Carbohydrate binding activities of Bradyrhizobium japonicum. II. Isolation and characterization of a galactose-specific lectin

Extracts of Bradyrhizobium japonicum were fractionated on Sepharose columns covalently derivatized with lactose. Elution of the material that was specifically bound to the affinity column with lactose yielded a protein of Mr approximately 38,000. Isoelectric focusing of this sample yielded two spots with pI values of 6.4 and 6.8. This protein specifically bound to galactose-containing glycoconjugates, but did not bind either to glucose or mannose. Derivatives of galactose at the C-2 position showed much weaker binding; there was an 18-fold difference in the relative binding affinities of galactose versus N-acetyl-D-galactosamine. These results indicate that we have purified a newly identified carbohydrate-binding protein from Bradyrhizobium japonicum, that can exquisitely distinguish galactose from its derivatives at the C-2 position.     

Carbohydrate binding activities of Bradyrhizobium japonicum. I. Saccharide-specific inhibition of homotypic and heterotypic adhesion

Bradyrhizobium japonicum (R110d) exhibited four saccharide-specific binding activities: (a) adsorption to Sepharose beads containing covalently coupled lactose; (b) homotypic agglutination through one pole of the cell (star formation); (c) heterotypic adhesion to the cultured soybean cell line, SB-1; and (d) attachment to roots of soybean plants. Each of these binding activities can be inhibited by the addition of galactose or lactose, but not by derivatives such as N-acetyl-D-galactosamine or melibiose. Treatment of wild-type bacteria with N-methyl-N'-nitro-N-nitrosoguanidine followed by selection on the basis of reduced binding to SB-1 cells, resulted in two specific mutants, designated N4 and N6. Compared to wild type, these two mutants also exhibited decreased binding activity in: (a) adsorption to lactose-Sepharose beads; (b) homotypic star formation; and (c) heterotypic attachment to roots of soybeans plants. These results suggest that all four of the saccharide-inhibitable binding activities of Bradyrhizobium japonicum may be mediated by the same mechanism(s) or molecular component(s).     

A two-component regulator mediates population-density-dependent expression of the Bradyrhizobium japonicum nodulation genes

Bradyrhizobium japonicum nod gene expression was previously shown to be population density dependent. Induction of the nod genes is highest at low culture density and repressed at high population densities. This repression involves both NolA and NodD2 and is mediated by an extracellular factor found in B. japonicum conditioned medium. NolA and NodD2 expression is maximal at high population densities. We demonstrate here that a response regulator, encoded by nwsB, is required for the full expression of the B. japonicum nodYABC operon. In addition, NwsB is also required for the population-density-dependent expression of both nolA and nodD2. Expression of nolA and nodD2 in the nwsB mutant remained at a basal level, even at high culture densities. The nwsB defect could be complemented by overexpression of a second response regulator, NodW. Consistent with the fact that NolA and NodD2 repress nod gene expression, the expression of a nodY-lacZ fusion in the nwsB mutant was unaffected by culture density. In plant assays with GUS fusions, nodules infected with the wild type showed no nodY-GUS expression. In contrast, nodY-GUS expression was not repressed in nodules infected with the nwsB mutant. Nodule competition assays between the wild type and the nwsB mutant revealed that the addition of conditioned medium resulted in a competitive advantage for the nwsB mutant.     

Feedback regulation of the Bradyrhizobium japonicum nodulation genes

Lipochitin Nod signals are produced by rhizobia and are required for the establishment of a nitrogen-fixing symbiosis with a legume host. The nodulation genes encode products required for the synthesis of this signal and are induced in response to plant-produced flavonoid compounds. The addition of chitin and lipo-chitin oligomers to Bradyrhizobium japonicum cultures resulted in a significant reduction in the expression of a nod–lacZ fusion. Intracellular expression of NodC, encoding a chitin synthase, also reduced nod gene expression. In contrast, expression of the ChiB chitinase increased nod gene expression. The chain length of the oligosaccharide was important in feedback regulation, with chitotetraose molecules the best modulators of nod gene expression. Feedback regulation is mediated by the induction of nolA by chitin, resulting in elevated levels of the repressor protein, NodD2.     

Aberrant nodulation response of Vigna umbellata to a Bradyrhizobium japonicum NodZ mutant and nodulation signals.

