Bradyrhizobium Japonicum USDA Research Articles

Silver nanoparticles inhibit nitrogen fixation in soybean (Glycine max) root nodules.

Antimicrobial silver nanoparticles (AgNPs) are popular in consumer and industrial products, leading to increasing concentrations in the environment. We tested whether exposure to AgNPs could be detrimental to a microbe, its host plant, and their symbiotic relationship. When subjected to 10µg/mL AgNPs, growth of Bradyrhizobium japonicum USDA 110 was halted. Axenic nitrogen-fertilized Glycine max seedlings were unaffected by 2.5µg/mL of 30nm AgNPs, but growth was inhibited with the same dose of 16nm AgNPs. With 2.5µg/mL AgNPs, biomass of inoculated plants was 50% of the control. Bacteroids were not found in nodules on plants treated with 2.5µg/mL AgNPs and plants given 0.5-2.5µg/mL AgNPs had 40-65% decreased nitrogen fixation. In conclusion, AgNPs not only interfere with general plant and bacterial growth but also inhibit nodule development and bacterial nitrogen fixation. We should be mindful of not releasing AgNPs to the environment or to agricultural land.

Agronomic Performance of Soybean with Bradyrhizobium Inoculation in Double-Cropped Farming

Land degradation is a serious problem in arid regions, including in Central Asian countries. Soybean symbiosis with rhizobia microbes has an essential role in improving crop productivity and sustaining soil fertility in an arid environment. An experiment was conducted in light straw-colored sierozem soils in the Syrdarya region of Uzbekistan (41.4° N, 64.6° E) under arid conditions over the 2016–2017 and 2017–2018 growing seasons. This study aimed to assess the beneficial N fixation (BNF) ability of soybean in association with the Bradyrhizobium R6 strain and the Bradyrhizobium japonicum USDA110 strains and their combined effect on soil fertility and crop yield. The residues of winter wheat and soybean improved soil structure, i.e., soil humus and N and P contents, significantly differing from those on the soybean followed by summer fallow treatment. Furthermore, soybean in association with dual inoculation had the highest N derived from the atmosphere (Ndfa) (62.9 kg N ha−1), followed by individual soybean treatments with the R6 and USDA110 strains at 51.9 and 40.6 kg N ha−1, respectively. Improved soil quality positively impacted crop output, increasing winter wheat and soybean yields by 36.5% and 34.6%, respectively. Likewise, the yield parameters, i.e., the number of pods, weight of grain per pods, and 1000 seeds were significantly higher in the inoculated treatment with the highest value observed in the dual-inoculated treatment. These results suggest the insertion of soybean with symbiotic bacteria into the cropping system has considerable potential to contribute to sustainable land management practices in arid zones.

Co-inoculation of rhizobacteria promotes growth, yield, and nutrient contents in soybean and improves soil enzymes and nutrients under drought conditions

Drought stress is the major abiotic factor limiting crop production. Co-inoculating crops with nitrogen fixing bacteria and plant growth-promoting rhizobacteria (PGPR) improves plant growth and increases drought tolerance in arid or semiarid areas. Soybean is a major source of high-quality protein and oil for humans. It is susceptible to drought stress conditions. The co-inoculation of drought-stressed soybean with nodulating rhizobia and root-colonizing, PGPR improves the root and the shoot growth, formation of nodules, and nitrogen fixation capacity in soybean. The present study was aimed to observe if the co-inoculation of soybean (Glycine max L. (Merr.) nodulating with Bradyrhizobium japonicum USDA110 and PGPR Pseudomonas putida NUU8 can enhance drought tolerance, nodulation, plant growth, and nutrient uptake under drought conditions. The results of the study showed that co-inoculation with B. japonicum USDA110 and P. putida NUU8 gave more benefits in nodulation and growth of soybean compared to plants inoculated with B. japonicum USDA110 alone and uninoculated control. Under drought conditions, co-inoculation of B. japonicum USDA 110 and P. putida NUU8 significantly enhanced the root length by 56%, shoot length by 33%, root dry weight by 47%, shoot dry weight by 48%, and nodule number 17% compared to the control under drought-stressed. Co-inoculation with B. japonicum, USDA 110 and P. putida NUU8 significantly enhanced plant and soil nutrients and soil enzymes compared to control under normal and drought stress conditions. The synergistic use of B. japonicum USDA110 and P. putida NUU8 improves plant growth and nodulation of soybean under drought stress conditions. The results suggested that these strains could be used to formulate a consortium of biofertilizers for sustainable production of soybean under drought-stressed field conditions.

