Cloning of a DNA region from Bradyrhizobium japonicum encoding pleiotropic functions in heme metabolism and respiration

Random and site-directed Tn5-induced mutagenesis of Bradyrhizobium japonicum yielded two mutations, one in strain 2960 and the other in strain 2606::Tn5-20, which mapped close to each other but in separate genes. The corresponding wild-type genes were cloned, and their approximate location on the cloned DNA was determined. Mutant 2960 was Fix^- and formed green nodules on soybean, whereas strain 2606::Tn5-20 had ca. 4% of wild-type Fix activity and formed white nodules. Cytochrome oxidase assays (Nadi tests) showed a negative reaction with both mutants, indicating a functional deficiency of cytochrome c or its terminal oxidase or both. However, the mutants grew well under aerobic conditions on minimal media with different carbon sources. Furthermore, mutant 2960 had a reduced activity in hydrogen uptake, was unable to grow anaerobically with nitrate as the terminal electron acceptor and 2960-infected soybean nodules contained little, if any, functional leghemoglobin. Southern blot analysis showed that a B. japonicum heme biosynthesis mutant [strain LO505: O'Brian MR, Kirshbom PM, Maier RJ (1987) Proc Natl Acad Sci USA 84: 8390–8393] had its mutation close to the Tn5 insertion site of our mutant 2606::Tn5-20. This finding, combined with the observed phenotypes, suggested that the genes affected in mutants 2960 and 2606::Tn5-20 were involved in some steps of heme biosynthesis thus explaining the pleiotropic respiratory deficiencies of the mutants. Similar to strain LO505, the mutant 2606::Tn5-20 (but not 2960) was defective in the activity of protoporphyrinogen IX oxidase which catalyzes the penultimate step in the heme biosynthesis pathway. This suggests that one of the two cloned genes may code for this enzyme.     

Isolation and characterization of the lipopolysaccharides from Bradyrhizobium japonicum.

The lipopolysaccharide (LPS) of Bradyrhizobium japonicum 61A123 was isolated and partially characterized. Phenol-water extraction of strain 61A123 yielded LPS exclusively in the phenol phase. The water phase contained low-molecular-weight glucans and extracellular or capsular polysaccharides. The LPSs from B. japonicum 61A76, 61A135, and 61A101C were also extracted exclusively into the phenol phase. The LPSs from strain USDA 110 and its Nod- mutant HS123 were found in both the phenol and water phases. The LPS from strain 61A123 was further characterized by polyacrylamide gel electrophoresis, composition analysis, and 1H and 13C nuclear magnetic resonance spectroscopy. Analysis of the LPS by polyacrylamide gel electrophoresis showed that it was present in both high- and low-molecular-weight forms (LPS I and LPS II, respectively). Composition analysis was also performed on the isolated lipid A and polysaccharide portions of the LPS, which were purified by mild acid hydrolysis and gel filtration chromatography. The major components of the polysaccharide portion were fucose, fucosamine, glucose, and mannose. The intact LPS had small amounts of 2-keto-3-deoxyoctulosonic acid. Other minor components were quinovosamine, glucosamine, 4-O-methylmannose, heptose, and 2,3-diamino-2,3-dideoxyhexose. The lipid A portion of the LPS contained 2,3-diamino-2,3-dideoxyhexose as the only sugar component. The major fatty acids were beta-hydroxymyristic, lauric, and oleic acids. A long-chain fatty acid, 27-hydroxyoctacosanoic acid, was also present in this lipid A. Separation and analysis of LPS I and LPS II indicated that glucose, mannose, 4-O-methylmannose, and small amounts of 2,2-diamino-2,3-dideozyhexose and heptose were components of the core region of the LPS, whereas fucose, fucosmine, mannose, and small amounts of quinovosamine and glucosamine were components of the LPS O-chain region.     

Isolation and expression of Rhizobium japonicum cloned DNA encoding an early soybean nodulation function.

A first visible step in the nodulation of legumes by Rhizobium spp. is the deformation and curling of root hairs. We have identified and cloned DNA sequences encoding this function from two strains of Rhizobium japonicum (USDA 122 and USDA 110) with a weakly homologous probe from Rhizobium meliloti. Root hair curling encoded by the cloned DNA fragments was examined on soybeans (Glycine soja ) after conjugative transfer of these sequences in broad-host-range vectors to various bacterial genera. Pseudomonas putida gave unambiguous expression of the root hair curling genes. This enabled us to identify the 8.7-kilobase EcoRI fragments encoding root hair curling from each strain. The phenotypes encoded by the plasmids pBS1 (derived from strain USDA 122) and pBS2 (derived from strain USDA 110) are distinct and represent a phenotype characteristic of their parent R. japonicum strains. Subclones of pBS1 and pBS2 were generated in single and multicopy vectors, and their expression was analyzed in P. putida. We established that a 4.2-kilobase internal Sa/I fragment of pBS1 and a 3.5-kilobase SstI -EcoRI fragment of pBS2 are sufficient to confer root hair curling on soybeans.     

