Methylated chalcones are required for rhizobial nod gene induction in the Medicago truncatula rhizosphere.

Legume nodulation requires the detection of flavonoids in the rhizosphere by rhizobia to activate their production of Nod factor countersignals. Here we investigated the flavonoids involved in nodulation of Medicago truncatula. We biochemically characterized five flavonoid-O-methyltransferases (OMTs) and a lux-based nod gene reporter was used to investigate the response of Sinorhizobium medicae NodD1 to various flavonoids. We found that chalcone-OMT 1 (ChOMT1) and ChOMT3, but not OMT2, 4, and 5, were able to produce 4,4'-dihydroxy-2'-methoxychalcone (DHMC). The bioreporter responded most strongly to DHMC, while isoflavones important for nodulation of soybean (Glycine max) showed no activity. Mutant analysis revealed that loss of ChOMT1 strongly reduced DHMC levels. Furthermore, chomt1 and omt2 showed strongly reduced bioreporter luminescence in their rhizospheres. In addition, loss of both ChOMT1 and ChOMT3 reduced nodulation, and this phenotype was strengthened by the further loss of OMT2. We conclude that: the loss of ChOMT1 greatly reduces root DHMC levels; ChOMT1 or OMT2 are important for nod gene activation in the rhizosphere; and ChOMT1/3 and OMT2 promote nodulation. Our findings suggest a degree of exclusivity in the flavonoids used for nodulation in M. truncatula compared to soybean, supporting a role for flavonoids in rhizobial host range.

RNA-Seq analysis of mung bean (Vigna radiata L.) roots shows differential gene expression and predicts regulatory pathways responding to taxonomically different rhizobia.

Symbiotic interaction among legume and rhizobia is a complex phenomenon which results in the formation of nitrogen-fixing nodules. Mung bean is promiscuous host however expression profile of this important legume plant in response to rhizobial infection was particularly lacking and urgently needed. We have demonstrated the pattern of gene expression of mung bean roots inoculated with two symbionts Bradyrhizobium yuanmingense Vr50 and Sinorhizobium (Ensifer) aridi Vr33 and non-inoculated control (CK). The RNA-Seq data analyzed at two growth stages i.e., 1-3 h and 10-16 days post inoculation revealed significantly higher number of differentially expressed genes (DEGs) at nodulation stage. The DEGs encoding receptor kinases identified at early stage might be involved in perception of Nod factors produced by different rhizobia. At nodulation stage important genes involved in plant hormone signal transduction, nitrogen and sulfur metabolism were identified. KEGG pathway enrichment analysis showed that metabolic pathways were most prominent in both groups (Group 1: Vr33 vs CK; Group 2: Vr50 vs CK), followed by biosynthesis of secondary metabolites, plant hormone signal transduction and biosynthesis of amino acids. Furthermore, DEGs involved in cell communication and plant hormone signal transduction were found to be different among two symbiotic systems while DEGs involved in carbon, nitrogen and sulfur metabolism were similar but their expression varied in response to two rhizobial strains. This study provides the first insight into the mechanisms underlying interactions of mung bean host with two taxonomically different symbionts (Bradyrhizobium and Sinorhizobium) and the candidate genes for better understanding the mechanisms of symbiotic host-specificity.

Symbiotic Nodule Development and Efficiency in the Medicago truncatula Mtefd-1 Mutant Is Highly Dependent on Sinorhizobium Strains.

Symbiotic nitrogen fixation (SNF) can play a key role in agroecosystems to reduce the negative impact of nitrogen fertilizers. Its efficiency is strongly affected by the combination of bacterial and plant genotypes, but the mechanisms responsible for the differences in the efficiency of rhizobium strains are not well documented. In Medicago truncatula, SNF has been mostly studied using model systems, such as M. truncatula A17 in interaction with Sinorhizobium meliloti Sm2011. Here we analyzed both the wild-type (wt) A17 and the Mtefd-1 mutant in interaction with five S. meliloti and two Sinorhizobium medicae strains. ETHYLENE RESPONSE FACTOR REQUIRED FOR NODULE DIFFERENTIATION (MtEFD) encodes a transcription factor, which contributes to the control of nodule number and differentiation in M. truncatula. We found that, in contrast to Sm2011, four strains induce functional (Fix+) nodules in Mtefd-1, although less efficient for SNF than in wt A17. In contrast, the Mtefd-1 hypernodulation phenotype is not strain-dependent. We compared the plant nodule transcriptomes in response to SmBL225C, a highly efficient strain with A17, versus Sm2011, in wt and Mtefd-1 backgrounds. This revealed faster nodule development with SmBL225C and early nodule senescence with Sm2011. These RNA sequencing analyses allowed us to identify candidate plant factors that could drive the differential nodule phenotype. In conclusion, this work shows the value of having a set of rhizobium strains to fully evaluate the biological importance of a plant symbiotic gene.

