Bradyrhizobium Diazoefficiens USDA110 Research Articles

Genome Sequence of Bacillus velezensis S141, a New Strain of Plant Growth-Promoting Rhizobacterium Isolated from Soybean Rhizosphere.

ABSTRACTBacillus velezensis strain S141 is a plant growth-promoting rhizobacterium isolated from soybean (Glycine max) rhizosphere that enhances soybean growth, nodulation, and N2 fixation efficiency by coinoculation with Bradyrhizobium diazoefficiens USDA110. The S141 genome was identified to comprise a 3,974,582-bp-long circular DNA sequence encoding at least 3,817 proteins.

Nonspecific Symbiosis Between Sophora flavescens and Different Rhizobia.

We explored the genetic basis of the promiscuous symbiosis of Sophora flavescens with diverse rhizobia. To determine the impact of Nod factors (NFs) on the symbiosis of S. flavescens, nodulation-related gene mutants of representative rhizobial strains were generated. Strains with mutations in common nodulation genes (nodC, nodM, and nodE) failed to nodulate S. flavescens, indicating that the promiscuous nodulation of this plant is strictly dependent on the basic NF structure. Mutations of the NF decoration genes nodH, nodS, nodZ, and noeI did not affect the nodulation of S. flavescens, but these mutations affected the nitrogen-fixation efficiency of nodules. Wild-type Bradyrhizobium diazoefficiens USDA110 cannot nodulate S. flavescens, but we obtained 14 Tn5 mutants of B. diazoefficiens that nodulated S. flavescens. This suggested that the mutations had disrupted a negative regulator that prevents nodulation of S. flavescens, leading to nonspecific nodulation. For Ensifer fredii CCBAU 45436 mutants, the minimal NF structure was sufficient for nodulation of soybean and S. flavescens. In summary, the mechanism of promiscuous symbiosis of S. flavescens with rhizobia might be related to its nonspecific recognition of NF structures, and the host specificity of rhizobia may also be controlled by currently unknown nodulation-related genes.

Arsenic toxicity in soybean alleviated by a symbiotic species of Bradyrhizobium

Arsenic (As) is a toxic metalloid that has gained special interest in the past years as a global environmental problem. Groundwater in Cordoba province (Argentina) presents high As concentrations which can be absorbed by plants or be used for artificial irrigation. The aim of this research was to elucidate the differential responses of symbiotic interactions established with three bacterial strains and soybean plants to realistic doses of arsenic. The reference strain Bradyrhizobium diazoefficiens USDA110 and the native isolate Bradyrhizobium sp. Per 3.64 were able to grow up to 13 mM As(V) whereas the native strain Bradyrhizobium sp. Per 3.61 grew up to 9.5 mM As(V). Metalloid addition did not modify the soybean plant growth at 6 μM As(V). Nevertheless, it was enough to induce oxidative stress as observed by an increase on lipid peroxidation. The soybean-Bradyrhizobium sp. assay at 6 μM As(V) showed no changes in growth variables (shoot and root dry weight) in plants inoculated with the reference microsymbiont or Bradyrhizobium sp. Per 3.61. Regarding As uptake by plants, metalloid accumulation followed the same distribution pattern among strains. Remarkably, at 6 μM As(V), soybean inoculation with Bradyrhizobium sp. Per 3.61 revealed a significantly lower translocation factor (TF) in comparison to other inoculated strains promoting As phytostabilization. At the highest As(V) concentration tested, only Bradyrhizobium diazoefficiens USDA110 was able to nodulate the legume, however, a significant decrease in the number and dry weight of nodules and nitrogen content was observed. In conclusion, the inoculation of soybean plants with the reference strain Bradyrhizobium diazoefficiens USDA110 exposed to high As(V) concentration represents an effective and promising symbiotic interaction that allows the development of the legume due to the minimal effects on plant growth. However, in low As(V) concentration environments, the native isolate Bradyrhizobium sp. Per 3.61, is shown to be the best inoculant among the tested strains, owing to the limitation of metalloid translocation and accumulation to edible parts of the legume, avoiding fruit contamination and human poisoning.

Complete Genome Sequence of Bradyrhizobium diazoefficiens USDA 122, a Nitrogen-Fixing Soybean Symbiont.

ABSTRACTWe report the complete genome sequence of Bradyrhizobium diazoefficiens USDA 122, a nitrogen-fixing soybean symbiont. The genome consists of a 9.1 Mb circular chromosome, and 8,551 coding sequences (CDSs) were predicted on the genome. The sequence will provide insight into the evolution of rhizobial genome, and the symbiotic compatibility with host plants.

Construction and simulation of the Bradyrhizobium diazoefficiens USDA110 metabolic network: a comparison between free-living and symbiotic states.

