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To successfully colonize legume root nodules, rhizobia must effectively evade host-generated reactive oxygen species (ROS). LsrB, a redox regulator from Sinorhizobium meliloti , is essential for symbiosis with alfalfa ( Medicago sativa ). The three cysteine residues in LsrB's substrate domain play distinct roles in activating downstream redox genes. The study found that LsrB's substrate-binding domain, dependent on the cysteine residue Cys146, is involved in oxidized glutathione (GSSG) resistance and alfalfa nodulation symbiosis. LsrB homologues from other rhizobia, with Cys172/Cys238 or Cys146, enhance GSSG resistance and complement lsrB mutant's symbiotic nodulation. Substituting amino acids in Azorhizobium caulinodans LsrB with Cys restores lsrB mutant phenotypes. The lsrB deletion mutant shows increased sensitivity to NCR247, suggesting an interaction with host plant-derived NCRs in alfalfa nodules. Our findings reveal that the key cysteine residue in the LsrB's substrate domain is vital for rhizobium-legume symbiosis.

Sinorhizobium meliloti senses nutrients and compounds exuded from alfalfa host roots and coordinates an excitation, termination, and adaptation pathway during chemotaxis. We investigated the role of the novel S. meliloti chemotaxis protein CheT. While CheT and the Escherichia coli phosphatase CheZ share little sequence homology, CheT is predicted to possess an α-helix with a DXXXQ phosphatase motif. Phosphorylation assays demonstrated that CheT dephosphorylates the phosphate-sink response regulator, CheY1~P by enhancing its decay two-fold but does not affect the motor response regulator CheY2~P. Isothermal Titration Calorimetry (ITC) experiments revealed that CheT binds to a phosphomimic of CheY1~P with a K D of 2.9 μM, which is 25-fold stronger than its binding to CheY1. Dissimilar chemotaxis phenotypes of the ΔcheT mutant and cheT DXXXQ phosphatase mutants led to the hypothesis that CheT exerts additional function(s). A screen for potential binding partners of CheT revealed that it forms a complex with the methyltransferase CheR. ITC experiments confirmed CheT/CheR binding with a K D of 19 μM, and a SEC-MALS analysis determined a 1:1 and 2:1 CheT/CheR complex formation. Although they did not affect each other's enzymatic activity, CheT binding to CheY1~P and CheR may serve as a link between signal termination and sensory adaptation., (© 2024 The Author(s). Molecular Microbiology published by John Wiley & Sons Ltd.)

In the understanding of the molecular interaction between plants and their microbiome, a key point is to identify simplified models of the microbiome including relevant bacterial and fungal partners which could also be effective in plant growth promotion. Here, as proof-of-concept, we aim to identify the possible molecular interactions between symbiotic nitrogen-fixing rhizobia and soil fungi (Trichoderma spp.), hence shed light on synergistic roles rhizospheric fungi could have in the biology of symbiotic nitrogen fixation bacteria. We selected 4 strains of the model rhizobium Sinorhizobium meliloti and 4 Trichoderma species (T. velutinum, T. tomentosum, T. gamsii and T. harzianum). In an experimental scheme of 4 ×4 strains x species combinations, we investigated the rhizobia physiological and transcriptomic responses elicited by fungal spent media, as well as spent media effects on rhizobia-host legume plant (alfalfa, Medicago sativa L.) symbiosis. Fungal spent media had large effects on rhizobia, specific for each fungal species and rhizobial strains combination, indicating a generalized rhizobia genotype x fungal genotype interaction, including synergistic, neutral and antagonistic effects on alfalfa symbiotic phenotypes. Differential expression of a high number of genes was shown in rhizobia strains with up to 25% of total genes differentially expressed upon treatment of cultures with fungal spent media. Percentages over total genes and type of genes differentially expressed changed according to both fungal species and rhizobial strain. To support the hypothesis of a relevant rhizobia genotype x fungal genotype interaction, a nested Likelihood Ratio Test indicated that the model considering the fungus-rhizobium interaction explained 23.4% of differentially expressed genes. Our results provide insights into molecular interactions involving nitrogen-fixing rhizobia and rhizospheric fungi, highlighting the panoply of genes and genotypic interactions (fungus, rhizobium, host plant) which may concur to plant symbiosis., (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Rhizobial phosphatidylcholine (PC) is thought to be a critical phospholipid for the symbiotic relationship between rhizobia and legume host plants. A PC-deficient mutant of Sinorhizobium meliloti overproduces succinoglycan, is unable to swim, and lacks the ability to form nodules on alfalfa (Medicago sativa) host roots. Suppressor mutants had been obtained which did not overproduce succinoglycan and regained the ability to swim. Previously, we showed that point mutations leading to altered ExoS proteins can reverse the succinoglycan and swimming phenotypes of a PC-deficient mutant. Here, we report that other point mutations leading to altered ExoS, ChvI, FabA, or RpoH1 proteins also revert the succinoglycan and swimming phenotypes of PC-deficient mutants. Notably, the suppressor mutants also restore the ability to form nodule organs on alfalfa roots. However, nodules generated by these suppressor mutants express only low levels of an early nodulin, do not induce leghemoglobin transcript accumulation, thus remain white, and are unable to fix nitrogen. Among these suppressor mutants, we detected a reduced function mutant of the 3-hydoxydecanoyl-acyl carrier protein dehydratase FabA that produces reduced amounts of unsaturated and increased amounts of shorter chain fatty acids. This alteration of fatty acid composition probably affects lipid packing thereby partially compensating for the previous loss of PC and contributing to the restoration of membrane homeostasis., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Importance: Root nodule endosymbioses between diazotrophic rhizobia and legumes provide the largest input of combined N to the biosphere, thus representing an alternative to harmful chemical fertilizers for sustainable crop production. Rhizobia have evolved intricate strategies to coordinate N assimilation for their own benefit with N 2 fixation to sustain plant growth. The rhizobial N status is transduced by the NtrBC two-component system, the seemingly ubiquitous form of N signal transduction in Proteobacteria. Here, we show that the regulatory sRNA NfeR1 (nodule formation efficiency RNA) of the alfalfa symbiont Sinorhizobium meliloti is transcribed from a complex promoter repressed by NtrC in a N-dependent manner and feedback silences ntrBC by complementary base-pairing. These findings unveil a more prominent role of NtrC as a transcriptional repressor than hitherto anticipated and a novel RNA-based mechanism for NtrBC regulation. The NtrBC-NfeR1 double-negative feedback loop accurately rewires symbiotic S. meliloti N metabolism and is likely conserved in α-rhizobia., Competing Interests: The authors declare no conflict of interest.

Bacterial surface components and extracellular compounds such as exopolysaccharides (EPSs) are crucial for interactions between cells, tolerance to different types of stress, and host colonization. Sinorhizobium meliloti produces two EPSs: Succinoglycan (EPS I), which is involved in the establishment of symbiosis with Medicago sativa, and galactoglucan (EPS II), associated with biofilm formation and the promotion of aggregation. Here, we aimed to assess their role in aggregative interactions between cells of the same strain of a given species (auto-aggregation), and between genetically different strains of the same or different species (intra- or intergeneric coaggregation). To do this, we used S. meliloti mutants which are defective in the production of EPS I, EPS II, or both. Macroscopic and microscopic coaggregation tests were performed with combinations or pairs of different bacterial strains. The EPS II-producing strains were more capable of coaggregation than those that cannot produce EPS II. This was true both for coaggregations between different S. meliloti strains, and between S. meliloti and other common rhizobacteria of agricultural relevance, such as Pseudomonas fluorescens and Azospirillum brasilense. The exogenous addition of EPS II strongly promoted coaggregation, thus confirming the polymer's importance for this phenotype. EPS II may therefore be a key factor in events of physiological significance for environmental survival, such as aggregative interactions and biofilm development. Furthermore, it might be a connecting molecule with relevant properties at an ecological, biotechnological, and agricultural level., (© 2023 Wiley-VCH GmbH.)

Sustainable effect of a nitrogen-fixing bacterium Sinorhizobium meliloti on nodulation and photosynthetic traits (phenomenological fluxes) in four leguminous plants species under low moisture stress (20-25% soil moisture content) environment was studied. Sinorhizobium  meliloti was isolated from fenugreek (Trigonella foenum-graecum) root nodules, and later, it was cultured and purified. Nodulation and photosynthetic ability in the presence of S.  meliloti were tested in four leguminous plant species, that is, kidney bean (cv. lobia-2000), black bean (cv. NM-97), mung bean (cv. NM-2006) and chickpea (cv. Pb-2008). Plants of each species were grown in sterilized soil that was previously treated with 25 ml suspension containing S.  meliloti at 41 × 10 6  CFU ml -1  kg -1 pot. One-month-old plants were subjected to low soil moisture stress conditions for 15 days, and soil moisture contents were maintained to 20-25% throughout the experimental period. The ability to fix nitrogen, nodule formation, and their subsequent effect on phenomenological fluxes in low moisture treated legumes were studied., (© 2021 The Society for Applied Microbiology.)

