Bradyrhizobium Species Research Articles

Studies of the Bradyrhizobium japonicum nodD1 promoter: a repeated structure for the nod box

Induction of nod genes in Rhizobium and Bradyrhizobium species is dependent on the presence of plant-produced flavonoids, the NodD protein, and the cis-acting nod box promoter sequence. Although the nodD (nodD1) gene in Rhizobium species is constitutively expressed, nodD1 expression in Bradyrhizobium japonicum is inducible by isoflavones in a manner similar to that of the nodYABC operon. A consensus nod box sequence is found 5' of the nodYABC operon, whereas a presumptive, nod box-like sequence is found 5' of the nodD1 gene. As an initial step toward examining the nodD1 promoter, the transcriptional start sites of the nodD1 and nodYABC operons were determined and found to be 44 and 28 bp, respectively, downstream of their respective nod box sequences. A series of deletions of the nodD1 promoter were constructed and fused to the lacZ gene. Analysis of the activity of these deletions clearly showed that the divergent nod box sequence was essential for nodD1 induction by isoflavones or soybean seed extract. The induction of nodD1 expression requires NodD1, as tested in B. japonicum and in a heterologous system, Agrobacterium tumefaciens. On the basis of these data, we analyzed the published nod box sequences and propose a new consensus sequence composed of paired 9-bp repeats. Analysis of the nodD1 nod box and synthetic constructs of the nocYABC nod box indicate that at least two 9-bp repeats are required for NodD1-mediated induction. Furthermore, insertions between the paired repeats of the nodYABC nod box suggest that orientation of the repeats on opposite faces of the DNA helix is essential for maximum nod gene expression.

Characterization of a soluble catalase-peroxidase hemoprotein b-590, previously identified as 'cytochrome alpha 1' from Bradyrhizobium japonicum bacteroids.

The cytochrome "a1" or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified. The water-soluble native hemoprotein has an Mr of 136,000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b-590 (Escherichia coli; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO. It has catalase and peroxidase activities, again resembling the E. coli b-590. Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish peroxidase reacts with oxygen to form such a complex. The hemoprotein, which we name hemoprotein b-590 (Bradyrhizobium japonicum), may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species. The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.

Characterization of a soluble catalase-peroxidase hemoproteinb-590, previously identified as ‘cytochromea1’ fromBradyrhizobium japonicumbacteroids

Abstract The cytochrome “ a 1 ” or P-428, previously proposed to be a high affinity terminal oxidase in nitrogen-fixing bacteroids of Bradyrhizobium japonicum has been purified. The water-soluble native hemoprotein has an M r of 136 000, lacks heme a and is a high-spin ferric protohemoprotein: It is slowly reduced with dithionite to give a species with an optical spectrum like that of hemoprotein b -590 ( Escherichia coli ; peak at 555 nm, shoulder at 590 nm), and which reacts slowly with CO. It has catalase and peroxidase activities, again resembling the E. coli b -590. Neither hemoprotein forms a stable oxy complex under conditions in which dithionite-reduced horseradish per-oxidase reacts with oxygen to form such a complex. The hemoprotein, which we name hemoprotein, b -590 ( Bradyrhizobium japonicum , may play a role in removal of peroxides generated during respiration in the bacteroids of several Rhizobium and Bradyrhizobium species. The high-affinity terminal oxidase under nitrogen-fixing conditions remains to be identified.

Contents of isoflavones and effect of isoflavones on root hair curling in non-nodulating-Rj1Rj1) soybean plant

Abstract Leguminous plants form symbiotic nitrogen-fixing nodules with specific bacterial species of Rhizobium or Bradyrhizobium. This symbiosis takes place by interaction of both partners. The non-nodulating mutants of the host plants are useful materials for physiological and genetical analyses of plant function in nodule formation. In a Rj 1 non-nodulating mutant of soybean, root hair curling was greatly reduced, whereas bacterial adhesion onto the root surface was not affected as compared to the nodulating soybean (Suganuma et al. 1990). Recently, plant flavonoids have been identified as specific-signals inducing the expression of Rhizobium nod ABC genes responsible for root hair curling (Firmin et al. 1986; Peters et al. 1986; Djordjevic et al. 1987; Zaat et al. 1987). The flavonoid secreted by the plant interacts with the product of the nodD gene which is constitutively expressed in the microsymbiont and induces the expression of several nodulation genes including the nod ABC genes (Rolfe and Gresshoff 1988). In the soybean/Bradyrhizobium symbiosis, two inducing compounds were isolated from soybean seedlings and were identified as 4,7-dihydroxyisoflavone (daidzein) and 4,5,7-trihydroxyisoflavone (genistein) (Kosslak et al. 1987). In the present study, we determined whether the lack of root hair curling in the Rj 1 soybean mutant was due to the absence or reduced contents of isoflavones.

