Metagenomic Analysis to Assess the Impact of Plant Growth-Promoting Rhizobacteria on Peanut (Arachis hypogaea L.) Crop Production and Soil Enzymes and Microbial Diversity.

Peanut production could be increased through plant growth-promoting rhizobacteria (PGPR). In this regard, the present field research aimed at elucidating the impact of PGPR on peanut yield, soil enzyme activity, microbial diversity, and structure. Three PGPR strains (Bacillus velezensis, RI3; Bacillus velezensis, SC6; Pseudomonas psychrophila, P10) were evaluated, along with Bradyrhizobium japonicum (BJ), taken as a control. PGPR increased seed yield by 8%, improving the radiation use efficiency (4-14%). PGPR modified soil enzymes (fluorescein diacetate activity by 17% and dehydrogenase activity by 28%) and microbial abundance (12%). However, PGPR did not significantly alter microbial diversity; nonetheless, it modified the relative abundance of key phyla (Actinobacteria > Proteobacteria > Firmicutes) and genera (Bacillus > Arthrobacter > Pseudomonas). PGPRs modified the relative abundance of genes associated with N-fixation and nitrification while increasing genes related to N-assimilation and N-availability. PGPR improved agronomic traits without altering rhizosphere diversity.

The distinct cell physiology of Bradyrhizobium at the population and cellular level.

The α-Proteobacteria belonging to Bradyrhizobium genus are microorganisms of extreme slow growth. Despite their extended use as inoculants in soybean production, their physiology remains poorly characterized. In this work, we produced quantitative data on four different isolates: B. diazoefficens USDA110, B. diazoefficiens USDA122, B. japonicum E109 and B. japonicum USDA6 which are representative of specific genomic profiles. Notably, we found conserved physiological traits conserved in all the studied isolates: (i) the lag and initial exponential growth phases display cell aggregation; (ii) the increase in specific nutrient concentration such as yeast extract and gluconate hinders growth; (iii) cell size does not correlate with culture age; and (iv) cell cycle presents polar growth. Meanwhile, fitness, cell size and in vitro growth widely vary across isolates correlating to ribosomal RNA operon number. In summary, this study provides novel empirical data that enriches the comprehension of the Bradyrhizobium (slow) growth dynamics and cell cycle.

Pleiotropic Effects of PhaR Regulator in Bradyrhizobium diazoefficiens Microaerobic Metabolism.

Bradyrhizobium diazoefficiens can live inside soybean root nodules and in free-living conditions. In both states, when oxygen levels decrease, cells adjust their protein pools by gene transcription modulation. PhaR is a transcription factor involved in polyhydroxyalkanoate (PHA) metabolism but also plays a role in the microaerobic network of this bacterium. To deeply uncover the function of PhaR, we applied a multipronged approach, including the expression profile of a phaR mutant at the transcriptional and protein levels under microaerobic conditions, and the identification of direct targets and of proteins associated with PHA granules. Our results confirmed a pleiotropic function of PhaR, affecting several phenotypes, in addition to PHA cycle control. These include growth deficiency, regulation of carbon and nitrogen allocation, and bacterial motility. Interestingly, PhaR may also modulate the microoxic-responsive regulatory network by activating the expression of fixK2 and repressing nifA, both encoding two transcription factors relevant for microaerobic regulation. At the molecular level, two PhaR-binding motifs were predicted and direct control mediated by PhaR determined by protein-interaction assays revealed seven new direct targets for PhaR. Finally, among the proteins associated with PHA granules, we found PhaR, phasins, and other proteins, confirming a dual function of PhaR in microoxia.

Revealing potential functions of hypothetical proteins induced by genistein in the symbiosis island of Bradyrhizobium japonicum commercial strain SEMIA 5079 (= CPAC 15).

