nif Gene expression in a Nif+, Fix−Bradyrhizobium japanicum variant

Abstract Bradyhizobium japonicum USDA 110 has been shown to contain several genetically similar, naturally occurring colony morphology variants. One of these variants, L2-110, although devoid of symbiotic nitrogen fixation, retains significant levels of explanta nitrogen fixation ability relative to other symbiotically competent USDA 110 variants (MN-110 and I-110). Interestingly, Northern blot analyses revealed that L2-110 nodules, despite their lack of symbiotic nitrogen fixation, contained 65% the level of mRNA for dinitrogenase ( nif DK) and 64% the level of dinitrogenase reductase ( nif H) mRNA relative to MN-110 nodules. Western blot analyses of tissue from the same nodules detected 32% the level of dinitrogenase and 31% the level of dinitrogenase reductase in L2-110 relative to MN-110. L2-110 appears to be a new class of mutant based on the complete absence of symbiotic nitrogen fixation (Fix⁻ and the presence of significant exanta nitrogen fixation (Nif⁺).     

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.     

Rhizobial siderophore as an iron source for clover

Iron uptake by clover plants ( Trifoliuin pratense L. cv. Hruszowska) was studied using radioactive ferric iron (⁵⁵FeCl₂). As shown by autoradiography of non-infected plants, purified rhizobial siderophore isolated from Rhizobium leguminosarum by, trifolii , stimulated the uptake and shoot transport of iron. Addition of rhizobial siderophore into the growth medium of nodulated clover did not affect the iron transport to the shoots. In the absence of the rhizobial siderophore, clover infected by either nitrogen-fixing (Nod⁻ Fix⁺) or nonfixing (Nod⁺ Fix⁻) R. L. trifolii strains took up and transported into the shoots more iron than the non-infected control plants. Nodulated clover reduced Fe(III) more efficiently than the non-infected control plants.     

Bradyrhizobium japonicum strain identification by RFLP analysis using the repeated sequence RSα

Twenty-one strains of Bradyrhizobium japonicum from different sources or isolated from commercial inoculants were compared and clustered using total DNA hybridization with the repeated sequence RSα, and two antisera. RSα fingerprinting provides a useful method for differentiating strains belonging to the same serogroup and to estimate genotypic relatedness among the strains.     

光敏生物素标记总DNA探针对大豆根瘤菌的检测

以光敏生物素标记慢生型大豆根瘤菌(Bradyrhizobium japonicum)USDA110总DNA作为探针,与快生型大豆根瘤菌杂交时,没有杂交斑点形成,而与慢生型大豆根瘤菌中的部分菌株能形成杂交斑点,表明该探针具有种和部分菌株特异性,用该探针与压碎的根瘤汁液进行DNA杂交,检测USDA110在不灭菌的盆栽土壤中的竞争结瘤能力,发现USDA110在大豆不同生育期的占瘤率为70%～90%。     

The pha gene cluster of Rhizobium meliloti involved in pH adaptation and symbiosis encodes a novel type of K+ efflux system

The fix-2 mutant of Rhizobium meliloti affected in the invasion of alfalfa root nodules (Inf⁻/Fix⁻) is K⁺ sensitive and unable to adapt to alkaline pH in the presence of K⁺. Using directed Tn 5 mutagenesis, we delimited a 6 kb genomic region in which mutations resulted in both Inf⁻/Fix⁻ and K⁺-sensitive phenotypes. In this DNA region, seven open reading frames (ORFs) were identified and the corresponding genes were designated phaA , B , C , D , E , F and G . The putative PhaABC proteins exhibit homology to the subunits of a Na⁺/H⁺ antiporter from an alkalophilic Bacillus strain. Moreover, PhaA and PhaD also show similarity to the ND5 and ND4 subunits of the proton-pumping NADH:ubiquinone oxidoreductase respectively. Computer analysis suggests that all seven proteins are highly hydrophobic with several possible transmembrane domains. Some of these domains were confirmed by generating active alkaline phosphatase fusions. Ion transport studies on phaA mutant cells revealed a defect in K⁺ efflux at alkaline pH after the addition of a membrane-permeable amine. These results suggest that the pha genes of R. meliloti encode for a novel type of K⁺ efflux system that is involved in pH adaptation and is required for the adaptation to the altered environment inside the plant.     

