Bacterial gasdermins reveal an ancient mechanism of cell death.

Gasdermin proteins form large membrane pores in human cells that release immune cytokines and induce lytic cell death. Gasdermin pore formation is triggered by caspase-mediated cleavage during inflammasome signaling and is critical for defense against pathogens and cancer. We discovered gasdermin homologs encoded in bacteria that defended against phages and executed cell death. Structures of bacterial gasdermins revealed a conserved pore-forming domain that was stabilized in the inactive state with a buried lipid modification. Bacterial gasdermins were activated by dedicated caspase-like proteases that catalyzed site-specific cleavage and the removal of an inhibitory C-terminal peptide. Release of autoinhibition induced the assembly of large and heterogeneous pores that disrupted membrane integrity. Thus, pyroptosis is an ancient form of regulated cell death shared between bacteria and animals.

Application of Recombinant Human scFv Antibody as a Powerful Tool to Monitor Nitrogen Fixing Biofertilizer in Rice and Legume.

Bradyrhizobium is an endophytic bacterium under investigation as an efficient biofertilizer for sustainable legume-rice rotational cropping system. Monitoring and bio-imaging of this nitrogen fixing bacterium is essential for the study of plant-microbe evolution, soil microbiome, as well as quality control in organic farming. While phage display antibody technology has been widely used to generate recombinant antibody for myriad medical purposes, so far, this technology has been minimally applied in the agricultural sector. In this study, single-chain variable fragments (scFv) against two Bradyrhizobium strains SUTN9-2 (yiN92-1e10) and DOA9 (yiDOA9-162) were isolated from a human phage display antibody library. Specific binding of scFv was demonstrated by ELISA and confocal-immunofluorescence imaging techniques. Bradyrhizobium localization in both endophytic and bacteroid forms could be observed inside rice tissue and plant nodule, respectively. Moreover, successful application of the recombinant antibody for the evaluation of nodule occupancy was also demonstrated in comparison with standard GUS-staining method. The results of this study showed for the first time the potential use of human phage display scFv antibody for imaging and monitoring of Bradyrhizobium biofertilizer and thus could be further applied for point-of-detection of bacterial inoculum in the legume-rice rotational crop system. IMPORTANCE Human scFv antibody generated from phage display technology was successfully used for the generation of specific recombinant antibodies: yiN92-1e10 and yiDOA9-162 for the detection of Bradyrhizobium strains SUTN9-2 and DOA9, respectively. These two recombinant scFv antibodies could be used for precise detection of the rhizobia both in symbiosis with legume and endophyte in rice tissue by ELISA and immunofluorescent staining, during legume-rice rotational cropping system in the field. This methodology can be further employed for the study of other plant-microbe interactions and monitoring of biofertilizer in diverse sustainable cropping systems as well as in precision agriculture.

Physiological and biochemical responses of soybean plants inoculated with Arbuscular mycorrhizal fungi and Bradyrhizobium under drought stress.

BACKGROUND: The present study aims to study the effects of biofertilizers potential of Arbuscular Mycorrhizal Fungi (AMF) and Bradyrhizobium japonicum (B. japonicum) strains on yield and growth of drought stressed soybean (Giza 111) plants at early pod stage (50 days from sowing, R3) and seed development stage (90 days from sowing, R5). RESULTS: Highest plant biomass, leaf chlorophyll content, nodulation, and grain yield were observed in the unstressed plants as compared with water stressed-plants at R3 and R5 stages. At soil rhizosphere level, AMF and B. japonicum treatments improved bacterial counts and the activities of the enzymes (dehydrogenase and phosphatase) under well-watered and drought stress conditions. Irrespective of the drought effects, AMF and B. japonicum treatments improved the growth and yield of soybean under both drought (restrained irrigation) and adequately-watered conditions as compared with untreated plants. The current study revealed that AMF and B. japonicum improved catalase (CAT) and peroxidase (POD) in the seeds, and a reverse trend was observed in case of malonaldehyde (MDA) and proline under drought stress. The relative expression of the CAT and POD genes was up-regulated by the application of biofertilizers treatments under drought stress condition. Interestingly a reverse trend was observed in the case of the relative expression of the genes involved in the proline metabolism such as P5CS, P5CR, PDH, and P5CDH under the same conditions. The present study suggests that biofertilizers diminished the inhibitory effect of drought stress on cell development and resulted in a shorter time for DNA accumulation and the cycle of cell division. There were notable changes in the activities of enzymes involved in the secondary metabolism and expression levels of GmSPS1, GmSuSy, and GmC-INV in the plants treated with biofertilizers and exposed to the drought stress at both R3 and R5 stages. These changes in the activities of secondary metabolism and their transcriptional levels caused by biofertilizers may contribute to increasing soybean tolerance to drought stress. CONCLUSIONS: The results of this study suggest that application of biofertilizers to soybean plants is a promising approach to alleviate drought stress effects on growth performance of soybean plants. The integrated application of biofertilizers may help to obtain improved resilience of the agro ecosystems to adverse impacts of climate change and help to improve soil fertility and plant growth under drought stress.

