Screening and Characterization of Two Extracellular Polysaccharide-Producing Bacteria from the Biocrust of the Mu Us Desert.

The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.

Plant transcriptome analysis reveals specific molecular interactions between alfalfa and its rhizobial symbionts below the species level.

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.

Heavy metal accumulation in Lathyrus sativus growing in contaminated soils and identification of symbiotic resistant bacteria.

In this study, two populations of leguminous plants Lathyrus sativus were grown in four soils that were collected from sites differently contaminated by heavy metals. Evaluations included basic soil properties, concentrations of major nutrients and four metals (copper, zinc, lead and cadmium) in these soils. Investigation of Lathyrus sativus response to contamination showed that the increase of heavy metal concentration in soils affected biomass of plant, number of nodules and plant metal uptake. Heavy metal tolerance of 46 isolated bacteria from the root nodules was evaluated and demonstrated that the maximum concentration of Cd, Pb, Cu and Zn tolerated by strains were 0.8, 2.5, 0.2, and 0.5 mM, respectively. Twenty-two isolates were tested for their effects on plant biomass production and nodule formation and showed that only R. leguminosarum nodulated Lathyrus sativus, while some bacteria improved the shoot and root dry biomass. Sequences of their 16S rDNA gene fragments were also obtained and evaluated for tentative identification of the isolates which revealed different bacterial genera represented by Rhizobium sp, Rhizobium leguminosarum, Sinorhizobium meliloti, Pseudomonas sp, Pseudomonas fluorescens, Luteibacter sp, Variovorax sp, Bacillus simplex and Bacillus megaterium. The existence of Pb- and Cd-resistant genes (PbrA and CadA) in these bacteria was determined by PCR, and it showed high homology with PbrA and CadA genes from other bacteria. The tested resistant population was able to accumulate high concentrations of Pb and Cd in all plant parts and, therefore, can be classified as a strong metal accumulator with suitable potential for phytoremediation of Pb and Cd polluted sites. Heavy metal resistant and efficient bacteria isolated from root nodules were chosen with Lathyrus sativus to form symbiotic associations for eventual bioremediation program, which could be tested to remove pollutants from contaminated sites.

Mining alfalfa (Medicago sativa L.) nodules for salinity tolerant non-rhizobial bacteria to improve growth of alfalfa under salinity stress.

There are fewer reports on plant growth promoting (PGP) bacteria living in nodules as helper to tolerance to abiotic stress such as salinity and drought. The study was conducted to isolate rhizobial and non-rhizobial drought and salinity tolerant bacteria from the surface sterilized root nodules of alfalfa, grown in saline soils, and evaluate the effects of effective isolates on plant growth under salt stress. Based on drought and salinity tolerance of bacterial isolates and having multiple PGP traits, two non-rhizobial endophytic isolates and one rhizobial endophytic isolate were selected for further identification and characterization. Based on partial sequences of 16 S rRNA genes, non-rhizobial isolates and rhizobial isolate were closely related to Klebsiella sp., Kosakonia cowanii, and Sinorhizobium meliloti, respectively. None of the two non-rhizobial strains were able to form nodules on alfalfa roots under greenhouse and in vitro conditions. Co-inoculation of alfalfa plant with Klebsiella sp. A36, K. cowanii A37, and rhizobial strain S. meliloti ARh29 had a positive effect on plant growth indices under salinity stress. In addition, the single inoculation of non-rhizobial strains without rhizobial strain resulted in an increase in alfalfa growth indices compared to the plants non-inoculated and the ones inoculated with S. meliloti ARh29 alone under salinity stress, indicating that nodule non-rhizobial strains have PGP potentials and may be a promising way for improving effectiveness of Rhizobium bio-fertilizers in salt-affected soils.

Biogeography of a Novel Ensifer meliloti Clade Associated with the Australian Legume Trigonella suavissima.