BACKGROUND: Bradyrhizobium japonicum strain SEMIA 5079 (= CPAC 15) is a nitrogen-fixing symbiont of soybean broadly used in commercial inoculants in Brazil. Its genome has about 50% of hypothetical (HP) protein-coding genes, many in the symbiosis island, raising questions about their putative role on the biological nitrogen fixation (BNF) process. This study aimed to infer functional roles to 15 HP genes localized in the symbiosis island of SEMIA 5079, and to analyze their expression in the presence of a nod-gene inducer. RESULTS: A workflow of bioinformatics tools/databases was established and allowed the functional annotation of the HP genes. Most were enzymes, including transferases in the biosynthetic pathways of cobalamin, amino acids and secondary metabolites that may help in saprophytic ability and stress tolerance, and hydrolases, that may be important for competitiveness, plant infection, and stress tolerance. Putative roles for other enzymes and transporters identified are discussed. Some HP proteins were specific to the genus Bradyrhizobium, others to specific host legumes, and the analysis of orthologues helped to predict roles in BNF. CONCLUSIONS: All 15 HP genes were induced by genistein and high induction was confirmed in five of them, suggesting major roles in the BNF process.

Multiple ways to evade the bacteriostatic action of glyphosate in rhizobia include the mutation of the conserved serine 90 of the nitrogenase subunit NifH to alanine.

The genome resequencing of spontaneous glyphosate-resistant mutants derived from the soybean inoculant E109 allowed identifying genes most likely associated with the uptake (gltL and cya) and metabolism (zigA and betA) of glyphosate, as well as with nitrogen fixation (nifH). Mutations in these genes reduce the lag phase and improve nodulation under glyphosate stress. In addition to providing glyphosate resistance, the amino acid exchange Ser90Ala in NifH increased the citrate synthase activity, growth rate and plant growth-promoting efficiency of E109 in the absence of glyphosate stress, suggesting roles for this site during both the free-living and symbiotic growth stages.

Whole-Genome Resequencing of Spontaneous Oxidative Stress-Resistant Mutants Reveals an Antioxidant System of Bradyrhizobium japonicum Involved in Soybean Colonization.

Soybean is the most inoculant-consuming crop in the world, carrying strains belonging to the extremely related species Bradyrhizobium japonicum and Bradyrhizobium diazoefficiens. Currently, it is well known that B. japonicum has higher efficiency of soybean colonization than B. diazoefficiens, but the molecular mechanism underlying this differential symbiotic performance remains unclear. In the present study, genome resequencing of four spontaneous oxidative stress-resistant mutants derived from the commercial strain B. japonicum E109 combined with molecular and physiological studies allowed identifying an antioxidant cluster (BjAC) containing a transcriptional regulator (glxA) that controls the expression of a catalase (catA) and a phosphohydrolase (yfbR) related to the hydrolysis of hydrogen peroxide and oxidized nucleotides, respectively. Integrated synteny and phylogenetic analyses supported the fact that BjAC emergence in the B. japonicum lineage occurred after its divergence from the B. diazoefficiens lineage. The transformation of the model bacterium B. diazoefficiens USDA110 with BjAC from E109 significantly increased its ability to colonize soybean roots, experimentally recapitulating the beneficial effects of the occurrence of BjAC in B. japonicum. In addition, the glxA mutation significantly increased the nodulation competitiveness and plant growth-promoting efficiency of E109. Finally, the potential applications of these types of non-genetically modified mutant microbes in soybean production worldwide are discussed.

