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Foreign AT-rich genes drive bacterial adaptation to new niches while challenging the existing regulation network. Here we report that MucR, a conserved regulator in α-proteobacteria, balances adaptation and regulatory integrity in Sinorhizobium fredii, a facultative microsymbiont of legumes. Chromatin immunoprecipitation sequencing coupled with transcriptomic data reveal that average transcription levels of both target and non-target genes, under free-living and symbiotic conditions, increase with their conservation levels. Targets involved in environmental adaptation and symbiosis belong to genus or species core and can be repressed or activated by MucR in a condition-dependent manner, implying regulatory integrations. However, most targets are enriched in strain-specific genes of lower expression levels and higher AT%. Within each conservation levels, targets have higher AT% and average transcription levels than non-target genes and can be further up-regulated in the mucR mutant. This is consistent with higher AT% of spacers between −35 and −10 elements of promoters for target genes, which enhances transcription. The MucR recruitment level linearly increases with AT% and the number of a flexible pattern (with periodic repeats of Ts) of target sequences. Collectively, MucR directly represses AT-rich foreign genes with predisposed high transcription potential while progressive erosions of its target sites facilitate regulatory integrations of foreign genes.

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.

Bacteria have evolved a series of mechanisms to maintain their survival and reproduction in changeable and stressful environments. In-depth understanding of these mechanisms can allow for better developing and utilizing of bacteria with various biological functions. In this study, we found that water-soluble humic materials (WSHM), a well-known environment-friendly plant growth biostimulant, significantly promoted the free-living growth and survival of Sinorhizobium fredii CCBAU45436 in a bell-shaped, dose-dependent manner, along with more-efficient carbon source consumption and relief of medium acidification. By using RNA-Seq analysis, a total of 1,136 genes significantly up-/downregulated by external addition of WSHM were identified under test conditions. These differentially expressed genes (DEGs) were enriched in functional categories related to carbon/nitrogen metabolism, cellular stress response, and genetic information processing. Further protein-protein interaction (PPI) network analysis and reverse genetic engineering indicated that WSHM might reprogram the transcriptome through inhibiting the expression of key hub gene rsh, which encodes a bifunctional enzyme catalyzing synthesis and hydrolysis of the “magic spot” (p)ppGpp. In addition, the root colonization and viability in soil of S. fredii CCBAU45436 were increased by WSHM. These findings provide us with new insights into how WSHM benefit bacterial adaptations and demonstrate great application value to be a unique inoculant additive. IMPORTANCE Sinorhizobium fredii CCBAU45436 is a highly effective, fast-growing rhizobium that can establish symbiosis with multiple soybean cultivars. However, it is difficult to maintain the high-density effective viable cells in the rhizobial inoculant for the stressful conditions during production, storage, transport, and application. Here, we showed that WSHM greatly increased the viable cells of S. fredii CCBAU45436 in culture, modulating metabolism and triggering stress defense. The root colonization and viability in soil of S. fredii CCBAU45436 were also increased by WSHM. Our results shed new insights into the effects of WSHM on bacteria and the importance of metabolism and stress defense during the bacteria’s whole life. In addition, the functional mechanism of WSHM may provide candidate genes for improving environmental adaptability and application potential of bacteria through genetic engineering.

International audience; Rhizobia are nitrogen-fixing soil bacteria that can infect legume plants to establish root nodules symbiosis. To do that, a complex exchange of molecular signals occurs between plants and bacteria. Among them, rhizobial Nops (Nodulation outer proteins), secreted by a type III secretion system (T3SS) determine the host-specificity for efficient symbiosis with plant roots. Little is known about the molecular function of secreted Nops (also called effectors (T3E)) and their role in the symbiosis process. We performed the structure-function characterization of NopAA, a T3E from Sinorhizobium fredii by using a combination of X-ray crystallography, biochemical and biophysical approaches. this work displays for the first time a complete structural and biochemical characterization of a symbiotic T3E. Our results showed that NopAA has a catalytic domain with xyloglucanase activity extended by a N-terminal unfolded secretion domain that allows its secretion. We proposed that these original structural properties combined with the specificity of NopAA toward xyloglucan, a key component of root cell wall which is also secreted by roots in the soil, can give NopAA a strategic position to participate in recognition between bacteria and plant roots and to intervene in nodulation process. Many bacterial pathogens use type III secretion systems (T3SS) to inject virulence factors, named effectors, directly into the cytoplasm of target eukaryotic cells. Most of the T3SS apparatus components are conserved among plant and animal pathogens, suggesting a common mechanism of recognition and secretion of effec-tors (for review see 1). Secretion of effectors depends on the presence of secretion signals composed by 20 to 30 non-conserved amino acids at the N-terminus. Most of T3SS effectors (T3E) are predicted to possess a N-terminal intrinsically disordered (ID) region enriched in serine residues and required for secretion 2. Many secreted proteins also depend on the interaction with a cytoplasmic specific T3S chaperone 3 that stabilize ID regions in bacterial cytoplasm prior translocation. For SopB, a T3E of Salmonella it has been proposed that its cognate chaperone SigE may be responsible of the formation of ring like hexamers, conformation that may be required for substrate recognition by the type three apparatus 4. In pathogenic bacteria, T3SS genes coding for the secretion apparatus, effectors and accessory proteins are clustered in pathogenicity islands (PAIs) and organized in operons. Interestingly, this secretion strategy is also used by non-pathogenic organisms contributing to symbiotic interactions with hosts as shown for rhizobia 5,6. Rhizobia are soil bacteria having the ability to establish a specific symbiotic association with leguminous host-plant roots. This interaction leads to the formation of root nodules by the plant, specialized organs in which bacteria differentiate into nitrogen fixing bacteroids able to convert atmospheric nitrogen into ammonia for the usage in plant growth 7. This process starts with the secretion of

The broad-host-range bacterium Sinorhizobium fredii HH103 cannot nodulate the model legume Lotus japonicus Gifu. This bacterium possesses a type III secretion system (T3SS), a specialized secretion apparatus used to deliver effector proteins (T3Es) into the host cell cytosol to alter host signaling and/or suppress host defence responses to promote infection. However, some of these T3Es are recognized by specific plant receptors and hence trigger a strong defence response to block infection. In rhizobia, T3Es are involved in nodulation efficiency and host-range determination, and in some cases directly activate host symbiosis signalling in a Nod factor-independent manner. In this work, we show that HH103 RifR T3SS mutants, unable to secrete T3Es, gain nodulation with L. japonicus Gifu through infection threads, suggesting that plant recognition of a T3E could block the infection process. To identify the T3E involved, we performed nodulation assays with a collection of mutants that affect secretion of each T3E identified in HH103 RifR so far. The nopC mutant could infect L. japonicus Gifu by infection thread invasion and switch the infection mechanism in Lotus burttii from intercellular infection to infection thread formation. Lotus japonicus gene expression analysis indicated that the infection-blocking event occurs at early stages of the symbiosis.