The (Brady)rhizobium nodulation gene products synthesize lipo-chitin oligosaccharide (LCO) signal molecules that induce nodule primordia on legume roots. In spot inoculation assays with roots of Vigna umbellata, Bradyrhizobium elkanii LCO and chemically synthesized LCO induced aberrant nodule structures, similar to the activity of these LCOs on Glycine soja (soybean). LCOs containing a pentameric chitin backbone and a reducing-end 2-O-methyl fucosyl moiety were active on V. umbellata. In contrast, the synthetic LCO-IV(C16:0), which has previously been shown to be active on G. soja, was inactive on V. umbellata. A B. japonicum NodZ mutant, which produces LCO without 2-O-methyl fucose at the reducing end, was able to induce nodule structures on both plants. Surprisingly, the individual, purified, LCO molecules produced by this mutant were incapable of inducing nodule formation on V. umbellata roots. However, when applied in combination, the LCOs produced by the NodZ mutant acted cooperatively to produce nodulelike structures on V. umbellata roots.     

Effect of organic N source on bacterial growth, lipo-chitooligosaccharide production, and early soybean nodulation by Bradyrhizobium japonicum

Production of Bradyrhizobium japonicum inoculants is problematic because high inoculation rates are necessary but expensive, while production of rhizobial Nod factors (lipo-chitooligosaccharides (LCOs)), key signal molecules in the establishment of legume-rhizobia symbioses, may be inhibited at high culture cell densities. We conducted experiments to determine the effects of growth medium N source on B. japonicum growth, LCO production, and early nodulation of soybean. We found that 1.57 mmol ammonium nitrate x L(-1) resulted in less rhizobial growth and rhizobial capacity to produce LCOs (on a per cell basis) than did 0.4 g yeast extract x L(-1), which contained the same amount of N as the ammonium nitrate. Increasing yeast extract to 0.8 g x L(-1) increased rhizobial growth and LCO production on a volume basis (per litre of culture) and did not affect cell capacity to produce LCOs; however, at 1.4 g yeast extract x L(-1) per cell, production was reduced. A mixture of 0.8 g yeast extract x L(-1) and 1.6 g casein hydrolysate x L(-1) resulted in the greatest bacterial growth and LCO production on a volume basis but reduced LCO production per cell. Changes in organic N level and source increased production of some of the measured LCOs more than others. LCO production was positively correlated with cell density when expressed on a volume basis; however, it was negatively correlated on a per cell basis. We conclude that although quorum sensing affected Nod factor production, increased levels of organic N, and specific compositions of organic N, increased LCO production on a volume basis. Greenhouse inoculation experiments showed that the medium did not modify nodule number and N fixation in soybean, suggesting that it could have utility in inoculant production.     

Soybean cultivars affect nodulation competition of Bradyrhizobium japonicum strains

The influence of five Thai soybean cultivars on nodulation competitiveness of four Bradyrhizobium japonicum strains was investigated. Cultures of B. japonicum strains THA5, THA6, USDA110 and SEMIA5019 were mixed with each other prior to inoculating germinated soybean seeds growing in Leonard jars with nitrogen-free nutrient solution. At harvest, nodule occupancy by each strain was determined by a fluorescent antibody technique. The term ‘general competitive ability’ was introduced to describe the average competitive nodule occupancy of a strain in paired co-inoculation with a number of strains on soybean. The nodule occupancies by an individual strain were directly correlated with the proportions of that strain in the inoculum mixtures. USDA110 showed higher nodulation competitiveness than the other strains on three of the five cultivars. The Thai strain THA6 appeared to be more competitive than USDA110 on cultivar SJ5. Thus, nodulation competitiveness of the B. japonicum strains was affected by the cultivars of soybean used. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chemical control of interstrain competition for soybean nodulation by Bradyrhizobium japonicum.

Previous research has shown that a significant limitation to the agricultural use of improved rhizobial inoculant strains is competition from the indigenous soil population. In this work, we sought to test whether chemical inhibitors of flavonoid-induced nod gene expression in Bradyrhizobium japonicum could be identified and utilized to affect interstrain competition for nodulation of soybeans. Approximately 1,000 structural and functional analogs of the known, natural inducers of nod gene expression were tested on six strains of B. japonicum containing a nodY-lacZ fusion. We successfully identified effective inhibitors of nodY expression. The addition of the inhibitor 7-hydroxy-5-methylflavone significantly inhibited nodulation by a sensitive strain and could be used to effectively manipulate the competition between strains for soybean nodulation. However, this work also uncovered significant limitations for the practical use of this methodology. For example, despite the almost universal induction response to the identified natural inducers, there was a wide variability among strains in their response to any specific inhibitor. Given this unexpected variability, the cost of registration of an agronomic chemical, and the potential for the development of resistant field populations, it is unlikely that chemical inhibitors can be successfully applied to a field situation.     

Isolation and characterization of the DNA region encoding nodulation functions in Bradyrhizobium japonicum.