Diversity of Rhizobium Bacteria Isolated from the Root Nodules of Leguminous Trees

Sixty rhizobial strains isolated from the root nodules of Acacia senegal and Prosopis chilensis in the Sudan were compared with 37 rhizobia isolated from woody legumes in other regions and with 25 representatives of recognized Rhizobium species by performing a numerical analysis of 115 phenotypic characteristics. Nineteen clusters were formed below the boundary level of 0.725 average distance, which was the level that separated the reference Rhizobium and Bradyrhizobium species. Our results indicated that tree rhizobia are very diverse with respect to their cross-nodulation patterns, as well as their physiological and biochemical properties, since 12 of the clusters formed consisted of tree rhizobia alone. Two distinctive features of tree rhizobia isolated in the Sudan were their high maximum growth temperature and their high salt tolerance.

The nifEN genes participating in FeMo cofactor biosynthesis and genes encoding dinitrogenase are part of the same operon in Bradyrhizobium species

The nucleotide sequences of genes homologous to the Klebsiella pneumoniae nifEN genes have been determined in Bradyrhizobium japonicum 110. The coding regions for the nifE and nifN consist, respectively, of 1641 and 1407 nucleotides. The nifD gene (coding for the beta-subunit of dinitrogenase) and nifE are linked, and separated by 95 nucleotides. In the region of 12 nucleotides that separates nifE from nifN the stop codon for nifE overlaps the putative ribosome binding site for nifN. In contrast to Klebsiella and Azotobacter vinelandii, the B. japonicum nifEN genes are linked to the nifDK genes in the same operon. Comparison of dinitrogenase polypeptides (nifDK products) and the polypeptides of the nifE and nifN genes reveals considerable homology between nifD and nifE, and between nifK and nifN. Several protein domains, containing highly conserved cysteine residues, are conserved among the gene products of nifD, nifK, nifE and nifN. This result allows us to propose a probable evolutionary pathway for the common origin of these genes.

Rhizobitoxine inhibition of hydrogenase synthesis in free-living Bradyrhizobium japonicum

Rhizobitoxine produced by Bradyrhizobium species strongly prevented derepression of hydrogenase expression in free-living Bradyrhizobium japonicum, although the toxin had no effect on the activity of cells which had already synthesized hydrogenase protein. Dihydrorhizobitoxine, a structural analog of rhizobitoxine, proved to be a less potent inhibitor of hydrogenase derepression. Rhizobitoxine did not cause cell death at a concentration sufficient to eliminate hydrogenase expression. The large subunit of hydrogenase was not detectable with antibody after derepression in the presence of rhizobitoxine. The general pattern of proteins synthesized from 14C-labeled amino acids during derepression was not significantly different in the presence or absence of rhizobitoxine. These results indicated that rhizobitoxine inhibited hydrogenase synthesis in free-living B. japonicum. Cystathionine and methionine strongly prevented the inhibition of hydrogenase derepression by rhizobitoxine, suggesting that the inhibition involves the level of sulfur-containing amino acids in the cell.

Common nodABC genes in Nod locus 1 of Azorhizobium caulinodans: Nucleotide sequence and plant-inducible expression

Azorhizobium caulinodans strain ORS571 induces nitrogen-fixing nodules on roots and stem-located root primordia of Sesbania rostrata. Two essential Nod loci have been previously identified in the bacterial genome, one of which (Nod locus 1) shows weak homology with the common nodC gene of Rhizobium meliloti. Here we present the nucleotide sequence of this region and show that it contains three contiguous open reading frames (ORFA, ORFB and ORFC) that are related to the nodABC genes of Rhizobium and Bradyrhizobium species. ORFC is followed by a fourth (ORF4) and probably a fifth (ORF5) open reading frame. ORF4 may be analogous to the nodI gene of R. leguminosarum, whereas ORF5 could be similar to the rhizobial nodF genes. Coordinated expression of this set of five genes seems likely from the sequence organization. There is no typical nod promoter consensus sequence (nod box) in the region upstream of the first gene (ORFA) and there is no nodD-like gene. LacZ fusions constructed with ORFA, ORFB, ORFC, and ORF4 showed inducible beta-galactosidase expression in the presence of S. rostrata seedlings as well as around stem-located root primordia. Among a series of phenolic compounds tested, the flavanone naringenin was the most efficient inducer of the expression of this ORS571 nod gene cluster.

