Phylogenetic analyses of symbiotic genes and characterization of functional traits of Mesorhizobium spp. strains associated with the promiscuous species Acacia seyal Del.

Aims: To assess the phenotypic, symbiotic and genotypic diversity scope of Mesorhizobium spp. strains associated with Acacia seyal (Del.) isolated from different agro‐ecological zones in Senegal, and uses of susceptible microbial inoculum in a reafforestation process. Methods and Results: A polyphasic approach including phenotypic and genotypic techniques was used to study the diversity and their relationships with other biovars and species of rhizobia. The geographical origins of the strains have limited effect on their phylogenetic and phenotypic classification. Nodulation tests indicated promiscuity of the strains studied, because they were capable of nodulating six woody legume species (Acacia auriculiformis, Acacia senegal, A. seyal, Acacia tortilis ssp. raddiana, Leucaena leucocephala and Prosopis juliflora). Sequencing and phylogenetic analyses of nodA, nodC and nifH genes pointed out that in contrast to nodA gene, the phylogenies of nodC and nifH genes were not consistent with that of 16S rRNA, indicating that these genes of the A. seyal‐nodulating rhizobia might have different origins. Microbial inoculation on nonsterile soil had significant effect on the nodules number and the growth of the seedlings, indicating that these strains of rhizobia might be used as inoculum. Conclusions: The results indicated that A. seyal is a nonselective host that can establish effective symbiosis with Mesorhizobium spp. strains from diverse genomic backgrounds and that the selected A. seyal‐nodulating rhizobia could enhance plant growth. Significance and Impact of the Study: These results showed the important role that A. seyal could play in the improvement of reafforestation process as a promiscuous host, which can establish effective symbiosis with rhizobia from diverse genomic backgrounds.     

Phloem‐derived γ‐aminobutyric acid (GABA) is involved in upregulating nodule N2 fixation efficiency in the model legume Medicago truncatula

Nitrogen fixation in legumes is downregulated through a whole plant N feedback mechanism, for example, when under stress. This mechanism is probably triggered by the impact of shoot‐borne, phloem‐delivered compounds. However, little is known about any whole‐plant mechanism that might upregulate nitrogen fixation, for example, under N deficiency. We induced emerging N‐deficiency through partial excision of nodules from Medicago truncatula plants. Subsequently, the activity and composition of the remaining nodules and shifts in concentration of free amides/amino acids in the phloem were monitored. Furthermore, we mimicked these shifts through artificial feeding of γ‐aminobutyric acid (GABA) into the phloem of undisturbed plants. As a result of increased specific activity of nodules, N₂ fixation per plant recovered almost completely 4–5 d after excision. The concentration of amino acids, sugars and organic acids increased strongly in the upregulated nodules. A concomitant analysis of the phloem revealed a significant increase in GABA concentration. Comparable with the effect of nodule excision, artificial GABA feeding into the phloem resulted in an increased activity and higher concentration of amino acids and organic acids in nodules. It is concluded that GABA might be involved in upregulating nodule activity, possibly because of its constituting part of a putative amino acid cycle between bacteroids and the cytosol.     

Legume species identity and soil nitrogen supply determine symbiotic nitrogen-fixation responses to elevated atmospheric [CO2

In nitrogen (N)-limited systems, the response of symbiotic N fixation to elevated atmospheric [CO2] may be an important determinant of ecosystem responses to this global change. Experimental tests of the effects of elevated [CO2] have not been consistent. Although rarely tested, differences among legume species and N supply may be important. In a field free-air CO2 enrichment (FACE) experiment, we determined, for four legume species, whether the effects of elevated atmospheric [CO2] on symbiotic N fixation depended on soil N availability or species identity. Natural abundance and pool-dilution 15N methods were used to estimate N fixation. Although N addition did, in general, decrease N fixation, contrary to theoretical predictions, elevated [CO2] did not universally increase N fixation. Rather, the effect of elevated [CO2] on N fixation was positive, neutral or negative, depending on the species and N addition. Our results suggest that legume species identity and N supply are critical factors in determining symbiotic N-fixation responses to increased atmospheric [CO2].     