Introduction of the mercury transposon Tn501 into Rhizobium japonicum strains 31 and 110

Abstract Transposon Tn 501 , which encodes resistance to mercuric ions, was introduced into Rhizobium japonicum 110 and 31 by conjugal transfer. The transposon donor plasmid (pMD100) was able to mobilize into R. japonicum , but could not be maintained. Hg²⁺-resistant colonies were recovered at a frequency of 1.9 × 10⁻⁸/recipient for strain 110, and 1.7 × 10⁻⁷/recipient for strain 31. Presence of Tn 501 in Hg-resistant isolates was verified by Southern analysis and demonstrating transposition of Hg resistance. Transposon mutagenesis has been used to generate auxotrophic mutations at low frequency.     

Restriction Endonuclease and nif Homology Patterns of Bradyrhizobium japonicum USDA 110 Derivatives With and Without Nitrogen Fixation Competence

DNAs from Bradyrhizobium japonicum USDA 110 derivatives that differ in nitrogen-fixing ability produced similar electrophoretic patterns with five different restriction enzymes. Our data support the hypothesis of common ancestry for these derivatives. Derivatives I-110 and L1-110 differed as much as 100-fold in acetylene reduction activity when they were tested with several soybean cultivars in both greenhouse and field experiments. While possessing nodulating ability, derivative L1-110 is deficient in symbiotic nitrogen-fixing ability, whereas derivative I-110 is symbiotically competent. Hybridization of nifDK and nifH probes from B. japonicum to Southern blots of restricted DNAs from strain USDA 110 derivatives produced similar patterns. This finding indicates similar structural gene organization for both derivative I-110 and derivative L1-110 and implies that the difference in symbiotic nitrogen fixation is probably not due to structural gene rearrangements. However, our hybridization data do not rule out the possibility of differences in expression of structural nif genes or alterations in the structure or expression of other genes required for symbiotic nitrogen fixation.     

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

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

Photosynthetic Bradyrhizobium Sp. strain ORS285 synthesizes 2-O-methylfucosylated lipochitooligosaccharides for nod gene? Dependent interaction with Aeschynomene plants

Bradyrhizobium sp. strain ORS285 is a photosynthetic bacterium that forms nitrogen-fixing nodules on the roots and stems of tropical aquatic legumes of the Aeschynomene genus. The symbiotic interaction of Bradyrhizobium sp. strain ORS285 with certain Aeschynomene spp. depends on the presence of nodulation (nod) genes whereas the interaction with other species is nod gene independent. To study the nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and Aeschynomene spp., we used a nodB-lacZ reporter strain to monitor the nod gene expression with various flavonoids. The flavanones liquiritigenin and naringenin were found to be the strongest inducers of nod gene expression. Chemical analysis of the culture supernatant of cells grown in the presence of naringenin showed that the major Nod factor produced by Bradyrhizobium sp. strain ORS285 is a modified chitin pentasaccharide molecule with a terminal N-C(18:1)-glucosamine and with a 2-O-methyl fucose linked to C-6 of the reducing glucosamine. In this respect, the Bradyrhizobium sp. strain ORS285 Nod factor is the same as the major Nod factor produced by the nonphotosynthetic Bradyrhizobium japonicum USDA110 that nodulates the roots of soybean. This suggests a classic nod gene-dependent molecular dialogue between Bradyrhizobium sp. strain ORS285 and certain Aeschynomene spp. This is supported by the fact that B. japonicum USDA110 is able to form N(2)-fixing nodules on both the roots and stems of Aeschynomene afraspera.     

The mntH gene encodes the major Mn2+ transporter in Bradyrhizobium japonicum and is regulated by manganese via the Fur protein

The bacterial Nramp family protein MntH is a divalent metal transporter, but mntH mutants have little or no phenotype in organisms where it has been studied. Here, we identify the mntH homologue of Bradyrhizobium japonicum , and demonstrate that it is essential for Mn²⁺ transport and for maintenance of cellular manganese homeostasis. Transport activity was induced under manganese deficiency, and Fe²⁺ did not compete with ⁵⁴Mn²⁺ for uptake by cells. The steady-state level of mntH mRNA was negatively regulated by manganese, but was unaffected by iron. Control of mntH expression and Mn²⁺ transport by manganese was lost in a fur strain, resulting in constitutively high activity. Fur protected a 35 bp region of the mntH promoter in DNase I footprinting analysis that includes three imperfect direct repeat hexamers that are needed for full occupancy. Mn²⁺ increased the affinity of Fur for the mntH promoter by over 50-fold, with a K _d value of 2.2 nM in the presence of metal. The findings identify MntH as the major Mn²⁺ transporter in B. japonicum , and show that Fur is a manganese-responsive regulator in that organism. Furthermore, Fe²⁺ is neither a substrate for MntH nor a regulator of mntH expression in vivo .     