Here, we describe a novel clade within Ensifer meliloti and consider how geographic and ecological isolation contributed to the limited distribution of this group. Members of the genus Ensifer are best known for their ability to form nitrogen-fixing symbioses with forage legumes of three related genera, Medicago L., Melilotus Mill., and Trigonella L., which are members of the tribe Trifolieae. These legumes have a natural distribution extending from the Mediterranean Basin through western Asia, where there is an unsurpassed number of species belonging to these genera. Trigonella suavissima L. is unusual in that it is the only species in the tribe Trifolieae that is native to Australia. We compared the genetic diversity and taxonomic placement of rhizobia nodulating T. suavissima with those of members of an Ensifer reference collection. Our goal was to determine if the T. suavissima rhizobial strains, like their plant host, are naturally limited to the Australian continent. We used multilocus sequence analysis to estimate the genetic relatedness of 56 T. suavissima symbionts to 28 Ensifer reference strains. Sequence data were partitioned according to the replicons in which the loci are located. The results were used to construct replicon-specific phylogenetic trees. In both the chromosomal and chromid trees, the Australian strains formed a distinct clade within E. meliloti The strains also shared few alleles with Ensifer reference strains from other continents. Carbon source utilization assays revealed that the strains are also unusual in their ability to utilize 2-oxoglutarate as a sole carbon source. A strategy was outlined for locating similar strains elsewhere.IMPORTANCE In this study, we employed a biogeographical approach to investigate the origins of a symbiotic relationship between an Australian legume and its nitrogen-fixing rhizobia. The question of the ancestral origins of these symbionts is based on the observation that the legume host is not closely related to other native Australian legumes. Previous research has shown that the legume host Trigonella suavissima is instead closely related to legumes native to the Mediterranean Basin and western Asia, suggesting that it may have been introduced in Australia from those regions. This led to the question of whether its rhizobia may have been introduced as well. In this study, we were unable to find persuasive evidence supporting this hypothesis. Instead, our results suggest either that the T. suavissima rhizobia are native to Australia or that our methods for locating their close relatives elsewhere are inadequate. A strategy to investigate the latter alternative is proposed.

Expanding the regulatory network that controls nitrogen fixation in Sinorhizobium meliloti: elucidating the role of the two-component system hFixL-FxkR.

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.

[Occurrence of islands in genomes of Sinorhizobium meliloti native isolates].

Genomes of 184 Sinorhizobium meliloti native isolates were studied to test the occurence of islands Sme21T, Sme19T, and Sme80S previously described in the model strain Rm1021. This analysis was conducted using PCR methodology involving specific primers. It was demonstrated that, in the examined geographically distinct populations of S. meliloti from the Northern Caucasus (NCG) and the Aral Sea region (PAG), the strains containing genomic islands were observed with similar frequency (0.55 and 0.57, respectively). Island Sme80S, denoted as an island of "environmental adaptivity," was identified predominantly (frequency of 0.38) in genomes of strains which exhibited a lower level of salt tolerance and was isolated in PAG, a modern center of introgressive hybridization of alfalfa subjected to salinity. Island Sme21T designated as "ancestral" was observed in genomes of strains isolated in NCG, the primary center of host-plant biodiversity, 10-fold more often than in strains from PAG. An island Sme19T, which predominantly carries genes encoding transposases, was observed in genomes of strains in both populations with average frequency of 0.10. The analysis of linkage disequilibrium (LD) based on the assessment of probability for detection of different islands combinations in genomes revealed an independent inheritance of islands in salt-sensitive strains of various geographic origin. In contrast, the absence of this trend was noted in the majority of the examined combinations of salt-tolerant strains. It was concluded that the structure of chromosome in PAG strains which predominantly possessed a salt-sensitive phenotype was subjected to active recombinant processes, which could predetermine the intensity of microevolutionary processes in bacterial populations and facilitate an adaptation of bacteria in adverse environmental effect.

[Root Nodule Bacteria Sinorhizobium meliloti: Tolerance to Salinity and Bacterial Genetic Determinants].

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.

[Sinorhizobium meliloti strains screening for efficient bactarization of Melilotus albus Medik].