Dual Control of Flagellar Synthesis and Exopolysaccharide Production by FlbD-FliX Class II Regulatory Proteins in Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, has two independent flagellar systems: a single subpolar flagellum and several lateral flagella. Each flagellum is a very complex organelle composed of 30 to 40 different proteins located inside and outside the cell whereby flagellar gene expression must be tightly controlled. Such control is achieved by a hierarchy of regulators that ensure the timing of synthesis and the allocation of the different flagellar substructures. Previously, we analyzed the gene organization, expression, and function of the lateral flagellar system. Here, we studied the role of the response regulator FlbD and its trans-acting regulator FliX in the regulation of subpolar flagellar genes. We found that the LP-ring, distal rod, and hook of the subpolar flagellum were tightly controlled by FlbD and FliX. Furthermore, we obtained evidence for the existence of cross-regulation between these gene products and the expression of LafR, the master regulator of lateral flagella. In addition, we observed that extracellular polysaccharide production and biofilm formation also responded to these flagellar regulators. In this regard, FlbD might contribute to the switch between the planktonic and sessile states.IMPORTANCE Most environmental bacteria switch between two free-living states: planktonic, in which individual cells swim propelled by flagella, and sessile, in which bacteria form biofilms. Apart from being essential for locomotion, the flagellum has accessory functions during biofilm formation. The synthesis of flagella is a highly regulated process, and coordination with accessory functions requires the interconnection of various regulatory networks. Here, we show the role of class II regulators involved in the synthesis of the B. diazoefficiens subpolar flagellum and their possible participation in cross-regulation with the lateral flagellar system and exopolysaccharide production. These findings highlight the coordination of the synthetic processes of external structures, such as subpolar and lateral flagella, with exopolysaccharides, which are the main component of the biofilm matrix.

Bradyrhizobium sp. sv. retamae nodulates Retama monosperma grown in a lead and zinc mine tailings in Eastern Morocco.

The aim of this work was to characterize and identify some bacteria isolated from the root nodules of Retama monosperma grown in Sidi Boubker lead and zinc mine tailings. Very few root nodules were obtained on the root nodules of R. monosperma grown in these soils. The three bacteria isolated from the root nodules were tolerant in vitro to different concentrations of heavy metals, including lead and zinc. The rep-PCR experiments showed that the three isolates have different molecular fingerprints and were considered as three different strains. The analysis of their 16S rRNA gene sequences proved their affiliation to the genus Bradyrhizobium. The analysis and phylogeny of the housekeeping genes atpD, glnII, gyrB, recA, and rpoB confirmed that the closest species was B. valentinum with similarity percentages of 95.61 to 95.82%. The three isolates recovered from the root nodules were slow-growing rhizobia capable to renodulate their original host plant in the presence of Pb-acetate. They were able to nodulate R. sphaerocarpa and Lupinus luteus also but not Glycine max or Phaseolus vulgaris. The phylogeny of the nodA and nodC nodulation genes as well as the nifH gene of the three strains showed that they belong to the symbiovar retamae of the genus Bradyrhizobium. The three strains isolated could be considered for use as inoculum for Retama plants before use in phytoremediation experiments.

Phylogenetic diversity analysis reveals Bradyrhizobium yuanmingense and Ensifer aridi as major symbionts of mung bean (Vigna radiata L.) in Pakistan.

The present study was carried out to evaluate the diversity of rhizobia associated with nodules of mung bean in Pakistan, because this information is necessary for inoculum development. Based on sequence analysis of 16S rRNA gene of thirty-one bacteria, 11 were assigned to genus Bradyrhizobium, 17 to Ensifer, and 3 to Rhizobium. Phylogenetic analyses on the basis of 16S-23S ITS region, atpD, recA, nifH, and nodA of representative strains revealed that B. yuanmingense is the predominant species distributed throughout different mung bean-growing areas. Among the fast-growing rhizobia, Ensifer aridi was predominant in Faisalabad, Layyah, and Rawalpindi, while E. meliloti in Thal desert. Sequence variations and phylogeny of nifH and nodA genes suggested that these genes might have been co-evolved with the housekeeping genes and maintained by vertical gene transfer in rhizobia detected in the present study. Host infectivity assay revealed the successful nodulation of host by rhizobia related to genera Bradyrhizobium, Ensifer and Rhizobium. Among all, Bradyrhizobium and Ensifer spp. inoculation exhibited a significantly higher number of nodules (11-34 nodules plant-1) and nitrogenase activity (nodule ARA 60-110 µmol g-1 h-1). Contrary to the previous studies, our data reveal that B. yuanmingense and E. aridi are predominant species forming effective nodules in mung bean in Pakistan. Furthermore, to the best of our knowledge, this is the first report showing the effective symbiosis of E. aridi, E. meliloti, and Rhizobium pusense with mung bean. The diversity of rhizobia in different habitats revealed in the present study will contribute towards designing site-specific inocula for mung bean.