Complete genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110.

The complete nucleotide sequence of the genome of a symbiotic bacterium Bradyrhizobium japonicum USDA110 was determined. The genome of B. japonicum was a single circular chromosome 9,105,828 bp in length with an average GC content of 64.1%. No plasmid was detected. The chromosome comprises 8317 potential protein-coding genes, one set of rRNA genes and 50 tRNA genes. Fifty-two percent of the potential protein genes showed sequence similarity to genes of known function and 30% to hypothetical genes. The remaining 18% had no apparent similarity to reported genes. Thirty-four percent of the B. japonicum genes showed significant sequence similarity to those of both Mesorhizobium loti and Sinorhizobium meliloti, while 23% were unique to this species. A presumptive symbiosis island 681 kb in length, which includes a 410-kb symbiotic region previously reported by G?ttfert et al., was identified. Six hundred fifty-five putative protein-coding genes were assigned in this region, and the functions of 301 genes, including those related to symbiotic nitrogen fixation and DNA transmission, were deduced. A total of 167 genes for transposases/104 copies of insertion sequences were identified in the genome. It was remarkable that 100 out of 167 transposase genes are located in the presumptive symbiotic island. DNA segments of 4 to 97 kb inserted into tRNA genes were found at 14 locations in the genome, which generates partial duplication of the target tRNA genes. These observations suggest plasticity of the B. japonicum genome, which is probably due to complex genome rearrangements such as horizontal transfer and insertion of various DNA elements, and to homologous recombination.     

Plant symbiotic mutants as a tool to analyse nitrogen nutrition and yield relationship in field-growth peas (Pisum sativum L.)

Pisum sativum L. is known for high seed and protein yields but also for.yield instability. Because legumes utilize two sources of nitrogen (atmospheric N₂ fixed in nodules and assimilation of soil mineral N), studies on their nitrogen nutrition is more complex than in other plants. In this work, pea symbiotic mutants (with no nodules at all ([Nod^-]), with inefficient nodules ([Nod⁺Fix^-]) or showing an hypernodulating and a ‘nitrate-tolerant symbiosis’ character ([Nod⁺⁺Nts]), their semi-leafless isogenic homologues and the parental control line cv Frisson were fertilized with three levels of mineral nitrogen (0, 25 or 50 g N m^-2) to generate a range of mineral nitrogen regimes in the same genetic background. Impact of the source and level of nitrogen nutrition was measured on reproductive development, growth, nitrogen accumulation and seed yield. It was shown that a N deficiency induced flowering termination. It also led to a large decrease in the number of seeds produced and the amount of N accumulated in forage and in seeds, when little effect was observed on the progression rates of reproductive stages along the stem. The single seed weight and the amount of dry matter accumulated in forage neither responded strongly to N deficiency. The source of nitrogen was shown to be of little importance to yield but the application of about 50 g N m^-2 was necessary to reach the yield of the control cv Frisson when exclusive assimilation was ensuring the N requirements of the plant. Despite the fact that the nitrate-tolerant and hypernodulating mutant P64 used in this study did not yield as well as the parent cv Frisson, it is proposed that [Nod⁺⁺Nts] characters could act as a yield regulating factor.     

Complete genome structure of the nitrogen-fixing symbiotic bacterium Mesorhizobium loti.

The complete nucleotide sequence of the genome of a symbiotic bacterium Mesorhizobium loti strain MAFF303099 was determined. The genome of M. loti consisted of a single chromosome (7,036,071 bp) and two plasmids, designated as pMLa (351,911 bp) and pMLb (208, 315 bp). The chromosome comprises 6752 potential protein-coding genes, two sets of rRNA genes and 50 tRNA genes representing 47 tRNA species. Fifty-four percent of the potential protein genes showed sequence similarity to genes of known function, 21% to hypothetical genes, and the remaining 25% had no apparent similarity to reported genes. A 611-kb DNA segment, a highly probable candidate of a symbiotic island, was identified, and 30 genes for nitrogen fixation and 24 genes for nodulation were assigned in this region. Codon usage analysis suggested that the symbiotic island as well as the plasmids originated and were transmitted from other genetic systems. The genomes of two plasmids, pMLa and pMLb, contained 320 and 209 potential protein-coding genes, respectively, for a variety of biological functions. These include genes for the ABC-transporter system, phosphate assimilation, two-component system, DNA replication and conjugation, but only one gene for nodulation was identified.     