Nitrogen is the most limiting macronutrient for plant growth, and rhizobia are important bacteria for agriculture because they can fix atmospheric nitrogen and make it available to legumes through the establishment of a symbiotic relationship with their host plants. In this work, we studied the nitrogen fixation process in the microsymbiont Sinorhizobium fredii at the genome level. A metabolic model was built using genome annotation and literature to reconstruct the symbiotic form of S. fredii. Genes controlling the nitrogen fixation process were identified by simulating gene knockouts. Additionally, the nitrogen-fixing capacities of S. fredii CCBAU45436 in symbiosis with cultivated and wild soybeans were evaluated. The predictions suggested an outperformance of S. fredii with cultivated soybean, consistent with published experimental evidence. The reconstruction presented here will help to understand and improve nitrogen fixation capabilities of S. fredii and will be beneficial for agriculture by reducing the reliance on fertilizer applications., Rhizobia are soil bacteria able to establish symbiosis with diverse host plants. Specifically, Sinorhizobium fredii is a soil bacterium that forms nitrogen-fixing root nodules in diverse legumes, including soybean. The strain S. fredii CCBAU45436 is a dominant sublineage of S. fredii that nodulates soybeans in alkaline-saline soils in the Huang-Huai-Hai Plain region of China. Here, we present a manually curated metabolic model of the symbiotic form of Sinorhizobium fredii CCBAU45436. A symbiosis reaction was defined to describe the specific soybean-microsymbiont association. The performance and quality of the reconstruction had a 70% score when assessed using a standardized genome-scale metabolic model test suite. The model was used to evaluate in silico single-gene knockouts to determine the genes controlling the nitrogen fixation process. One hundred forty-one of 541 genes (26%) were found to influence the symbiotic process, wherein 121 genes were predicted as essential and 20 others as having a partial effect. Transcriptomic profiles of CCBAU45436 were used to evaluate the nitrogen fixation capacity in cultivated versus in wild soybean inoculated with the microsymbiont. The model quantified the nitrogen fixation activities of the strain in these two hosts and predicted a higher nitrogen fixation capacity in cultivated soybean. Our results are consistent with published data demonstrating larger amounts of ureides and total nitrogen in cultivated soybean than in wild soybean. This work presents the first metabolic network reconstruction of S. fredii as an example of a useful tool for exploring the potential benefits of microsymbionts to sustainable agriculture and the ecosystem. IMPORTANCE Nitrogen is the most limiting macronutrient for plant growth, and rhizobia are important bacteria for agriculture because they can fix atmospheric nitrogen and make it available to legumes through the establishment of a symbiotic relationship with their host plants. In this work, we studied the nitrogen fixation process in the microsymbiont Sinorhizobium fredii at the genome level. A metabolic model was built using genome annotation and literature to reconstruct the symbiotic form of S. fredii. Genes controlling the nitrogen fixation process were identified by simulating gene knockouts. Additionally, the nitrogen-fixing capacities of S. fredii CCBAU45436 in symbiosis with cultivated and wild soybeans were evaluated. The predictions suggested an outperformance of S. fredii with cultivated soybean, consistent with published experimental evidence. The reconstruction presented here will help to understand and improve nitrogen fixation capabilities of S. fredii and will be beneficial for agriculture by reducing the reliance on fertilizer applications.

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., 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.

Rhizobia are soil bacteria that form important symbiotic associations with legumes, and rhizobial surface polysaccharides, such as K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS), might be important for symbiosis. Previously, we obtained a mutant of Sinorhizobium fredii HH103, rkpA, that does not produce KPS, a homopolysaccharide of a pseudaminic acid derivative, but whose LPS electrophoretic profile was indistinguishable from that of the WT strain. We also previously demonstrated that the HH103 rkpLMNOPQ operon is responsible for 5-acetamido-3,5,7,9-tetradeoxy-7-(3-hydroxybutyramido)-L-glyc-ero-L-manno-nonulosonic acid [Pse5NAc7(3OHBu)] production and is involved in HH103 KPS and LPS biosynthesis and that an HH103 rkpM mutant cannot produce KPS and displays an altered LPS structure. Here, we analyzed the LPS structure of HH103 rkpA, focusing on the carbohydrate portion, and found that it contains a highly heterogeneous lipid A and a peculiar core oligosaccharide composed of an unusually high number of hexuronic acids containing b-configured Pse5NAc7(3OHBu). This pseudaminic acid derivative, in its a-configuration, was the only structural component of the S. fredii HH103 KPS and, to the best of our knowledge, has never been reported from any other rhizobial LPS. We also show that Pse5NAc7(3OHBu) is the complete or partial epitope for a mAb, NB6-228.22, that can recognize the HH103 LPS, but not those of most of the S. fredii strains tested here. We also show that the LPS from HH103 rkpM is identical to that of HH103 rkpA but devoid of any Pse5NAc7(3OHBu) residues. Notably, this rkpM mutant was severely impaired in symbiosis with its host, Macroptilium atropurpureum. Fil: Di Lorenzo, Flaviana. Università degli Studi di Napoli Federico II; Italia Fil: Speciale, Immacolata. Università degli Studi di Napoli Federico II; Italia Fil: Silipo, Alba. Università degli Studi di Napoli Federico II; Italia Fil: Alías Villegas, Cynthia. Universidad de Sevilla; España Fil: Acosta Jurado, Sebastián. Universidad de Sevilla; España Fil: Rodríguez Carvajal, Miguel Ángel. Universidad de Sevilla; España Fil: Dardanelli, Marta Susana. Universidad Nacional de Río Cuarto. Facultad de Ciencias Exactas Fisicoquímicas y Naturales. Instituto de Biotecnología Ambiental y Salud - Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Biotecnología Ambiental y Salud; Argentina Fil: Palmigiano, Angelo. Consiglio Nazionale delle Ricerche; Italia Fil: Garozzo, Domenico. Consiglio Nazionale delle Ricerche; Italia Fil: Ruiz Sainz, José Enrique. Universidad de Sevilla; España Fil: Molinaro, Antonio. University of Naples Federico II; Italia Fil: Vinardell, José María. Universidad de Sevilla; España

