Rhizobium Sinorhizobium Meliloti Research Articles

Genotype-by-genotype interkingdom cross-talk between symbiotic nitrogen fixing Sinorhizobium meliloti strains and Trichoderma species

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.

Promotion effect of AM fungi and rhizobia co-inoculations on alfalfa (Medicago Sativa L.) growth

Arbuscular mycorrhizal fungi (AMF) and rhizobia have been widely used in agriculture in recent years to increase production, reduce fertilizer consumption and control plant diseases. However, it is unclear whether there are differences in the effects of AMF and rhizobia inoculation alone and in combination. In this study, alfalfa seedlings were inoculated with AMF Glomus mosseae (G.m), Glomus intraradices (G.i) and co-inoculated with AMF and rhizobia Sinorhizobium meliloti (S.m) before being transplanted outdoors. The growth characteristics, fine root yield and fine root death of alfalfa were measured throughout the experiment, and the results showed that co-inoculation with AMF and rhizobia promoted alfalfa growth, while increasing plant height, branch number and total biomass. However, the growth promoting effects of AMF and rhizobia differed; AMF inoculation displayed no advantage over S.m inoculation in increasing aboveground biomass (P > 0.05) but did lead to an increase in underground biomass (P < 0.05). Compared with that observed following the single inoculations with G.m or S.m, the annual fine root production following co-inoculation with G.m and S.m increased by 15.38% and 47.06%, respectively, although there was less effect following co-inoculation with G. i and S.m. These results indicated that there was mutual selectivity and interaction between AMF and rhizobia. G.m and S.m inoculation treatment has a positive effect on alfalfa growth, on the one hand, it is because that AMF-Rhizobia synergically promoted alfalfa root system to absorb nutrients (N), then improve the plant growth, on the other hand, their vaccination can reduce the death of alfalfa fine roots, significantly improve the alfalfa biomass of underground part.

Impact of co-inoculation with plant-growth-promoting rhizobacteria and rhizobium on the biochemical responses of alfalfa-soil system in copper contaminated soil

The effects and regulatory mechanisms of co-inoculation of plant-growth-promoting rhizobacteria (PGPRs) and rhizobium in plant-soil systems remain unclear, despite numerous reports that PGPRs or rhizobium can alleviate metal toxicity. We used the co-inoculation of the PGPR Paenibacillus mucilaginosus and the metal-resistant rhizobium Sinorhizobium meliloti for exploring the physiological and biochemical responses of the plant-soil system in metal-contaminated soil. The co-inoculation with the PGPR and rhizobium significantly increased the nutrient (N, P, and K) contents in plant tissues and promoted plant growth in soil contaminated with copper (Cu). Stress from Cu-induced reactive oxygen species and lipid peroxidation were largely attenuated by the co-inoculation by increasing the activities of antioxidant enzymes. The contents and uptake of Cu in plant tissues increased significantly in the co-inoculation treatment compared with the uninoculated control and individual inoculation treatment. Co-inoculation with PGPR and rhizobium significantly increased soil microbial biomass, enzymatic activities, total nitrogen, available phosphorus, and soil organic matter contents compared with the uninoculated control. Interestingly, co-inoculation also affected the composition of the rhizospheric microbial community, and slightly increased rhizospheric microbial diversity. These improvements of the soil fertility and biological activity also had a beneficial impact on plant growth under Cu stress. Our results suggested that alfalfa co-inoculated with PGPR and rhizobium could increase plant growth and Cu uptake in metal-contaminated soil by alleviating plant Cu stress and improving soil biochemical properties. These results indicate that the co-application of PGPR and rhizobium can have a positive effect on the biochemical responses of alfalfa-soil systems in soil contaminated by heavy metals and can provide an efficient strategy for the phytomanagement of metal-contaminated land.

Co-inoculations of arbuscular mycorrhizal fungi and rhizobia under salinity in alfalfa

Alfalfa (Medicago sativa L.) is cultivated in arid and semi-arid regions where salinity is one of the main limiting factors for its production. Thus, this experiment was conducted to evaluate the efficacy of arbuscular mycorrhizal fungus (AMF), Glomus mosseae, alfalfa rhizobia Sinorhizobium meliloti (R) seed inoculation in the development of salinity tolerance of different alfalfa cultivars (Rehnani, Pioneer and Bami) under a variety of salinity levels. The results revealed that under non-stress condition, root mycorrhizal infection, nodulation (the number and weight of nodules per plant), potassium (K), calcium (Ca), phosphorus (P), zinc (Zn), copper (Cu) and magnesium (Mg) contents of the root and shoot, the value of the K/Na ratio, protein [calculated from the nitrogen (N) content] and proline contents of the shoot and the alfalfa yield were found to be the highest while Na contents of the root and shoot were seen to be the lowest when seeds were double inoculated followed by mycorrhizae, rhizobium and control treatments, respectively. Similarly, under salinity condition, the greatest amounts of mycorrhizal infection, nodulation, root and shoot P contents, the value of K/Na ratio, the shoot proline content and the root Ca content were enhanced with the least amount of leaf Na content related to the cases of seeds which were double inoculated, followed by mycorrhizae, rhizobium and control treatments respectively. The results suggested that inoculation of alfalfa seed with AMF or R, especially double inoculation, causes a considerable increase in alfalfa yield under both saline and non-saline conditions by increasing colonization, nodulation and nutrient uptake.