Strains of Mesorhizobium amorphae and Mesorhizobium tianshanense, carrying symbiotic genes of common chickpea endosymbiotic species, constitute a novel biovar (ciceri) capable of nodulating Cicer arietinum

AIMS: To identify several strains of Mesorhizobium amorphae and Mesorhizobium tianshanense nodulating Cicer arietinum in Spain and Portugal, and to study the symbiotic genes carried by these strains. METHODS AND RESULTS: The sequences of 16S-23S intergenic spacer (ITS), 16S rRNA gene and symbiotic genes nodC and nifH were analysed. According to their 16S rRNA gene and ITS sequences, the strains from this study were identified as M. amorphae and M. tianshanense. The type strains of these species were isolated in China from Glycyrrhiza pallidiflora and Amorpha fruticosa nodules, respectively, and are not capable of nodulating chickpea. These strains carry symbiotic genes, phylogenetically divergent from those of the chickpea isolates, whose nodC and nifH genes showed more than 99% similarity with respect to those from Mesorhizobium ciceri and Mesorhizobium mediterraneum, the two common chickpea nodulating species in Spain and Portugal. CONCLUSIONS: The results from this study showed that different symbiotic genes have been acquired by strains from the same species during their coevolution with different legumes in distinct geographical locations. SIGNIFICANCE AND IMPACT OF THE STUDY: A new infrasubspecific division named biovar ciceri is proposed within M. amorphae and M. tianshanense to include the strains able to effectively nodulate Cicer arietinum.     

Nitrogen fixation is synchronized with carbon metabolism in Lotus japonicus and Medicago truncatula nodules under salt stress

Abstract

In the present work, the response to NaCl applied at the vegetative stage to Medicago truncatula and Lotus japonicus has been evaluated in order to ascertain whether the effect of salt stress on nitrogen fixation is due to a limitation on nodular carbon metabolism. Results show maximum sucrose synthase (SS) and alkaline invertase (AI) activities were obtained at the vegetative stage, when maximum nitrogenase activity was detected in both species. SS activity decreased with the salt treatment, providing evidence of the regulatory role of this enzyme for the carbon supply to the bacteroids. Phosphoenolpyruvate carboxylase (PEPC) and malate dehydrogenase (MDH) activities could account for higher nitrogen fixation efficiency detected in L. japonicus nodules and isocitrate dehydrogenase (ICDH) activity compensated for the carbon limitations that occur under salt stress. These results support that nitrogenase inhibition in nodules experiencing salt stress is doubt to a carbon flux shortage, as result of carbon metabolism enzymes activities down-regulation.     

Occurrence and activity of hydrogenase in symbiotic Frankia from field-collected Alnus incana

Occurrence and activity of the hydrogen uptake enzyme were studied in root nodule homogenates made from plants of Alnus incana (L.) Moench collected from field sites in the northern part of Sweden. Nitrogenase (EC 1.7.99.2) activity (estimated by acetylene reduction) and hydrogen evolution were studied in excised nodules. All Frankia sources showed acetylene reduction activity, and possessed a hydrogen uptake system. Hydrogen uptake in nodule homogenates from the Frankia sources measured at 23.8 μM H₂ ranged from 0.04 to 5.0 μmol H₂ (g fresh weight nodule)⁻¹ h⁻¹. The H₂ uptake capacity of nodule homogenates from one of the Frankia sources was almost 8 times higher than the hydrogen evolution from nitrogenase, both expressed on a nodule fresh weight basis. Frankia sources from field sites 6 and 11 showed K_m for H₂ of 13.0 and 23.6 μM H₂, respectively. This indicates similarities in the hydrogen uptake enzymes in the two Frankia sources. It is concluded that hydrogen uptake is a common characteristic in Frankia.     