The data presents about analytical selection of root nodule bacteria of Melilotus to obtain bacterial fertilizer under sweet clover, presowing inoculation of it seeds and form a legume-rhizobial effective symbiosis. From natural melilot population a number of new strains had been allocated, inoculation of them was contributed to an increase of height. biomass Melilotus albus Medik., and nitrogenase activity in comparison to the influence of the existing production strains. The identification of most effective strains Sinorhizobium meliloti had been determined.

Biogeography of Sinorhizobium meliloti nodulating alfalfa in different Croatian regions.

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.

Nodulation competitiveness of Ensifer meliloti alfalfa nodule isolates and their potential for application as inoculants.

Alfalfa (Medicago sativa) is a widely cultivated legume, which enters into nitrogen-fixing symbiosis with Ensifer (Sinorhizobium) spp. In this study, an autochthonous rhizobial population of Ensifer sp. occupying alfalfa nodules grown in arable soil was used as the basis for selection of potential inoculants. Alfalfa nodule isolates were identified as Ensifer meliloti by partial 16S rDNA, recA, atpD and nodC nucleotide sequencing. The sampled isolates displayed different symbiotic performance and diversity in the number of plasmids and molecular weight. Isolates that were the most efficient in symbiotic nitrogen fixation were tagged with a constitutively expressed gusA gene carried by a stable plasmid vector pJBA21Tc and used in competition experiments in soil under greenhouse conditions. Two E. meliloti strains LU09 and LU12, which effectively competed with indigenous soil rhizobia, were selected. The metabolic profiles of these selected strains showed differences in the use of carbon and energy sources. In addition, the LU09 strain exhibited bacteriocin production and LU12 mineral phosphate solubilization, which are valuable traits for soil survival. These strains may be considered as potential biofertilizers for alfalfa cultivation.

Definition and evolution of a new symbiovar, sv. rigiduloides, among Ensifer meliloti efficiently nodulating Medicago species.

Understanding functional diversity is one of the main goals of microbial ecology, and definition of new bacterial ecotypes contributes significantly to this objective. Nitrogen-fixing bacteria provide a good system for investigation of ecotypes/biovars/symbiovars, as they present different specific associations with several host plants. This specific symbiosis is reflected both in the nodulation and fixation efficiency and in genetic characters of the bacteria, and several biovars have already been described in the bacterial species Ensifer meliloti. In the present study, the species affiliation of E. meliloti strains trapped from nodules sampled from Medicago rigiduloïdes roots was analyzed using housekeeping recA genes and DNA-DNA hybridization. The genetic diversity of these isolates was also investigated using several symbiotic markers: nodulation (nodA, nodB, nodC) and nitrogen fixation (nifH) genes, as well as the performance of phenotypic tests of nodulation capacity and nitrogen fixation efficiency. These analyses led to the proposal of a new bacterial symbiovar, E. meliloti sv. rigiduloides, that fixed nitrogen efficiently on M. rigiduloïdes, but not on Medicago truncatula. Using phylogenetic reconstructions, including the different described symbiovars, several hypotheses of lateral gene transfer and gene loss are proposed to explain the emergence of symbiovars within this species. The widespread geographical distribution of this symbiovar around the Mediterranean Basin, in contrast to restriction of M. rigiduloïdes to Eastern European countries, suggests that these isolates might also be associated with other plant species. The description of a new symbiovar within E. meliloti confirms the need for accurate bacterial ecological classification, especially for analysis of bacterial populations.

Use of electrophoretic techniques and MALDI-TOF MS for rapid and reliable characterization of bacteria: analysis of intact cells, cell lysates, and "washed pellets".