The rhizosphere and rhizoplane are nutrient-rich but selective environments for the root microbiome. Here, we deciphered a posttranscriptional network regulated by the homologous trans -small RNAs (sRNAs) AbcR1 and AbcR2, which rewire the metabolism of the nitrogen-fixing α-rhizobium Sinorhizobium meliloti during preinfection stages of symbiosis with its legume host alfalfa. The LysR-type regulator LsrB, which transduces the cell redox state, is indispensable for AbcR1 expression in actively dividing bacteria, whereas the stress-induced transcription of AbcR2 depends on the alternative σ factor RpoH1. MS2 affinity purification coupled with RNA sequencing unveiled exceptionally large and overlapping AbcR1/2 mRNA interactomes, jointly representing ⁓6% of the S. meliloti protein-coding genes. Most mRNAs encode transport/metabolic proteins whose translation is silenced by base pairing to two distinct anti-Shine Dalgarno motifs that function independently in both sRNAs. A metabolic model-aided analysis of the targetomes predicted changes in AbcR1/2 expression driven by shifts in carbon/nitrogen sources, which were confirmed experimentally. Low AbcR1/2 levels in some defined media anticipated overexpression growth phenotypes linked to the silencing of specific mRNAs. As a proof of principle, we confirmed AbcR1/2-mediated downregulation of the l-amino acid AapQ permease. AbcR1/2 interactomes are well represented in rhizosphere-related S. meliloti transcriptomic signatures. Remarkably, a lack of AbcR1 specifically compromised the ability of S. meliloti to colonize the root rhizoplane. The AbcR1 regulon likely ranks the utilization of available substrates to optimize metabolism, thus conferring on S. meliloti an advantage for efficient rhizosphere/rhizoplane colonization. AbcR1 regulation is predicted to be conserved in related α-rhizobia, which opens unprecedented possibilities for engineering highly competitive biofertilizers. IMPORTANCE Nitrogen-fixing root nodule symbioses between rhizobia and legume plants provide more than half of the combined nitrogen incorporated annually into terrestrial ecosystems, rendering plant growth independent of environmentally unfriendly chemical fertilizers. The success of symbiosis depends primarily on the capacity of rhizobia to establish competitive populations in soil and rhizosphere environments. Here, we provide insights into the regulation and architecture of an extensive RNA posttranscriptional network that fine-tunes the metabolism of the alfalfa symbiont S. meliloti, thereby enhancing the ability of this beneficial bacterium to colonize nutrient-rich but extremely selective niches, such as the rhizosphere of its host plant. This pervasive RNA regulation of metabolism is a major adaptive mechanism, predicted to operate in diverse rhizobial species. Because RNA regulation relies on modifiable base-pairing interactions, our findings open unexplored avenues for engineering the legumes rhizobiome within sustainable agricultural practices.

Sinorhizobium meliloti is a soil bacterium of great agricultural importance because of its ability to fix atmospheric nitrogen in symbiotic association with alfalfa ( Medicago sativa ) roots. We looked into the involvement of exopolysaccharides (EPS) in its survival when exposed to different environmental stressors, as well as in bacteria-bacteria and bacteria-substrate interactions. The strains used were wild-type Rm8530 and two strains that are defective in the biosynthesis of EPS II: wild-type Rm1021, which has a non-functional expR locus, and mutant Rm8530 expA . Under stress by water deficiency, Rm8530 remained viable and increased in number, whereas Rm1021 and Rm8530 expA did not. These differences could be due to Rm8530's ability to produce EPS II. Survival experiments under saline stress showed that viability was reduced for Rm1021 but not for Rm8530 or Rm8530 expA , which suggests the existence of some regulating mechanism dependent on a functional expR that is absent in Rm1021. The results of salinity-induced stress assays regarding biofilm-forming capacity (BFC) and autoaggregation indicated the protective role of EPS II. As a whole, our observations demonstrate that EPS play major roles in rhizobacterial survival.

Background: Leguminous plants alter patterns of gene expression in response to symbiotic colonization and infection by their cognate rhizobial bacteria, but the extent of the transcriptomic response has rarely been examined below the species level. Here we describe the identification of 12 rhizobial biotypes of Ensifer meliloti, which form nitrogen-fixing nodules in the roots of alfalfa (Medicago sativa L.), followed by a comparative RNA-seq analysis of four alfalfa cultivars each inoculated with two E. meliloti strains varying in symbiotic performance and phylogenetic relatedness., Results: Rhizobial biotypes were identified on the basis of their symbiotic performance, particularly shoot dry weight. Differentially expressed genes (DEGs) and metabolic pathways were determined by comparing the RNA-seq data with that of the uninoculated control plant. Significant differences were found between DEGs generated in each cultivar with the inoculation of two rhizobial strains in comparison (P < 0.01). A total of 8111 genes was differentially expressed, representing ~ 17.1% of the M. sativa genome. The proportion of DEGs ranges from 0.5 to 12.2% for each alfalfa cultivar. Interestingly, genes with predicted roles in flavonoid biosynthesis and plant-pathogen interaction (NBS-LRR) were identified as the most significant DEGs. Other DEGs include Medsa002106 and genes encoding nodulins and NCR peptides whose expression is specifically induced during the development of nitrogen-fixing nodules. More importantly, strong significant positive correlations were observed between plant transcriptomes (DEGs and KEGG pathways) and phylogenetic distances between the two rhizobial inoculants., Conclusions: Alfalfa expresses significantly distinct sets of genes in response to infection by different rhizobial strains at the below-species levels (i.e. biotype or strain). Candidate genes underlying the specific interactions include Medsa002106 and those encoding nodulins and NCR peptides and proteins in the NBS-LRR family.

In nitrogen-fixing rhizobia, emerging evidence shows significant roles for polyamines in growth and abiotic stress resistance. In this work we show that a polyamine-deficient ornithine decarboxylase null mutant ( odc2 ) derived from Sinorhizobium meliloti Rm8530 had significant phenotypic differences from the wild-type, including greatly reduced production of exopolysaccharides (EPS; ostensibly both succinoglycan and galactoglucan), increased sensitivity to oxidative stress and decreased swimming motility. The introduction of the odc2 gene borne on a plasmid into the odc2 mutant restored wild-type phenotypes for EPS production, growth under oxidative stress and swimming. The production of calcofluor-binding EPS (succinoglycan) by the odc2 mutant was also completely or mostly restored in the presence of exogenous spermidine (Spd), norspermidine (NSpd) or spermine (Spm). The odc2 mutant formed about 25 % more biofilm than the wild-type, and its ability to form biofilm was significantly inhibited by exogenous Spd, NSpd or Spm. The odc2 mutant formed a less efficient symbiosis with alfalfa, resulting in plants with significantly less biomass and height, more nodules but less nodule biomass, and 25 % less nitrogen-fixing activity. Exogenously supplied Put was not able to revert these phenotypes and caused a similar increase in plant height and dry weight in uninoculated plants and in those inoculated with the wild-type or odc2 mutant. We discuss ways in which polyamines might affect the phenotypes of the odc2 mutant.

Bacterial surface molecules have an important role in the rhizobia-legume symbiosis. Ensifer meliloti (previously, Sinorhizobium meliloti), a symbiotic Gram-negative rhizobacterium, produces two different exopolysaccharides (EPSs), termed EPS I (succinoglycan) and EPS II (galactoglucan), with different functions in the symbiotic process. Accordingly, we undertook a study comparing the potential differences in alfalfa nodulation by E. meliloti strains with differences in their EPSs production. Strains recommended for inoculation as well as laboratory strains and native strains isolated from alfalfa fields were investigated. This study concentrated on EPS-II production, which results in mucoid colonies that are dependent on the presence of an intact expR gene. The results revealed that although the studied strains exhibited different phenotypes, the differences did not affect alfalfa nodulation itself. However, subtle changes in timing and efficacy to the effects of inoculation with the different strains may result because of other as-yet unknown factors. Thus, additional research is needed to determine the most effective inoculant strains and the best conditions for improving alfalfa production under agricultural conditions.