Impact of sewage sludges on Medicago truncatula symbiotic proteome

The effects of sewage sludges were investigated on the symbiotic interactions between the model plant Medicago truncatula and the arbuscular mycorrhizal fungus Glomus mosseae or the rhizobial bacteria Sinorhizobium meliloti. By comparison to a control sludge showing positive effects on plant growth and root symbioses, sludges enriched with polycylic aromatic hydrocarbons or heavy metals were deleterious. Symbiosis-related proteins were detected and identified by two-dimensional electrophoresis and matrix-assisted laser desorption ionization mass spectrometry, and image analysis was used to study the effects of sewage sludges on M. truncatula symbiotic proteome.     

Recombination and selection shape the molecular diversity pattern of nitrogen-fixing Sinorhizobium sp. associated to Medicago

We investigate the genetic structure and molecular selection pattern of a sympatric population of Sinorhizobium meliloti and Sinorhizobium medicae. These bacteria fix nitrogen in association with plants of the genus Medicago. A set of 116 isolates were obtained from a soil sample, from root nodules of three groups of plants representing among-species, within-species and intraline diversity in the Medicago genus. Bacteria were characterized by sequencing at seven loci evenly distributed along the genome of both Sinorhizobium species, covering the chromosome and the two megaplasmids. We first test whether the diversity of host plants influence the bacterial diversity recovered. Using the same data set, we then analyse the selective pattern at each locus. There was no relationship between the diversity of Medicago plants that were used for sampling and the diversity of their symbionts. However, we found evidence of selection within each of the two main symbiotic regions, located on the two different megaplasmids. Purifying selection or a selective sweep was found to occur in the nod genomic region, which includes genes involved in nodulation specificity, whereas balancing selection was detected in the exo region, close to genes involved in exopolysaccharide production. Such pattern likely reflects the interaction between host plants and bacterial symbionts, with a possible conflict of interest between plants and cheater bacterial genotypes. Recombination appears to occur preferentially within and among loci located on megaplasmids, rather than within the chromosome. Thus, recombination may play an important role in resolving this conflict by allowing different selection patterns at different loci.     

Sequential evolution of a symbiont inferred from the host: Wolbachia and Drosophila simulans

This study aims to unravel the biogeography of a model symbiont/host system by exploiting the prediction that a symbiont will leave a signature of infection on the host. Specifically, a global sample of 1,442 Drosophila simulans from 33 countries and 64 sampling localities was employed to infer the phylogeography of the maternally inherited alpha-proteobacteria Wolbachia. Phylogenetic analyses, from three symbiont genes and 24 mtDNA genomes (excluding the A + T-rich region), showed that each of four Wolbachia strains infected D. simulans once. The global distribution and abundance of the Wolbachia strains and the three mtDNA haplogroups (D. simulans siI, siII and siIII) was then determined. Finally, network analyses of variable regions within siI (584 bp from seven additional lines) and siII (1,701 bp from 383 lines) facilitated a detailed biogeographic discussion. There is little variation in siIII and the haplogroup is restricted in its distribution. These data show how the history of an infection can be mapped by combining data from the symbiont and the host. They say little about the organismal history of the host because the mtDNA genome is a biased representation of the whole genome.     

Regulation of symbiotic nitrogen fixation in root nodules of alfalfa (Medicago sativa) infected with Rhizobium meliloti

Symbiotic nitrogen fixation of Rhizobium meliloti bacteroids in Medicago sativa root nodules was suppressed by several inorganic nitrogen sources. Amino acids like glutamine, glutamic acid and aspartic acid, which can serve as sole nitrogen sources for the unnodulated plant did not influence nitrogenase activity of effective nodules, even at high concentrations.Ammonia and nitrate suppressed symbiotic nitrogen fixation in vivo only at concentrations much higher than those needed for suppression of nitrogenase activity in free living nitrogen fixing bacteria. The kinetics of suppression were slow compared with that of free living nitrogen fixing bacteria. On the other hand, nitrite, which acts as a direct inhibitor of nitrogenase, suppressed very quickly and at low concentrations. Glutamic acid and glutamine enhanced the effect of ammonia dramatically, while the suppression by nitrate was enhanced only slightly.     