Quorum sensing (QS) is a bacterial cell-to-cell signaling mechanism that collectively regulates and synchronizes behaviors by means of small diffusible chemical molecules. In rhizobia, QS systems usually relies on the synthesis and detection of N-acyl-homoserine lactones (AHLs). In the model bacterium Sinorhizobium meliloti functions regulated by the QS systems TraI-TraR and SinI-SinR(-ExpR) include plasmid transfer, production of surface polysaccharides, motility, growth rate and nodulation. These systems are also present in other bacteria of the Sinorhizobium genus, with variations at the species and strain level. In Sinorhizobium fredii NGR234 phenotypes regulated by QS are plasmid transfer, growth rate, sedimentation, motility, biofilm formation, EPS production and copy number of the symbiotic plasmid (pSym). The analysis of the S. fredii HH103 genomes reveal also the presence of both QS systems. In this manuscript we characterized the QS systems of S. fredii HH103, determining that both TraI and SinI AHL-synthases proteins are responsible of the production of short- and long-chain AHLs, respectively, at very low and not physiological concentrations. Interestingly, the main HH103 luxR-type genes, expR and traR, are split into two ORFs, suggesting that in S. fredii HH103 the corresponding carboxy-terminal proteins, which contain the DNA-binding motives, may control target genes in an AHL-independent manner. The presence of a split traR gene is common in other S. fredii strains.

The symbiosis between Sinorhizobium fredii HH103 and its host legumes is influenced by the type 3 secretion system (T3SS), which delivers proteins (effectors) directly into the host cells to promote infection. GunA, one of the predicted HH103 effectors, potentially codes for a cellulase. In this work we tried to characterise GunA and elucidate its role in symbosis with soybean and cowpea. A GunA::HA fusion protein was constructed to study T3SS-dependent secretion. Cellulase activity of GunA was measured and gunA::uidA-GFP and gunA::cyA fusions were constructed to monitor gunA expression in nodules and to study translocation to the host cells, respectively. Finally, the symbiotic performance of a gunA mutant was studied in soybean and cowpea. GunA from S. fredii HH103 shows cellulase activity and is secreted through the T3SS in response to the inducer flavonoid genistein. Interestingly, at the beggining of the symbiotic process, GunA was partially responsible for the induction of the expression of the soybean GmPR1 gene, a gene used as a marker for plant defense responses. However, GunA was also detected in soybean and cowpea developed nodules. Finally, nodulation assays indicate that GunA is beneficial for symbiosis with soybean but detrimental with cowpea. Secretion of GunA through the S. fredii HH103 T3SS clearly and differentially impacts the symbiotic performance of this strain with soybean and cowpea. GunA, or its cellulase activity, is recognised by soybean root cells very early in the symbiotic process but, curiously, its secretion can also be detected in mature nodules. This suggests different symbiotic roles at different symbiotic stages that need to be further elucidated.

Phosphate homeostasis is tightly modulated in all organisms, including bacteria, which harbor both high- and low-affinity transporters acting under conditions of fluctuating phosphate levels. It was thought that nitrogen-fixing rhizobia, named bacteroids, inhabiting root nodules of legumes are not phosphate limited. Here, we show that the high-affinity phosphate transporter PstSCAB, rather than the low-affinity phosphate transporter Pit, is essential for effective nitrogen fixation of Sinorhizobium fredii in soybean nodules. Symbiotic and growth defects of the pst mutant can be effectively restored by knocking out PhoB, the transcriptional repressor of pit. The pst homologs of representative rhizobia were actively transcribed in bacteroids without terminal differentiation in nodules of diverse legumes (soybean, pigeonpea, cowpea, common bean, and Sophora flavescens) but exhibited a basal expression level in terminally differentiated bacteroids (alfalfa, pea, and peanut). Rhizobium leguminosarum bv. viciae Rlv3841 undergoes characteristic nonterminal and terminal differentiations in nodules of S. flavescens and pea, respectively. The pst mutant of Rlv3841 showed impaired adaptation to the nodule environment of S. flavescens but was indistinguishable from the wild-type strain in pea nodules. Taken together, root nodule rhizobia can be either phosphate limited or nonlimited regarding the rhizobial differentiation fate, which is a host-dependent feature.

Receiving nodulation and nitrogen fixation genes does not guarantee rhizobia an effective symbiosis with legumes. Here, variations in gene content were determined for three Sinorhizobium species showing contrasting symbiotic efficiency on soybeans. A nitrate-reduction gene cluster absent in S. sojae was found to be essential for symbiotic adaptations of S. fredii and S. sp. III. In S. fredii, the deletion mutation of the nap (nitrate reductase), instead of nir (nitrite reductase) and nor (nitric oxide reductase), led to defects in nitrogen-fixation (Fix- ). By contrast, none of these core nitrate-reduction genes were required for the symbiosis of S. sp. III. However, within the same gene cluster, the deletion of hemN1 (encoding oxygen-independent coproporphyrinogen III oxidase) in both S. fredii and S. sp. III led to the formation of nitrogen-fixing (Fix+ ) but ineffective (Eff- ) nodules. These Fix+ /Eff- nodules were characterized by significantly lower enzyme activity of glutamine synthetase indicating rhizobial modulation of nitrogen-assimilation by plants. A distant homologue of HemN1 from S. sojae can complement this defect in S. fredii and S. sp. III, but exhibited a more pleotropic role in symbiosis establishment. These findings highlighted the lineage-dependent optimization of symbiotic functions in different rhizobial species associated with the same host.