The DNA region encoding early nodulation functions of Bradyrhizobium japonicum 3I1b110 (I110) was isolated by its homology to the functionally similar region from Rhizobium meliloti. Isolation of a number of overlapping recombinant clones from this region allowed the construction of a restriction map of the region. The identified nodulation region of B. japonicum shows homology exclusively to those regions of R. meliloti and Rhizobium leguminosarum DNA known to encode early nodulation functions. The region of homology with these two fast-growing Rhizobium species was narrowed to an 11.7-kilobase segment. A nodulation-defective mutant of Rhizobium fredii USDA 201, strain A05B-2, was isolated and found to be defective in the ability to curl soybean root hairs. Some of the isolated recombinant DNA clones of B. japonicum were found to restore wild-type nodulation function to this mutant. Analysis of the complementation results allows the identification of a 1.8-kilobase region as essential for restoration of Hac function.     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant.

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbohydrate binding activities ofBradyrhizobium japonicum: IV. Effect of lactose and flavones on the expression of the lectin,BJ38

BJ38 is a galactose/lactose-specific lectin (M _r 38000) found at one pole ofBradyrhizobium japonicum. It has been implicated in mediating the adhesion of the bacteria to soybean roots, leading to the establishment of a nitrogen-fixing symbiosis. When the ligand lactose is added to cultures of the bacteria for at least 1 h prior to harvesting the cells for BJ38 isolation, the yield of the protein was found to be elevated in a dose-dependent fashion. Half maximal stimulation was observed at 50 µm; the effect was saturated at 1mm, where a 10-fold higher yield of BJ38 was obtained. Saccharides with a lower affinity for BJ38 than lactose yielded a correspondingly smaller induction effect when compared at a concentration of 1mm. The higher level of BJ38 induced by lactose is also manifested by an elevated amount of BJ38 detectable at the cell surface and by a higher number ofB. japonicum cells adsorbed onto soybean cells. Surprisingly, the induction of BJ38 expression seen with lactose was also observed with certain, but not all, flavonoids that induce thenod genes of the bacteria; genistein mimicked the induction observed with lactose, whereas luteolin failed to stimulate BJ38 production.     

nodZ, a unique host-specific nodulation gene, is involved in the fucosylation of the lipooligosaccharide nodulation signal of Bradyrhizobium japonicum.

The nodulation genes of rhizobia are regulated by the nodD gene product in response to host-produced flavonoids and appear to encode enzymes involved in the production of a lipo-chitose signal molecule required for infection and nodule formation. We have identified the nodZ gene of Bradyrhizobium japonicum, whose product is required for the addition of a 2-O-methylfucose residue to the terminal reducing N-acetylglucosamine of the nodulation signal. This substitution is essential for the biological activity of this molecule. Mutations in nodZ result in defective nodulation of siratro. Surprisingly, although nodZ clearly codes for nodulation function, it is not regulated by NodD and, indeed, shows elevated expression in planta. Therefore, nodZ represents a unique nodulation gene that is not under the control of NodD and yet is essential for the synthesis of an active nodulation signal.     

Chemical control of interstrain competition for soybean nodulation by Bradyrhizobium japonicum

Previous research has shown that a significant limitation to the agricultural use of improved rhizobial inoculant strains is competition from the indigenous soil population. In this work, we sought to test whether chemical inhibitors of flavonoid-induced nod gene expression in Bradyrhizobium japonicum could be identified and utilized to affect interstrain competition for nodulation of soybeans. Approximately 1,000 structural and functional analogs of the known, natural inducers of nod gene expression were tested on six strains of B. japonicum containing a nodY-lacZ fusion. We successfully identified effective inhibitors of nodY expression. The addition of the inhibitor 7-hydroxy-5-methylflavone significantly inhibited nodulation by a sensitive strain and could be used to effectively manipulate the competition between strains for soybean nodulation. However, this work also uncovered significant limitations for the practical use of this methodology. For example, despite the almost universal induction response to the identified natural inducers, there was a wide variability among strains in their response to any specific inhibitor. Given this unexpected variability, the cost of registration of an agronomic chemical, and the potential for the development of resistant field populations, it is unlikely that chemical inhibitors can be successfully applied to a field situation.     

Suppression of nodulation in soybeans by superoptimal inoculation with Bradyrhizobium japonicum

Symbiotic nodulation of the primary roots of soybeans ( Glycine max L. Merrill cv. Pride 216) is regulated by the plant, and is suppressed in response to a high inoculum dose of Bradyrhizobium japonicum USDA strain I–110 (ARS)⁺ applied at one time to the root. If an optimal dose is followed 10 h later by a superoptimal dose, nodules from the first inoculum near the base of the primary root are suppressed in a dose-dependent way similar to that observed after single inoculations. The nodules which appear are probably derived from infections initiated by the bacteria in both inocula.     