Disproof of the Proposed Structures of Bradyoxetin, a Putative Bradyrhizobium japonicum Signaling Molecule, and HMCP, a Putative Ralstonia solanacearum Quorum-Sensing Molecule.

First, we revisited the reported NMR data of bradyoxetin, a putative cell density factor of Bradyrhizobium japonicum, and found some inconsistencies in the proposed structure. To elucidate the correct structure, we synthesized model oxetane compounds and confirmed that the NMR data of the synthetic compounds did not match those of the reported bradyoxetin. After reinterpreting the reported NMR data, we concluded that bradyoxetin must be chloramphenicol. Next, some derivatives of 2-hydroxy-4-((methylamino)(phenyl)methyl)cyclopentanone (HMCP), which is a putative quorum-sensing molecule of Ralstonia solanacearum, were synthesized. The NMR spectra of the synthesized compounds were completely different from those of the reported natural products. Based on theoretical studies, including the estimation of 1H and 13C NMR chemical shifts using density functional theory calculations, we confirmed the correctness of the structure of the synthesized compound. These results strongly suggest that the proposed structure of HMCP could be incorrect.

Reverse protein engineering of a novel 4-domain copper nitrite reductase reveals functional regulation by protein-protein interaction.

Cu-containing nitrite reductases that convert NO2- to NO are critical enzymes in nitrogen-based energy metabolism. Among organisms in the order Rhizobiales, we have identified two copies of nirK, one encoding a new class of 4-domain CuNiR that has both cytochrome and cupredoxin domains fused at the N terminus and the other, a classical 2-domain CuNiR (Br2D NiR). We report the first enzymatic studies of a novel 4-domain CuNiR from Bradyrhizobium sp. ORS 375 (BrNiR), its genetically engineered 3- and 2-domain variants, and Br2D NiR revealing up to ~ 500-fold difference in catalytic efficiency in comparison with classical 2-domain CuNiRs. Contrary to the expectation that tethering would enhance electron delivery by restricting the conformational search by having a self-contained donor-acceptor system, we demonstrate that 4-domain BrNiR utilizes N-terminal tethering for downregulating enzymatic activity instead. Both Br2D NiR and an engineered 2-domain variant of BrNiR (Δ(Cytc-Cup) BrNiR) have 3 to 5% NiR activity compared to the well-characterized 2-domain CuNiRs from Alcaligenes xylosoxidans (AxNiR) and Achromobacter cycloclastes (AcNiR). Structural comparison of Δ(Cytc-Cup) BrNiR and Br2D NiR with classical 2-domain AxNiR and AcNiR reveals structural differences of the proton transfer pathway that could be responsible for the lowering of activity. Our study provides insights into unique structural and functional characteristics of naturally occurring 4-domain CuNiR and its engineered 3- and 2-domain variants. The reverse protein engineering approach utilized here has shed light onto the broader question of the evolution of transient encounter complexes and tethered electron transfer complexes. ENZYME: Copper-containing nitrite reductase (CuNiR) (EC 1.7.2.1). DATABASE: The atomic coordinate and structure factor of Δ(Cytc-Cup) BrNiR and Br2D NiR have been deposited in the Protein Data Bank (http://www.rcsb.org/) under the accession code 6THE and 6THF, respectively.

Identification of soybean Bradyrhizobium strains used in commercial inoculants in Brazil by MALDI-TOF mass spectrometry.