Heterologous production and functional characterization of Bradyrhizobium japonicum copper-containing nitrite reductase and its physiological redox partner cytochrome c550.

Two domain copper-nitrite reductases (NirK) contain two types of copper centers, one electron transfer (ET) center of type 1 (T1) and a catalytic site of type 2 (T2). NirK activity is pH-dependent, which has been suggested to be produced by structural modifications at high pH of some catalytically relevant residues. To characterize the pH-dependent kinetics of NirK and the relevance of T1 covalency in intraprotein ET, we studied the biochemical, electrochemical, and spectroscopic properties complemented with QM/MM calculations of Bradyrhizobium japonicum NirK (BjNirK) and of its electron donor cytochrome c550 (BjCycA). BjNirK presents absorption spectra determined mainly by a S(Cys)3pπ → Cu2+ ligand-to-metal charge-transfer (LMCT) transition. The enzyme shows low activity likely due to the higher flexibility of a protein loop associated with BjNirK/BjCycA interaction. Nitrite is reduced at high pH in a T1-decoupled way without T1 → T2 ET in which proton delivery for nitrite reduction at T2 is maintained. Our results are analyzed in comparison with previous results found by us in Sinorhizobium meliloti NirK, whose main UV-vis absorption features are determined by S(Cys)3pσ/π → Cu2+ LMCT transitions.

Impact of pesticides in properties of Bradyrhizobium spp. and in the symbiotic performance with soybean.

Soybean [Glycine max (L.) Merr.] has great economic and nutritional importance mainly due to its high protein content. All plant's N needs can be met by the symbiosis with elite Bradyrhizobium strains applied as inoculants to the seeds at sowing time; however, the increasing use of pesticides in seed treatments can impair the contribution of the biological nitrogen fixation. In this study, we report decreases in cell survival of two strains, B. japonicum SEMIA 5079 and B. elkanii SEMIA 587 in seeds inoculated and treated with StandakTop™, composed of the fungicides pyraclostrobin and thiophanate-methyl and the insecticide fipronil, the pesticides most used in soybean seed treatment in several countries. Cell death was enhanced with the time of exposure to the pesticides, and B. elkanii was less tolerant, with almost no detectable viable cells after 15 days. Change in colony morphology with smaller colonies was observed in the presence of the pesticides, being more drastic with the time of exposure, and attributed to an adaptive response towards survival in the presence of the abiotic stress. However, morphological changes were reversible after elimination of the stressing agent and symbiotic performance under controlled greenhouse conditions was similar between strains that had been or not exposed to the pesticides. In addition, no changes in DNA profiles (BOX-PCR) of both strains were observed after the contact with the pesticides. In two field experiments, impacting effects of the pesticides were observed mainly on the total N accumulated in grains of plants relying on both N2-fixation and N-fertilizer. Our data indicate that StandakTop® affects parameters never reported before, including colony morphology of Bradyrhizobium spp. and N metabolism and/or N remobilization to soybean grains.

Characterization of FliL Proteins in Bradyrhizobium diazoefficiens: Lateral FliL Supports Swimming Motility, and Subpolar FliL Modulates the Lateral Flagellar System.