Polyamines are ubiquitous molecules containing two or more amino groups that fulfill varied and often essential physiological and regulatory roles in all organisms. In the symbiotic nitrogen-fixing bacteria known as rhizobia, putrescine and homospermidine are invariably produced while spermidine and norspermidine synthesis appears to be restricted to the alfalfa microsymbiont Sinorhizobium meliloti. Studies with rhizobial mutants deficient in the synthesis of one or more polyamines have shown that these compounds are important for growth, stress resistance, motility, exopolysaccharide production and biofilm formation. In this review, we describe these studies and examine how polyamines are synthesized and regulated in rhizobia., (© FEMS 2019.)

Symbiotic nitrogen fixation (SNF) in the interaction between the soil bacteria Sinorhizobium meliloti and legume plant Medicago sativa is carried out in specialized root organs called nodules. During nodule development, each symbiont must drastically alter their proteins, transcripts, and metabolites in order to support nitrogen fixation. Moreover, bacteria within the nodules are under stress, including challenges by plant antimicrobial peptides, low pH, limited oxygen availability, and strongly reducing conditions, all of which challenge proteome integrity. S. meliloti stress adaptation, proteome remodeling, and quality control are controlled in part by the large oligomeric protease complexes HslUV and ClpXP1. To improve understanding of the roles of S. meliloti HslUV and ClpXP1 under free-living conditions and in symbiosis with M. sativa , we generated Δ hslU , Δ hslV , Δ hslUV , and Δ clpP1 knockout mutants. The shoot dry weight of M. sativa plants inoculated with each deletion mutant was significantly reduced, suggesting a role in symbiosis. Further, slower free-living growth of the Δ hslUV and Δ clpP1 mutants suggests that HslUV and ClpP1 were involved in adapting to heat stress, the while Δ hslU and Δ clpP1 mutants were sensitive to kanamycin. All deletion mutants produced less exopolysaccharide and succinoglycan, as shown by replicate spot plating and calcofluor binding. We also generated endogenous C-terminal enhanced green fluorescent protein (eGFP) fusions to HslU, HslV, ClpX, and ClpP1 in S. meliloti Using anti-eGFP antibodies, native coimmunoprecipitation experiments with proteins from free-living and nodule tissues were performed and analyzed by mass spectrometry. The results suggest that HslUV and ClpXP were closely associated with ribosomal and proteome quality control proteins, and they identified several novel putative protein-protein interactions. IMPORTANCE Symbiotic nitrogen fixation (SNF) is the primary means by which biologically available nitrogen enters the biosphere, and it is therefore a critical component of the global nitrogen cycle and modern agriculture. SNF is the result of highly coordinated interactions between legume plants and soil bacteria collectively referred to as rhizobia, e.g., Medicago sativa and S. meliloti , respectively. Accomplishing SNF requires significant proteome changes in both organisms to create a microaerobic environment suitable for high-level bacterial nitrogenase activity. The bacterial protease systems HslUV and ClpXP are important in proteome quality control, in metabolic remodeling, and in adapting to stress. This work shows that S. meliloti HslUV and ClpXP are involved in SNF, in exopolysaccharide production, and in free-living stress adaptation., (Copyright © 2019 American Society for Microbiology.)

Sinorhizobium meliloti is a soil-dwelling endosymbiont of alfalfa that has eight chemoreceptors to sense environmental stimuli during its free-living state. The functions of two receptors have been characterized, with McpU and McpX serving as general amino acid and quaternary ammonium compound sensors, respectively. Both receptors use a dual Cache ( ca lcium channels and che motaxis receptors) domain for ligand binding. We identified that the ligand-binding periplasmic region (PR) of McpV contains a single Cache domain. Homology modeling revealed that McpV PR is structurally similar to a sensor domain of a chemoreceptor with unknown function from Anaeromyxobacter dehalogenans , which crystallized with acetate in its binding pocket. We therefore assayed McpV for carboxylate binding and S. meliloti for carboxylate sensing. Differential scanning fluorimetry identified 10 potential ligands for McpV PR Nine of these are monocarboxylates with chain lengths between two and four carbons. We selected seven compounds for capillary assay analysis, which established positive chemotaxis of the S. meliloti wild type, with concentrations of peak attraction at 1 mM for acetate, propionate, pyruvate, and glycolate, and at 100 mM for formate and acetoacetate. Deletion of mcpV or mutation of residues essential for ligand coordination abolished positive chemotaxis to carboxylates. Using microcalorimetry, we determined that dissociation constants of the seven ligands with McpV PR were in the micromolar range. An McpV PR variant with a mutation in the ligand coordination site displayed no binding to isobutyrate or propionate. Of all the carboxylates tested as attractants, only glycolate was detected in alfalfa seed exudates. This work examines the relevance of carboxylates and their sensor to the rhizobium-legume interaction. IMPORTANCE Legumes share a unique association with certain soil-dwelling bacteria known broadly as rhizobia. Through concerted interorganismal communication, a legume allows intracellular infection by its cognate rhizobial species. The plant then forms an organ, the root nodule, dedicated to housing and supplying fixed carbon and nutrients to the bacteria. In return, the engulfed rhizobia, differentiated into bacteroids, fix atmospheric N 2 into ammonium for the plant host. This interplay is of great benefit to the cultivation of legumes, such as alfalfa and soybeans, and is initiated by chemotaxis to the host plant. This study on carboxylate chemotaxis contributes to the understanding of rhizobial survival and competition in the rhizosphere and aids the development of commercial inoculants., (Copyright © 2018 American Society for Microbiology.)

The importance of root nodule bacteria in biotechnology is determined by their distinctive feature: symbiotic nitrogen fixation resulting in the production of organic nitrogen-containing compounds. While interacting with host legume plants, the cells of these bacteria undergo global changes at all levels of expression of genetic information leading to the formation in root nodules of so-called bacteroids functioning as nitrogen fixation factories. The molecular mechanisms underlying plant-microbial symbiosis are actively investigated, and one of the most interesting and poorly studied aspects of this problem is the species-specificity of interaction between root nodule bacteria and host plants. In this work we have performed the proteomic analysis of the Sinorhizobium meliloti bacteroids isolated from two legume species: alfalfa (Medicago sativa L.) and yellow sweet clover (Melilotus officinalis L.). It has been shown that the S. meliloti bacteroids produce a lot of proteins (many of them associated with symbiosis) in a host-specific manner, i.e., only in certain host plant species. It has been demonstrated for the first time that the levels of expression in bacteroids of the genes encoding the ExoZ and MscL proteins responsible for the synthesis of surface lipopolysaccha-rides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features.

Agrobacterium tumefaciens infects and causes crown galls in dicot plants by transferring T-DNA from the Ti plasmid to the host plant via a type IV secretion system. This process requires appropriate environmental conditions, certain plant secretions, and bacterial regulators. In our previous work, a member of the LysR family of transcriptional regulators (LsrB) in Sinorhizobium meliloti was found to modulate its symbiotic interactions with the host plant alfalfa. However, the function of its homolog in A. tumefaciens remains unclear. In this study, we show that the LsrB protein of A. tumefaciens is required for efficient transformation of host plants. A lsrB deletion mutant of A. tumefaciens exhibits a number of defects, including in succinoglycan production, attachment, and resistance to oxidative stress and iron limitation. RNA-sequencing analysis indicated that 465 genes were significantly differentially expressed (upregulation of 162 genes and downregulation of 303 genes) in the mutant, compared with the wild-type strain, including those involved in succinoglycan production, iron transporter, and detoxification enzymes for oxidative stress. Moreover, expression of the lsrB gene from S. meliloti, Brucella abortus, or A. tumefaciens rescued the defects observed in the S. meliloti or A. tumefaciens lsrB deletion mutant. Our findings suggest that a conserved mechanism of LsrB function exists in symbiotic and pathogenic bacteria of the family Rhizobiaceae.