Biological nitrogen fixation (BNF) with the soybean crop probably represents the major sustainable technology worldwide, saving billions of dollars in N fertilizers and decreasing water pollution and the emission of greenhouse gases. Accordingly, the identification of strains occupying nodules under field conditions represents a critical step in studies that are aimed at guaranteeing increased BNF contribution. Current methods of identification are mostly based on serology, or on DNA profiles. However, the production of antibodies is restricted to few laboratories, and to obtain DNA profiles of hundreds of isolates is costly and time-consuming. Conversely, the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS technique might represent a golden opportunity for replacing serological and DNA-based methods. However, MALDI-TOF databases of environmental microorganisms are still limited, and, most importantly, there are concerns about the discrimination of protein profiles at the strain level. In this study, we investigated four soybean rhizobial strains carried in commercial inoculants used in over 35 million hectares in Brazil and also in other countries of South America and Africa. A supplementary MALDI-TOF database with the protein profiles of these rhizobial strains was built and allowed the identification of unique profiles statistically supported by multivariate analysis and neural networks. To test this new database, the nodule occupancy by Bradyrhizobium strains in symbiosis with soybean was characterized in a field experiment and the results were compared with serotyping of bacteria by immuno-agglutination. The results obtained by both techniques were highly correlated and confirmed the viability of using the MALDI-TOF MS technique to effectively distinguish bacteria at the strain level.

Structural analysis of a novel N-carbamoyl-d-amino acid amidohydrolase from a Brazilian Bradyrhizobium japonicum strain: In silico insights by molecular modelling, docking and molecular dynamics.

In this work we performed several in silico analyses to describe the relevant structural aspects of an enzyme N-Carbamoyl-d-amino acid amidohydrolase (d-NCAase) encoded on the genome of the Brazilian strain CPAC 15 (=SEMIA 5079) of Bradyrhizobium japonicum, a nonpathogenic species belonging to the order Rhizobiales. d-NCAase has wide applications particularly in the pharmaceutical industry, since it catalyzes the production of d-amino acids such as D-p-hydroxyphenylglycine (D-HPG), an intermediate in the synthesis of ß-lactam antibiotics. We applied a homology modelling approach and 50 ns of molecular dynamics simulations to predict the structure and the intersubunit interactions of this novel d-NCAase. Also, in order to evaluate the substrate binding site, the model was subjected to 50 ns of molecular dynamics simulations in the presence of N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) (a d-NCAase canonical substrate) and water-protein/water-substrate interactions analyses were performed. Overall, the structural analysis and the molecular dynamics simulations suggest that d-NCAase of B. japonicum CPAC-15 has a homodimeric structure in solution. Here, we also examined the substrate specificity of the catalytic site of our model and the interactions with water molecules into the active binding site were comprehensively discussed. Also, these simulations showed that the amino acids Lys123, His125, Pro127, Cys172, Asp174 and Arg176 are responsible for recognition of ligand in the active binding site through several chemical associations, such as hydrogen bonds and hydrophobic interactions. Our results show a favourable environment for a reaction of hydrolysis that transforms N-Carbamoyl-d-p-hydroxyphenylglycine (Cp-HPG) into the active compound D-p-hydroxyphenylglycine (D-HPG). This work envisage the use of d-NCAase from the Brazilian Bradyrhizobium japonicum strain CPAC-15 (=SEMIA 5079) for the industrial production of D-HPG, an important intermediate for semi-synthesis of ß-lactam antibiotics such as penicillins, cephalosporins and amoxicillin.

Interaction studies of lead(II) ion with cyclic ß-(1→3),(1→6) glucans extracted from Bradyrhizobium japonicum based on 'chelation enhanced fluorescence' (CHEF) effect.

Cyclic ß-(1→3),(1→6) glucans (CBGs) isolated from Bradyrhizobium japonicum bacteria are the periplasmic oligosaccharides having cyclic structures. This paper presents the isolation of CBGs from the bacteria cultured using optimized medium that improved yields to 350-450 mg per gram of cellular dry weight along with analytical interaction with lead(II) ions in the range 33.0-2.0 ppm with CBG as a binding ligand, using constant wavelength synchronous fluorescence spectroscopy. The binding ability of CBGs towards lead(II) is clearly evident from the scanning electron microscopy (SEM) images. The theoretical calculations using HEX 8.0 gave an insight about the interaction between CBG and lead(II) to be in the ratio of 3:1. The method displayed the sensitivity and selectivity towards lead(II) ions up to 2.0 ppm. This observed property of CBGs can potentially hold an application in bioremediation of the soil contaminated with lead.

Microbial cyclic ß-(1→3),(1→6)-glucans as potential drug carriers: Interaction studies between cyclic ß-glucans isolated from Bradyrhizobium japonicum and betulinic acid.