Cryptic diversity of the symbiotic cyanobacterium Synechococcus spongiarum among sponge hosts

Cyanobacteria are common members of sponge-associated bacterial communities and are particularly abundant symbionts of coral reef sponges. The unicellular cyanobacterium Synechococcus spongiarum is the most prevalent photosynthetic symbiont in marine sponges and inhabits taxonomically diverse hosts from tropical and temperate reefs worldwide. Despite the global distribution of S. spongiarum , molecular analyses report low levels of genetic divergence among 16S ribosomal RNA (rRNA) gene sequences from diverse sponge hosts, resulting either from the widespread dispersal ability of these symbionts or the low phylogenetic resolution of a conserved molecular marker. Partial 16S rRNA and entire 16S–23S rRNA internal transcribed spacer (ITS) genes were sequenced from cyanobacteria inhabiting 32 sponges (representing 18 species, six families and four orders) from six geographical regions. ITS phylogenies revealed 12 distinct clades of S. spongiarum that displayed 9% mean sequence divergence among clades and less than 1% sequence divergence within clades. Symbiont clades ranged in specificity from generalists to specialists, with most (10 of 12) clades detected in one or several closely related hosts. Although multiple symbiont clades inhabited some host sponges, symbiont communities appear to be structured by both geography and host phylogeny. In contrast, 16S rRNA sequences were highly conserved, exhibiting less than 1% sequence divergence among symbiont clades. ITS gene sequences displayed much higher variability than 16S rRNA sequences, highlighting the utility of ITS sequences in determining the genetic diversity and host specificity of S. spongiarum populations among reef sponges. The genetic diversity of S. spongiarum revealed by ITS sequences may be correlated with different physiological capabilities and environmental preferences that may generate variable host–symbiont interactions.     

Symbiosis between the root-nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions

Two hundred forty-three isolates of alfalfa root-nodule bacteria (Sinorhizobium meliloti) were obtained from nodules and soils sampled in the northern Aral region, experiencing secondary salinization. Isolates obtained from nodules (N isolates) were significantly more salt-tolerant than those from soils (T isolates) when grown in a liquid medium with 3.5% NaCl. It was found that wild species of alfalfa, melilot, and trigonella preferably formed symbioses with salt-tolerant root-nodule bacteria in both salinized and nonsalinized soils. Only two alfalfa species, Medicago falcata and M. trautvetteri, formed efficient symbioses in soils contrasting in salinity. The formation of efficient symbiosis with alfalfa in the presence of 0.6% NaCl was studied in 36 isolates (N and T) differing in salt tolerance and symbiotic efficiency. Fifteen isolates formed efficient symbioses in the presence of salt. The increase in the dry weight of the plants was 25–68% higher than in the control group. The efficiency of symbiotic interaction under salinization conditions depended on the symbiotic efficiency of the isolates under standard conditions but did not correlate with the source of root-nodule bacteria (soil or nodule) or their salt tolerance. The results indicate that the strains of root-nodule bacteria forming efficient symbioses under salinization conditions can be found.     

Phylogenetic perspectives on nodulation: evolving views of plants and symbiotic bacteria

Phylogenetic studies are contributing greatly to our knowledge of relationships on both sides of the plant–bacteria nodulation symbiosis. Multiple origins of nodulation (perhaps even within the legume family) appear likely. However, all nodulating flowering plants are more closely related than previously suspected, suggesting that the predisposition to nodulate might have arisen only once. Phylogenies of 16S rRNA genes highlight the evolutionary diversity of symbiotic bacteria and appear to rule out any broad coevolution with their plant hosts, but high levels of gene transfer might obscure the relevant pattern. The origins of nodulation, and the extent to which developmental programs are conserved in nodules remain unclear, but an improved understanding of the relationships between nodulin genes is providing some clues.