Surface motility and biofilm formation are behaviours which enable bacteria to infect their hosts and are controlled by different chemical signals. In the plant symbiotic alpha-proteobacterium Sinorhizobium meliloti, the lack of long-chain fatty acyl-coenzyme A synthetase activity (FadD) leads to increased surface motility, defects in biofilm development and impaired root colonization. In this study, analyses of lipid extracts and volatiles revealed that a fadD mutant accumulates 2-tridecanone (2-TDC), a methylketone (MK) known as a natural insecticide. Application of pure 2-TDC to the wild-type strain phenocopies the free-living and symbiotic behaviours of the fadD mutant. Structural features of the MK determine its ability to promote S. meliloti surface translocation, which is mainly mediated by a flagella-independent motility. Transcriptomic analyses showed that 2-TDC induces differential expression of iron uptake, redox and stress-related genes. Interestingly, this MK also influences surface motility and impairs biofilm formation in plant and animal pathogenic bacteria. Moreover, 2-TDC not only hampers alfalfa nodulation but also the development of tomato bacterial speck disease. This work assigns a new role to 2-TDC as an infochemical that affects important bacterial traits and hampers plant-bacteria interactions by interfering with microbial colonization of plant tissues., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The chemosensory system in Sinorhizobium meliloti has several important deviations from the widely studied enterobacterial paradigm. To better understand the differences between the two systems and how they are optimally tuned, we determined the cellular stoichiometry of the methyl-accepting chemotaxis proteins (MCPs) and the histidine kinase CheA in S. meliloti Quantitative immunoblotting was used to determine the total amount of MCPs and CheA per cell in S. meliloti The MCPs are present in the cell in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY and McpZ), whereas McpT was below the detection limit. The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 23.5:1, highly similar to that seen in Bacillus subtilis (23:1) and about 10 times higher than that in Escherichia coli (3.4:1). Different from E. coli , the high-abundance receptors in S. meliloti are lacking the carboxy-terminal NWETF pentapeptide that binds the CheR methyltransferase and CheB methylesterase. Using transcriptional lacZ fusions, we showed that chemoreceptors are positively controlled by the master regulators of motility, VisNR and Rem. In addition, FlbT, a class IIA transcriptional regulator of flagellins, also positively regulates the expression of most chemoreceptors except for McpT and McpY, identifying chemoreceptors as class III genes. Taken together, these results demonstrate that the chemosensory complex and the adaptation system in S. meliloti deviates significantly from the established enterobacterial paradigm but shares some similarities with B. subtilis IMPORTANCE The symbiotic soil bacterium Sinorhizobium meliloti is of great agricultural importance because of its nitrogen-fixing properties, which enhances growth of its plant symbiont, alfalfa. Chemotaxis provides a competitive advantage for bacteria to sense their environment and interact with their eukaryotic hosts. For a better understanding of the role of chemotaxis in these processes, detailed knowledge on the regulation and composition of the chemosensory machinery is essential. Here, we show that chemoreceptor gene expression in S. meliloti is controlled through the main transcriptional regulators of motility. Chemoreceptor abundance is much lower in S. meliloti than in Escherichia coli and Bacillus subtilis Moreover, the chemoreceptor-to-kinase CheA ratio is different from that of E. coli but similar to that of B. subtilis ., (Copyright © 2018 American Society for Microbiology.)

Glutathione (l-γ-glutamyl-l-cysteinylglycine) (GSH), one of the key antioxidants in Sinorhizobium meliloti , is required for the development of alfalfa ( Medicago sativa ) nitrogen-fixing nodules. Glutathione exists as either reduced glutathione (GSH) or oxidized glutathione (GSSG), and its content is regulated by two pathways in S. meliloti The first pathway is the de novo synthesis of glutathione from its constituent amino acids, namely, Glu, Cys, and Gly, catalyzed by γ-glutamylcysteine synthetase (GshA) and glutathione synthetase (GshB). The second pathway is the recycling of GSSG via glutathione reductase (GR). However, whether the S. meliloti GR functions similarly to GshA and GshB1 during symbiotic interactions with alfalfa remains unknown. In this study, a plasmid insertion mutation of the S. meliloti gor gene, which encodes GR, was constructed, and the mutant exhibited delayed alfalfa nodulation, with 75% reduction in nitrogen-fixing capacity. The gor mutant demonstrated increased accumulation of GSSG and a decreased GSH/GSSG ratio in cells. The mutant also showed defective growth in rich broth and minimal broth and was more sensitive to the oxidants H 2 O 2 and sodium nitroprusside. Interestingly, the expression of gshA , gshB1 , katA , and katB was induced in the mutant. These findings reveal that the recycling of glutathione is important for S. meliloti to maintain redox homeostasis and to interact symbiotically with alfalfa. IMPORTANCE The antioxidant glutathione is regulated by its synthetase and reductase in cells. In the symbiotic bacterium S. meliloti , the de novo synthesis of glutathione is essential for alfalfa nodulation and nitrogen fixation. In this study, we observed that the recycling of glutathione from GSSG not only was required for redox homeostasis and oxidative stress protection in S. meliloti cells but also contributed to alfalfa nodule development and competition capacity. Our findings demonstrate that the recycling of glutathione plays a key role in nitrogen fixation symbiosis., (Copyright © 2018 American Society for Microbiology.)

In the N2-fixing symbiont of alfalfa root nodules, Sinorhizobium meliloti 2011, the mmgR gene encodes a 77 nt small untranslated RNA (sRNA) that negatively regulates the accumulation of polyhydroxybutyrate (PHB) when the bacterium is grown under conditions of surplus carbon (C) in relation to nitrogen (N). We previously showed that the expression of mmgR is primarily controlled at the transcriptional level and that it depends on the cellular N status, although the regulatory mechanism and the factors involved were unknown. In this study, we provide experimental data supporting that: (a) mmgR is induced upon N limitation with the maximum expression found at the highest tested C/N molar ratio in the growth medium; (b) a conserved heptamer TTGTGCA located between the -35 and -10 mmgR promoter elements is necessary and sufficient for induction by N limitation; (c) induction of mmgR requires the N-status regulator NtrC; (d) under C limitation, mmgR transcription is repressed by AniA, a global regulator of C flow; (e) the mmgR promoter contains a conserved dyadic motif (TGC[N3]GCA) partially overlapping the heptamer TTGTGCA, which was also found in the promoters of the PHB-related genes phaP1, phaP2, phaZ and phaR (aniA) of S. meliloti and other alpha-proteobacteria. Taken together, these results suggest that the mmgR promoter would integrate signals from the metabolism of C and N through - at least - the global regulators NtrC and AniA, to provide an optimal level of the MmgR sRNA to fine-tune gene expression post-transcriptionally according to varying C and N availability.

Sinorhizobium meliloti is a Gram-negative alphaproteobacterium that can enter into a symbiotic relationship with Medicago sativa and Medicago truncatula Previous work determined that a mutation in the tkt2 gene, which encodes a putative transketolase, could prevent medium acidification associated with a mutant strain unable to metabolize galactose. Since the pentose phosphate pathway in S. meliloti is not well studied, strains carrying mutations in either tkt2 and tal , which encodes a putative transaldolase, were characterized. Carbon metabolism phenotypes revealed that both mutants were impaired in growth on erythritol and ribose. This phenotype was more pronounced for the tkt2 mutant strain, which also displayed auxotrophy for aromatic amino acids. Changes in pentose phosphate pathway metabolite concentrations were also consistent with a mutation in either tkt2 or tal The concentrations of metabolites in central carbon metabolism were also found to shift dramatically in strains carrying a tkt2 mutation. While the concentrations of proteins involved in central carbon metabolism did not change significantly under any conditions, the levels of those associated with iron acquisition increased in the wild-type strain with erythritol induction. These proteins were not detected in either mutant, resulting in less observable rhizobactin production in the tkt2 mutant. While both mutants were impaired in succinoglycan synthesis, only the tkt2 mutant strain was unable to establish symbiosis with alfalfa. These results suggest that tkt2 and tal play central roles in regulating the carbon flow necessary for carbon metabolism and the establishment of symbiosis. IMPORTANCE Sinorhizobium meliloti is a model organism for the study of plant-microbe interactions and metabolism, especially because it effects nitrogen fixation. The ability to derive the energy necessary for nitrogen fixation is dependent on an organism's ability to metabolize carbon efficiently. The pentose phosphate pathway is central in the interconversion of hexoses and pentoses. This study characterizes the key enzymes of the nonoxidative branch of the pentose phosphate pathway by using defined genetic mutations and shows the effects the mutations have on the metabolite profile and on physiological processes such as the biosynthesis of exopolysaccharide, as well as the ability to regulate iron acquisition., (Copyright © 2017 American Society for Microbiology.)

argC encodes N-acetyl-gamma-glutamyl phosphate reductase, the enzyme that catalyzes the high-energy-consuming third step in the arginine synthesis pathway. A comparative analysis revealed two translation start sites in argC from Sinorhizobium meliloti. To determine whether both protein versions are synthesized in the organism and their functional role, we obtained genetic constructs with one (1S) or two (2S) start sites, with promoters of low (pspeB) or high (plac) transcriptional rate. The constructs were transferred to the S. meliloti 1021 derivative argC mutant strain. Both protein versions were found in the free-living proteomes, but only ArgC 1S showed post-translational modification. Expression levels from argC 1S were five times higher than those of 2S, when transcribed by plac, and in concordance, its protein activity was 3-fold greater. The overexpression of both versions under plac delayed cellular growth. Inoculation of Medicago sativa plants with the S. meliloti strain harboring the argC 1S under plac induced nodulation but not nitrogen fixation. However, the strain with the argC 2S under the same promoter had a positive phenotype. Overproduction of ArgC protein for the synthesis of arginine induced physiological and symbiotic effects., (© FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