Betulinic acid (BA), a pentacyclic triterpenoid, is a very promising therapeutic drug with varied medicinal properties but it has low water solubility and consequentially low bioavailability. Cyclic ß-(1→3),(1→6)-glucans (CBG), microbial cyclooligosaccharides produced by Bradyrhizobium japonicum ATCC 10324 having a cavity structure and good solubility in water have been tested for their ability to encapsulate betulinic acid and drug-binding interactions of CBG and BA were studied. First, in silico approach was employed to study the scope of any interaction between the CBG and BA. Then, the cyclic glucan-betulinic acid complexes were prepared in three compositions of 1:1, 1:2 and 1:3 CBG:BA. The complexes were analysed using UV-VIS spectroscopy, IR spectroscopy, powder XRD, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA) to confirm the computational results and consequently the encapsulation efficiency was found to be 9.53%.

Evaluation of MALDI-TOF mass spectrometry for the competitiveness analysis of selected indigenous cowpea (Vigna unguiculata L. Walp.) Bradyrhizobium strains from Kenya.

Cowpea N2 fixation and yield can be enhanced by selecting competitive and efficient indigenous rhizobia. Strains from contrasting agro-ecologies of Kilifi and Mbeere (Kenya) were screened. Two pot experiments were established consisting of 13 Bradyrhizobium strains; experiment 1 (11 Mbeere + CBA + BK1 from Burkina Faso), experiment 2 (12 Kilifi + CBA). Symbiotic effectiveness was assessed (shoot biomass, SPAD index and N uptake). Nodule occupancy of 13 simultaneously co-inoculated strains in each experiment was analyzed by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) to assess competitiveness. Strains varied in effectiveness and competitiveness. The four most efficient strains were further evaluated in a field trial in Mbeere during the 2014 short rains. Strains from bacteroids of cowpea nodules from pot and field experiments were accurately identified as Bradyrhizobium by MALDI-TOF based on the SARAMIS™ database. In the field, abundant indigenous populations 7.10 × 103 rhizobia g-1 soil, outcompeted introduced strains. As revealed by MALDI-TOF, indigenous strains clustered into six distinct groups (I, II, III, IV, V and VI), group III were most abundant occupying 80% of nodules analyzed. MALDI-TOF was rapid, affordable and reliable to identify Bradyrhizobium strains directly from nodule suspensions in competition pot assays and in the field with abundant indigenous strains thus, its suitability for future competition assays. Evaluating strain competitiveness and then symbiotic efficacy is proposed in bioprospecting for potential cowpea inoculant strains.

Biophysical investigation of type A PutAs reveals a conserved core oligomeric structure.

Many enzymes form homooligomers, yet the functional significance of self-association is seldom obvious. Herein, we examine the connection between oligomerization and catalytic function for proline utilization A (PutA) enzymes. PutAs are bifunctional enzymes that catalyze both reactions of proline catabolism. Type A PutAs are the smallest members of the family, possessing a minimal domain architecture consisting of N-terminal proline dehydrogenase and C-terminal l-glutamate-γ-semialdehyde dehydrogenase modules. Type A PutAs form domain-swapped dimers, and in one case (Bradyrhizobium japonicum PutA), two of the dimers assemble into a ring-shaped tetramer. Whereas the dimer has a clear role in substrate channeling, the functional significance of the tetramer is unknown. To address this question, we performed structural studies of four-type A PutAs from two clades of the PutA tree. The crystal structure of Bdellovibrio bacteriovorus PutA covalently inactivated by N-propargylglycine revealed a fold and substrate-channeling tunnel similar to other PutAs. Small-angle X-ray scattering (SAXS) and analytical ultracentrifugation indicated that Bdellovibrio PutA is dimeric in solution, in contrast to the prediction from crystal packing of a stable tetrameric assembly. SAXS studies of two other type A PutAs from separate clades also suggested that the dimer predominates in solution. To assess whether the tetramer of B. japonicum PutA is necessary for catalytic function, a hot spot disruption mutant that cleanly produces dimeric protein was generated. The dimeric variant exhibited kinetic parameters similar to the wild-type enzyme. These results implicate the domain-swapped dimer as the core structural and functional unit of type A PutAs. ENZYMES: Proline dehydrogenase (EC 1.5.5.2); l-glutamate-γ-semialdehyde dehydrogenase (EC 1.2.1.88). DATABASES: The atomic coordinates and structure factor amplitudes have been deposited in the Protein Data Bank under accession number 5UR2. The SAXS data have been deposited in the SASBDB under the following accession codes: SASDCP3 (BbPutA), SASDCQ3 (DvPutA 1.5 mg·mL-1 ), SASDCX3 (DvPutA 3.0 mg·mL-1 ), SASDCY3 (DvPutA 4.5 mg·mL-1 ), SASDCR3 (LpPutA 3.0 mg·mL-1 ), SASDCV3 (LpPutA 5.0 mg·mL-1 ), SASDCW3 (LpPutA 8.0 mg·mL-1 ), SASDCS3 (BjPutA 2.3 mg·mL-1 ), SASDCT3 (BjPutA 4.7 mg·mL-1 ), SASDCU3 (BjPutA 7.0 mg·mL-1 ), SASDCZ3 (R51E 2.3 mg·mL-1 ), SASDC24 (R51E 4.7 mg·mL-1 ), SASDC34 (R51E 7.0 mg·mL-1 ).