Global Transcriptional Repression of Diguanylate Cyclases by MucR1 Is Essential for Sinorhizobium-Soybean Symbiosis.

The ubiquitous bacterial second messenger c-di-GMP is intensively studied in pathogens but less so in mutualistic bacteria. Here, we report a genome-wide investigation of functional diguanylate cyclases (DGCs) synthesizing c-di-GMP from two molecules of GTP in Sinorhizobium fredii CCBAU45436, a facultative microsymbiont fixing nitrogen in nodules of diverse legumes, including soybean. Among 25 proteins harboring a putative GGDEF domain catalyzing the biosynthesis of c-di-GMP, eight functional DGCs were identified by heterogenous expression in Escherichia coli in a Congo red binding assay. This screening result was further verified by in vitro enzymatic assay with purified full proteins or the GGDEF domains from representative functional and nonfunctional DGCs. In the same in vitro assay, a functional EAL domain catalyzing the degradation of c-di-GMP into pGpG was identified in a protein that has an inactive GGDEF domain but with an active phosphodiesterase (PDE) function. The identified functional DGCs generally exhibited low transcription levels in soybean nodules compared to free-living cultures, as revealed in transcriptomes. An engineered upregulation of a functional DGC in nodules led to a significant increase of c-di-GMP level and symbiotic defects, which were not observed when a functional EAL domain was upregulated at the same level. Further transcriptional analysis and gel shift assay demonstrated that these functional DGCs were all transcriptionally repressed in nodules by a global pleiotropic regulator, MucR1, that is essential in Sinorhizobium-soybean symbiosis. These findings shed novel insights onto the systematic regulation of c-di-GMP biosynthesis in mutualistic symbiosis. IMPORTANCE The ubiquitous second messenger c-di-GMP is well-known for its role in biofilm formation and host adaptation of pathogens, whereas it is less investigated in mutualistic symbioses. Here, we reveal a cocktail of eight functional diguanylate cyclases (DGCs) catalyzing the biosynthesis of c-di-GMP in a broad-host-range Sinorhizobium that can establish nitrogen-fixing nodules on soybean and many other legumes. These functional DGCs are generally transcribed at low levels in soybean nodules compared to free-living conditions. The engineered nodule-specific upregulation of DGC can elevate the c-di-GMP level and cause symbiotic defects, while the upregulation of a phosphodiesterase that quenches c-di-GMP has no detectable symbiotic defects. Moreover, eight functional DGCs located on two different replicons are all directly repressed in nodules by a global silencer, MucR1, that is essential for Sinorhizobium-soybean symbiosis. These findings represent a novel mechanism of a strategic regulation of the c-di-GMP biosynthesis arsenal in prokaryote-eukaryote interactions.

Sinorhizobium medicae WSM419 Genes That Improve Symbiosis between Sinorhizobium meliloti Rm1021 and Medicago truncatula Jemalong A17 and in Other Symbiosis Systems.