In this study electrophoretic and mass spectrometric analysis of three types of bacterial sample (intact cells, cell lysates, and "washed pellets") were used to develop an effective procedure for the characterization of bacteria. The samples were prepared from specific bacterial strains. Five strains representing different species of the family Rhizobiaceae were selected as model microorganisms: Rhizobium leguminosarum bv. trifolii, R. leguminosarum bv. viciae, R. galegae, R. loti, and Sinorhizobium meliloti. Samples of bacteria were subjected to analysis by four techniques: capillary zone electrophoresis (CZE), capillary isoelectric focusing (CIEF), gel IEF, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). These methods are potential alternatives to DNA-based methods for rapid and reliable characterization of bacteria. Capillary electrophoretic (CZE and CIEF) analysis of intact cells was suitable for characterization of different bacterial species. CIEF fingerprints of "washed pellets" and gel IEF of cell lysates helped to distinguish between closely related bacterial species that were not sufficiently differentiated by capillary electrophoretic analysis of intact cells. MALDI-TOF MS of "washed pellets" enabled more reliable characterization of bacteria than analysis of intact cells or cell lysates. Electrophoretic techniques and MALDI-TOF MS can both be successfully used to complement standard methods for rapid characterization of bacteria.

Taxonomic and symbiotic diversity of bacteria isolated from nodules of Acacia tortilis subsp. raddiana in arid soils of Tunisia.

A collection of rhizobia isolated from Acacia tortilis subsp. raddiana nodules from various arid soils in Tunisia was analyzed for their diversity at both taxonomic and symbiotic levels. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at 40 °C. Genetic characterization emphasized that most of the strains (42/50) belong to the genus Ensifer, particularly the species Ensifer meliloti, Ensifer garamanticus, and Ensifer numidicus. Symbiotic properties of isolates showed diversity in their capacity to nodulate their host plant and to fix atmospheric nitrogen. The most effective isolates were closely related to E. garamanticus. Nodulation tests showed that 3 strains belonging to Mesorhizobium genus failed to renodulate their host plant, which is surprising for symbiotic rhizobia. Furthermore, our results support the presence of non-nodulating endophytic bacteria belonging to the Acinetobacter genus in legume nodules.

A positive correlation between bacterial autoaggregation and biofilm formation in native Sinorhizobium meliloti isolates from Argentina.

Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodule formation on roots of alfalfa plants. S. meliloti produces two exopolysaccharides (EPSs), termed EPS I and EPS II, that are both able to promote symbiosis. EPS I and EPS II are secreted in two major fractions that reflect differing degrees of subunit polymerization, designated high- and low-molecular-weight fractions. We reported previously that EPSs are crucial for autoaggregation and biofilm formation in S. meliloti reference strains and isogenic mutants. However, the previous observations were obtained by use of "domesticated" laboratory strains, with mutations resulting from successive passages under unnatural conditions, as has been documented for reference strain Rm1021. In the present study, we analyzed the autoaggregation and biofilm formation abilities of native S. meliloti strains isolated from root nodules of alfalfa plants grown in four regions of Argentina. 16S rRNA gene analysis of all the native isolates revealed a high degree of identity with reference S. meliloti strains. PCR analysis of the expR gene of all the isolates showed that, as in the case of reference strain Rm8530, this gene is not interrupted by an insertion sequence (IS) element. A positive correlation was found between autoaggregation and biofilm formation abilities in these rhizobia, indicating that both processes depend on the same physical adhesive forces. Extracellular complementation experiments using mutants of the native strains showed that autoaggregation was dependent on EPS II production. Our results indicate that a functional EPS II synthetic pathway and its proper regulation are essential for cell-cell interactions and surface attachment of S. meliloti.

Draft genome sequence of Sinorhizobium meliloti CCNWSX0020, a nitrogen-fixing symbiont with copper tolerance capability isolated from lead-zinc mine tailings.

Sinorhizobium meliloti CCNWSX0020 was isolated from Medicago lupulina plants growing in lead-zinc mine tailings, which can establish a symbiotic relationship with Medicago species. Also, the genome of this bacterium contains a number of protein-coding sequences related to metal tolerance. We anticipate that the genomic sequence provides valuable information to explore environmental bioremediation.

The complete genome sequence of the dominant Sinorhizobium meliloti field isolate SM11 extends the S. meliloti pan-genome.

