, USDA 127, or USDA 129 versus CB1809, USDA 123 versus USDA 127, USDA 123 versus USDA 129, and USDA 127 versus USDA 129. PI 377578 severely reduced the nodulation and competitiveness of USDA 123 and USDA 127, while PI 417566 similarly affected the nodulation and competitiveness of USDA 129. Thus, the two soybean genotypes can reduce the nodulation and competitiveness of each of the three serocluster 123 serotype strains. Our results indicate that host control of restricted nodulation and reduced competitiveness is quite specific and effectively discriminates between B. japonicum strains which are serologically related.     

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.