Regulation of nitrous oxide reductase genes by NasT-mediated transcription antitermination in Bradyrhizobium diazoefficiens.

In Bradyrhizobium diazoefficiens, maximal expression of the nitrous oxide reductase gene (nosZ) requires oxygen limitation and the presence of a nitrogen oxide. The putative transcription antiterminator NasT is a positive regulator of nosZ; but in the absence of nitrate, NasT is counteracted by the nitrate sensor NasS. Here, we examined the NasT-mediated mechanism of nosRZDFYLX gene cluster expression. We mapped two transcription start sites of nosR and identified two potential hairpins, H1 and H2, within the 5'-leader of nosR transcripts. Electrophoretic mobility shift assay showed that NasT specifically bound the nosR-leader RNA and deletion of H1 abolished such binding. Under aerobic nitrate-deficient conditions, deletion of H1 or H2 increased the level of nosRZD transcripts. Under denitrifying conditions (anaerobiosis with nitrate supply), the level of nosRZD transcripts was severely impaired in the nasT mutant; in the nasT background, deletions of either hairpin led to increased level of nosRZD transcripts. In contrast to nosRZD coding region, nosR-leader transcript level was not affected by nasS or nasT mutations under aerobic or denitrifying conditions respectively. These results suggest that the two-hairpin RNA structure acts for transcription termination upstream of nosR and the binding of NasT to H1 facilitates read-through transcription to induce nos expression.

Synthesis of Bradyrhizose Oligosaccharides Relevant to the Bradyrhizobium O-Antigen.

The unique α-(1→7)-bradyrhizoside linkages are constructed for the first time via judicious choice of the glycosylation partners and conditions, thus tetra- and penta-bradyrhizosides relevant to the peculiar O-antigen of Bradyrhizobium are synthesized, which are shown to adopt the defined right-handed helical conformations and to be unable to induce innate immune responses in plants.

Structural characterization of the thermostable Bradyrhizobium japonicumD-sorbitol dehydrogenase.

Bradyrhizobium japonicum sorbitol dehydrogenase is NADH-dependent and is active at elevated temperatures. The best substrate is D-glucitol (a synonym for D-sorbitol), although L-glucitol is also accepted, giving it particular potential in industrial applications. Crystallization led to a hexagonal crystal form, with crystals diffracting to 2.9 Šresolution. In attempts to phase the data, a molecular-replacement solution based upon PDB entry 4nbu (33% identical in sequence to the target) was found. The solution contained one molecule in the asymmetric unit, but a tetramer similar to that found in other short-chain dehydrogenases, including the search model, could be reconstructed by applying crystallographic symmetry operations. The active site contains electron density consistent with D-glucitol and phosphate, but there was not clear evidence for the binding of NADH. In a search for the features that determine the thermostability of the enzyme, the Tm for the orthologue from Rhodobacter sphaeroides, for which the structure was already known, was also determined, and this enzyme proved to be considerably less thermostable. A continuous ß-sheet is formed between two monomers in the tetramer of the B. japonicum enzyme, a feature not generally shared by short-chain dehydrogenases, and which may contribute to thermostability, as may an increased Pro/Gly ratio.

Polysaccharide-based bioflocculant template of a diazotrophic Bradyrhizobium japonicum 36 for controlled assembly of AgCl nanoparticles.