Bradyrhizobium diazoefficiens is a soil alphaproteobacterium that possesses two evolutionarily distinct flagellar systems, a constitutive subpolar flagellum and inducible lateral flagella that, depending on the carbon source, may be expressed simultaneously in liquid medium and used interactively for swimming. In each system, more than 30 genes encode the flagellar proteins, most of which are well characterized. Among the exceptions is FliL, which has been scarcely studied in alphaproteobacteria and whose function in other bacterial classes is somewhat controversial. Because each B. diazoefficiens flagellar system contains its own fliL paralog, we obtained the respective deletions ΔfliLS (subpolar) and ΔfliLL (lateral) to study their functions in swimming. We determined that FliLL was essential for lateral flagellum-driven motility. FliLS was dispensable for swimming in either liquid or semisolid medium; however, it was found to play a crucial role in upregulation of the lateral flagellum regulon under conditions of increased viscosity/flagellar load. Therefore, although FliLS seems to be not essential for swimming, it may participate in a mechanosensor complex that controls lateral flagellum induction.IMPORTANCE Bacterial motility propelled by flagella is an important trait in most environments, where microorganisms must explore the habitat toward beneficial resources and evade toxins. Most bacterial species have a unique flagellar system, but a few species possess two different flagellar systems in the same cell. An example is Bradyrhizobium diazoefficiens, the N2-fixing symbiont of soybean, which uses both systems for swimming. Among the less-characterized flagellar proteins is FliL, a protein typically associated with a flagellum-driven surface-based collective motion called swarming. By using deletion mutants in each flagellar system's fliL, we observed that one of them (lateral) was required for swimming, while the other (subpolar) took part in the control of lateral flagellum synthesis. Hence, this protein seems to participate in the coordination of activity and production of both flagellar systems.

Brazilian-adapted soybean Bradyrhizobium strains uncover IS elements with potential impact on biological nitrogen fixation.

Bradyrhizobium diazoefficiens CPAC 7 and Bradyrhizobium japonicum CPAC 15 are broadly used in commercial inoculants in Brazil, contributing to most of the nitrogen required by the soybean crop. These strains differ in their symbiotic properties: CPAC 7 is more efficient in fixing nitrogen, whereas CPAC 15 is more competitive. Comparative genomics revealed many transposases close to genes associated with symbiosis in the symbiotic island of these strains. Given the importance that insertion sequences (IS) elements have to bacterial genomes, we focused on identifying the local impact of these elements in the genomes of these and other related Bradyrhizobium strains to further understand their phenotypic differences. Analyses were performed using bioinformatics approaches. We found IS elements disrupting and inserted at regulatory regions of genes involved in symbiosis. Further comparative analyses with 21 Bradyrhizobium genomes revealed insertional polymorphism with distinguishing patterns between B. diazoefficiens and B. japonicum lineages. Finally, 13 of these potentially impacted genes are differentially expressed under symbiotic conditions in B. diazoefficiens USDA 110. Thus, IS elements are associated with the diversity of Bradyrhizobium, possibly by providing mechanisms for natural variation of symbiotic effectiveness.

Impact of double inoculation with Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39 on soybean plants grown under arsenic stress.

Inoculation practice with plant growth-promoting bacteria (PGPB) has been proposed as a good biotechnological tool to enhance plant performance and alleviate heavy metal/metalloid stress. Soybean is often cultivated in soil with high arsenic (As) content or irrigated with As-contaminated groundwater, which causes deleterious effects on its growth and yield, even when it was inoculated with rhizobium. Thus, the effect of double inoculation with known PGPB strains, Bradyrhizobium japonicum E109 and Azospirillum brasilense Az39 was evaluated in plants grown in pots under controlled conditions and treated with As. First, the viability of these co-cultivated bacteria was assayed using a flow cytometry analysis using SYTO9 and propidium iodide (PI) dyes. This was performed in vitro to evaluate the bacterial population dynamic under 25 µM AsV and AsIII treatment. A synergistic effect was observed when bacteria were co-cultured, since mortality diminished, compared to each growing alone. Indole acetic acid (IAA) produced by A. brasilense Az39 would be one of the main components involved in B. japonicum E109 mortality reduction, mainly under AsIII treatment. Regarding in vivo assays, under As stress, plant growth improvement, nodule number and N content increase were observed in double inoculated plants. Furthermore, double inoculation strategy reduced As translocation to aerial parts thus improving As phytostabilization potential of soybean plants. These results suggest that double inoculation with B. japonicum E109 and A. brasilense Az39 could be a safe and advantageous practice to improve growth and yield of soybean exposed to As, accompanied by an important metalloid phytostabilization.