Effect of salt stress on soybean growth and nodulation under inoculation with soybean rhizobia

ABSTRACT Salinity is one of the major problems when it comes to soil fertility and agricultural productivity. In this regard, this study aimed to elucidate the influence of four salinity levels (0, 20, 50, 100 mM NaCl) on the soybean growth and the nodulation by rhizobia inoculated on the soybean variety CNS. Additionally, the compatibility of soybean varieties with the seven representative strains of rhizobia was also investigated. Through the compatibility test, it was revealed that the Sinorhizobium (Ensifer) fredii USDA 191 and the three bradyrhizobia (Bradyrhizobium japonicum USDA 6T, B. elkanii USDA 31, and B. diazoefficiens USDA 110T) showed compatible nodulation with the Rfg1 genotype soybean, whereas, the S. fredii USDA 192, 193, and 205T showed incompatible nodulation. The inoculation tests with bradyrhizobia (USDA 31, 110T) and sinorhizobia (USDA 191, 192, 193) indicated a better growth of soybean for bradyrhizobia than the sinorhizobia until 50 mM NaCl. Note that even though the symbiotic efficiency at each strain was not different among 0, 20, and 50 mM NaCl treatments, the symbiotic efficiency of bradyrhizobia was higher than the sinorhizobia at each NaCl treatment. The result of the mix inoculation test indicated that the nodule occupancy rate of strain USDA 191 increased significantly along with the NaCl concentration but strain USDA 110T showed an opposite response. These results indicate a positive relationship between the soybean growth and the nitrogen fixation ability of the inoculant under salt-stress conditions. Furthermore, the results obtained from the mix inoculation test partly describe the dominance of sinorhizobia under saline conditions.

Co-Inoculation of Rhizobacteria and Biochar Application Improves Growth and Nutrientsin Soybean and Enriches Soil Nutrients and Enzymes

Gradual depletion in soil nutrients has affected soil fertility, soil nutrients, and the activities of soil enzymes. The applications of multifarious rhizobacteria can help to overcome these issues, however, the effect of co-inoculation of plant-growth promoting rhizobacteria (PGPR) and biochar on growth andnutrient levelsin soybean and on the level of soil nutrients and enzymes needs in-depth study. The present study aimed to evaluate the effect of co-inoculation of multifarious Bradyrhizobium japonicum USDA 110 and Pseudomonas putida TSAU1 and different levels (1 and 3%) of biochar on growth parameters and nutrient levelsin soybean and on the level of soil nutrients and enzymes. Effect of co-inoculation of rhizobacteria and biochar (1 and 3%) on the plant growth parameters and soil biochemicals were studied in pot assay experiments under greenhouse conditions. Both produced good amounts of indole-acetic acid; (22 and 16 µg mL−1), siderophores (79 and 87%SU), and phosphate solubilization (0.89 and 1.02 99 g mL−1). Co-inoculation of B. japonicum with P. putida and 3% biochar significantly improved the growth and nutrient content ofsoybean and the level of nutrients and enzymes in the soil, thus making the soil more fertile to support crop yield. The results of this research provide the basis of sustainable and chemical-free farming for improved yields and nutrients in soybean and improvement in soil biochemical properties.