A simple and green method was developed for the biosynthesis of silver chloride nanoparticles, free from silver nanoparticles, using polysaccharide-based bioflocculant of a diazotrophic rhizobacteria Bradyrhizobium japonicum 36 strain. The synthesized silver chloride nanoparticles were characterized by UV-vis, XRD, FT-IR and TEM. The concentration-dependent and controllable method for silver chloride nanoparticles was developed. The biosynthesized silver chloride nanoparticles exhibited strong antimicrobial activity towards pathogenic microorganisms such as Escherichia coli, Staphylococcus aureus and Candida albicans. The synthesized silver chloride nanoparticles can be exploited as a promising new biocide bionanocomposite against pathogenic microorganisms.

Rhizoleucinoside, a Rhamnolipid-Amino Alcohol Hybrid from the Rhizobial Symbiont Bradyrhizobium sp. BTAi1.

Rhizoleucinoside (1), a unique rhamnolipid-amino alcohol hybrid, was isolated from the rhizobial symbiont bacterium Bradyrhizobium sp. BTAi1. Compound 1 features a rare rhamnolipid core attached to an unprecedented leucinol moiety. Its structure and absolute configuration were determined by spectroscopic analysis, tandem mass spectrometry, chemical degradation, and application of the Marfey's method. Compound 1 possesses moderate cytotoxicity to BV-2 murine microglia and highly aggressive proliferating immortalized (HAPI) rat microglia cells.

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.

An enzyme captured in two conformational states: crystal structure of S-adenosyl-L-homocysteine hydrolase from Bradyrhizobium elkanii.

S-Adenosyl-L-homocysteine hydrolase (SAHase) is involved in the enzymatic regulation of S-adenosyl-L-methionine (SAM)-dependent methylation reactions. After methyl-group transfer from SAM, S-adenosyl-L-homocysteine (SAH) is formed as a byproduct, which in turn is hydrolyzed to adenosine (Ado) and homocysteine (Hcy) by SAHase. The crystal structure of BeSAHase, an SAHase from Bradyrhizobium elkanii, which is a nitrogen-fixing bacterial symbiont of legume plants, was determined at 1.7 Šresolution, showing the domain organization (substrate-binding domain, NAD(+) cofactor-binding domain and dimerization domain) of the subunits. The protein crystallized in its biologically relevant tetrameric form, with three subunits in a closed conformation enforced by complex formation with the Ado product of the enzymatic reaction. The fourth subunit is ligand-free and has an open conformation. The BeSAHase structure therefore provides a unique snapshot of the domain movement of the enzyme induced by the binding of its natural ligands.

A Putative Type III Secretion System Effector Encoded by the MA20_12780 Gene in Bradyrhizobium japonicum Is-34 Causes Incompatibility with Rj4 Genotype Soybeans.

The nodulation of Bradyrhizobium japonicum Is-34 is restricted by Rj4 genotype soybeans (Glycine max). To identify the genes responsible for this incompatibility, Tn5 mutants of B. japonicum Is-34 that were able to overcome this nodulation restriction were obtained. Analysis of the Tn5 mutants revealed that Tn5 was inserted into a region containing the MA20_12780 gene. In addition, direct disruption of this gene using marker exchange overcame the nodulation restriction by Rj4 genotype soybeans. The MA20_12780 gene has a tts box motif in its upstream region, indicating a possibility that this gene encodes a type III secretion system (T3SS) effector protein. Bioinformatic characterization revealed that the MA20_12780 protein contains the small ubiquitin-like modifier (SUMO) protease domain of the C48 peptidase (ubiquitin-like protease 1 [Ulp1]) family. The results of the present study indicate that a putative T3SS effector encoded by the MA20_12780 gene causes the incompatibility with Rj4 genotype soybeans, and they suggest the possibility that the nodulation restriction of B. japonicum Is-34 may be due to Rj4 genotype soybeans recognizing the putative T3SS effector (MA20_12780 protein) as a virulence factor.

Synthesis of bradyrhizose, a unique inositol-fused monosaccharide relevant to a Nod-factor independent nitrogen fixation.

The symbiosis of Bradyrhizobium sp. BTAi1 with its host plant Aeschynomene indica relies on a Nod-factor independent mechanism, wherein the Bradyrhizobium O-antigen is regarded as a key factor. This O-antigen polysaccharide is composed of a unique C10 monosaccharide, namely bradyrhizose, which has a galactose-inositol trans-fused scaffold, via a homogeneous α-(1 → 7)-linkage. Herein, we report the first synthesis of bradyrhizose. The scalable synthesis requires 26 steps in a high overall yield of 9%, with the inositol scaffold being constructed effectively via a Ferrier II rearrangement from a fully functionalized C2 and C4 branched pyranose derivative.