Sinorhizobium fredii is a dominant rhizobium on alkaline-saline land that can induce nitrogen-fixing symbiotic root nodules in soybean. Two S. fredii strains, CCBAU25509 and CCBAU45436, were used in this study to facilitate in-depth analyses of this species and its interactions with soybean. We have previously completed the full assembly of the genomes and detailed transcriptomic analyses for these two S. fredii strains, CCBAU25509 and CCBAU45436, that exhibit differential compatibility toward some soybean hosts. In this work, we performed high-throughput Orbitrap analyses of the whole proteomes and secretomes of CCBAU25509 and CCBAU45436 at different growth stages. Our proteomic data cover coding sequences in the chromosome, chromid, symbiotic plasmid, and other accessory plasmids. In general, we found higher levels of protein expression by genes in the chromosomal genome, whereas proteins encoded by the symbiotic plasmid were differentially accumulated in bacteroids. We identified secreted proteins from the extracellular medium, including seven and eight Nodulation Outer Proteins (Nops) encoded by the symbiotic plasmid of CCBAU25509 and CCBAU45436, respectively. Differential host restriction of CCBAU25509 and CCBAU45436 is regulated by the allelic type of the soybean Rj2(Rfg1) protein. Using sequencing data from this work and available in public databases, our analysis confirmed that the soybean Rj2(Rfg1) protein has three major allelic types (Rj2/rfg1, rj2/Rfg1, rj2/rfg1) that determine the host restriction of some Bradyrhizobium diazoefficiens and S. fredii strains. A mutant defective in the type 3 protein secretion system (T3SS) in CCBAU25509 allowed this strain to nodulate otherwise-incompatible soybeans carrying the rj2/Rfg1 allelic type, probably by disrupting Nops secretion. The allelic forms of NopP and NopI in S. fredii might be associated with the restriction imposed by Rfg1. By swapping the NopP between CCBAU25509 and CCBAU45436, we found that only the strains carrying NopP from CCBAU45436 could nodulate soybeans carrying the rj2/Rfg1 allelic type. However, no direct interaction between either forms of NopP and Rfg1 could be observed.

Sinorhizobium fredii HH103 RifR is a broad host-range rhizobial strain able to nodulate with soybean and Lotus burttii, but it is ineffective with L. japonicus. Here, we study the role of the HH103 RifR SyrM protein in the regulation of gene expression and its relevance in symbiosis with those three legumes. RNAseq analyses show that HH103 SyrM is an important transcriptional regulator not only in the presence of inducer flavonoids but also in its absence. Lack of SyrM increases Nod factors production and decreases genistein-mediated repression of exopolysaccharide production in HH103. In symbiosis, mutation of syrM partially impaired interaction with soybean but improves effectiveness with L. burttii and extends the host-rage to L. japonicus Gifu. In addition, HH103 syrM mutants enter in both Lotus species by infection threads, whereas HH103 uses the more primitive intercellular infection to enter into L. burttii roots These symbiotic phenotypes were previously observed in two other HH103 mutants affected in symbiotic regulators, nodD2 and nolR, revealing that in S. fredii HH103 numerous transcriptional regulators finely modulate symbiotic gene expression.

Sinorhizobium fredii HH103 Rif R , a broad-host-range rhizobial strain, forms ineffective nodules with Lotus japonicus but induces nitrogen-fixing nodules in Lotus burttii roots that are infected by intercellular entry. Here we show that HH103 Rif R nolR or nodD2 mutants gain the ability to induce infection thread formation and to form nitrogen-fixing nodules in L. japonicus Gifu. Microscopy studies showed that the mode of infection of L. burttii roots by the nodD2 and nolR mutants switched from intercellular entry to infection threads (ITs). In the presence of the isoflavone genistein, both mutants overproduced Nod-factors. Transcriptomic analyses showed that, in the presence of Lotus japonicus Gifu root exudates, genes related to Nod factors production were overexpressed in both mutants in comparison to HH103 Rif R . Complementation of the nodD2 and nolR mutants provoked a decrease in Nod-factor production, the incapacity to form nitrogen-fixing nodules with L. japonicus Gifu and restored the intercellular way of infection in L. burttii. Thus, the capacity of S. fredii HH103 Rif R nodD2 and nolR mutants to infect L. burttii and L. japonicus Gifu by ITs and fix nitrogen L. japonicus Gifu might be correlated with Nod-factor overproduction, although other bacterial symbiotic signals could also be involved.

Sinorhizobium fredii HH103 is a rhizobial strain showing a broad host range of nodulation. In addition to the induction of bacterial nodulation genes, transition from a free-living to a symbiotic state requires complex genetic expression changes with the participation of global regulators. We have analyzed the role of the zinc-finger transcriptional regulator MucR1 from S.fredii HH103 under both free-living conditions and symbiosis with two HH103 host plants, Glycine max and Lotus burttii. Inactivation of HH103 mucR1 led to a severe decrease in exopolysaccharide (EPS) biosynthesis but enhanced production of external cyclic glucans (CG). This mutant also showed increased cell aggregation capacity as well as a drastic reduction in nitrogen-fixation capacity with G. max and L. burttii. However, in these two legumes, the number of nodules induced by the mucR1 mutant was significantly increased and decreased, respectively, with respect to the wild-type strain, indicating that MucR1 can differently affect nodulation depending on the host plant. RNA-Seq analysis carried out in the absence and the presence of flavonoids showed that MucR1 controls the expression of hundreds of genes (including some related to EPS production and CG transport), some of them being related to the nod regulon. © 2016 The American Phytopathological Society., This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-30229) and the Andalusia Government (P07-CVI-07500). SAJ and PNG are recipients of PhD grants from the VPPI of the University of Seville. We thank the Biology, Microscopy, NMR, and Mass Spectrometry Services of the Centro de Investigación, Tecnología e Innovación (CITIUS) of the University of Seville.

The lack of an appropriate genomic platform has precluded the use of gain-of-function approaches to study the rhizobium-legume symbiosis, preventing the establishment of the genes necessary and sufficient for symbiotic nitrogen fixation (SNF) and potentially hindering synthetic biology approaches aimed at engineering this process. Here, we describe the development of an appropriate system by reverse engineering Sinorhizobium meliloti. Using a novel in vivo cloning procedure, the engA-tRNA-rmlC (ETR) region, essential for cell viability and symbiosis, was transferred from Sinorhizobium fredii to the ancestral location on the S. meliloti chromosome, rendering the ETR region on pSymB redundant. A derivative of this strain lacking both the large symbiotic replicons (pSymA and pSymB) was constructed. Transfer of pSymA and pSymB back into this strain restored symbiotic capabilities with alfalfa. To delineate the location of the single-copy genes essential for SNF on these replicons, we screened a S. meliloti deletion library, representing > 95% of the 2900 genes of the symbiotic replicons, for their phenotypes with alfalfa. Only four loci, accounting for