Bradyrhizobium diazoefficiens is a rhizobium able to convert atmospheric nitrogen into ammonium by establishing mutualistic symbiosis with soybean. It has been recognized as an important parent strain for microbial agents and is widely applied in agricultural and environmental fields. In order to study the metabolic properties of symbiotic nitrogen fixation and the differences between a free-living cell and a symbiotic bacteroid, a genome-scale metabolic network of B. diazoefficiens USDA110 was constructed and analyzed. The metabolic network, iYY1101, contains 1031 reactions, 661 metabolites, and 1101 genes in total. Metabolic models reflecting free-living and symbiotic states were determined by defining the corresponding objective functions and substrate input sets, and were further constrained by high-throughput transcriptomic and proteomic data. Constraint-based flux analysis was used to compare the metabolic capacities and the effects on the metabolic targets of genes and reactions between the two physiological states. The results showed that a free-living rhizobium possesses a steady state flux distribution for sustaining a complex supply of biomass precursors while a symbiotic bacteroid maintains a relatively condensed one adapted to nitrogen-fixation. Our metabolic models may serve as a promising platform for better understanding the symbiotic nitrogen fixation of this species.

Bradyrhizobium elkanii nod regulon: insights through genomic analysis.

A successful symbiotic relationship between soybean [Glycine max (L.) Merr.] and Bradyrhizobium species requires expression of the bacterial structural nod genes that encode for the synthesis of lipochitooligosaccharide nodulation signal molecules, known as Nod factors (NFs). Bradyrhizobium diazoefficiens USDA 110 possesses a wide nodulation gene repertoire that allows NF assembly and modification, with transcription of the nodYABCSUIJnolMNOnodZ operon depending upon specific activators, i.e., products of regulatory nod genes that are responsive to signaling molecules such as flavonoid compounds exuded by host plant roots. Central to this regulatory circuit of nod gene expression are NodD proteins, members of the LysR-type regulator family. In this study, publicly available Bradyrhizobium elkanii sequenced genomes were compared with the closely related B. diazoefficiens USDA 110 reference genome to determine the similarities between those genomes, especially with regards to the nod operon and nod regulon. Bioinformatics analyses revealed a correlation between functional mechanisms and key elements that play an essential role in the regulation of nod gene expression. These analyses also revealed new genomic features that had not been clearly explored before, some of which were unique for some B. elkanii genomes.

Growth Rate of and Gene Expression in Bradyrhizobium diazoefficiens USDA110 due to a Mutation in blr7984, a TetR Family Transcriptional Regulator Gene.

Previous transcriptome analyses have suggested that a gene cluster including a transcriptional regulator (blr7984) of the tetracycline repressor family was markedly down-regulated in symbiosis. Since blr7984 is annotated to be the transcriptional repressor, we hypothesized that it is involved in the repression of genes in the genomic cluster including blr7984 in symbiotic bacteroids. In order to examine the function and involvement of the blr7984 gene in differentiation into bacteroids, we compared the free-living growth/symbiotic phenotype and gene expression between a blr7984-knockout mutant and the wild-type strain of Bradyrhizobium diazoefficiens USDA110. The mutant transiently increased the cell growth rate under free-living conditions and nodule numbers over those with the wild-type strain USDA110. The expression of three genes adjacent to the disrupted blr7984 gene was strongly up-regulated in the mutant in free-living and symbiotic cells. The mutant also induced the expression of genes for glutathione S-transferase, cytochrome c oxidases, ABC transporters, PTS sugar transport systems, and flagella synthesis under free-living conditions. bll7983 encoding glutathione S-transferase was up-regulated the most by the blr7984 disruption. Since redox regulation by glutathione is known to be involved in cell division in prokaryotes and eukaryotes, the strong expression of glutathione S-transferase encoded by the bll7983 gene may have caused redox changes in mutant cells, which resulted in higher rates of cell division.

The tight-adhesion proteins TadGEF of Bradyrhizobium diazoefficiens USDA 110 are involved in cell adhesion and infectivity on soybean roots

Adhesion of symbiotic bacteria to host plants is an essential early step of the infection process that leads to the beneficial interaction. In the Bradyrhizobium diazoefficiens-soybean symbiosis few molecular determinants of adhesion are known. Here we identified the tight-adhesion gene products TadGEF in the open-reading frames blr3941–blr3943 of the B. diazoefficiens USDA 110 complete genomic sequence. Predicted structure of TadG indicates a transmembrane domain and two extracytosolic domains, from which the C-terminal has an integrin fold. TadE and TadF are also predicted as bearing transmembrane segments. Mutants in tadG or the small cluster tadGEF were impaired in adhesion to soybean roots, and the root infection was delayed. However, nodule histology was not compromised by the mutations, indicating that these effects were restricted to the earliest contact of the B. diazoefficiens and root surfaces. Knowledge of preinfection determinants is important for development of inoculants that are applied to soybean crops worldwide.

Stable Fluorescent and Enzymatic Tagging of Bradyrhizobium diazoefficiens to Analyze Host-Plant Infection and Colonization.