Isolation, characterization, and complementation of Rhizobium meliloti 104A14 mutants that lack glutamine synthetase II activity

The glutamine synthetase (GS)-glutamate synthase pathway is the primary route used by members of the family Rhizobiaceae to assimilate ammonia. Two forms of glutamine synthetase, GSI and GSII, are found in Rhizobium and Bradyrhizobium species. These are encoded by the glnA and glnII genes, respectively. Starting with a Rhizobium meliloti glnA mutant as the parent strain, we isolated mutants unable to grow on minimal medium with ammonia as the sole nitrogen source. For two auxotrophs that lacked any detectable GS activity, R. meliloti DNA of the mutated region was cloned and partially characterized. Lack of cross-hybridization indicated that the cloned regions were not closely linked to each other or to glnA; they therefore contain two independent genes needed for GSII synthesis or activity. One of the cloned regions was identified as glnII. An R. meliloti glnII mutant and an R. meliloti glnA glnII double mutant were constructed. Both formed effective nodules on alfalfa. This is unlike the B. japonicum-soybean symbiosis, in which at least one of these GS enzymes must be present for nitrogen-fixing nodules to develop. However, the R. meliloti double mutant was not a strict glutamine auxotroph, since it could grow on media that contained glutamate and ammonia, an observation that suggests that a third GS may be active in this species.

Nitrogen Partitioning in Cowpea as Influenced by Rhizobial Strain and Mineral Nitrogen

AbstractPartitioning of nitrogen into the pods of legumes is an important yield determinant. The ability of cowpea [Vigna unguiculata (L.) Walp.] ‘Bush Purple Hull’ to partition N from dinitrogen fixation and assimilated nitrate into pods was measured with the aid of 15N. Cowpea was inoculated with four strains of Bradyrhizobium species and grown in a glasshouse to maturity. Two nitrate treatments were included; one provided an initial dose of starter N (40 mg plant−1) and the other provided split applications of N throughout the growth period (total of 240 mg plant−1). With starter N only, the results indicated that the strain of rhizobia did not influence partitioning of biologically fixed dinitrogen or nitrate N into pods. However when inoculated with either of two strains, cowpea partitioned approximately 63% of the total assimilated nitrate from the continuous N treatment into the pods, whereas another strain partitioned approximately 52% into pods. Of the total N from dinitrogen fixation, 53 and 58% was partitioned into the pods for the starter N and continuous N treatments respectively. The strains of rhizobia were similar in the quantity of nodule tissue formed on the roots, but the quantity of N contained in the nodule tissue varied from 40 to 20 mg N plant−1 for the starter N treatment. It appears that strains of rhizobia should be considered when undertaking experiments involving N partitioning.

Identification of Bradyrhizobium nod genes involved in host-specific nodulation

Three loci important for soybean nodulation by Bradyrhizobium japonicum were delimited by Tn5 mutagenesis on a 5.3-kilobase EcoRI fragment adjacent to the nodABC genes. Results of hybridization studies suggested that this region is conserved in Bradyrhizobium species but absent in all Rhizobium species. lacZ translational fusions of two of the loci contained in this region were found to be inducible by host-produced flavonoid chemicals via a mechanism requiring a functional nodD gene product. A mutation in one of the loci was found to result in an alteration of the host range of B. japonicum. This mutation appears to block nodulation at the step at which plant root cortical cell division is induced.

Interspecies homology of nodule development genes inRhizobiumandBradyrhizobiumspp.

DNA fragments representatives of ndvA and ndvB have been used as probes against genomic DNAs from different Rhizobium and Bradyrhizobium species. ndvA and ndvB homologues were found in all species, indicating extensive conservation of these genes. All Rhizobium species show chromosomal localization of ndvA and ndvB homologues.

Lipid A with 2,3-diamino-2,3-dideoxy-glucose in lipopolysaccharides from slow-growing members of Rhizobiaceae and from "Pseudomonas carboxydovorans".