We here characterized the stress-tolerant alfalfa microsymbiont Sinorhizobium meliloti B401. B401-treated plants showed high nitrogen fixation rates under humid and semiarid environments. The production of glycine betaine in isolated bacteroids positively correlated with low precipitation levels, suggesting that this compound acts as a critical osmoprotectant under field conditions. Genome analysis revealed that strain B401 contains alternative pathways for the biosynthesis and uptake of glycine betaine and its precursors. Such genomic information will offer substantial insight into the environmental physiology of this biotechnologically valuable nitrogen-fixing bacterium., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rhizobia are α- and ß-proteobacteria that associate with legumes in symbiosis to fix atmospheric nitrogen. The chemical communication between roots and rhizobia begins in the rhizosphere. Using signature-tagged-Tn5 mutagenesis (STM) we performed a genome-wide screening for Ensifer meliloti genes that participate in colonizing the rhizospheres of alfalfa and other legumes. The analysis of ca. 6,000 mutants indicated that genes relevant for rhizosphere colonization account for nearly 2% of the rhizobial genome and that most (ca. 80%) are chromosomally located, pointing to the relevance and ancestral origin of the bacterial ability to colonize plant roots. The identified genes were related to metabolic functions, transcription, signal transduction, and motility/chemotaxis among other categories; with several ORFs of yet-unknown function. Most remarkably, we identified a subset of genes that impacted more severely the colonization of the roots of alfalfa than of pea. Further analyses using other plant species revealed that such early differential phenotype could be extended to other members of the Trifoliae tribe (Trigonella, Trifolium), but not the Fabeae and Phaseoleae tribes. The results suggest that consolidation of E. meliloti into its current symbiotic state should have occurred in a rhizobacterium that had already been adapted to rhizospheres of the Trifoliae tribe., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Sinorhizobium meliloti can translocate over surfaces. However, little is known about the regulatory mechanisms that control this trait and its relevance for establishing symbiosis with alfalfa plants. To gain insights into this field, we isolated Tn5 mutants of S. meliloti GR4 with impaired surface motility. In mutant strain GRS577, the transposon interrupted the ntrY gene encoding the sensor kinase of the NtrY/NtrX two-component regulatory system. GRS577 is impaired in flagella synthesis and overproduces succinoglycan, which is responsible for increased biofilm formation. The mutant also shows altered cell morphology and higher susceptibility to salt stress. GRS577 induces nitrogen-fixing nodules in alfalfa but exhibits decreased competitive nodulation. Complementation experiments indicate that both ntrY and ntrX account for all the phenotypes displayed by the ntrY::Tn5 mutant. Ectopic overexpression of VisNR, the motility master regulator, was sufficient to rescue motility and competitive nodulation of the transposant. A transcriptome profiling of GRS577 confirmed differential expression of exo and flagellar genes, and led to the demonstration that NtrY/NtrX allows for optimal expression of denitrification and nifA genes under microoxic conditions in response to nitrogen compounds. This study extends our knowledge of the complex role played by NtrY/NtrX in S. meliloti.

Small non-coding RNAs (sRNAs) are expected to have pivotal roles in the adaptive responses underlying symbiosis of nitrogen-fixing rhizobia with legumes. Here, we provide primary insights into the function and activity mechanism of the Sinorhizobium meliloti trans-sRNA NfeR1 (Nodule Formation Efficiency RNA). Northern blot probing and transcription tracking with fluorescent promoter-reporter fusions unveiled high nfeR1 expression in response to salt stress and throughout the symbiotic interaction. The strength and differential regulation of nfeR1 transcription are conferred by a motif, which is conserved in nfeR1 promoter regions in α-proteobacteria. NfeR1 loss-of-function compromised osmoadaptation of free-living bacteria, whilst causing misregulation of salt-responsive genes related to stress adaptation, osmolytes catabolism and membrane trafficking. Nodulation tests revealed that lack of NfeR1 affected competitiveness, infectivity, nodule development and symbiotic efficiency of S. meliloti on alfalfa roots. Comparative computer predictions and a genetic reporter assay evidenced a redundant role of three identical NfeR1 unpaired anti Shine-Dalgarno motifs for targeting and downregulation of translation of multiple mRNAs from transporter genes. Our data provide genetic evidence of the hyperosmotic conditions of the endosymbiotic compartments. NfeR1-mediated gene regulation in response to this cue could contribute to coordinate nutrient uptake with the metabolic reprogramming concomitant to symbiotic transitions., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

The bacterium Sinorhizobium meliloti is attracted to seed exudates of its host plant alfalfa (Medicago sativa). Since quaternary ammonium compounds (QACs) are exuded by germinating seeds, we assayed chemotaxis of S. meliloti towards betonicine, choline, glycine betaine, stachydrine and trigonelline. The wild type displayed a positive response to all QACs. Using LC-MS, we determined that each germinating alfalfa seed exuded QACs in the nanogram range. Compared to the closely related nonhost species, spotted medic (Medicago arabica), unique profiles were released. Further assessments of single chemoreceptor deletion strains revealed that an mcpX deletion strain displayed little to no response to these compounds. Differential scanning fluorimetry showed interaction of the isolated periplasmic region of McpX (McpX PR and McpX 34-306 ) with QACs. Isothermal titration calorimetry experiments revealed tight binding to McpX PR with dissociation constants (K d ) in the nanomolar range for choline and glycine betaine, micromolar K d for stachydrine and trigonelline and a K d in the millimolar range for betonicine. Our discovery of S. meliloti chemotaxis to plant-derived QACs adds another role to this group of compounds, which are known to serve as nutrient sources, osmoprotectants and cell-to-cell signalling molecules. This is the first report of a chemoreceptor that mediates QACs taxis through direct binding., (© 2016 John Wiley & Sons Ltd.)

We report that the smb20752 gene of the alfalfa symbiont Sinorhizobium meliloti is a novel symbiotic gene required for full N 2 -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&#95;b20860 gene of Sinorhizobium fredii NGR234. A ngr&#95;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&#95;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&#95;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., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Specific amino acids have protective functions in plants under stress conditions. This study assessed the effects of rhizobial strains on the amino acid composition in alfalfa under salt stress. Two alfalfa cultivars (Medicago sativa L. cv Apica and salt-tolerant cv Halo) in association with two Sinorhizobium meliloti strains with contrasting growth under salt stress (strain A2 and salt-tolerant strain Rm1521) were exposed to different levels of NaCl (0, 20, 40, 80 or 160 mM NaCl) under controlled conditions. We compared root and shoot biomasses, as well as root:shoot ratio for each association under these conditions as indicators of the salt tolerance of the symbiosis. Amino acid concentrations were analyzed in nodules, leaves and roots. The total concentration of free amino acids in nodules was mostly rhizobial-strain dependent while in leaves and roots it was mostly responsive to salt stress. For both cultivars, total and individual concentrations of amino acids including asparagine, proline, glutamine, aspartate, glutamate, γ-aminobutyric acid (GABA), histidine and ornithine were higher in Rm1521 nodules than in A2 nodules. Conversely, lysine and methionine were more abundant in A2 nodules than in Rm1521 nodules. Proline, glutamine, arginine, GABA and histidine substantially accumulated in salt-stressed nodules, suggesting an enhanced production of amino acids associated with osmoregulation, N storage or energy metabolism to counteract salt stress. Combining the salt-tolerant strain Rm1521 and the salt-tolerant cultivar Halo enhanced the root:shoot ratios and amino acid concentrations involved in plant protection which could be in part responsible for the enhancement of salt tolerance in alfalfa., (Crown Copyright © 2016. Published by Elsevier Masson SAS. All rights reserved.)