Comparative evaluation of Bradyrhizobium japonicum USDA110 transferors for greener and economic soybean (Glycine max L.) agronomy in Southwestern Nigeria

Comparative evaluation of Bradyrhizobium japonicum USDA110 transferors for greener and economic soybean (Glycine max L.) agronomy in Southwestern Nigeria

Direct Stimulation by Methanol Addition on the Cultured Medium for Methanol Dehydrogenase Protein Purification from Bradyrhizobium japonicum USDA110

Methanol dehydrogenase (MDH) enzyme was purified from Bradyrhizobium japonicum USDA110 cell-free extract. The bacteria were grown in a culture medium with direct 0.5% methanol addition aimed to stimulates the MDH catalytic enzyme activation. Bradyrhizobium japonicum USDA110 MDH enzyme was purified by using 25 mM 2-(N-morpholine) ethanesulfonic acid/MES pH 5.5 buffer and 1 M sodium chloride/NaCl which separated into three columns, the first column was PD-10 for buffer exchange; the second column was HiTrap Sepharose HP to obtain unbonded fraction in the column; and the third column was Mono S 5/50 GL integrated with two pumps HPLC (high-performance liquid chromatography) to obtain pure MDH enzyme for serial changing of 1 M NaCl-25mM MES pH 5.5 with the flow rate at 1 ml/min. The protein concentration and MDH catalytic enzyme activity were observed on each purification process starting from the cell-free extract to pure MDH enzyme. The pure MDH enzyme was obtained by Mono S 5/50 GL-HPLC purification which showed a single band on SDS PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis). The MDH enzyme purification from Bradyrhizobium japonicum USDA110 showed 90-fold purification, a sub-molecular weight of 63 kDa, specific activity at 2.69 U/mg, and optimum activity at a 35oC temperature and pH 9.

Bacterial exopolysaccharides as a modern biotechnological tool for modification of fungal laccase properties and metal ion binding

Four bacterial EPSs extracted from Rhizobium leguminosarum bv. trifolii Rt24.2, Sinorhizobium meliloti Rm1021, Bradyrhizobium japonicum USDA110, and Bradyrhizobium elkanii USDA76 were determined towards their metal ion adsorption properties and possible modification of Cerrena unicolor laccase properties. The highest magnesium and iron ion-sorption capacity (~ 42 and ~ 14.5%, respectively) was observed for EPS isolated from B. japonicum USDA110. An evident influence of EPSs on the stability of laccase compared to the control values (without EPSs) was shown after 30-day incubation at 25 °C. The residual activity of laccases was obtained in the presence of Rh76EPS and Rh1021EPS, i.e., 49.5 and 41.5% of the initial catalytic activity, respectively. This result was confirmed by native PAGE electrophoresis. The EPS effect on laccase stability at different pH (from 3.8 to 7.0) was also estimated. The most significant changes at the optimum pH value (pH 5.8) was observed in samples of laccase stabilized by Rh76EPS and Rh1021EPS. Cyclic voltamperometry was used for analysis of electrochemical parameters of laccase stabilized by bacterial EPS and immobilized on single-walled carbon nanotubes (SWCNTs) with aryl residues. Laccases with Rh76EPS and Rh1021EPS had an evident shift of the value of the redox potential compared to the control without EPS addition. In conclusion, the results obtained in this work present a new potential use of bacterial EPSs as a metal-binding component and a modulator of laccase properties especially stability of enzyme activity, which can be a very effective tool in biotechnology and industrial applications.

Transcriptomic Studies of the Effect of nod Gene-Inducing Molecules in Rhizobia: Different Weapons, One Purpose.