The nitrogen phosphotransferase system (PTS Ntr ) consists of EI Ntr , NPr, and EIIA Ntr . The active phosphate moiety derived from phosphoenolpyruvate is transferred through EI Ntr and NPr to EIIA Ntr . Sinorhizobium fredii can establish a nitrogen-fixing symbiosis with the legume crops soybean (as determinate nodules) and pigeonpea (as indeterminate nodules). In this study, S. fredii strains with mutations in ptsP and ptsO (encoding EI Ntr and NPr, respectively) formed ineffective nodules on soybeans, while a strain with a ptsN mutation (encoding EIIA Ntr ) was not defective in symbiosis with soybeans. Notable reductions in the numbers of bacteroids within each symbiosome and of poly-β-hydroxybutyrate granules in bacteroids were observed in nodules infected by the ptsP or ptsO mutant strains but not in those infected with the ptsN mutant strain. However, these defects of the ptsP and ptsO mutant strains were recovered in ptsP ptsN and ptsO ptsN double-mutant strains, implying a negative role of unphosphorylated EIIA Ntr in symbiosis. Moreover, the symbiotic defect of the ptsP mutant was also recovered by expressing EI Ntr with or without the GAF domain, indicating that the putative glutamine-sensing domain GAF is dispensable in symbiotic interactions. The critical role of PTS Ntr in symbiosis was also observed when related PTS Ntr mutant strains of S. fredii were inoculated on pigeonpea plants. Furthermore, nodule occupancy and carbon utilization tests suggested that multiple outputs could be derived from components of PTS Ntr in addition to the negative role of unphosphorylated EIIA Ntr .

In rhizobial species that nodulate inverted repeat-lacking clade (IRLC) legumes, such as the interaction between Sinorhizobium meliloti and Medicago, bacteroid differentiation is driven by an endoreduplication event that is induced by host nodule-specific cysteine rich (NCR) antimicrobial peptides and requires the participation of the bacterial protein BacA. We have studied bacteroid differentiation of Sinorhizobium fredii HH103 in three host plants: Glycine max, Cajanus cajan and the IRLC legume Glycyrrhiza uralensis. Flow cytometry, microscopy analyses and viability studies of bacteroids as well as confocal microscopy studies carried out in nodules showed that S. fredii HH103 bacteroids, regardless of the host plant, had deoxyribonucleic acid (DNA) contents, cellular sizes and survival rates similar to those of free-living bacteria. Contrary to S. meliloti, S. fredii HH103 showed little or no sensitivity to Medicago NCR247 and NCR335 peptides. Inactivation of S. fredii HH103 bacA neither affected symbiosis with Glycyrrhiza nor increased bacterial sensitivity to Medicago NCRs. Finally, HH103 bacteroids isolated from Glycyrrhiza, but not those isolated from Cajanus or Glycine, showed an altered lipopolysaccharide. Our studies indicate that, in contrast to the S. meliloti-Medicago model symbiosis, bacteroids in the S. fredii HH103-Glycyrrhiza symbiosis do not undergo NCR-induced and bacA-dependent terminal differentiation.

Sinorhizobium fredii indigenous populations are prevalent in provinces of Central China whereas Bradyrhizobium species (Bradyrhizobium japonicum, B. diazoefficiens, B. elkanii, and others) are more abundant in northern and southern provinces. The symbiotic properties of different soybean rhizobia have been investigated with 40 different wild soybean (Glycine soja) accessions from China, Japan, Russia, and South Korea. Bradyrhizobial strains nodulated all the wild soybeans tested, albeit efficiency of nitrogen fixation varied considerably among accessions. The symbiotic capacity of S. fredii HH103 with wild soybeans from Central China was clearly better than with the accessions found elsewhere. S. fredii NGR234, the rhizobial strain showing the broadest host range ever described, also formed nitrogen-fixing nodules with different G. soja accessions from Central China. To our knowledge, this is the first report describing an effective symbiosis between S. fredii NGR234 and G. soja. Mobilization of the S. fredii HH103 symbiotic plasmid to a NGR234 pSym-cured derivative (strain NGR234C) yielded transconjugants that formed ineffective nodules with G. max cv. Williams 82 and G. soja accession CH4. By contrast, transfer of the symbiotic plasmid pNGR234a to a pSym-cured derivative of S. fredii USDA193 generated transconjugants that effectively nodulated G. soja accession CH4 but failed to nodulate with G. max cv. Williams 82. These results indicate that intra-specific transference of the S. fredii symbiotic plasmids generates new strains with unpredictable symbiotic properties, probably due to the occurrence of new combinations of symbiotic signals. España, Junta de Andalucía P11-CVI-7500 España Ministerio de Economía y Competitividad BIO2016-78409-R

Lotus japonicus is a well-studied nodulating legume and a model organism for the investigation of plant-microbe interactions. The majority of legume transcriptome studies have focused on interactions with compatible symbionts, whereas responses to non-adapted rhizobia and pathogenic bacteria have not been well-characterized. In this study, we first characterized the transcriptomic response of L. japonicus to its compatible symbiont, Mesorhizobium loti R7A, through RNA-seq analysis of various plant tissues. Early symbiotic signaling was largely Nod factor-dependent and enhanced within root hairs, and we observed large-scale transcriptional reprogramming in nodule primordia and mature nitrogen-fixing nodules. We then characterized root transcriptional responses to a spectrum of L. japonicus interacting bacteria ranging from semi-compatible symbionts to pathogens. M. loti R7A and the semi-compatible strain Sinorhizobium fredii HH103 showed remarkably similar responses, allowing us to identify a small number of genes potentially involved in differentiating between fully and semi-compatible symbionts. The incompatible symbiont Bradyrhizobium elkanii USDA61 induced a more attenuated response, but the weakest response was observed for the foliar pathogen Pseudomonas syringae pv. tomato DC3000, where the affected genes also responded to other tested bacteria, pointing to a small set of common bacterial response genes. In contrast, the root pathogen Ralstonia solanacearum JS763 induced a pronounced and distinct transcriptomic pathogen response, which we compared to the results of the other treatments. This comparative analysis did not support the concept that an early defense-like response is generally evoked by compatible rhizobia during establishment of symbiosis.