Some soil bacteria, called rhizobia, can interact symbiotically with legumes, in which they form nodules on the plant roots, where they can reduce atmospheric dinitrogen to ammonia, a form of nitrogen that can be used by growing plants. Rhizobium-plant combinations can differ in how successful this symbiosis is: for example, Sinorhizobium meliloti Rm1021 forms a relatively ineffective symbiosis with Medicago truncatula Jemalong A17, but Sinorhizobium medicae WSM419 is able to support more vigorous plant growth. Using proteomic data from free-living and symbiotic S. medicae WSM419, we previously identified a subset of proteins that were not closely related to any S. meliloti Rm1021 proteins and speculated that adding one or more of these proteins to S. meliloti Rm1021 would increase its effectiveness on M. truncatula A17. Three genes, Smed_3503, Smed_5985, and Smed_6456, were cloned into S. meliloti Rm1021 downstream of the E. coli lacZ promoter. Strains with these genes increased nodulation and improved plant growth, individually and in combination with one another. Smed_3503, renamed iseA (increased symbiotic effectiveness), had the largest impact, increasing M. truncatula biomass by 61%. iseA homologs were present in all currently sequenced S. medicae strains but were infrequent in other Sinorhizobium isolates. Rhizobium leguminosarum bv. viciae 3841 containing iseA led to more nodules on pea and lentil. Split-root experiments with M. truncatula A17 indicated that S. meliloti Rm1021 carrying the S. medicae iseA is less sensitive to plant-induced resistance to rhizobial infection, suggesting an interaction with the plant's regulation of nodule formation. IMPORTANCE Legume symbiosis with rhizobia is highly specific. Rhizobia that can nodulate and fix nitrogen on one legume species are often unable to associate with a different species. The interaction can be more subtle. Symbiotically enhanced growth of the host plant can differ substantially when nodules are formed by different rhizobial isolates of a species, much like disease severity can differ when conspecific isolates of pathogenic bacteria infect different cultivars. Much is known about bacterial genes essential for a productive symbiosis, but less is understood about genes that marginally improve performance. We used a proteomic strategy to identify Sinorhizobium genes that contribute to plant growth differences that are seen when two different strains nodulate M. truncatula A17. These genes could also alter the symbiosis between R. leguminosarum bv. viciae 3841 and pea or lentil, suggesting that this approach identifies new genes that may more generally contribute to symbiotic productivity.

Decreased coevolutionary potential and increased symbiont fecundity during the biological invasion of a legume-rhizobium mutualism.

Although most invasive species engage in mutualism, we know little about how mutualism evolves as partners colonize novel environments. Selection on cooperation and standing genetic variation for mutualism traits may differ between a mutualism's invaded and native ranges, which could alter cooperation and coevolutionary dynamics. To test for such differences, we compare mutualism traits between invaded- and native-range host-symbiont genotype combinations of the weedy legume, Medicago polymorpha, and its nitrogen-fixing rhizobium symbiont, Ensifer medicae, which have coinvaded North America. We find that mutualism benefits for plants are indistinguishable between invaded- and native-range symbioses. However, rhizobia gain greater fitness from invaded-range mutualisms than from native-range mutualisms, and this enhancement of symbiont fecundity could increase the mutualism's spread by increasing symbiont availability during plant colonization. Furthermore, mutualism traits in invaded-range symbioses show lower genetic variance and a simpler partitioning of genetic variance between host and symbiont sources, compared to native-range symbioses. This suggests that biological invasion has reduced mutualists' potential to respond to coevolutionary selection. Additionally, rhizobia bearing a locus (hrrP) that can enhance symbiotic fitness have more exploitative phenotypes in invaded-range than in native-range symbioses. These findings highlight the impacts of biological invasion on the evolution of mutualistic interactions.

Minimal gene set from Sinorhizobium (Ensifer) meliloti pSymA required for efficient symbiosis with Medicago.

Reduction of N2 gas to ammonia in legume root nodules is a key component of sustainable agricultural systems. Root nodules are the result of a symbiosis between leguminous plants and bacteria called rhizobia. Both symbiotic partners play active roles in establishing successful symbiosis and nitrogen fixation: while root nodule development is mostly controlled by the plant, the rhizobia induce nodule formation, invade, and perform N2 fixation once inside the plant cells. Many bacterial genes involved in the rhizobia-legume symbiosis are known, and there is much interest in engineering the symbiosis to include major nonlegume crops such as corn, wheat, and rice. We sought to identify and combine a minimal bacterial gene complement necessary and sufficient for symbiosis. We analyzed a model rhizobium, Sinorhizobium (Ensifer) meliloti, using a background strain in which the 1.35-Mb symbiotic megaplasmid pSymA was removed. Three regions representing 162 kb of pSymA were sufficient to recover a complete N2-fixing symbiosis with alfalfa, and a targeted assembly of this gene complement achieved high levels of symbiotic N2 fixation. The resulting gene set contained just 58 of 1,290 pSymA protein-coding genes. To generate a platform for future synthetic manipulation, the minimal symbiotic genes were reorganized into three discrete nod, nif, and fix modules. These constructs will facilitate directed studies toward expanding the symbiosis to other plant partners. They also enable forward-type approaches to identifying genetic components that may not be essential for symbiosis, but which modulate the rhizobium's competitiveness for nodulation and the effectiveness of particular rhizobia-plant symbioses.