Bradyrhizobium diazoefficiens USDA 110 (formerly named Bradyrhizobium japonicum) can fix dinitrogen when living as an endosymbiont in root nodules of soybean and some other legumes. Formation of a functional symbiosis relies on a defined developmental program mediated by controlled gene expression in both symbiotic partners. In contrast to other well-studied Rhizobium-legume model systems that have been thoroughly examined by means of genetically tagged strains, analysis of B. diazoefficiens host infection has been impaired due to the lack of suitable tagging systems. Here, we describe the construction of B. diazoefficiens strains constitutively expressing single-copy genes for fluorescent proteins (eBFP2, mTurquoise2, GFP+, sYFP2, mCherry, HcRed) and enzymes (GusA, LacZ). For stable inheritance, the constructs were recombined into the chromosome. Effectiveness and versatility of the tagged strains was demonstrated in plant infection assays. (i) The infection process was followed from root-hair attachment to colonization of nodule cells with epifluorescent microscopy. (ii) Monitoring mixed infections with two strains producing different fluorescent proteins allowed rapid analysis of nodule occupancy and revealed that the majority of nodules contained clonal populations. (iii) Microscopic analysis of nodules induced by fluorescent strains provided evidence for host-dependent control of B. diazoefficiens bacteroid morphology in nodules of Aeschynomene afraspera and Arachis hypogaea (peanut), as deduced from their altered morphology compared with bacteroids in soybean nodules.

Proposed some interactions at molecular level of PGPR coinoculated with Bradyrhizobium diazoefficiens USDA110 and B.japonicum THA6 on soybean symbiosis and its potential of field application

The objectives of this research were to select the appropriate plant growth promoting rhizobacteria (PGPR) and evaluate their influence in promoting nodulation and N2-fixing efficiency of soybean (Glycine max) by coinoculation with Bradyrhizobium diazoefficiens USDA110 and B. japonicum THA6 strains. Selected 12 PGPR performed a significant capability of promoting N2-fixation, nodule number, nodule and plant dry weight with both of the commercial bradyrhizobial strains, USDA110 and THA6 (P<0.05). Furthermore, isolates S141 and S222, which are closely related to Bacillus subtilis and Staphylococcus sp., were selected for coinoculation with USDA110 and THA6. The effective coinoculation doses of PGPR:Bradyrhizobium on soybean were 106:106 colony forming unit (CFU)ml−1seed−1. The expression levels of soybean and Bradyrhizobium related genes including Glyma17g07330, otsA, phbC, dctA and nifH in nodule discontinuously triggered both up- and down-regulation at different time frames (2–7 WAI). The transmission electron microscopy (TEM) micrograph of coinoculated soybean nodule showed the compact cluster of bacteroids which was densely packed with poly-ß-hydroxybutyrate (PHB) granules. The amounts of PHBs remained in mature nodule of coinoculation treatments whilst single inoculation nodules were senescence. The induction of soybean root subsequently increased the nodulation signaling and then activated the trehalose accumulation and the transport of carbon that represented an increase in PHB accumulation, resulting in the enhancement of the nodulation and N2-fixation in soybean. These results were accordingly related with phenotypic characters in Leonard’s jar experiment in terms of enhancing the nodulation and N2-fixation in soybean. The effect of coinoculation experiment under field condition could increase 9.7–43.6% of seed yield per hectare which was higher than those of uninoculation or single inoculation of PGPR or Bradyrhizobium. Therefore, the efficiency to enhance soybean N2-fixation by coinoculation of S141 and S222 with Bradyrhizobium strategy could be developed for supreme soybean inoculants.

Biotechnological potential of rhizobial metabolites to enhance the performance of Bradyrhizobium spp. and Azospirillum brasilense inoculants with soybean and maize.

Agricultural sustainability may represent the greatest encumbrance to increasing food production. On the other hand, as a component of sustainability, replacement of chemical fertilizers by bio-fertilizers has the potential to lower costs for farmers, to increase yields, and to mitigate greenhouse-gas emissions and pollution of water and soil. Rhizobia and plant-growth-promoting rhizobacteria (PGPR) have been broadly used in agriculture, and advances in our understanding of plant-bacteria interactions have been achieved; however, the use of signaling molecules to enhance crop performance is still modest. In this study, we evaluated the effects of concentrated metabolites (CM) from two strains of rhizobia—Bradyrhizobium diazoefficiens USDA 110T (BD1) and Rhizobium tropici CIAT 899T (RT1)—at two concentrations of active compounds (10–8 and 10–9 M)—on the performances of two major plant-microbe interactions, of Bradyrhizobium spp.-soybean (Glycine max (L.) Merr.) and Azospirillum brasilense-maize (Zea mays L.). For soybean, one greenhouse and two field experiments were performed and effects of addition of CM from the homologous and heterologous strains, and of the flavonoid genistein were investigated. For maize, three field experiments were performed to examine the effects of CM from RT1. For soybean, compared to the treatment inoculated exclusively with Bradyrhizobium, benefits were achieved with the addition of CM-BD1; at 10–9 M, grain yield was increased by an average of 4.8%. For maize, the best result was obtained with the addition of CM-RT1, also at 10–9 M, increasing grain yield by an average of 11.4%. These benefits might be related to a combination of effects attributed to secondary compounds produced by the rhizobial strains, including exopolysaccharides (EPSs), plant hormones and lipo-chitooligosaccharides (LCOs). The results emphasize the biotechnological potential of using secondary metabolites of rhizobia together with inoculants containing both rhizobia and PGPR to improve the growth and yield of grain crops.