Sinorhizobium fredii is well known for its ability to establish symbiosis with diverse legumes such as Glycine max (soybean, determinate nodules) and Cajanus cajan (pigeon pea, indeterminate nodules). In order to make screening of S. fredii genes related to symbiosis cost-effective, we constructed a large Tn5 insertion mutant library of S. fredii CCBAU45436 using the signature-tagged mutagenesis (STM) technique. This STM library contains a total of 25,500 independent mutants distributed in 17 sublibraries tagged by corresponding distinct DNA bar-code sequences. After the pilot screening of 255 mutants in 15 batches, Tag85-4, Tag4-17, Tag4-11 and Tag10-13 were found to have attenuated competitiveness (0-30 % in nodule occupation) compared to the wild-type strain when inoculated on soybean. Further characterization of these mutants suggests that Tag4-11 (a pyrC mutant) and Tag10-13 (a nrdJ mutant) are defective in establishing symbiosis with soybean. The pyrC mutant induced uninfected pseudonodules while the nrdJ mutant formed significantly more nodules containing bacteroids with poor persistence ability. When these two mutants were tested on pigeon pea, host-specific symbiotic defects were found. These results demonstrated the STM library as a valuable resource for identifying S. fredii genes relevant to symbiosis.

© 2015 The American Phytopathological Society. Plants that interact with pathogenic bacteria in their natural environments have developed barriers to block or contain the infection. Phytopathogenic bacteria have evolved mechanisms to subvert these defenses and promote infection. Thus, the type 3 secretion system (T3SS) delivers bacterial effectors directly into the plant cells to alter host signaling and suppress defenses, providing an appropriate environment for bacterial multiplication. Some rhizobial strains possess a symbiotic T3SS that seems to be involved in the suppression of host defenses to promote nodulation and determine the host range. In this work, we show that the inactivation of the Sinorhizobium (Ensifer) fredii HH103 T3SS negatively affects soybean nodulation in the early stages of the symbiotic process, which is associated with a reduction of the expression of early nodulation genes. This symbiotic phenotype could be the consequence of the bacterial triggering of soybean defense responses associated with the production of salicylic acid (SA) and the impairment of the T3SS mutant to suppress these responses. Interestingly, the early induction of the transcription of GmMPK4, which negatively regulates SA accumulation and defense responses in soybean via WRKY33, could be associated with the differential defense responses induced by the parental and the T3SS mutant strain.

The bacterium Sinorhizobium meliloti Rm2011 forms N2-fixing root nodules on alfalfa and other leguminous plants. The pSymB chromid contains a 110-kb region (the ETR region) showing high synteny to a chromosomally located region in Sinorhizobium fredii NGR234 and related rhizobia. We recently introduced the ETR region from S. fredii NGR234 into the S. meliloti chromosome. Here, we report that, unexpectedly, the S. fredii NGR234 ETR region did not complement deletion of the S. meliloti ETR region in symbiosis with Medicago sativa. This phenotype was due to the bacA gene of NGR234 not being functionally interchangeable with the S. meliloti bacA gene during M. sativa symbiosis. Further analysis revealed that, whereas bacA genes from S. fredii or Rhizobium leguminosarum bv. viciae 3841 failed to complement the Fix− phenotype of a S. meliloti bacA mutant with M. sativa, they allowed for further developmental progression prior to a loss of viability. In contrast, with Melilotus alba, bacA from S. fredii and R. leguminosarum supported N2 fixation by a S. meliloti bacA mutant. Additionally, the S. meliloti bacA gene can support N2 fixation of a R. leguminosarum bacA mutant during symbiosis with Pisum sativum. A phylogeny of BacA proteins illustrated that S. meliloti BacA has rapidly diverged from most rhizobia and has converged toward the sequence of pathogenic genera Brucella and Escherichia. These data suggest that the S. meliloti BacA has evolved toward a specific interaction with Medicago and highlights the limitations of using a single model system for the study of complex biological topics.

et al., [Background]: Rhizobial surface polysaccharides are important molecular determinants required for successful symbiosis with legumes. In Sinorhizobium (Ensifer) meliloti Rm41, the rkp-2 region is involved in the biosynthesis of K-antigen polysaccharide (KPS) and lipopolysaccharide (LPS). This region is composed of two genes, lpsL and rkpK, which are respectively responsible for the production of galacturonic and glucuronic acid. [Results]: In this work, we show that in S. (Ensifer) fredii HH103 these genes do not form a transcriptional unit and that the transcriptional rate of rkpK is much higher than that of lpsL. Inactivation of each of these genes resulted in alterations in LPS, but did not affect KPS production, which is in agreement with the lack of uronic acids in S. fredii HH103 KPS. Mutation of rkpK also impaired HH103 exopolysaccharide (EPS) production, most probably due to the presence of glucuronic acid in HH103 EPS, as well as increased bacterial autoaggregation and osmosensitivity and decreased biofilm formation on plastic surfaces. Inactivation of rkpK affected negatively symbiosis with cowpea but not with soybean. Mutation of lpsL led to a complete symbiotic impairment with cowpea, whereas soybean plants inoculated with this mutant only formed pseudonodules. In both plants, the lpsL mutant showed defects in root infection. [Conclusion]: These results confirm the symbiotic importance of HH103 LPS in symbiosis with legumes., This work was supported by grants from the Spanish Ministry of Science and Innovation (BIO2011-30229 and BIO2016-78409-R) and the Andalusia Government (P07-CVI07500). SAJ and PNG are recipients of PhD grants from the VPPI of the University of Seville.

Endophytic bacteria from roots and leaves of rice plants were isolated and identified in order to select the diazotrophs and improve their nitrogen-fixing abilities. The nitrogen-fixing endophytes were identified by PCR amplification of the nifH gene fragment. For this purpose, two isolates, Enterobacter cloacae RCA25 and Klebsiella variicola RCA26, and two model bacteria (Herbaspirillum seropedicae z67 and Sinorhizobium fredii NGR234) were transformed to increase the biosynthesis of the main plant auxin indole-3-acetic acid (IAA). A significant increase in the production of IAA was observed for all strains. When the expression of nifH gene and the activity of the nitrogenase enzyme were analyzed in liquid cultures, we found that they were positively affected in the IAA-overproducing endophytes as compared to the wild-type ones. Rice plants inoculated with these modified strains showed a significant upregulation of the nitrogenase activity when plants infected with the wild-type strains were used as reference. Similar results were obtained too with common bean plants infected with the S. fredii NGR234 strain. These findings suggest that IAA overproduction improves nitrogen-fixing apparatus of endophytic bacteria both in liquid cultures and in inoculated host plants. The present study highlights new perspectives to enhance nitrogen-fixing ability in non-legume crops. These strains could be used as bioinoculants to improve the growth and the yield of agricultural crops, offering an alternative to the use of chemical nitrogen fertilizers.