Simultaneous quantification of transcripts of the whole bacterial genome allows the analysis of the global transcriptional response under changing conditions. RNA-seq and microarrays are the most used techniques to measure these transcriptomic changes, and both complement each other in transcriptome profiling. In this review, we exhaustively compiled the symbiosis-related transcriptomic reports (microarrays and RNA sequencing) carried out hitherto in rhizobia. This review is specially focused on transcriptomic changes that takes place when five rhizobial species, Bradyrhizobium japonicum (=diazoefficiens) USDA 110, Rhizobium leguminosarum biovar viciae 3841, Rhizobium tropici CIAT 899, Sinorhizobium (=Ensifer) meliloti 1021 and S. fredii HH103, recognize inducing flavonoids, plant-exuded phenolic compounds that activate the biosynthesis and export of Nod factors (NF) in all analysed rhizobia. Interestingly, our global transcriptomic comparison also indicates that each rhizobial species possesses its own arsenal of molecular weapons accompanying the set of NF in order to establish a successful interaction with host legumes.

Nodulation and Symbiotic Nitrogen Fixation in the Biofuel Legume Tree Pongamia pinnata

The legume tree Pongamia pinnata (also called Millettia pinnata) is a non-food crop that can grow on marginal land not destined for the cultivation of food crops. It is an important candidate for the production of biofuel (bio-oil, biodiesel and aviation biofuel) from its oil-rich seeds. An important trait is its ability to grow in marginal, nitrogen-limited soils. This growth ability is attributed to the nitrogen-fixation activity of root nodules. Cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks. These parameters were reduced when both cotyledons were removed; plants were visibly yellow. Surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. To isolate rhizobial inoculants from soil, samples were collected from Meandu Mine, a coal-mining site near Kingaroy, Queensland (Australia), where pongamia is currently grown to rehabilitate the soil. Pongamia nodules were also collected from Mt. Coot-tha Botanic Gardens (Brisbane), where pongamia was introduced decades ago. Established ‘Baiting technique’ and ‘One-drop-one-nodule technique’ were applied to isolate potential rhizobia from the soil samples and nodules. All isolates were screened and characterised for symbiotic effectiveness and nitrogenase activity. A total of 21 putative rhizobial samples were isolated – five from soil samples and 16 from pongamia nodules. Out of these iso lates, seven were slow-growers while 14 were fast-growers. These isolates were coded as PR-UQ. Among the isolates, the fast-growers PR-UQ-03 and PR-UQ-05 were considered as the superior strains compared to the slow-growers PR-UQ-01 and PR-UQ-04, which were more effective than the rest of the isolates. The new isolates enhanced nodule number, shoot length and total plant dry weight and resulted in better plant growth than the available Bradyrhizobium japonicum strains CB1809, USDA110 and CB564. Acetylene reduction assay (short term) and Nitrogen Difference Analysis (long term) further confirmed that pongamia fixed more nitrogen upon inoculation with these rhizobia. Based on 16S rDNA sequence analysis, PR-UQ-01 and PR-UQ-04 are closely related to Bradyrhizobium elkanii and Bradyrhizobium pachyrizi, while PR-UQ-03 and PR-UQ-05 are both related to Rhizobium mesoamericanum. Cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. The newly isolated, fast-growing rhizobia PR-UQ-03 and PR-UQ-05 and the slow-growing PR-UQ-01 and PR-UQ-04 are related to Rhizobium and Bradyrhizobium species and aid in the nodulation, nitrogen fixation and seedling/sapling growth of Pongamia pinnata.