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 < 12% of pSymA and pSymB, were essential for SNF. These regions will serve as our preliminary target of the minimal set of horizontally acquired genes necessary and sufficient for SNF., (© 2016 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Background: Malic enzymes decarboxylate the tricarboxylic acid (TCA) cycle intermediate malate to the glycolytic end-product pyruvate and are well positioned to regulate metabolic flux in central carbon metabolism. Despite the wide distribution of these enzymes, their biological roles are unclear in part because the reaction catalyzed by these enzymes can be by-passed by other pathways. The N2-fixing alfalfa symbiont Sinorhizobium meliloti contains both a NAD(P)-malic enzyme (DME) and a separate NADP-malic enzyme (TME) and to help understand the role of these enzymes, we investigated growth, metabolomic, and transcriptional consequences resulting from loss of these enzymes in free-living cells., Results: Loss of DME, TME, or both enzymes had no effect on growth with the glycolytic substrate, glucose. In contrast, the dme mutants, but not tme, grew slowly on the gluconeogenic substrate succinate and this slow growth was further reduced upon the addition of glucose. The dme mutant strains incubated with succinate accumulated trehalose and hexose sugar phosphates, secreted malate, and relative to wild-type, these cells had moderately increased transcription of genes involved in gluconeogenesis and pathways that divert metabolites away from the TCA cycle. While tme mutant cells grew at the same rate as wild-type on succinate, they accumulated the compatible solute putrescine., Conclusions: NAD(P)-malic enzyme (DME) of S. meliloti is required for efficient metabolism of succinate via the TCA cycle. In dme mutants utilizing succinate, malate accumulates and is excreted and these cells appear to increase metabolite flow via gluconeogenesis with a resulting increase in the levels of hexose-6-phosphates and trehalose. For cells utilizing succinate, TME activity alone appeared to be insufficient to produce the levels of pyruvate required for efficient TCA cycle metabolism. Putrescine was found to accumulate in tme cells growing with succinate, and whether this is related to altered levels of NADPH requires further investigation.

In Sinorhizobium meliloti, the nodG gene is located in the nodFEG operon of the symbiotic plasmid. Although strong sequence similarity (53% amino acid identities) between S. meliloti NodG and Escherichia coli FabG was reported in 1992, it has not been determined whether S. meliloti NodG plays a role in fatty acid synthesis. We report that expression of S. meliloti NodG restores the growth of the E. coli fabG temperature-sensitive mutant CL104 under nonpermissive conditions. Using in vitro assays, we demonstrated that NodG is able to catalyze the reduction of the 3-oxoacyl-ACP intermediates in E. coli fatty acid synthetic reaction. Moreover, although deletion of the S. meliloti nodG gene does not cause any growth defects, upon overexpression of nodG from a plasmid, the S. meliloti fabG gene encoding the canonical 3-oxoacyl-ACP reductase (OAR) can be disrupted without any effects on growth or fatty acid composition. This indicates that S. meliloti nodG encodes an OAR and can play a role in fatty acid synthesis when expressed at sufficiently high levels. Thus, a bacterium can simultaneously possess two or more OARs that can play a role in fatty acid synthesis. Our data also showed that, although SmnodG increases alfalfa nodulation efficiency, it is not essential for alfalfa nodulation.

In Sinorhizobium meliloti, nitrogen fixation is regulated in response to oxygen concentration through the FixL-FixJ two-component system (TCS). Besides this conserved TCS, the field isolate SM11 also encodes the hFixL-FxkR TCS, which is responsible for the microoxic response in Rhizobium etli. Through genetic and physiological assays, we evaluated the role of the hFixL-FxkR TCS in S. meliloti SM11. Our results revealed that this regulatory system activates the expression of a fixKf orthologue (fixKa), in response to low oxygen concentration. Null mutations in either hFixL or FxkR promote upregulation of fixK1, a direct target of FixJ. Furthermore, the absence of this TCS translates into higher nitrogen fixation values as well as higher expression of fixN1 in nodules. Individual mutations in each of the fixK-like regulators encoded in the S. meliloti SM11 genome do not completely restrict fixN1 or fixN2 expression, pointing towards redundancy among these regulators. Both copies of fixN are necessary to achieve optimal levels of nitrogen fixation. This work provides evidence that the hFixL-FxkR TCS is activated in response to low oxygen concentration in S. meliloti SM11 and that it negatively regulates the expression of fixK1, fixN1 and nitrogen fixation.

Small non-coding regulatory RNAs (sRNAs) are key players in post-transcriptional regulation of gene expression. Hundreds of sRNAs have been identified in Sinorhizobium meliloti, but their biological function remains unknown for most of them. In this study, we characterized the expression pattern of the gene encoding the 77-nt sRNA MmgR in S. meliloti strain 2011. A chromosomal transcriptional reporter fusion (PmmgR-gfp) showed that the mmgR promoter is active along different stages of the interaction with alfalfa roots. In pure cultures, PmmgR-gfp activity paralleled the sRNA abundance indicating that mmgR expression is primarily controlled at the level of transcriptional initiation. PmmgR-gfp activity was higher during growth in rhizobial defined medium (RDM) than in TY medium. Furthermore, PmmgR-gfp was induced at 60 min after shifting growing cells from TY to RDM medium, i.e. shorter than the cell doubling time. In defined RDM medium containing NO3 (-), both PmmgR-gfp and MmgR level were repressed by the addition of tryptone or single amino acids, suggesting that mmgR expression depends on the cellular nitrogen (N) status. In silico analysis failed to detect conserved motifs upstream the promoter RNA polymerase binding site, but revealed a strongly conserved motif centered at -28 that may be linked to the observed regulatory pattern by the N source., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Unlabelled: The stringent response, mediated by the (p)ppGpp synthetase RelA and the RNA polymerase-binding protein DksA, is triggered by limiting nutrient conditions. For some bacteria, it is involved in regulation of virulence. We investigated the role of two DksA-like proteins from the Gram-negative nitrogen-fixing symbiont Sinorhizobium meliloti in free-living culture and in interaction with its host plant Medicago sativa The two paralogs, encoded by the genes SMc00469 and SMc00049, differ in the constitution of two major domains required for function in canonical DksA: the DXXDXA motif at the tip of a coiled-coil domain and a zinc finger domain. Using mutant analyses of single, double, and triple deletions for SMc00469(designated dksA),SMc00049, and relA, we found that the ΔdksA mutant but not the ΔSMc00049 mutant showed impaired growth on minimal medium, reduced nodulation on the host plant, and lower nitrogen fixation activity in early nodules, while its nod gene expression was normal. The ΔrelA mutant showed severe pleiotropic phenotypes under all conditions tested. Only S. meliloti dksA complemented the metabolic defects of an Escherichia coli dksA mutant. Modifications of the DXXDXA motif in SMc00049 failed to establish DksA function. Our results imply a role for transcriptional regulator DksA in the S. meliloti-M. sativa symbiosis., Importance: The stringent response is a bacterial transcription regulation process triggered upon nutritional stress.Sinorhizobium meliloti, a soil bacterium establishing agriculturally important root nodule symbioses with legume plants, undergoes constant molecular adjustment during host interaction. Analyzing the components of the stringent response in this alphaproteobacterium helps understand molecular control regarding the development of plant interaction. Using mutant analyses, we describe how the lack of DksA influences symbiosis with Medicago sativa and show that a second paralogous S. meliloti protein cannot substitute for this missing function. This work contributes to the field by showing the similarities and differences of S. meliloti DksA-like proteins to orthologs from other species, adding information to the diversity of the stringent response regulatory system., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Strigolactones (SLs) are multifunctional molecules acting as modulators of plant responses under nutrient deficient conditions. One of the roles of SLs is to promote beneficial association with arbuscular mycorrhizal (AM) fungi belowground under such stress conditions, mainly phosphorus shortage. Recently, a role of SLs in the Rhizobium-legume symbiosis has been also described. While SLs' function in AM symbiosis is well established, their role in the Rhizobium-legume interaction is still emerging. Recently, SLs have been suggested to stimulate surface motility of rhizobia, opening the possibility that they could also act as molecular cues. The possible effect of SLs in the motility in the alfalfa symbiont Sinorhizobium meliloti was investigated, showing that the synthetic SL analogue GR24 stimulates swarming motility in S. meliloti in a dose-dependent manner. On the other hand, it is known that SL production is regulated by nutrient deficient conditions and by AM symbiosis. Using the model alfalfa-S. meliloti, the impact of phosphorus and nitrogen deficiency, as well as of nodulation on SL production was also assessed. The results showed that phosphorus starvation promoted SL biosynthesis, which was abolished by nitrogen deficiency. In addition, a negative effect of nodulation on SL levels was detected, suggesting a conserved mechanism of SL regulation upon symbiosis establishment., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

In Gram-negative bacteria, tyrosine phosphorylation has been shown to play a role in the control of exopolysaccharide (EPS) production. This study demonstrated that the chromosomal ORF SMc02309 from Sinorhizobium meliloti 2011 encodes a protein with significant sequence similarity to low molecular mass protein-tyrosine phosphatases (LMW-PTPs), such as the Escherichia coli Wzb. Unlike other well-characterized EPS biosynthesis gene clusters, which contain neighbouring LMW-PTPs and kinase, the S. meliloti succinoglycan (EPS I) gene cluster located on megaplasmid pSymB does not encode a phosphatase. Biochemical assays revealed that the SMc02309 protein hydrolyses p-nitrophenyl phosphate (p-NPP) with kinetic parameters similar to other bacterial LMW-PTPs. Furthermore, we show evidence that SMc02309 is not the LMW-PTP of the bacterial tyrosine-kinase (BY-kinase) ExoP. Nevertheless, ExoN, a UDP-glucose pyrophosphorylase involved in the first stages of EPS I biosynthesis, is phosphorylated at tyrosine residues and constitutes an endogenous substrate of the SMc02309 protein. Additionally, we show that the UDP-glucose pyrophosphorylase activity is modulated by SMc02309-mediated tyrosine dephosphorylation. Moreover, a mutation in the SMc02309 gene decreases EPS I production and delays nodulation on Medicago sativa roots.