Lipid A's from two Bradyrhizobium species and from the phylogenetically closely related species "Pseudomonas carboxydovorans" were found to contain 2,3-diamino-2,3-dideoxy-glucose as lipid A backbone sugar. In contrast, three representatives of the genus Rhizobium, as well as the phylogenetically related species Agrobacterium tumefaciens, contain solely glucosamine as lipid A backbone sugar. These findings support independent studies on the phylogenetical relatedness based on 16S rRNA-data of the genus Bradyrhizobium with "Pseudomonas carboxydovorans" and Rhodopseudomonas palustris, which form a tight phylogenetical cluster and which all contain the 2,3-diamino-2,3-dideoxy-glucose-containing lipid A. The relatedness of these species to the glucosamine-containing species of the genus Rhizobium and to Agrobacterium tumefaciens is rather distant as documented by 16S rRNA studies.

Rhizobia in tropical legumes: Environmental factors and the reduction of nitrogen

Existing methods for measuring environmental effects on nitrogen assimilation were applied to tropical legumes. Gas-exchange data showed that optimal conditions for photosynthesis of Centrosema pubsecens and Vigna unguiculata were: a temperature of 30°C; a relative humidity of 60–80%; and a light intensity between 1000 and 1200 μE m −2 s −1. Experiments in nutrient solution demonstrated that the optimal temperature for root growth was also 30°C, while watering plants with 5–10mmol nitrate (either as KNO 3 or NH 4NO 3) promoted vegetative growth of the legumes and inhibited nodulation. Under optimal environmental conditions, the C 2H 2-reduction method gave average ratios of mol C 2H 2 reduced to mol nitrogen fixed of 3.01:1. Constitutive nitrate reductase activity was found in all nodules examined and every Bradyrhizobium species studied reduced C 2H 2 to C 2H 4 under free-living conditions. Negative correlations existed between bacteroid nitrate reductase activities and C 2H 2-reduction. Nitrate reductase of Rhizobium sp. NGR234 was partially purified and shown to be stable—perhaps explaining its high activity in old nodules. No correlation between nitrogenase and nitrate reductase activities in free-living rhizobia could be found.

DNA Homologies between Rhizobium fredii, Rhizobia That Nodulate Galega sp., and Other Rhizobium and Bradyrhizobium Species

The relationships between Rhizobium fredii and the rhizobia that nodulate Galega officinalis and Galega orientalis (goat’s rue) and recognized species of Rhizobium and Bradyrhizobium were investigated by using deoxyribonucleic acid (DNA)-DNA hybridization, legume nodulation tests, and phage typing. The R. fredii strains formed a distinct DNA homology group which could be divided into two subgroups. The mean levels of relative homology at 65°C of 11 strains of R. fredii with R. fredii reference strains USDA 208 and USDA 191 were 86 and 80%, respectively. These values were significantly higher (Student’s t test, P < 0.001) than the mean levels of relative homology with DNAs from other groups of rhizobia. The Galega-nodulating rhizobia also formed a distinct DNA homology group. The mean levels of relative homology at 65°C of DNAs from 11 strains with DNAs from reference strains gall and galNW3, which effectively nodulate G. officinalis, were 79 and 85%, respectively. These values were also significantly higher (Student’s t test, P < 0.001) than the values obtained with DNAs from other groups of rhizobia. These results correlated with cross-inoculation and phage-typing results and indicated that the two groups are genetically distinct. Genomic DNA was used as a probe in a modified colony hybridization autoradiographic procedure for the identification of DNAs from Rhizobium leguminosarum, R. fredii, and Rhizobium sp. (Galega) in colonies and from nodules.

Intra- and Intergeneric Similarities between the Ribosomal Ribonucleic Acid Cistrons of Rhizobium and Bradyrhizobium Species and Some Related Bacteria