Strain selection for improvement of Bradyrhizobium japonicum competitiveness for nodulation of soybean

A Bradyrhizobium japonicum USDA 110-derived strain able to produce wider halos in soft-agar medium than its parental strain was obtained by recurrent selection. It was more chemotactic than the wild type towards mannitol and three amino acids. When cultured in minimal medium with mannitol as a single carbon-source, it had one thick subpolar flagellum as the wild type, plus several other flagella that were thinner and sinusoidal. Root adsorption and infectivity in liquid media were 50-100% higher for the selected strain, but root colonization in water-unsaturated vermiculite was similar to the wild type. A field experiment was then carried out in a soil with a naturalized population of 1.8 x 10(5) soybean-nodulating rhizobia g of soil(-1). Bradyrhizobium japonicum strains were inoculated either on the soybean seeds or in the sowing furrows. Nodule occupation was doubled when the strains were inoculated in the sowing furrows with respect to seed inoculation (significant with P<0.05). On comparing strains, nodule occupation with seed inoculation was 6% or 10% for the wild type or selected strains, respectively, without a statistically significant difference, while when inoculated in the sowing furrows, nodule occupation increased to 12% and 22%, respectively (differences significant with P<0.05).     

Effect of lectin on nodulation by wild-type Bradyrhizobium japonicum and a nodulation-defective mutant

The nodulation characteristics of wild-type Bradyrhizobium japonicum USDA 110 and mutant strain HS111 were examined. Mutant strain HS111 exhibits a delayed-nodulation phenotype, a result of its inability to initiate successful nodulation promptly following inoculation of the soybean root. Previously, we showed that the defect in initiation of infection leading to subsequent nodulation which is found in HS111 can be phenotypically reversed by pretreatment with soybean root exudate or soybean seed lectin. This effect is not seen after pretreatment with root exudates and lectins obtained from other plant species. Treatment of strain HS111 with as little as 10 soybean seed lectin molecules per bacterium (3.3 X 10 (-12) M) resulted in enhancement of nodule formation. Pretreatment of wild-type B. japonicum USDA 110 with soybean root exudate or seed lectin increased nodule numbers twofold on 6-week-old plants. Wild-type strain USDA 110 cells inoculated at 10(4) cells per seedling exhibited a delay in initiation of infection leading to subsequent nodulation. Wild-type cells pretreated in soybean root exudates or seed lectin did not exhibit a delay in nodulation at this cell concentration. Mutant strain HS111 pretreated in seed lectin for 0 or 1 h, followed by washing with the hapten D-galactose to remove the lectin, exhibited a delay in initiation of nodulation. Phenotypic reversal of the delayed-nodulation phenotype exhibited by strain HS111 was seen if incubation was continued for an additional 71 h in plant nutrient solution following 1 h of lectin pretreatment. Reversal of the delayed-nodulation phenotype of HS111 through lectin pretreatment was prevented by chloramphenicol or rifampin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and use of actinomycetes for enhanced nodulation of soybean co-inoculated with Bradyrhizobium japonicum

The utilization of actinomycetes as potential soybean (Glycine max (L.)) co-inoculants was evaluated. Soil samples from Carbondale and Belleville, Ill., were used to inoculate pre-germinated soybean plants to determine antibiotic sensitivity in the native Bradyrhizobium japonicum population. Sensitivity was in the order kanamycin > tetracycline > oxytetracycline > rifampicin > neomycin. Antagonism by five actinomycete cultures toward seven test strains of B. japonicum was also assessed. The ranking average inhibition (across all seven B. japonicum strains) by these actino mycetes was Streptomyces kanamyceticus = Streptomyces coeruleoprunus > Streptomyces rimosus > Streptomyces sp. > Amy colatopsis mediterranei. Ten antibiotic combinations were used to isolate antibiotic-resistant mutants of B. japonicum I-110 and 3I1B-110 via successive cycles of mutation. Eighty-one antibiotic-resistant strains were isolated and tested for symbiotic competency; nine of which were selected for further characterization in a greenhouse pot study. Few differences in nodule number were caused by these treatments. Nodule occupancy varied from 0% to 18.3% when antibiotic-resistant strains of B. japonicum were used as the sole inoculants. However, when three mutant strains of B. japonicum were co-inoculated with S. kanamyceticus, significant increases in nodule occupancy (up to 55%) occurred. Increases in shoot nitrogen composition (27.1%-40.9%) were also caused by co-inoculation with S. kanamyceticus.