Analysis of the denitrification pathway and greenhouse gases emissions in Bradyrhizobium sp. strains used as biofertilizers in South America.

AIMS: Greenhouse gases are considered as potential atmospheric pollutants, with agriculture being one of the main emission sources. The practice of inoculating soybean seeds with Bradyrhizobium sp. might contribute to nitrous oxide (N2 O) emissions. We analysed this capacity in five of the most used strains of Bradyrhizobium sp. in South America. METHODS AND RESULTS: We analysed the denitrification pathway and N2 O production by Bradyrhizobium japonicum E109 and CPAC15, Bradyrhizobium diazoefficiens CPAC7 and B. elkanii SEMIA 587 and SEMIA 5019, both in free-living conditions and in symbiosis with soybean. The in silico analysis indicated the absence of nosZ genes in B. japonicum and the presence of all denitrification genes in B. diazoefficiens strains, as well as the absence of nirK, norC and nosZ genes in B. elkanii. The in planta analysis confirmed N2 O production under saprophytic conditions or symbiosis with soybean root nodules. In the case of symbiosis, up to 26.1 and 18.4 times higher in plants inoculated with SEMIA5019 and E109, respectively, than in those inoculated with USDA110. CONCLUSIONS: The strains E109, SEMIA 5019, CPAC15 and SEMIA 587 showed the highest N2 O production both as free-living cells and in symbiotic conditions in comparison with USDA110 and CPAC7, which do have the nosZ gene. Although norC and nosZ could not be identified in silico or in vitro in SEMIA 587 and SEMIA 5019, these strains showed the capacity to produce N2 O in our experimental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report to analyse and confirm the incomplete denitrification capacity and N2 O production in four of the five most used strains of Bradyrhizobium sp. for soybean inoculation in South America.

Bradyrhizobium and Pseudomonas strains obtained from coal-mining areas nodulate and promote the growth of Calopogonium muconoides plants used in the reclamation of degraded areas.

AIMS: The objective of this work was to isolate and characterize indigenous rhizobia from coal-mining areas able to efficiently nodulate and fix nitrogen in association with Calopogonium mucunoides (calopo). METHODS AND RESULTS: Isolation, authentication and morphological, biochemical and molecular characterization of the autochthonous rhizobia were performed and their symbiotic efficiency (SE) evaluated. Efficient rhizobial isolates suitable for the inoculation of calopo in coal-mining regions were obtained. A total of 30 isolates were obtained after nodulation authentication, of which five presented high SE with plant-growth promoting traits such as indole-3-acetic acid production, phosphate solubilization and biofilm formation. These isolates were identified as belonging to Bradyrhizobium, Pseudomonas and Rhizobium. CONCLUSIONS: Bradyrhizobium sp. A2-10 and Pseudomonas sp. A6-05 were able to promote calopo plant growth using soil obtained from coal-mining degraded areas, thus indicating their potential as inoculants aiming at land reclamation. SIGNIFICANCE AND IMPACT OF THE STUDY: To our knowledge, this is the first report of Pseudomonas nodule formation in calopo. Furthermore, the results demonstrated that autochthonous rhizobia obtained from degraded soils presented high SE in calopo and possess a wide range of plant-growth promoting traits. Ultimately, they may all contribute to an increased leguminous plant growth under stress conditions. The selected rhizobia strains may be used as inoculants and present a valuable role in the development of strategies aiming to recover coal-mining degraded areas. Bacterial inoculants would greatly reduce the use of often harmful nitrogen fertilizers vastly employed in revegetation programmes of degraded areas.

New insights into auxin metabolism in Bradyrhizobium japonicum.