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

Sulfur is an essential element for rhizobia, such as sulfated modified Nod factors and nitrogenase. To investigate the role of sulfur metabolism in Rhizobium-Soybean symbiosis, a transponson random insertional mutants' library was constructed and a sulfur assimilation-related gene was isolated and characterized. A mutant strain unable to utilized sulfate was screened from 11,000 random insertional mutants of Sinorhizobium fredii WGF03. Sequencing analysis showed that a sulfate assimilation-related gene (cysDN) was inserted by the Tn transponson. Mutants inactivated in cysD and cysN (SMcysDF and SMcysNF) were constructed by homologous recombination using the suicide plasmid pK18mob. The mutants SMcysDF and SMcysNF could no longer utilize sulfate as sulfur source. Phenotype analysis revealed that mutation of cysDN had multiple effects on S. fredii WGF03. Root hair deformation assay showed that the activity of Nod factors secreted by mutants SMcysDR and SMcysNR elicited minimal hair initiation only. Soybean plant tests indicated that the mutant strains delayed 1-2 days to nodulate and exhibited lower nodulation efficiency and symbiotic efficiency than the wild-type strain. The complementary strain of cysD and cysN (HcysDF and HcysNF) could restore the nodulation efficiency.

Many bacteria regulate their gene expression in response to changes in their population density in a process called quorum sensing (QS), which involves communication between cells mediated by small diffusible signal molecules termed autoinducers. n -acyl-homoserine-lactones (AHLs) are the most common autoinducers in proteobacteria. QS-regulated genes are involved in complex interactions between bacteria of the same or different species and even with some eukaryotic organisms. Eukaryotes, including plants, can interfere with bacterial QS systems by synthesizing molecules that interfere with bacterial QS systems. In this work, the presence of AHL-mimic QS molecules in diverse Oryza sativa (rice) and Phaseolus vulgaris (bean) plant-samples were detected employing three biosensor strains. A more intensive analysis using biosensors carrying the lactonase enzyme showed that bean and rice seed-extract contain molecules that lack the typical lactone ring of AHLs. Interestingly, these molecules specifically alter the QS-regulated biofilm formation of two plant-associated bacteria, Sinorhizobium fredii SMH12 and Pantoea ananatis AMG501, suggesting that plants are able to enhance or to inhibit the bacterial QS systems depending on the bacterial strain. Further studies would contribute to a better understanding of plant–bacteria relationships at the molecular level.

Sinorhizobium fredii HH103-Rifr, a broad host range rhizobial strain, induces nitrogen-fixing nodules in Lotus burttii but ineffective nodules in L. japonicus. Confocal microscopy studies showed that Mesorhizobium loti MAFF303099 and S. fredii HH103-Rifr invade L. burttii roots through infection threads or epidermal cracks, respectively. Infection threads in root hairs were not observed in L. burttii plants inoculated with S. fredii HH103-Rifr. A S. fredii HH103-Rifr nodA mutant failed to nodulate L. burttii, demonstrating that Nod factors are strictly necessary for this crack-entry mode, and a noeL mutant was also severely impaired in L. burttii nodulation, indicating that the presence of fucosyl residues in the Nod factor is symbiotically relevant. However, significant symbiotic impacts due to the absence of methylation or to acetylation of the fucosyl residue were not detected. In contrast S. fredii HH103-Rifr mutants showing lipopolysaccharide alterations had reduced symbiotic capacity, while mutants affected in production of either exopolysaccharides, capsular polysaccharides, or both were not impaired in nodulation. Mutants unable to produce cyclic glucans and purine or pyrimidine auxotrophic mutants formed ineffective nodules with L. burttii. Flagellin-dependent bacterial mobility was not required for crack infection, since HH103-Rifr fla mutants nodulated L. burttii. None of the S. fredii HH103-Rifr surface-polysaccharide mutants gained effective nodulation with L. japonicus.

Plant-released flavonoids induce the transcription of symbiotic genes in rhizobia and one of the first bacterial responses is the synthesis of so called Nod factors. They are responsible for the initial root hair curling during onset of root nodule development. This signal exchange is believed to be essential for initiating the plant symbiosis with rhizobia affiliated with the Alphaproteobacteria. Here, we provide evidence that in the broad host range strain Sinorhizobium fredii NGR234 the complete lack of quorum sensing molecules results in an elevated copy number of its symbiotic plasmid (pNGR234a). This in turn triggers the expression of symbiotic genes and the production of Nod factors in the absence of plant signals. Therefore, increasing the copy number of specific plasmids could be a widespread mechanism of specialized bacterial populations to bridge gaps in signalling cascades.

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N2 -fixation. Deletion of smb20752 resulted in lower nitrogenase activity and smaller nodules without impacting overall nodule morphology. Orthologs of smb20752 were present in all alpha and beta rhizobia, including the ngr_b20860 gene of Sinorhizobium fredii NGR234. A ngr_b20860 mutant formed Fix- determinate nodules that developed normally to a late stage of the symbiosis on the host plants Macroptilium atropurpureum and Vigna unguiculata. However an early symbiotic defect was evident during symbiosis with Leucaena leucocephala, producing Fix- indeterminate nodules. The smb20752 and ngr_b20860 genes encode putative 3-hydroxyisobutyryl-CoA (HIB-CoA) hydrolases. HIB-CoA hydrolases are required for l-valine catabolism and appear to prevent the accumulation of toxic metabolic intermediates, particularly methacrylyl-CoA. Evidence presented here and elsewhere (Curson et al., , PLoS ONE 9:e97660) demonstrated that Smb20752 and NGR_b20860 can also prevent metabolic toxicity, are required for l-valine metabolism, and play an undefined role in 3-hydroxybutyrate catabolism. We present evidence that the symbiotic defect of the HIB-CoA hydrolase mutants is independent of the inability to catabolize l-valine and suggest it relates to the toxicity resulting from metabolism of other compounds possibly related to 3-hydroxybutyric acid.