Biochemical and symbiotic properties of histidine-requiring mutants of Rhizobium leguminosarum biovar trifolii

A perturbation of the histidine biosynthetic pathway in legume microsymbionts can abolish their symbiotic competence. Twenty-one histidine-requiring (His⁻) mutants were isolated from berseem clover-nodulating, symbiotically-competent (Nod⁺, Fix⁺) Rhizobium leguminosarum bv. ' trifolii ' strain RTH 48 Sm^r by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) mutagenesis followed by enrichment. These mutants were analysed for their biochemical defect and the corresponding effect, if any, on their symbiotic abilities. Cross-feeding, supplementation and enzymatic studies identified three types of mutants. Group 1 mutants, His-2 and His-12, grew with histidine supplementation but not with the addition of either L -histidinol or L -histidinol phosphate to the medium ; they lacked histidinol dehydrogenase (EC 1.1.1.23) activity and consequently formed only ineffective, or 'non-fixing' nodules. Group 2 mutant, His-17, grew when supplemented with either L -histidinol or L -histidine, had low histidinol phosphate phosphatase (EC 3.1.3.15) activity (37% of wild-type), and consequently failed to nodulate berseem clover. Group 3, the remaining 18 mutants, grew when supplemented with L -histidinol phosphate, L -histidinol or histidine, and did not nodulate. Typically, reversion rates were between 10⁻⁷ and 10⁻⁸. Defects in early steps of the pathway abolished nodulating ability, whereas lesions in the last step did not. The last step, however, was required for symbiotic nitrogen fixation. It is hypothesized that histidine may be supplied by the host in sufficient quantity for nodulation by histidinol dehydrogenase mutants to occur, whereas the amount provided in the nodule may be insufficient to support bacteroid development and nitrogen fixation.     

Expression of nir, nor and nos denitrification genes from Bradyrhizobium japonicum in soybean root nodules

Expression of Bradyrhizobium japonicum wild-type strain USDA110 nirK , norC and nosZ denitrification genes in soybean root nodules was studied by in situ histochemical detection of β -galactosidase activity. Similarly, P_nirK- lacZ , P_norC- lacZ , and P_nosZ- lacZ fusions were also expressed in bacteroids isolated from root nodules. Levels of β -galactosidase activity were similar in both bacteroids and nodule sections from plants that were solely N₂-dependent or grown in the presence of 4 m M KNO₃. These findings suggest that oxygen, and not nitrate, is the main factor controlling expression of denitrification genes in soybean nodules. In plants not amended with nitrate, B. japonicum mutant strains GRK308, GRC131, and GRZ25, that were altered in the structural nirK , norC and nosZ genes, respectively, showed a wild-type phenotype with regard to nodule number and nodule dry weight as well as plant dry weight and nitrogen content. In the presence of 4 m M KNO₃, plants inoculated with either GRK308 or GRC131 showed less nodules, and lower plant dry weight and nitrogen content, relative to those of strains USDA110 and GRZ25. Taken together, the present results revealed that although not essential for nitrogen fixation, mutation of either the structural nirK or norC genes encoding respiratory nitrite reductase and nitric oxide reductase, respectively, confers B. japonicum reduced ability for nodulation in soybean plants grown with nitrate. Furthermore, because nodules formed by each the parental and mutant strains exhibited nitrogenase activity, it is possible that denitrification enzymes play a role in nodule formation rather than in nodule function.     

Novel methylated triterpenoids of the gammacerane series from the nitrogen-fixing bacterium Bradyrhizobium japonicum USDA 110.

The nitrogen-fixing, symbiotic root-nodule forming bacterium Bradyrhizobium japonicum USDA 110 contained gammacerane derivatives next to triterpenoids of the hopane series. Diploptene, diplopterol, 2 beta-methyldiplopterol, aminobacteriohopanetriol and adenosylhopane were accompanied by tetrahymanol and the corresponding novel methylated homologues 2 beta-methyltetrahymanol, 20 alpha-methyltetrahymanol, and 2 beta,20 alpha-dimethyltetrahymanol. Incorporation of [(2)H(3)]methyl-L-methionine indicated that the additional methyl groups originated from methionine, probably with S-adenosylmethionine acting as methyl donor, with retention of the three deuterium atoms. The simultaneous presence of hopane and gammacerane derivatives seems a characteristic feature of the genus Bradyrhizobium and the phylogenetically closely related Rhodopseudomonas palustris.     