Symbiotic and Nonsymbiotic Members of the Genus Ensifer (syn. Sinorhizobium) Are Separated into Two Clades Based on Comparative Genomics and High-Throughput Phenotyping.

Rhizobium-legume symbioses serve as paradigmatic examples for the study of mutualism evolution. The genus Ensifer (syn. Sinorhizobium) contains diverse plant-associated bacteria, a subset of which can fix nitrogen in symbiosis with legumes. To gain insights into the evolution of symbiotic nitrogen fixation (SNF), and interkingdom mutualisms more generally, we performed extensive phenotypic, genomic, and phylogenetic analyses of the genus Ensifer. The data suggest that SNF likely emerged several times within the genus Ensifer through independent horizontal gene transfer events. Yet, the majority (105 of 106) of the Ensifer strains with the nodABC and nifHDK nodulation and nitrogen fixation genes were found within a single, monophyletic clade. Comparative genomics highlighted several differences between the "symbiotic" and "nonsymbiotic" clades, including divergences in their pangenome content. Additionally, strains of the symbiotic clade carried 325 fewer genes, on average, and appeared to have fewer rRNA operons than strains of the nonsymbiotic clade. Initial characterization of a subset of ten Ensifer strains identified several putative phenotypic differences between the clades. Tested strains of the nonsymbiotic clade could catabolize 25% more carbon sources, on average, than strains of the symbiotic clade, and they were better able to grow in LB medium and tolerate alkaline conditions. On the other hand, the tested strains of the symbiotic clade were better able to tolerate heat stress and acidic conditions. We suggest that these data support the division of the genus Ensifer into two main subgroups, as well as the hypothesis that pre-existing genetic features are required to facilitate the evolution of SNF in bacteria.

Co-catabolism of arginine and succinate drives symbiotic nitrogen fixation.

Biological nitrogen fixation emerging from the symbiosis between bacteria and crop plants holds promise to increase the sustainability of agriculture. One of the biggest hurdles for the engineering of nitrogen-fixing organisms is an incomplete knowledge of metabolic interactions between microbe and plant. In contrast to the previously assumed supply of only succinate, we describe here the CATCH-N cycle as a novel metabolic pathway that co-catabolizes plant-provided arginine and succinate to drive the energy-demanding process of symbiotic nitrogen fixation in endosymbiotic rhizobia. Using systems biology, isotope labeling studies and transposon sequencing in conjunction with biochemical characterization, we uncovered highly redundant network components of the CATCH-N cycle including transaminases that interlink the co-catabolism of arginine and succinate. The CATCH-N cycle uses N2 as an additional sink for reductant and therefore delivers up to 25% higher yields of nitrogen than classical arginine catabolism-two alanines and three ammonium ions are secreted for each input of arginine and succinate. We argue that the CATCH-N cycle has evolved as part of a synergistic interaction to sustain bacterial metabolism in the microoxic and highly acid environment of symbiosomes. Thus, the CATCH-N cycle entangles the metabolism of both partners to promote symbiosis. Our results provide a theoretical framework and metabolic blueprint for the rational design of plants and plant-associated organisms with new properties to improve nitrogen fixation.

Biochemical, functional and molecular characterization of pigeon pea rhizobia isolated from semi-arid regions of India.

Pigeon pea (Cajanus cajan (L.) Millspaugh) is among the top ten legumes grown globally not only having high tolerance to environmental stresses along, but also has the high biomass and productivity with optimal nutritional profiles. In the present study, 55 isolates of rhizobia were identified from 22 nodule samples of pigeon pea collected from semi-arid regions of India on the basis of morphological, biochemical, plant growth promoting activities and their ability to tolerate the stress conditions viz. pH, salt, temperature and drought stress. Amongst all the 55 isolates, 37 isolates showed effective nodulation under in vitro conditions in pigeon pea. Further, five isolates having multiple PGP activities and high in vitro symbiotic efficiency were subjected to 16S rRNA sequencing and confirmed their identities as Rhizobium, Mesorhizobium, Sinorhizobium sp. Further these 37 isolates were characterized at molecular level using ARDRA and revealed significant molecular diversity. Based on UPGMA clustering analysis, these isolates showed significant molecular diversity. The high degree of molecular diversity is due to mixed cropping of legumes in the region. The assessment of genetic diversity and molecular characterization of novel strains is a very important tool for the replacement of ineffective rhizobial strains with the efficient strains for the improvement in the nodulation and pigeon pea quality. The pigeon pea isolates with multiple PGPR activities could be further used for commercial production.