Isolates of the symbiotic nitrogen-fixing species Sinorhizobium meliloti usually contain a chromosome and two large megaplasmids encoding functions that are absolutely required for the specific interaction of the microsymbiont with corresponding host plants leading to an effective symbiosis. The complete genome sequence, including the megaplasmids pSmeSM11c (related to pSymA) and pSmeSM11d (related to pSymB), was established for the dominant, indigenous S. meliloti strain SM11 that had been isolated during a long-term field release experiment with genetically modified S. meliloti strains. The chromosome, the largest replicon of S. meliloti SM11, is 3,908,022bp in size and codes for 3785 predicted protein coding sequences. The size of megaplasmid pSmeSM11c is 1,633,319bp and it contains 1760 predicted protein coding sequences whereas megaplasmid pSmeSM11d is 1,632,395bp in size and comprises 1548 predicted coding sequences. The gene content of the SM11 chromosome is quite similar to that of the reference strain S. meliloti Rm1021. Comparison of pSmeSM11c to pSymA of the reference strain revealed that many gene regions of these replicons are variable, supporting the assessment that pSymA is a major hot-spot for intra-specific differentiation. Plasmids pSymA and pSmeSM11c both encode unique genes. Large gene regions of pSmeSM11c are closely related to corresponding parts of Sinorhizobium medicae WSM419 plasmids. Moreover, pSmeSM11c encodes further novel gene regions, e.g. additional plasmid survival genes (partition, mobilisation and conjugative transfer genes), acdS encoding 1-aminocyclopropane-1-carboxylate deaminase involved in modulation of the phytohormone ethylene level and genes having predicted functions in degradative capabilities, stress response, amino acid metabolism and associated pathways. In contrast to Rm1021 pSymA and pSmeSM11c, megaplasmid pSymB of strain Rm1021 and pSmeSM11d are highly conserved showing extensive synteny with only few rearrangements. Most remarkably, pSmeSM11b contains a new gene cluster predicted to be involved in polysaccharide biosynthesis. Compilation of the S. meliloti SM11 genome sequence contributes to an extension of the S. meliloti pan-genome.

Characterization of a copper-resistant symbiotic bacterium isolated from Medicago lupulina growing in mine tailings.

A root nodule bacterium, Sinorhizobium meliloti CCNWSX0020, resistant to 1.4 mM Cu2+ was isolated from Medicago lupulina growing in mine tailings. In medium supplied with copper, this bacterium showed cell deformation and aggregation due to precipitation of copper on the cell surface. Genes similar to the copper-resistant genes, pcoR and pcoA from Escherichia coli, were amplified by PCR from a 1.4-Mb megaplasmid. Inoculation with S. meliloti CCNWSX0020 increased the biomass of M. lupulina grown in medium added 0 and 100 mg Cu2+ kg(-1) by 45.8% and 78.2%, respectively, and increased the copper concentration inside the plant tissues grown in medium supplied with 100 µM Cu2+ by 39.3%, demonstrating that it is a prospective symbiotic system for bioremediation purposes.

Partner choice in Medicago truncatula-Sinorhizobium symbiosis.

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula-Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.

Exopolysaccharide production by nitrogen-fixing bacteria within nodules of Medicago plants exposed to chronic radiation in the Chernobyl exclusion zone.

Nitrogen-fixing bacteria isolated from root nodules of Medicago plants growing in the 10 km zone around the Chernobyl nuclear power plant were screened for the production of new water-soluble acidic exopolysaccharides (EPSs). The different strains belonged to the Enteriobacteriaceae family (Enterobacter ludwigii, Raoultella terrigena, Klebsiella oxytoca), except for one which belonged to the Rhizobiaceae family (Sinorhizobium meliloti). All of the bacteria produced highly viscous EPS with an average molecular weight comprised between 1 x 10(6) and 3 x 10(6) Da. Five different compositions of EPS were characterized by physico-chemical analyses and (1)H NMR spectroscopy: galactose/mannose (2/1), galactose/glucose (1/1), galactose/glucose/mannose (1/2/1), fucose/galactose/glucose (2/1/1) and fucose/galactose/glucose/mannose (2/2/1/1 or 1/1/2/4). Glucuronic acid, a charged monosaccharide, was also recovered in most of the different EPSs.