Conserved small mRNA with an unique, extended Shine-Dalgarno sequence

ABSTRACTUp to now, very small protein-coding genes have remained unrecognized in sequenced genomes. We identified an mRNA of 165 nucleotides (nt), which is conserved in Bradyrhizobiaceae and encodes a polypeptide with 14 amino acid residues (aa). The small mRNA harboring a unique Shine-Dalgarno sequence (SD) with a length of 17 nt was localized predominantly in the ribosome-containing P100 fraction of Bradyrhizobium japonicum USDA 110. Strong interaction between the mRNA and 30S ribosomal subunits was demonstrated by their co-sedimentation in sucrose density gradient. Using translational fusions with egfp, we detected weak translation and found that it is impeded by both the extended SD and the GTG start codon (instead of ATG). Biophysical characterization (CD- and NMR-spectroscopy) showed that synthesized polypeptide remained unstructured in physiological puffer. Replacement of the start codon by a stop codon increased the stability of the transcript, strongly suggesting additional posttranscriptional regulation at the ribosome. Therefore, the small gene was named rreB (ribosome-regulated expression in Bradyrhizobiaceae). Assuming that the unique ribosome binding site (RBS) is a hallmark of rreB homologs or similarly regulated genes, we looked for similar putative RBS in bacterial genomes and detected regions with at least 16 nt complementarity to the 3′-end of 16S rRNA upstream of sORFs in Caulobacterales, Rhizobiales, Rhodobacterales and Rhodospirillales. In the Rhodobacter/Roseobacter lineage of α-proteobacteria the corresponding gene (rreR) is conserved and encodes an 18 aa protein. This shows how specific RBS features can be used to identify new genes with presumably similar control of expression at the RNA level.

Studies of rhizobial competitiveness for nodulation in soybean using a non-destructive split-root system.

Split-root systems (SRS) constitute an appropriate methodology for studying the relevance of both local and systemic mechanisms that participate in the control of rhizobia-legume symbioses. In fact, this kind of approach allowed to demonstrate the autoregulation of nodulation (AON) systemic response in soybean in the 1980s. In SRS, the plant main root is cut and two lateral roots that emerge from the seedlings after root-tip removal are confined into separate compartments. After several days of growth, these plants have two separate roots that can be inoculated with the same or with different bacteria, at the same or at different times. In this work, we have used a non-destructive SRS to study two different competitiveness relations between rhizobial strains in soybean roots. One of them is the competition for nodulation between two soybean-nodulating rhizobia: the slow-grower Bradyrhizobium japonicum USDA110 and the fast-grower Sinorhizobium fredii HH103. The second is the competitive blocking of S. fredii 257DH4 nodulation in the American soybean Osumi by Sinorhizobium fredii USDA257, which is unable to nodulate American soybeans. Our results showed that the competitiveness relationships studied in this work are mitigated or even avoided when the competitive strains are spatially separated in different compartments containing half-roots from the same plant, suggesting that competitive relations are more related to local plant responses. In our opinion, split-root systems are an appropriate approach to further study competitive relations among rhizobial strains.

Coordination between Bradyrhizobium and Pseudomonas alleviates salt stress in soybean through altering root system architecture

ABSTRACTIt is a well accepted strategy to improve plant salt tolerance through inoculation with beneficial microorganisms. However, its underlying mechanisms still remain unclear. In the present study, hydroponic experiments were conducted to evaluate the effects of Bradyrhizobium japonicum USDA 110 with salt-tolerant Pseudomonas putida TSAU1 on growth, protein content, nitrogen, and phosphorus uptake as well as root system architecture of soybean (Glycine max L.) under salt stress. The results indicated that the combined inoculation with USDA 110 and TSAU1 significantly improved plant growth, nitrogen and phosphorus contents, and contents of soluble leaf proteins under salt stress compared to the inoculation with the symbiont alone or compared to un-inoculated ones. The root architectural traits, like root length, surface area, project area, and root volume; as well as nodulation traits were also significantly increased by co-inoculation with USDA 110 and TSAU1. The plant-growth promoting rhizobacteria (PGPR) P. putida strain TSAU1 could improve the symbiotic interaction between the salt-stressed soybean and B. japonicum USDA 110. In conclusion, inoculation with B. japonicum and salt-tolerant P. putida synergistically improved soybean salt tolerance through altering root system architecture facilitating nitrogen and phosphorus acquisition, and nodule formation.