Characterization of Denitrifying Community for Application in Reducing Nitrogen: a Comparison of nirK and nirS Gene Diversity and Abundance.

Studies have shown that the addition of biochar to agricultural soils has the potential to mitigate climate change by decreasing nitrous oxide (N2O) emissions resulting from denitrification. Rice paddy field soils have been known to have strong denitrifying activity, but the response of microbes to biochar for weakening denitrification in rice paddy field soils is not well known. In this work, compared with the chemical fertilizer alone, the chemical fertilizer + 20 t hm-2 biochar fertilizer slightly decreased denitrifying the nitrite reductase activity (S-NiR) and N2O emission without statistic difference, whereas the chemical fertilizer + 40 t hm-2 biochar significantly boosted them. The abundance of nir-denitrifiers contributed to S-NiR and N2O emission, especially nirS-denitrifiers, rather than the variation of community structure. Pearson correlation analysis showed that NO2--N was a key factor for controlling the abundance of nir-denitrifiers, S-NiR and N2O emission. The biochar addition fertilization treatments strongly shaped the community structure of nirK-denitrifiers, while the community structure of nirS-denitrifiers remained relatively stable. In addition, Paracoccus and Sinorhizobium were revealed to be as the predominant lineage of nirS- and nirK-containing denitrifiers, respectively. Distance-based redundancy analysis (db-RDA) showed that changes in the nir-denitrifier community structure were significantly related to soil organic carbon, NO3--N, and total phosphorus. Our findings suggest that, although the nirS- and nirK-denitrifiers are both controlling nitrite reductase, their responses to biochar addition fertilization treatments showed significant discrepancies of diversity, abundance, and contribution to N2O and S-NiR in a paddy soil.

Modulation of Symbiotic Compatibility by Rhizobial Zinc Starvation Machinery.

Pathogenic bacteria need high-affinity zinc uptake systems to counteract the nutritional immunity exerted by infected hosts. However, our understanding of zinc homeostasis in mutualistic systems such as the rhizobium-legume symbiosis is limited. Here, we show that the conserved high-affinity zinc transporter ZnuABC and accessory transporter proteins (Zip1, Zip2, and c06450) made cumulative contributions to nodulation of the broad-host-range strain Sinorhizobium fredii CCBAU45436. Zur acted as a zinc-dependent repressor for the znuC-znuB-zur operon, znuA, and c06450 by binding to the associated Zur box, but did not regulate zip1 and zip2 ZnuABC was the major zinc transporter. Combined mutants lacking znuA and one of the three accessory genes had more severe defects in nodulation and growth under zinc starvation conditions than the znuA mutant, though rhizoplane colonization by these mutants was not impaired. In contrast to the elite strain CCBAU45436, more drastic symbiotic defects were observed for the znuA mutants of other Sinorhizobium strains, which lack at least one of the three accessory genes in their genomes and are characterized by their limited host range and geographical distribution. The znu-derived mutants showed a higher expression level of nod genes involved in Nod factor biosynthesis and a reduced expression of genes encoding a type three secretion system and its effector NopP, which can interfere with the host immune system. Application of exogenous zinc restored the nodulation ability of these znu-derived mutants. Therefore, the conserved ZnuABC and accessory components in the zinc starvation machinery play an important role in modulating symbiotic compatibility.IMPORTANCE The rhizobium-legume symbiosis contributes around 65% of biological nitrogen fixation in agriculture systems and is critical for sustainable agriculture by reducing the amount of chemical nitrogen fertilizer being used. Rhizobial inocula have been commercialized for more than 100 years, but the efficiency of inoculation can vary among legume cultivars, field sites, and years. These long-lasting challenging problems impede the establishment of a sustainable agriculture, particularly in developing countries. Here, we report that rhizobial zinc starvation machinery containing a conserved high-affinity zinc transporter and accessory components makes cumulative contributions to modulating rhizobial symbiotic compatibility. This work highlights a critical role of largely unexplored nutritional immunity in the rhizobium-legume symbiosis, which makes zinc starvation machinery an attractive target for improving rhizobial symbiotic compatibility.

Genomic insight into the origins and evolution of symbiosis genes in Phaseolus vulgaris microsymbionts.