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

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

The PhyR-σEcfG signalling cascade is involved in stress response and symbiotic efficiency in Bradyrhizobium japonicum

PhyR is an unusual type of response regulator consisting of a receiver domain and an extracytoplasmic function (ECF) sigma factor-like domain. It was recently described as a master regulator of general stress response in Methylobacterium extorquens . Orthologues of this regulator are present in essentially all free-living Alphaproteobacteria. In most of them, phyR is genetically closely linked to a gene encoding an ECF σ factor. Here, we investigate the role of these two regulators in the soybean symbiont Bradyrhizobium japonicum USDA110. Using deletion mutants and phenotypic assays, we showed that PhyR and the ECF σ factor σ^EcfG are involved in heat shock and desiccation resistance upon carbon starvation. Both mutants had symbiotic defects on the plant hosts Glycine max (soybean) and Vigna radiata (mungbean). They induced fewer nodules than the wild type and these nodules were smaller, less pigmented, and their specific nitrogenase activity was drastically reduced 2 or 3 weeks after inoculation. Four weeks after infection, soybean nodule development caught up to a large extent whereas most mungbean nodules remained defective even 5 weeks after infection. Remarkably, both mutants triggered aberrant nodules on the different host plants with ectopically emerging roots. Microarray analysis revealed that PhyR and σ^EcfG control congruent regulons suggesting both regulators are part of the same signalling cascade. This finding was further substantiated by in vitro protein–protein interaction studies which are in line with a partner-switching mechanism controlling gene regulation triggered by phosphorylation of PhyR. The large number of genes of unknown function present in the PhyR/σ^EcfG regulon and the conspicuous symbiotic phenotype suggest that these regulators are involved in the Bradyrhizobium –legume interaction via yet undisclosed mechanisms.     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

Characterization of a Mannitol-Utilizing, Nitrogen-Fixing Bradyrhizobium japonicum USDA 110 Derivative

We have isolated a colonial derivative of Bradyrhizobium japonicum USDA 110 (designated MN-110) that is both mannitol utilizing and N(2) fixing. Derivative MN-110 showed growth on mannitol and glucose similar to that of non-N(2)-fixing, mannitol-utilizing L2-110. Derivative MN-110 showed high constitutive and induced d-mannitol dehydrogenase activity (similar to L2-110) relative to N(2)-fixing, non-mannitol-utilizing I-110. Hybridization to EcoRI and HindIII total DNA digests with cloned USDA 110 nif DK and nif H genes revealed similar patterns for non-N(2)-fixing mannitol-utilizing derivative L1-110 and derivative MN-110. Symbiotic tests with soybean cultivars Ransom and Lee indicate MN-110 to be a superior N(2)-fixing derivative compared with derivative I-110 and the parent strain USDA 110. However, these differences were not revealed when comparing 28-day-old soybean-B. japonicum associations but were apparent in 49-day-old associations. It was apparent from this work that mannitol utilization was not necessarily correlated to symbiotic effectiveness in B. japonicum and that gene rearrangements were not responsible for differences in N(2) fixation between L1-110 or L2-110 and MN-110.     

The Glycine-Glomus-Bradyhizobium symbiosis. VIII. Phosphorususe efficiency of CO2 and N2 fixation in mycorrhizal soybean

Soybean [ Glycine max (L.) Merr. cv. Hobbit] plants nodulated by Bradyhizobium japonicum strain USDA 110 were grown in pot cultures in severely P- and N-deficient soil and either colonized by the vesicular-arbuscular mycorrhizal (VAM) fungus Glomus mosseae (Nicol. & Gerd.) Gerd. and Trappe or fertilized with a high (HP) or low (LP) level of KH₂PO₄ (0.6 or 0.3 m M , respectively), After 7 weeks of growth, nodule and chloroplast activities (C₂H₂ reduction and CO₂ exchange rate) were determined. Photosynthetic P-use efficiency of CO₂ fixation was significantly higher in VAM than in HP plants, while that of nitrogenase activity was lower. The LP plants were intermediate in both respects. The ratio of nodule to chloroplast activity [mol C₂H₂ reduced (mol CO₂ fixed)⁻¹] was highest in HP and lowest in VAM plants. Root colonization by the VAM fungus significantly increased nodule number and dry weight and reduced nodule specific mass and activity in comparison to HP plants. In spite of lower nodule activity, VAM plants were significantly larger and had higher N concentrations than the HP plants. The results suggest nonnutritional. VAM-elicited and host-mediated effects on the symbiotic functions of the legume association.