Nodulation and Symbiotic Nitrogen Fixation in the Biofuel Legume Tree Pongamia pinnata

The legume tree Pongamia pinnata (also called Millettia pinnata) is a non-food crop that can grow on marginal land not destined for the cultivation of food crops. It is an important candidate for the production of biofuel (bio-oil, biodiesel and aviation biofuel) from its oil-rich seeds. An important trait is its ability to grow in marginal, nitrogen-limited soils. This growth ability is attributed to the nitrogen-fixation activity of root nodules. Cutting both cotyledons in halves had little effect on shoot dry weight, nodule number and weight per plant at 8 weeks. These parameters were reduced when both cotyledons were removed; plants were visibly yellow. Surprisingly when no nutrients were supplied and with both cotyledons intact, plants grew as well as those with nutrients. To isolate rhizobial inoculants from soil, samples were collected from Meandu Mine, a coal-mining site near Kingaroy, Queensland (Australia), where pongamia is currently grown to rehabilitate the soil. Pongamia nodules were also collected from Mt. Coot-tha Botanic Gardens (Brisbane), where pongamia was introduced decades ago. Established ‘Baiting technique’ and ‘One-drop-one-nodule technique’ were applied to isolate potential rhizobia from the soil samples and nodules. All isolates were screened and characterised for symbiotic effectiveness and nitrogenase activity. A total of 21 putative rhizobial samples were isolated – five from soil samples and 16 from pongamia nodules. Out of these iso lates, seven were slow-growers while 14 were fast-growers. These isolates were coded as PR-UQ. Among the isolates, the fast-growers PR-UQ-03 and PR-UQ-05 were considered as the superior strains compared to the slow-growers PR-UQ-01 and PR-UQ-04, which were more effective than the rest of the isolates. The new isolates enhanced nodule number, shoot length and total plant dry weight and resulted in better plant growth than the available Bradyrhizobium japonicum strains CB1809, USDA110 and CB564. Acetylene reduction assay (short term) and Nitrogen Difference Analysis (long term) further confirmed that pongamia fixed more nitrogen upon inoculation with these rhizobia. Based on 16S rDNA sequence analysis, PR-UQ-01 and PR-UQ-04 are closely related to Bradyrhizobium elkanii and Bradyrhizobium pachyrizi, while PR-UQ-03 and PR-UQ-05 are both related to Rhizobium mesoamericanum. Cotyledons supply the seedling with nutrients for a considerable period of early seedling growth. The newly isolated, fast-growing rhizobia PR-UQ-03 and PR-UQ-05 and the slow-growing PR-UQ-01 and PR-UQ-04 are related to Rhizobium and Bradyrhizobium species and aid in the nodulation, nitrogen fixation and seedling/sapling growth of Pongamia pinnata.

Small Open Reading Frames, Non-Coding RNAs and Repetitive Elements in Bradyrhizobium japonicum USDA 110.

Small open reading frames (sORFs) and genes for non-coding RNAs are poorly investigated components of most genomes. Our analysis of 1391 ORFs recently annotated in the soybean symbiont Bradyrhizobium japonicum USDA 110 revealed that 78% of them contain less than 80 codons. Twenty-one of these sORFs are conserved in or outside Alphaproteobacteria and most of them are similar to genes found in transposable elements, in line with their broad distribution. Stabilizing selection was demonstrated for sORFs with proteomic evidence and bll1319_ISGA which is conserved at the nucleotide level in 16 alphaproteobacterial species, 79 species from other taxa and 49 other Proteobacteria. Further we used Northern blot hybridization to validate ten small RNAs (BjsR1 to BjsR10) belonging to new RNA families. We found that BjsR1 and BjsR3 have homologs outside the genus Bradyrhizobium, and BjsR5, BjsR6, BjsR7, and BjsR10 have up to four imperfect copies in Bradyrhizobium genomes. BjsR8, BjsR9, and BjsR10 are present exclusively in nodules, while the other sRNAs are also expressed in liquid cultures. We also found that the level of BjsR4 decreases after exposure to tellurite and iron, and this down-regulation contributes to survival under high iron conditions. Analysis of additional small RNAs overlapping with 3’-UTRs revealed two new repetitive elements named Br-REP1 and Br-REP2. These REP elements may play roles in the genomic plasticity and gene regulation and could be useful for strain identification by PCR-fingerprinting. Furthermore, we studied two potential toxin genes in the symbiotic island and confirmed toxicity of the yhaV homolog bll1687 but not of the newly annotated higB homolog blr0229_ISGA in E. coli. Finally, we revealed transcription interference resulting in an antisense RNA complementary to blr1853, a gene induced in symbiosis. The presented results expand our knowledge on sORFs, non-coding RNAs and repetitive elements in B. japonicum and related bacteria.