BACKGROUND: Phaseolus vulgaris (common bean) microsymbionts belonging to the bacterial genera Rhizobium, Bradyrhizobium, and Ensifer (Sinorhizobium) have been isolated across the globe. Individual symbiosis genes (e.g., nodC) of these rhizobia can be different within each genus and among distinct genera. Little information is available about the symbiotic structure of indigenous Rhizobium strains nodulating introduced bean plants or the emergence of a symbiotic ability to associate with bean plants in Bradyrhizobium and Ensifer strains. Here, we sequenced the genomes of 29 representative bean microsymbionts (21 Rhizobium, four Ensifer, and four Bradyrhizobium) and compared them with closely related reference strains to estimate the origins of symbiosis genes among these Chinese bean microsymbionts. RESULTS: Comparative genomics demonstrated horizontal gene transfer exclusively at the plasmid level, leading to expanded diversity of bean-nodulating Rhizobium strains. Analysis of vertically transferred genes uncovered 191 (out of the 2654) single-copy core genes with phylogenies strictly consistent with the taxonomic status of bacterial species, but none were found on symbiosis plasmids. A common symbiotic region was wholly conserved within the Rhizobium genus yet different from those of the other two genera. A single strain of Ensifer and two Bradyrhizobium strains shared similar gene content with soybean microsymbionts in both chromosomes and symbiotic regions. CONCLUSIONS: The 19 native bean Rhizobium microsymbionts were assigned to four defined species and six putative novel species. The symbiosis genes of R. phaseoli, R. sophoriradicis, and R. esperanzae strains that originated from Mexican bean-nodulating strains were possibly introduced alongside bean seeds. R. anhuiense strains displayed distinct host ranges, indicating transition into bean microsymbionts. Among the six putative novel species exclusive to China, horizontal transfer of symbiosis genes suggested symbiosis with other indigenous legumes and loss of originally symbiotic regions or non-symbionts before the introduction of common bean into China. Genome data for Ensifer and Bradyrhizobium strains indicated symbiotic compatibility between microsymbionts of common bean and other hosts such as soybean.

Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

Illumina sequencing of 16S rRNA tag shows disparity in rhizobial and non-rhizobial diversity associated with root nodules of mung bean (Vigna radiata L.) growing in different habitats in Pakistan.

In Rhizobium-legume symbiosis, the nodule is the most frequently studied compartment, where the endophytic/symbiotic microbiota demands critical investigation for development of specific inocula. We identified the bacterial diversity within root nodules of mung bean from different growing areas of Pakistan using Illumina sequencing of 16S rRNA gene. We observed specific OTUs related to specific site where Bradyrhizobium was found to be the dominant genus comprising of 82-94% of total rhizobia in nodules with very minor fraction of sequences from other rhizobia at three sites. In contrast, Ensifer (Sinorhizobium) was single dominant genus comprising 99.9% of total rhizobial sequences at site four. Among non-rhizobial sequences, the genus Acinetobacter was abundant (7-18% of total sequences), particularly in Bradyrhizobium-dominated nodule samples. Rhizobia and non-rhizobial PGPR isolated from nodule samples include Ensifer, Bradyrhizobium, Acinetobacter, Microbacterium and Pseudomonas strains. Co-inoculation of multi-trait PGPR Acinetobacter sp. VrB1 with either of the two rhizobia in field exhibited more positive effect on nodulation and plant growth than single-strain inoculation which favors the use of Acinetobacter as an essential component for development of mung bean inoculum. Furthermore, site-specific dominance of rhizobia and non-rhizobia revealed in this study may contribute towards decision making for development and application of specific inocula in different habitats.

Ensifer fredii symbiovar vachelliae nodulates endemic Vachellia gummifera in semiarid Moroccan areas.

The purpose of this work was to study the genetic diversity of the nodule-forming bacteria associated with native populations of Vachellia gummifera growing wild in Morocco. The nearly complete 16S rRNA gene sequences from three selected strains, following ARDRA and REP-PCR results, revealed they were members of the genus Ensifer and the sequencing of the housekeeping genes recA, gyrB, dnaK and rpoB, and their concatenated phylogenetic analysis, showed that the 3 strains belong to the species E. fredii. Based on the nodC and nodA phylogenies, the 3 strains clearly diverged from the type and other reference strains of E. fredii and formed a clearly separated cluster. The strains AGA1, AGA2 and AGB23 did not form nodules on Glycine max, Phaseolus vulgaris and Medicago truncatula, and effectively nodulated V. gummifera, Acacia cyanophylla, Prosopis chilensis and Leucaena leucocephala. Based on similarities of the nodC and nodA symbiotic genes and differences in the host range, the strains isolated from Moroccan endemic V. gummifera may form a different symbiovar within Ensifer species, for which the name "vachelliae" is proposed.