Twenty-seven and twenty-four soybean rhizobial isolates from Java and Sumatra, respectively,were characterized. Based on cross-inoculation, eight isolates from Java and nine from Sumatra could be grouped as soybean specific rhizobial species , while 19 isolates from Java and 15 from Sumatra were promiscuous. ARDRA of intergenic spacer region of 16S-23S rDNA showed that the isolates from Java were different from those from Sumatra. Six soybean specific isolates from Java and one from Sumatra were in the same cluster with the reference strain, Bradyrhizobium japonicum USDA 110, thus could be classified as B . japonicum . One soybean specific isolate from Java’s has a distinct position, while the other soybean specific isolate from Java was placed in another group dominated by isolates from Sumatra. The nineteen promiscuous isolates from Java were clustered in a different group. This group, together with the isolate with distinct position and the other group that were dominated by isolates from Sumatra, were distinct from B. japonicum USDA 110. Therefore it is tempting to speculate that they represent indigenous soybean rhizobial strains. Based on complete sequencing of the amplified 16S rDNA of 21 selected isolates, these isolates could be divided into three groups consisting of twelve Bradyrhizobium elkanii , eight Bradyrizobium japonicum and one Sinorhizobium fredii . Most of the B. elkanii strains were isolated from acid soils at Sitiung, West-Sumatra, while only two isolates were obtained from Java. Four isolates from Java, two isolates from Sitiung, and two isolates from Bukit Tinggi were identified as B. japonicum . One isolate from Java with a distinct position on the ARDRA was identified as S. fredii .

Sinorhizobium fredii USDA257 and S. fredii USDA191 are fast-growing rhizobia that form nitrogen-fixing nodules on soybean roots. In contrast to USDA191, USDA257 exhibits cultivar specificity and can form nodules only on primitive soybean cultivars. In response to flavonoids released from soybean roots, these two rhizobia secrete n odulation o uter p roteins (Nop) to the extracellular milieu through a type III secretion system. In spite of the fact that Nops are known to regulate legume nodulation in a host-specific manner, very little is known about the differences in the compositions of Nops and surface appendages elaborated by USDA191 and USDA257. In this study we compared the Nop profiles of USDA191 and USDA257 by one-dimensional (1D) and 2D gel electrophoresis and identified several of these proteins by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and liquid chromatography-tandem MS (LC-MS/MS). Examination of the surface appendages elaborated by these two strains of soybean symbionts by transmission electron microscopy revealed distinct differences in their morphologies. Even though the flagella produced by USDA191 and USDA257 were similar in their morphologies, they differed in their flagellin composition. USDA257 pili resembled long thin filaments, while USDA191 pili were short, rod shaped, and much thinner than the flagella. 2D gel electrophoresis of pilus-like appendages of USDA191 and USDA257 followed by mass spectrometry resulted in the identification of several of the Nops along with some proteins previously undetected in these strains. Some of the newly identified proteins show homology to putative zinc protease and a LabA-like protein from Bradyrhizobium sp. ORS278, fimbrial type 4 assembly proteins from Ralstonia solanacearum , and the type III effector Hrp-dependent protein from Rhizobium leguminosarum bv. trifolii .

Legume-nodulating rhizobia use N-acyl homoserine lactones (AHLs) to regulate several physiological traits related to the symbiotic plant–microbe interaction. In this work, we show that Sinorhizobium fredii SMH12, Rhizobium etli ISP42 and Rhizobium sullae IS123, three rhizobial strains with different nodulation ranges, produced a similar pattern of AHL molecules, sharing, in all cases, production of N-octanoyl homoserine lactone and its 3-oxo and/or 3-hydroxy derivatives. Interestingly, production of AHLs was enhanced when these three rhizobia were grown in the presence of their respective nod-gene-inducing flavonoid, while a new molecule, C14-HSL, was produced by S. fredii SMH12 upon genistein induction. In addition, expression of AHL synthesis genes traI from S. fredii SMH12 and cinI and raiI from R. etli ISP42 increased when induced with flavonoids, as demonstrated by qRT-PCR analysis.

A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S–23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study.

Rhizobia are a disparate collection of soil bacteria capable of reducing atmospheric nitrogen in symbiosis with legumes (Fix phenotype). Synthesis of the nitrogenase and its accessory components is under the transcriptional control of the key regulator NifA and is generally restricted to the endosymbiotic forms of rhizobia known as bacteroids. Amongst studied rhizobia, Sinorhizobium fredii strain NGR234 has the remarkable ability to fix nitrogen in association with more than 130 species in 73 legume genera that form either determinate, indeterminate or aeschynomenoid nodules. Hence, NGR234 is a model organism to study nitrogen fixation in association with a variety of legumes. The symbiotic plasmid pSfrNGR234a carries more than 50 genes that are under the transcriptional control of NifA. To facilitate the functional analysis of NifA-regulated genes a new transposable element, TnEKm-PwA, was constructed. This transposon combines the advantages of in vitro mutagenesis of cloned DNA fragments with a conditional read-out promoter from NGR234 (PwA) that reinitiates NifA-dependent transcription downstream of transposition sites. To test the characteristics of the new transposon, the nifQdctA1y4vGHIJ operon was mutated using either the Omega interposon or TnEKm-PwA. The symbiotic phenotypes on various hosts as well as the transcriptional characteristics of these mutants were analysed in detail and compared with the ineffective (Fix−) phenotype of strain NGRΔnifA, which lacks a functional copy of nifA. De novo transcription from inserted copies of TnEKm-PwA inside bacteroids was confirmed by qRT-PCR. Unexpectedly, polar mutants in dctA1 and nifQ were Fix+ on all of the hosts tested, indicating that none of the six genes of the nifQ operon of NGR234 is essential for symbiotic nitrogen fixation on plants that form nodules of either determinate or indeterminate types.

Sinorhizobium fredii is a nitrogen-fixing legume symbiont that stimulates the formation of root nodules. S. fredii nodulation of roots is influenced by Nop proteins, which are secreted through a type III secretion system (T3SS). We demonstrate that S. fredii injects NopP into Vigna unguiculata nodules in a T3SS-dependent manner.

Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters.

Here we analyzed carboxymethylcellulase (CMCase; EC 3, 2, 1, 4), one of the key enzymes in the early symbiotic process, in Rhizobium. Specific immunogold labeling of electron microscopy was confirmed in Sinorhizobium fredii BCRC15769, ATCC35423, Sinorhizobium meliloti ATCC9930, and barely detected in Bradyrhizobium japonicum BCRC13528, ATCC10324 and Rhizobium rhizogenes ATCC11325. Non-specific labeling was detected in Rhizobium leguminosarum bv. viceae ATCC10004, Rhizobium leguminosarum bv. trifolii ATCC10328, and Mesorhizobium loti ATCC33669. Treatment of S. fredii BCRC15769 in the early log phase with the flavonoid genistein caused relocalization of CMCase. Together our data suggests a role for CMCase in early symbiosis.