The Bradyrhizobium japonicum Ferrous Iron Transporter FeoAB Is Required for Ferric Iron Utilization in Free Living Aerobic Cells and for Symbiosis

The bacterium Bradyrhizobium japonicum USDA110 does not synthesize siderophores for iron utilization in aerobic environments, and the mechanism of iron uptake within symbiotic soybean root nodules is unknown. An mbfA bfr double mutant defective in iron export and storage activities cannot grow aerobically in very high iron medium. Here, we found that this phenotype was suppressed by loss of function mutations in the feoAB operon encoding ferrous (Fe(2+)) iron uptake proteins. Expression of the feoAB operon genes was elevated under iron limitation, but mutants defective in either gene were unable to grow aerobically over a wide external ferric (Fe(3+)) iron (FeCl3) concentration range. Thus, FeoAB accommodates iron acquisition under iron limited and iron replete conditions. Incorporation of radiolabel from either (55)Fe(2+) or (59)Fe(3+) into cells was severely defective in the feoA and feoB strains, suggesting Fe(3+) reduction to Fe(2+) prior to traversal across the cytoplasmic membrane by FeoAB. The feoA or feoB deletion strains elicited small, ineffective nodules on soybean roots, containing few bacteria and lacking nitrogen fixation activity. A feoA(E40K) mutant contained partial iron uptake activity in culture that supported normal growth and established an effective symbiosis. The feoA(E40K) strain had partial iron uptake activity in situ within nodules and in isolated cells, indicating that FeoAB is the iron transporter in symbiosis. We conclude that FeoAB supports iron acquisition under limited conditions of soil and in the iron-rich environment of a symbiotic nodule.

Rhizobium strains differ considerably in outer membrane permeability and polymyxin B resistance.

Six rhizobium (Rhizobium leguminosarum bv. Trifolii TA1, Sinorhizobium meliloti 1021, Mesorhizobium huakuii IFO 15243(T), Ochrobactrum lupini LUP 21(T), Bradyrhizobium japonicum USDA110 and B. elkanii USDA 76) and two Escherichia coli strains (E. coli ATCC 25922 and E. coli HB 101) were compared in respect to polymyxin B and EDTA resistance, as well as bacterial outer membrane (OM) permeability to a fluorescent hydrophobic agent (N-phenyl-1-naphthylamine - NPN). TEM (Transmission Electron Microscopy) and a microbial test demonstrated that all the rhizobia were much more resistant to polymyxin B in comparison with E. coli strains. EDTA and polymyxin B enhance permeability of B. japonicum and O. lupini OM. Other rhizobia incorporated NPN independently of the presence of membrane-deteriorating agents; however, the level of fluorescence (measured as NPN absorption) was strain dependent.

Genome-wide transcription start site mapping of Bradyrhizobium japonicum grown free-living or in symbiosis - a rich resource to identify new transcripts, proteins and to study gene regulation.