Integrating T7 RNA Polymerase and Its Cognate Transcriptional Units for a Host-Independent and Stable Expression System in Single Plasmid.

Metabolic engineering and synthetic biology usually require universal expression systems for stable and efficient gene expression in various organisms. In this study, a host-independent and stable T7 expression system had been developed by integrating T7 RNA polymerase and its cognate transcriptional units in single plasmid. The expression of T7 RNA polymerase was restricted below its lethal threshold using a T7 RNA polymerase antisense gene cassette, which allowed long periods of cultivation and protein production. In addition, by designing ribosome binding sites, we further tuned the expression capacity of this novel T7 system within a wide range. This host-independent expression system efficiently expressed genes in five different Gram-negative strains and one Gram-positive strain and was also shown to be applicable in a real industrial d- p-hydroxyphenylglycine production system.

Adaptive evolution of rhizobial symbiotic compatibility mediated by co-evolved insertion sequences.

Mutualism between bacteria and eukaryotes has essential roles in the history of life, but the evolution of their compatibility is poorly understood. Here we show that different Sinorhizobium strains can form either nitrogen-fixing nodules or uninfected pseudonodules on certain cultivated soybeans, while being all effective microsymbionts of some wild soybeans. However, a few well-infected nodules can be found on a commercial soybean using inocula containing a mixed pool of Tn5 insertion mutants derived from an incompatible strain. Reverse genetics and genome sequencing of compatible mutants demonstrated that inactivation of T3SS (type three secretion system) accounted for this phenotypic change. These mutations in the T3SS gene cluster were dominated by parallel transpositions of insertion sequences (ISs) other than the introduced Tn5. This genetic and phenotypic change can also be achieved in an experimental evolution scenario on a laboratory time scale using incompatible wild-type strains as inocula. The ISs acting in the adaptive evolution of Sinorhizobium strains exhibit broader phyletic and replicon distributions than other ISs, and prefer target sequences of low GC% content, a characteristic feature of symbiosis plasmid where T3SS genes are located. These findings suggest an important role of co-evolved ISs in the adaptive evolution of rhizobial compatibility.

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance.

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342mM NaCl as well as 127 or 99mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44°C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.

Retrieved 16S rRNA and nifH sequences reveal co-dominance of Bradyrhizobium and Ensifer (Sinorhizobium) strains in field-collected root nodules of the promiscuous host Vigna radiata (L.) R. Wilczek.

In the present study, the relative distribution of endophytic rhizobia in field-collected root nodules of the promiscuous host mung bean was investigated by sequencing of 16S ribosomal RNA (rRNA) and nifH genes, amplified directly from the nodule DNA. Co-dominance of the genera Bradyrhizobium and Ensifer was indicated by 32.05 and 35.84% of the total retrieved 16S rRNA sequences, respectively, and the sequences of genera Mesorhizobium and Rhizobium comprised only 0.06 and 2.06% of the recovered sequences, respectively. Sequences amplified from rhizosphere soil DNA indicated that only a minor fraction originated from Bradyrhizobium and Ensifer strains, comprising about 0.46 and 0.67% of the total retrieved sequences, respectively. 16S rRNA gene sequencing has also identified the presence of several non-rhizobial endophytes from phyla Proteobacteria, Actinobacteria, Bacteroides, and Firmicutes. The nifH sequences obtained from nodules also confirmed the co-dominance of Bradyrhizobium (39.21%) and Ensifer (59.23%) strains. The nifH sequences of the genus Rhizobium were absent, and those of genus Mesorhizobium comprised only a minor fraction of the sequences recovered from the nodules and rhizosphere soil samples. Two bacterial isolates, identified by 16S rRNA gene sequence analysis as Bradyrhizobium strain Vr51 and Ensifer strain Vr38, successfully nodulated the original host (mung bean) plants. Co-dominance of Bradyrhizobium and Ensifer strains in the nodules of mung bean indicates the potential role of the host plant in selecting specific endophytic rhizobial populations. Furthermore, successful nodulation of mung bean by the isolates showed that strains of both the genera Bradyrhizobium and Ensifer can be used for production of inoculum.