Queuosine Biosynthesis Is Required for Sinorhizobium meliloti-Induced Cytoskeletal Modifications on HeLa Cells and Symbiosis with Medicago truncatula

Rhizobia are symbiotic soil bacteria able to intracellularly colonize legume nodule cells and form nitrogen-fixing symbiosomes therein. How the plant cell cytoskeleton reorganizes in response to rhizobium colonization has remained poorly understood especially because of the lack of an in vitro infection assay. Here, we report on the use of the heterologous HeLa cell model to experimentally tackle this question. We observed that the model rhizobium Sinorhizobium meliloti, and other rhizobia as well, were able to trigger a major reorganization of actin cytoskeleton of cultured HeLa cells in vitro. Cell deformation was associated with an inhibition of the three major small RhoGTPases Cdc42, RhoA and Rac1. Bacterial entry, cytoskeleton rearrangements and modulation of RhoGTPase activity required an intact S. meliloti biosynthetic pathway for queuosine, a hypermodifed nucleoside regulating protein translation through tRNA, and possibly mRNA, modification. We showed that an intact bacterial queuosine biosynthetic pathway was also required for effective nitrogen-fixing symbiosis of S. meliloti with its host plant Medicago truncatula, thus indicating that one or several key symbiotic functions of S. meliloti are under queuosine control. We discuss whether the symbiotic defect of que mutants may originate, at least in part, from an altered capacity to modify plant cell actin cytoskeleton.

Transcriptomic insights into mechanisms of symbiotic cooperation

Intraspecific genetic variation can affect community structure and ecosystem processes (Bolnick etal. 2011). It can also influence phenotypic expression by genotypes within other species to produce genotype-by-genotype (G×G) interaction (Falconer & Mackay 1996). Evolution of one species drives correlated evolution of others when it causes G×G for fitness (Thompson 2005). However, the mechanisms by which species interact also influence evolutionary outcomes (Kummel & Salant 2006; Golubski & Klausmeier 2010; Akçay & Simms 2011; Grman etal. 2012). To identify genes and putative functional mechanisms underlying G×G interactions, Heath etal. (2012) analysed natural variation in the symbiotic transcriptome of the mutualistic nutritional symbiosis between a legume host Medicago truncatula and the facultative endosymbiotic rhizobium Sinorhizobium meliloti. Using twelve microarrays, the authors simultaneously measured host and symbiont gene expression in root nodules from four factorial pairings of host and symbiont genotypes that produced G×G in host fitness (Fig.1, upper panel). Rhizobium gene expression was influenced by rhizobium and plant genotype and the G×G interaction (Fig.1, lower panel), whereas plant gene expression was influenced primarily by plant genotype. The authors identified rhizobium genes that might contribute to G×G in host plant fitness. Heath etal. (2012) have moved beyond the constraints of single organism analysis towards a more realistic understanding of plants and bacteria as organisms inextricably linked with symbioses that affect even basic patterns of gene expression.

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

The proline analogue cis-4-hydroxy-L-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP's retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from L-proline by coexpression of L-proline cis-4-hydroxylases converting L-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its L-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing L-proline produced more N-acetyl CHOP than LB medium containing L-proline. E. coli ΔputA cells accumulated L-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and L-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the L-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.

Resting forms of Sinorhizobium meliloti

The ability of the symbiotrophic rhizobium Sinorhizobium meliloti P221 to produce cells having all the properties of resting forms (RFs) during the development cycles of the culture or after addition of the threshold concentrations of anabiosis autoinducers was demonstrated. The numbers, properties, and ultra-structure of S. meliloti resting forms depended on the conditions of growth and poststationary-phase incubation. In the four-month poststationary-phase, cultures grown in media deficient in some nutrient elements and energy sources (nitrogen, phosphorus, or oxygen), numerous cells (24–76% of the number of CFUs in the stationary-phase cultures) exhibiting a high degree of heat resistance and reversibly inhibited metabolic activity (the absence of endogenous respiration) were detected. According to their ultrastructure, all the resting forms detected in starving cultures were divided into three groups: (1) cystlike resting cells (CRCs) with thick cell envelopes and compacted nucleoids, (2) CRCs containing numerous (up to three-quarters of their volumes) polyhydroxyalkanoate inclusions, and (3) RFs similar to Azotobacter cysts. The resting forms obtained in the culture grown at high concentrations (5 × 10−5 M) of C12-AHB, a chemical analogue of microbial anabiosis autoregulators, were incapable of endogenous respiration and retained the colony-forming ability. The CFU number after plating of these resting forms was twice as high as in the control culture; the heat resistance of these cells (55°C, 10 min) was an order of magnitude higher. The bacterial cells obtained from the resting forms either had a mixed (Swa+Gri+) type of motility in semisolid agar, typical of the dominant phenotype of the parent cells, or switched to the Gri+ type. Emergence of different motility phenotypes depended on the conditions of RF formation. More severe stress conditions of RF formation induced the emergence of the Gri+ type of cell motility. The results obtained can be used for development of a new generation of bacterial preparations based on bacterial CRCs which are able to preserve their viability for a long time and are highly resistant to stress impacts.

Symbiotic activity of alfalfa rhizobia (Sinorhizobium meliloti) strains with genetically modified transport of dicarboxylic acids

Inactivation of the genes involved in dicarboxylic acid transport in the alfalfa rhizobia Sinorhizobium meliloti—the structural gene of succinate permease dctA and its transcriptional regulators dctBD, nifA, and ntrA—leads to complete or partial loss of N2-fixing activity, while amplification of these genes leads to an increase in this activity. This considerably elevates the accumulation of nitrogen and carbon in alfalfa; however, the plant weight increases to a lesser degree, which is associated with an incomplete translocation of nitrogen to the aboveground organs. Factor analysis has demonstrated that the amplification of dctABD genes significantly influences these characteristics of symbiotic efficiency in all the performed experiments, while the effects of dctA, nifA, and ntrA depend on the plant cultivar and vegetation conditions.