BackgroundDifferential RNA-sequencing (dRNA-seq) is indispensable for determination of primary transcriptomes. However, using dRNA-seq data to map transcriptional start sites (TSSs) and promoters genome-wide is a bioinformatics challenge. We performed dRNA-seq of Bradyrhizobium japonicum USDA 110, the nitrogen-fixing symbiont of soybean, and developed algorithms to map TSSs and promoters.ResultsA specialized machine learning procedure for TSS recognition allowed us to map 15,923 TSSs: 14,360 in free-living bacteria, 4329 in symbiosis with soybean and 2766 in both conditions. Further, we provide proteomic evidence for 4090 proteins, among them 107 proteins corresponding to new genes and 178 proteins with N-termini different from the existing annotation (72 and 109 of them with TSS support, respectively). Guided by proteomics evidence, previously identified TSSs and TSSs experimentally validated here, we assign a score threshold to flag 14 % of the mapped TSSs as a class of lower confidence. However, this class of lower confidence contains valid TSSs of low-abundant transcripts. Moreover, we developed a de novo algorithm to identify promoter motifs upstream of mapped TSSs, which is publicly available, and found motifs mainly used in symbiosis (similar to RpoN-dependent promoters) or under both conditions (similar to RpoD-dependent promoters). Mapped TSSs and putative promoters, proteomic evidence and updated gene annotation were combined into an annotation file.ConclusionsThe genome-wide TSS and promoter maps along with the extended genome annotation of B. japonicum represent a valuable resource for future systems biology studies and for detailed analyses of individual non-coding transcripts and ORFs. Our data will also provide new insights into bacterial gene regulation during the agriculturally important symbiosis between rhizobia and legumes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2602-9) contains supplementary material, which is available to authorized users.

Shelf-life of legume inoculants in different carrier materials available in East Africa

Adoption of legume inoculation with rhizobia by small-scale farmers in East Africa, and the resultant increase in biological nitrogen fixation requires that quality inoculants meet minimum standards. BIOFIX is one of the commercially available rhizobia/legume inoculants in East Africa, whose standard is at least 109 rhizobia g-1. We examined the effect of carrier material and storage conditions on the populations of two industry standard rhizobia, Bradyrhizobium japonicum USDA 110 for soybean (Glycine max) and Rhizobium tropici CIAT 899 for common bean (Phaseolus vulgaris) over 165 days, using the drop plate method on Congo Red Yeast Extract Mannitol Agar. Viable populations of rhizobia differed significantly between carriers and rhizobia strains (P<0.05). Rhizobium tropici CIAT899, prepared with filter mud carrier, achieved a shelf-life of 135 days and B. japonicum USDA110 contained over 109 cells g-1 for 105 days. Both of these results fall below the stated six months expiry period of BIOFIX. Replacing filter mud carrier with vermiculite, resulted in an inferior product; although, both more thorough sterilisation and refrigerated storage, after a 14 day curing stage, improved the shelf-life thizobia in the inoculant packet.Keywords: BIOFIX, CIAT 899, Kenya, USDA 110

Efficient synthesis of the intermediate of abacavir and carbovir using a novel (+)-γ-lactamase as a catalyst.

The enantiomers of 2-azabicyclo[2.2.1]hept-5-en-3-one (γ-lactam) are key chiral synthons in the synthesis of antiviral drugs such as carbovir and abacavir. (+)-γ-Lactamase can be used as a catalyst in the enzymatic preparation of optically pure (-)-γ-lactam. Here, a (+)-γ-lactamase discovered from Bradyrhizobium japonicum USDA 6 by sequence-structure guided genome mining was cloned, purified and characterized. The enzyme possesses a significant catalytic activity towards γ-lactam. The active site of the (+)-γ-lactamase was studied by homologous modeling and molecular docking, and the accuracy of the prediction was confirmed by site-specific mutagenesis. The (+)-γ-lactamase reveals the great practical potential as an enzymatic method for the efficient production of carbocyclic nucleosides of pharmaceutical interest.