Isolation and characterization of rhizobitoxine mutants of Bradyrhizobium japonicum.

To explore the role of rhizobitoxine in Bradyrhizobium-legume symbiosis, 11 rhizobitoxine mutants of B. japonicum USDA61 were isolated on the basis of their inability to synthesize the toxin in culture. Each mutant is prototrophic and symbiotically effective on soybean, cowpea, siratro, and Glycine soja. The rhizobitoxine mutants differ in their chlorosis phenotypes and rhizobitoxine production in planta. As expected, one group of mutant fail to make toxin in planta, resulting in the absence of chlorosis. Another group of mutants causes severe chlorosis on all cultivars of soybean tested. Surprisingly, this group of mutants makes more rhizobitoxine in soybean nodules than the wild-type strain does. This phenotype is only observed on soybean and not on other hosts such as cowpea, siratro, or G. soja. The remaining mutants all produce rhizobitoxine in planta but vary in the amount of toxin they produce and the severity of chlorosis they induce in soybean plants. Biochemical analysis of mutants demonstrates that one mutant is unable to synthesize serinol, a molecule hypothesized to be an intermediate in rhizobitoxine biosynthesis. By using these mutants, it was found that rhizobitoxine plays no apparent role in the nodulation of rj1 soybeans. Recently, it was found that inhibition of ethylene biosynthesis allows Rhizobium meliloti to overcome nitrate inhibition of nodule formation on alfalfa. Because rhizobitoxine also inhibits ethylene biosynthesis, we tested the ability of mutants which accumulate high levels of toxin in planta to overcome nitrate inhibition of nodule formation on soybean plants and found that the nodule formation induced by the wild type and that induced by mutant strains were equally suppressed in the presence of nitrate.     

A locus encoding host range is linked to the common nodulation genes of Bradyrhizobium japonicum.

By using cloned Rhizobium meliloti, Rhizobium leguminosarum, and Rhizobium sp. strain MPIK3030 nodulation (nod) genes as hybridization probes, homologous regions were detected in the slow-growing soybean symbiont Bradyrhizobium japonicum USDA 110. These regions were found to cluster within a 25-kilobase (kb) region. Specific nod probes from R. meliloti were used to identify nodA-, nodB-, nodC-, and nodD-like sequences clustered on two adjacent HindIII restriction fragments of 3.9 and 5.6 kb. A 785-base-pair sequence was identified between nodD and nodABC. This sequence contained an open reading frame of 420 base pairs and was oriented in the same direction as nodABC. A specific nod probe from R. leguminosarum was used to identify nodIJ-like sequences which were also contained within the 5.6-kb HindIII fragment. A nod probe from Rhizobium sp. strain MPIK3030 was used to identify hsn (host specificity)-like sequences essential for the nodulation of siratro (Macroptilium atropurpureum) on a 3.3-kb HindIII fragment downstream of nodIJ. A transposon Tn5 insertion within this region prevented the nodulation of siratro, but caused little or no delay in the nodulation of soybean (Glycine max).     

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.     

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.     

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.     

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.     

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.     

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)     

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.     

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.     

Efficient DNA transformation of Bradyrhizobium japonicum by electroporation.

Intact cells of Bradyrhizobium japonicum USDA 110 were transformed with a 30-kilobase plasmid to efficiencies of 10(6) to 10(7) transformants per microgram by high-voltage electroporation. The technique was reliable and simple, with single colonies arising from transformed cells within 5 days of antibiotic selection. Plasmid DNA from B. japonicum transformed the Bradyrhizobium (Arachis) sp. with high efficiency, while the same plasmid extracted from Escherichia coli transformed B. japonicum at very low efficiency. The electrical conditions that resulted in the highest efficiencies were high voltage (10.5 to 12.5 kV/cm) and short pulse length (6 to 7 ms). A linear increase in the number of transformants was observed as DNA concentration was increased over 4 orders of magnitude; saturation appeared to begin between 120 ng/ml and 1.2 micrograms/ml. This novel method of transformation should enhance B. japonicum genetic research by providing a valuable alternative to conjugal mating, which is currently the only efficient, widely used means of introducing DNA into this organism.