In order to effectively manipulate rhizobium-legume symbioses for our benefit, it is crucial to first gain a complete understanding of the underlying genetics and metabolism. Studies with rhizobium auxotrophs have provided insight into the requirement for amino acid biosynthesis during the symbiosis; however, a paucity of available L-proline auxotrophs has limited our understanding of the role of L-proline biosynthesis. Here, we examined the symbiotic phenotypes of a recently described Sinorhizobium meliloti L-proline auxotroph. Proline auxotrophy was observed to result in a host-plant-specific phenotype. The S. meliloti auxotroph displayed reduced symbiotic capability with alfalfa (Medicago sativa) due to a decrease in nodule mass formed and therefore a reduction in nitrogen fixed per plant. However, the proline auxotroph formed nodules on white sweet clover (Melilotus alba) that failed to fix nitrogen. The rate of white sweet clover nodulation by the auxotroph was slightly delayed, but the final number of nodules per plant was not impacted. Examination of white sweet clover nodules by confocal microscopy and transmission electron microscopy revealed the presence of the S. meliloti proline auxotroph cells within the host legume cells, but few differentiated bacteroids were identified compared with the bacteroid-filled plant cells of WT nodules. Overall, these results indicated that L-proline biosynthesis is a general requirement for a fully effective nitrogen-fixing symbiosis, likely due to a transient requirement during bacteroid differentiation.

ZnO nanoparticles (NPs) are reported as potentially phytotoxic in hydroponic and soil media. However, studies on ZnO NPs toxicity in a plant inoculated with bacterium in soil are limited. In this study, ZnO NPs, bulk ZnO, and ZnCl₂ were exposed to the symbiotic alfalfa (Medicago sativa L.)-Sinorhizobium meliloti association at concentrations ranging from 0 to 750 mg/kg soil. Plant growth, Zn bioaccumulation, dry biomass, leaf area, total protein, and catalase (CAT) activity were measured in 30 day-old plants. Results showed 50% germination reduction by bulk ZnO at 500 and 750 mg/kg and all ZnCl₂ concentrations. ZnO NPs and ionic Zn reduced root and shoot biomass by 80% and 25%, respectively. Conversely, bulk ZnO at 750 mg/kg increased shoot and root biomass by 225% and 10%, respectively, compared to control. At 500 and 750 mg/kg, ZnCl₂ reduced CAT activity in stems and leaves. Total leaf protein significantly decreased as external ZnCl₂ concentration increased. STEM-EDX imaging revealed the presence of ZnO particles in the root, stem, leaf, and nodule tissues. ZnO NPs showed less toxicity compared to ZnCl₂ and bulk ZnO found to be growth enhancing on measured traits. These findings are significant to reveal the toxicity effects of different Zn species (NPs, bulk, and ionic Zn) into environmentally important plant-bacterial system in soil., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The theoretical and experimental data on salt tolerance of root nodule bacteria Sinorhizobium meliloti (Ensifer meliloti), an alfalfa symbiont, and on genetic determination of this feature are reviewed. Extensive data on the genes affecting adaptation of proteobacteria are provided, as well as on the groups of genes with activity depending on the osmolarity of the medium. Structural and functional polymorphism of the bet genes involved in betaine synthesis and transport in S. meliloti is discussed. The phenotypic and. genotypic polymorphism in 282 environmental rhizobial strains isolated from the centers of alfalfa diversity affected by aridity and salinity is discussed. The isolates from the Aral Sea area and northern Caucasus were shown to possess the betC gene represented by two types of alleles: the dominant A-type allele found in Rm 1021 and the less common divergent E-type allele, which was revealed in regions at the frequencies at the frequencies of 0.35 and 0.48, respectively. In the isolates with the salt-tolerant phenotype, which were isolated from root nodules and subsequently formed less effective symbioses with alfalfa, the frequency of E-type alleles was 2.5 times higher. Analysis of the nucleotide and amino acid sequences of the E-type allele of the betC gene revealed that establishment of this allele in the population was a result of positive selection. It is concluded that diversification of the functionally diverse bet genes occurring in S. meliloti affects the salt tolerance and symbiotic effectivity of rhizobia.

Background: Swarming motility and biofilm formation are opposite, but related surface-associated behaviors that allow various pathogenic bacteria to colonize and invade their hosts. In Sinorhizobium meliloti, the alfalfa endosymbiont, these bacterial processes and their relevance for host plant colonization are largely unexplored. Our previous work demonstrated distinct swarming abilities in two S. meliloti strains (Rm1021 and GR4) and revealed that both environmental cues (iron concentration) and bacterial genes (fadD, rhb, rirA) play crucial roles in the control of surface motility in this rhizobial species. In the current study, we investigate whether these factors have an impact on the ability of S. meliloti to establish biofilms and to colonize host roots., Results: We found that strain GR4, which is less prone to translocate on solid surfaces than strain Rm1021, is more efficient in developing biofilms on glass and plant root surfaces. High iron conditions, known to prevent surface motility in a wild-type strain of S. meliloti, promote biofilm development in Rm1021 and GR4 strains by inducing the formation of more structured and thicker biofilms than those formed under low iron levels. Moreover, three different S. meliloti mutants (fadD, rhb, and rirA) that exhibit an altered surface translocation behavior compared with the wild-type strain, establish reduced biofilms on both glass and alfalfa root surfaces. Iron-rich conditions neither rescue the defect in biofilm formation shown by the rhb mutant, which is unable to produce the siderophore rhizobactin 1021 (Rhb1021), nor have any impact on biofilms formed by the iron-response regulator rirA mutant. On the other hand, S. meliloti FadD loss-of-function mutants do not establish normal biofilms irrespective of iron levels., Conclusions: Our studies show that siderophore Rhb1021 is not only required for surface translocation, but also for biofilm formation on glass and root surfaces by strain Rm1021. In addition, we present evidence for the existence of control mechanisms that inversely regulate swarming and biofilm formation in S. meliloti, and that contribute to efficient plant root colonization. One of these mechanisms involves iron levels and the iron global regulator RirA. The other mechanism involves the participation of the fatty acid metabolism-related enzyme FadD.

An artificial increase of cyclic diguanylate (c-di-GMP) levels in Sinorhizobium meliloti 8530, a bacterium that does not carry known cellulose synthesis genes, leads to overproduction of a substance that binds the dyes Congo red and calcofluor. Sugar composition and methylation analyses and NMR studies identified this compound as a linear mixed-linkage (1 → 3)(1 → 4)-β-D-glucan (ML β-glucan), not previously described in bacteria but resembling ML β-glucans found in plants and lichens. This unique polymer is hydrolyzed by the specific endoglucanase lichenase, but, unlike lichenan and barley glucan, it generates a disaccharidic → 4)-β-D-Glcp-(1 → 3)-β-D-Glcp-(1 → repeating unit. A two-gene operon bgsBA required for production of this ML β-glucan is conserved among several genera within the order Rhizobiales, where bgsA encodes a glycosyl transferase with domain resemblance and phylogenetic relationship to curdlan synthases and to bacterial cellulose synthases. ML β-glucan synthesis is subjected to both transcriptional and posttranslational regulation. bgsBA transcription is dependent on the exopolysaccharide/quorum sensing ExpR/SinI regulatory system, and posttranslational regulation seems to involve allosteric activation of the ML β-glucan synthase BgsA by c-di-GMP binding to its C-terminal domain. To our knowledge, this is the first report on a linear mixed-linkage (1 → 3)(1 → 4)-β-glucan produced by a bacterium. The S. meliloti ML β-glucan participates in bacterial aggregation and biofilm formation and is required for efficient attachment to the roots of a host plant, resembling the biological role of cellulose in other bacteria.