Bacterial metabolism of phytohormones includes several processes such as biosynthesis, catabolism, conjugation, hydrolysis and homeostatic regulation. However, only biosynthesis and occasionally catabolism are studied in depth in microorganisms. In this work, we evaluated and reconsidered IAA metabolism in Bradyrhizobiumjaponicum E109, one of the most widely used strains for soybean inoculation around the world. The genomic analysis of the strain showed the presence of several genes responsible for IAA biosynthesis, mainly via indole-3-acetonitrile (IAN), indole-3-acetamide (IAM) and tryptamine (TAM) pathways. However; in vitro experiments showed that IAA is not accumulated in the culture medium in significant amounts. On the contrary, a strong degradation activity was observed after exogenous addition of 0.1 mM of IAA, IBA or NAA to the medium. B. japonicum E109 was not able to grow in culture medium containing IAA as a sole carbon source. In YEM medium, the bacteria degraded IAA and hydrolyzed amino acid auxin conjugates with alanine (IAAla), phenylalanine (IAPhe), and leucine (IAPhe), releasing IAA which was quickly degraded. Finally, the presence of exogenous IAA induced physiological changes in the bacteria such as increased biomass and exopolysaccharide production, as well as infection effectiveness and symbiotic behavior in soybean plants.

Nodulation and Delayed Nodule Senescence: Strategies of Two Bradyrhizobium Japonicum Isolates with High Capacity to Fix Nitrogen.

The purpose of this work was to study further two Bradyrhizobium japonicum strains with high nitrogen-fixing capacity that were identified within a collection of approximately 200 isolates from the soils of Argentina. Nodulation and nitrogen-fixing capacity and the level of expression of regulatory as well as structural genes of nitrogen fixation and the 1-aminocyclopropane-1-carboxylate (ACC) deaminase gene of the isolates were compared with that of E109-inoculated plants. Both isolates of B. japonicum, 163 and 366, were highly efficient to fix nitrogen compared to commercial strain E109. Isolate 366 developed a higher number and larger biomass of nodules and because of this fixed more nitrogen. Isolate 163 developed the same number and nodule biomass than E109. However, nodules developed by isolate 163 had red interiors for a longer period, had a higher leghemoglobin content, and presented high levels of expression of acdS gene, that codes for an ACC deaminase. In conclusion, naturalized rhizobia of the soils of Argentina hold a diverse population that might be the source of highly active nitrogen-fixing rhizobia, a process that appears to be based on different strategies.

Genome sequence of Bradyrhizobium embrapense strain CNPSo 2833T, isolated from a root nodule of Desmodium heterocarpon

Abstract Bradyrhizobium embrapense CNPSo 2833T is a nitrogen-fixing symbiont of the legume pasture Desmodium. Its draft genome contains 8,267,832 bp and 7876 CDSs. The symbiotic island includes nodulation and nitrogen fixation genes resembling the operon organization of B. japonicum. Several CDSs related to secretion proteins and stress tolerance were also identified.

Dissecting the role of NtrC and RpoN in the expression of assimilatory nitrate and nitrite reductases in Bradyrhizobium diazoefficiens.

Bradyrhizobium diazoefficiens, a nitrogen-fixing endosymbiont of soybeans, is a model strain for studying rhizobial denitrification. This bacterium can also use nitrate as the sole nitrogen (N) source during aerobic growth by inducing an assimilatory nitrate reductase encoded by nasC located within the narK-bjgb-flp-nasC operon along with a nitrite reductase encoded by nirA at a different chromosomal locus. The global nitrogen two-component regulatory system NtrBC has been reported to coordinate the expression of key enzymes in nitrogen metabolism in several bacteria. In this study, we demonstrate that disruption of ntrC caused a growth defect in B. diazoefficiens cells in the presence of nitrate or nitrite as the sole N source and a decreased activity of the nitrate and nitrite reductase enzymes. Furthermore, the expression of narK-lacZ or nirA-lacZ transcriptional fusions was significantly reduced in the ntrC mutant after incubation under nitrate assimilation conditions. A B. diazoefficiens rpoN 1/2 mutant, lacking both copies of the gene encoding the alternative sigma factor σ54, was also defective in aerobic growth with nitrate as the N source as well as in nitrate and nitrite reductase expression. These results demonstrate that the NtrC regulator is required for expression of the B. diazoefficiens nasC and nirA genes and that the sigma factor RpoN is also involved in this regulation.