14C-labeled ribosomal ribonucleic acids (rRNAs) were prepared from Bradyrhizobium japonicum NZP 5549T(T = type strain) and Rhodopseudomonas palustris DSM 130, and [3H]rRNAs were prepared from Rhizobium meliloti NZP 4009 and Rhizobium loti NZP 2037. Labeled rRNA from Agrobacterium tumefaciens ICPB TT111 (a member of Agrobacterium cluster 2) was also used. These rRNAs were hybridized under optimal conditions with filter-fixed deoxyribonucleic acids (DNAs) from 24 strains of rhizobia representing nine previously identified DNA-DNA homology groups and with DNAs from various other organisms. Each hybrid was described by the following two parameters: the temperature at which 50% of the hybrid was denatured (Tm(e) ) and percentage of rRNA binding. From rRNA similarity maps and a Tm(e) dendrogram the following conclusions were drawn. (i) There are at least three genetically distinct groups in the present genus Rhizobium. Group I includes Rhizobium meliloti, Rhizobium fredii, and Rhizobium leguminosarum. Group II is represented by Rhizobium loti, and group III includes rhizobia isolated from Galega officinalis and Galega orientalis (goatsrue.). (ii) The rRNA cistrons of group I strains resemble those of Agrobacterium cluster 2 strains. (iii) The rRNA cistrons of Rhizobium fredii resemble those of Rhizobium meliloti more closely than the rRNA cistrons of Rhizobium leguminosarum or Agrobacterium cluster 2 do. (iv) The rRNA cistrons of Rhizobium loti resemble the rRNA cistrons of Rhizobium meliloti less than the rRNA cistrons of strains belonging to either Agrobacterium cluster 1 or Agrobacterium cluster 2. (v) The rRNA cistrons of Galega rhizobia resemble the rRNA cistrons of Rhizobium meliloti about as much as the rRNA cistrons of Agrobacterium cluster 1 and resemble the rRNA cistrons of Rhizobium loti less. (vi) The rRNA cistrons of Rhizobium and Bradyrhizobium resemble one another much less than do the rRNA cistrons of Rhizobium and other genera presently included in the family Rhizobiaceae (Agrobacterium and Phyllobacterium). (vii) Bradyrhizobium strains belonging to four DNA-DNA homology groups all contain rRNA cistrons which closely resembled the rRNA cistrons of Bradyrhizobium japonicum NZP 5549T. (viii) B. japonicum contains rRNA cistrons which have diverged from the rRNA cistrons of Rhodopseudomonas palustris to about the same extent as the rRNA cistrons of Rhizobium group I and Agrobacterium; on this basis we suggest that the photosynthetic ancestor of Bradyrhizobium may have been a rhodopseudomonad and that a phylogenetic classification should group these genera together. (ix) Stemnodulating strain ORS 571, from Sesbania rostrata belongs to the Rhodopseudomonas palustris rRNA branch; it is quite distinct from B. japonicum and Rhodopseudomonas palustris. Two representative strains which nodulate roots belong in the group containing the fast-growing rhizobia.

Rhizobium fredii sp. nov., a Fast-Growing Species That Effectively Nodulates Soybeans

A new species, Rhizobium fredii, is proposed for fast-growing root nodule bacteria isolated from soybeans. The type strain was isolated from a root nodule of Glycine max growing in Honan Province, China, and is designated strain USDA 205 (= ATCC 35423 = PRC 205). This new species is differentiated from currently recognized Rhizobium and Bradyrhizobium species by deoxyribonucleic acid hybridization comparisons, plant specificity, generation times, antibiotic resistance, and serology. The strains of R. fredii are differentiated into two proposed chemovars, R. fredii chemovar fredii chemovar nov. and R. fredii chemovar siensis chemovar nov., by deoxyribonucleic acid hybridization tests, growth in the presence of erythromycin (20 μg/ml), final pH of yeast extract-mannitol medium, and serology.

Antifungal Factor Produced by <i>Pseudomonas fluorescens</i> against an Endophytic Fungus

Growth characteristics of Piriformospora indica an endophytic fungus with plant growth promoting rhizobacteria in an aspergillus medium were studied. P. indica showed promotional effects with species of Azotobacter chroococcum, Azospirillum brasilensis, Bradyrhizobium spp., and inhibitory effect with Pseudomonas fluorescens. The biochemical and molecular level of inhibitory factor produced by P. fluorescence suggests that the antifungal substance was thermolabile and the protease test showed it to be protease resistant in nature. Dialysis test indicated that this substance had a molecular weight of more than 12,000 kDa. The inhibitory substance turns out to be 'fungistatic' as well as 'fungicidal' in nature. Scanning electron microscopy (SEM) and Transmission electron microscopy (TEM) studies showed the cell wall in damaged form suggesting the high potency of this antifungal substance. The inhibitory compound produced by P. fluorescence was suspected to be pyoverdine, a kind of siderophore, an iron containing substance. Key words: Pseudomonas fluorescens; Piriformospora indica; siderophore; rhizobacteria; TEM; SEM DOI: 10.3126/njst.v9i0.3167 Nepal Journal of Science and Technology 9 (2008) 65-71