Delftia sp. strain JD2 is a plant-growth-promoting bacterium that enhances legume nodulation and growth, acting as nodule-assisting bacterium during the co-inoculation of plants with rhizobial strains. In this work, we evaluate how the co-inoculation of alfalfa with Sinorhizobium meliloti U143 and JD2 increases plant yield under greenhouse conditions and we analyze the pattern of secreted bioactive compounds which may be involved in the microbe-plant communication. The chemical composition of extracellular cultures (EC) produced in hydroponic conditions (collected 4, 7, and 14 days after bacterial treatment) were characterized using different chromatographic and elucidation techniques. In addition, we assessed the effect that plant irrigation with cell-free EC, produced during co-inoculation experiments, would have on plant yield. Results showed increased alfalfa shoot and root matter, suggesting that U143-JD2 co-inoculation might be a beneficial agricultural practice. The pattern of secreted secondary metabolites among treatments showed important differences. Qualitative and quantitative changes in phenolic compounds (including flavonoids), organic acids, and volatile compounds were detected during the early microbe-plant interaction, suggesting that the production of some molecules positively affects the microbe-plant association. Finally, the irrigation of co-inoculated plants with cell-free EC under greenhouse conditions increased plant yield over agronomic expectations. This effect might be attributed to the bioactive secondary metabolites incorporated during the irrigation.

In this study we investigated effects of Zn supply on germination, growth, inorganic solutes (Zn, Ca, Fe, and Mg) partitioning and nodulation of Medicago sativa This plant was cultivated with and without Zn (2 mM). Treatments were plants without (control) and with Zn tolerant strain (S532), Zn intolerant strain (S112) and 2 mM urea nitrogen fertilisation. Results showed that M. sativa germinates at rates of 50% at 2 mM Zn. For plants given nitrogen fertilisation, Zn increased plant biomass production. When grown with symbionts, Zn supply had no effect on nodulation. Moreover, plants with S112 showed a decrease of shoot and roots biomasses. However, in symbiosis with S532, an increase of roots biomass was observed. Plants in symbiosis with S. meliloti accumulated more Zn in their roots than nitrogen fertilised plants. Zn supply results in an increase of Ca concentration in roots of fertilised nitrogen plants. However, under Zn supply, Fe concentration decreased in roots and increased in nodules of plants with S112. Zn supply showed contrasting effects on Mg concentrations for plants with nitrogen fertilisation (increase) and plants with S112 (decrease). The capacity of M. sativa to accumulate Zn in their nodulated roots encouraged its use in phytostabilisation processes.

The symbiotic nitrogen-fixing soil bacterium Sinorhizobium meliloti carries a large number of toxin-antitoxin (TA) modules both on the chromosome and megaplasmids. One of them, the vapBC-5 module that belongs to the type II systems was characterized here. It encodes an active toxin vapC-5, and was shown to be controlled negatively by the complex of its own proteins. Different mutants of the vapBC-5 genes exhibited diverse effects on symbiotic efficiency during interaction with the host plant Medicago sativa. The absence of the entire vapBC-5 region had no influence on nodule formation and nitrogen fixation properties. The strain carrying an insertion in the antitoxin gene showed a reduced nitrogen fixation capacity resulting in a lower plant yield. In contrast, when the toxin gene was mutated, the strain developed more efficient symbiosis with the host plant. The nitrogen fixing root nodules had a delayed senescent phenotype and contained elevated level of plant-derived molecules characteristic of later steps of nodule development. The longer bacteroid viability and abundance of active nitrogen fixing zone resulted in increased production of plant material. These data indicate that modification of the toxin/antitoxin production may influence bacteroid metabolism and may have an impact on the adaptation to changing environmental conditions., (© 2014 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Sinorhizobium meliloti strains unable to utilize galactose as a sole carbon source, due to mutations in the De-Ley Doudoroff pathway (dgoK), were previously shown to be more competitive for nodule occupancy. In this work, we show that strains carrying this mutation have galactose-dependent exopolysaccharide (EPS) phenotypes that were manifested as aberrant Calcofluor staining as well as decreased mucoidy when in an expR(+) genetic background. The aberrant Calcofluor staining was correlated with changes in the pH of the growth medium. Strains carrying dgoK mutations were subsequently demonstrated to show earlier acidification of their growth medium that was correlated with an increase expression of genes associated with succinoglycan biosynthesis as well as increased accumulation of high and low molecular weight EPS in the medium. In addition, it was shown that the acidification of the medium was dependent on the inability of S. meliloti strains to initiate the catabolism of galactose. To more fully understand why strains carrying the dgoK allele were more competitive for nodule occupancy, early nodulation phenotypes were investigated. It was found that strains carrying the dgoK allele had a faster rate of nodulation. In addition, nodule competition experiments using genetic backgrounds unable to synthesize either succinoglycan or EPSII were consistent with the hypothesis that the increased competition phenotype was dependent upon the synthesis of succinoglycan. Fluorescent microscopy experiments on infected root-hair cells, using the acidotropic dye Lysotracker Red DND-99, provide evidence that the colonized curled root hair is an acidic compartment.

Sinorhizobium meliloti is a nitrogen-fixing rhizobium symbiont of legumes, widespread in many temperate environments the high genetic diversity of which enables it to thrive as a symbiont of host legumes and free-living in soil. Soil type, together with geographic differences and host plant genotype, seem to be prominent factors in shaping rhizobial genetic diversity. While a large body of research supports the idea that the genetic structure of free-living microbial taxa exhibits a clear biogeographic pattern, few investigations have been performed on the biogeographic pattern of S. meliloti genotypes in a restricted geographic range. In the present study, a collection of 128 S. meliloti isolates from three different regions in Croatia was investigated to analyze the relationship between genetic diversity, geographic distribution, soil features and isolate phenotypes by using amplified fragment length polymorphism (AFLP) as a genome-wide scanning method. Results obtained led to the conclusion that the genotypes of isolates cluster according to the region of origin and that the differentiation of S. meliloti populations can be mainly ascribed to geographic isolation following an isolation-by-distance model, with a strong distance-decay relationship of genetic similarity with distance, in which local soil conditions are not the major component influencing the isolate phenotypes or their genomic differentiation., (Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.)

Rhizobia induce nitrogen-fixing nodules on host legumes, which is important in agriculture and ecology. Lipopolysaccharide (LPS) produced by rhizobia is required for infection or bacteroid survival in host cells. Genes required for LPS biosynthesis have been identified in several Rhizobium species. However, the regulation of their expression is not well understood. Here, Sinorhizobium meliloti LsrB, a member of the LysR family of transcriptional regulators, was found to be involved in LPS biosynthesis by positively regulating the expression of the lrp3-lpsCDE operon. An lsrB in-frame deletion mutant displayed growth deficiency, sensitivity to the detergent sodium dodecyl sulfate, and acidic pH compared to the parent strain. This mutant produced slightly less LPS due to lower expression of the lrp3 operon. Analysis of the transcriptional start sites of the lrp3 and lpsCDE gene suggested that they constitute one operon. The expression of lsrB was positively autoregulated. The promoter region of lrp3 was specifically precipitated by anti-LsrB antibodies in vivo. The promoter DNA fragment containing TN11A motifs was bound by the purified LsrB protein in vitro. These new findings suggest that S. meliloti LsrB is associated with LPS biosynthesis, which is required for symbiotic nitrogen fixation on some ecotypes of alfalfa plants., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Copper is an important element in host-microbe interactions, acting both as a catalyst in enzymes and as a potential toxin. Cu(+)-ATPases drive cytoplasmic Cu(+) efflux and protect bacteria against metal overload. Many pathogenic and symbiotic bacteria contain multiple Cu(+)-ATPase genes within particular genetic environments, suggesting alternative roles for each resulting protein. This hypothesis was tested by characterizing five homologous Cu(+)-ATPases present in the symbiotic organism Sinorhizobium meliloti. Mutation of each gene led to different phenotypes and abnormal nodule development in the alfalfa host. Distinct responses were detected in free-living S. meliloti mutant strains exposed to metal and redox stresses. Differential gene expression was detected under Cu(+), oxygen or nitrosative stress. These observations suggest that CopA1a maintains the cytoplasmic Cu(+) quota and its expression is controlled by Cu(+) levels. CopA1b is also regulated by Cu(+) concentrations and is required during symbiosis for bacteroid maturation. CopA2-like proteins, FixI1 and FixI2, are necessary for the assembly of two different cytochrome c oxidases at different stages of bacterial life. CopA3 is a phylogenetically distinct Cu(+)-ATPase that does not contribute to Cu(+) tolerance. It is regulated by redox stress and required during symbiosis. We postulated a model where non-redundant homologous Cu(+)-ATPases, operating under distinct regulation, transport Cu(+) to different target proteins., (© 2014 The Authors.)