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The rhizosphere community represents an “ecological interface” between plant and soil, providing the plant with a number of advantages. Despite close connection and mutual influence in this system, the knowledge about the connection of plant and rhizosphere diversity is still controversial. One of the most valuable factors of this uncertainty is a rough estimation of plant diversity. NGS sequencing can make the estimations of the plant community more precise than classical geobotanical methods. We investigate fallow and crop sites, which are similar in terms of environmental conditions and soil legacy, yet at the same time are significantly different in terms of plant diversity. We explored amplicons of both the plant root mass (ITS1 DNA) and the microbial communities (16S rDNA), determined alpha- and beta-diversity indices and their correlation, and performed differential abundance analysis. In the analysis, there is no correlation between the alpha-diversity indices of plants and the rhizosphere microbial communities. The beta-diversity between rhizosphere microbial communities and plant communities is highly correlated (R = 0.866, p = 0.01). ITS1 sequencing is effective for the description of plant root communities. There is a connection between rhizosphere communities and the composition of plants, but on the alpha-diversity level we found no correlation. In the future, the connection of alpha-diversities should be explored using ITS1 sequencing, even in more similar plant communities—for example, in different synusia.

Compared to free-living relatives, bacterial symbionts of plants exhibit higher rates of population diversification (microevolution), as well as of speciation and formation of super-species taxa (macroevolution). Analysis of rhizobia, the N2-fixing symbionts of leguminous plants, suggests that both evolutionary modes are determined by the hosts, which have mutagenic and recombinogenic effects on their microsymbionts, apart from activating the selective processes that determine the fate of newly emerged genotypes in their populations. Migration induced by leguminous plants plays a key role in the evolution of rhizobia, including: (1) joint movement of the symbionts and their hosts into new ecological zones and (2) local circulation of the symbionts in plant‒soil systems. In the first case, allopatric speciation occurs in rhizobia due to transfer of the symbiotically specialized (sym) genes from the introduced strains into local bacteria, which are transformed into novel symbionts. In the second case, a sympatric divergence of Rhizobium populations occurs, resulting in formation of intraspecies taxa (biovars and symbiovars) contrasting in their host ranges. In the evolutionarily advanced rhizobia, Rhizobium leguminosarum and Neorhizobium galegae interacting with galegoid legumes (tribes Fabeae, Galegae, and Trifolieae), these processes are associated with diversification of the sym genes representing the accessory genome parts due to the interaction between bacteria and the hosts differing in their symbiotic affinities. In parallel, formation of new cryptic (genomic) species was revealed in local rhizobia populations, which is associated with divergence of the core genome parts. It may be caused either by genetic drift or by selection for resistance against local edaphic stresses, which in combination with disruptive selection can sufficiently enhance the rhizobia diversity and change the structures of their pangenomes.

The rhizosphere community represents an “ecological interface” between plant and soil, providing the plant with a number of advantages. Close connection and mutual influence in this communication allow to talk about the self-adjusting “plant-rhizosphere community” system, which should be be studied in connection. Diversity estimation is one of the ways of describing both bacterial and plant communities. Based on the literature, there are two assumptions of how the diversity of plant communities related to the diversity of bacterial communities: 1) an increase in the species richness of plants leads to an increase in the number of available micro-niches, and increasing of microbial diversity, 2) an increase in the species richness of plants is accompanied by the predominant development of bacteria from highly productive specific taxa and decreasing in the diversity of microorganisms. Experimental studies show controversial results.We analyzed field sites (rye crop field and two fallow sites), using DNA isolation of both the plant root mass (followed by sequencing of the ITS1 region) and rhizosphere microorganisms (followed by sequencing of the 16s rDNA V4 region). This allowed us to 1) accurately determine the abundance and taxonomic position of plant communities; 2) extract information about both plant and microbial communities from the same sample.There was no correlation between alpha-diversity indices of plants and rhizosphere communities. Alpha-diversity connection should be explored in similar plant communities, such as synusia. We hypothesize, that the significant differences in plant abundances lead to significant changes in exudation profiles, and the loss of diversity connection. The beta-diversity between rhizosphere communities and plant communities is highly correlated, in particular in terms of the abundance of taxa. This can be explained by a potential correlation (as reported in the literature) or by the presence of statistical artifacts.

Rhizobia represent a diverse group of gram-negative bacteria capable of fixing atmospheric nitrogen in symbiosis with leguminous plants. Mechanisms of symbiotic efficiency are important to study not only to reveal the “fine tuning” of the host–symbiont supra-organismal genetic system emergence, but also to develop agriculture with minimal environmental risks. In this paper we demonstrate that among seven genes whose inactivation by Tn5 insertions results in an increased efficiency of rhizobia (Sinorhizobium meliloti) symbiosis with alfalfa (Eff++ phenotype), six genes are involved in the metabolism of small molecules. One of them (SMc04399) encodes for acetate-CoA transferase catalyzing the formation of acetyl-CoA from acyl-CoA. Since acetyl-CoA is required for operation of the Krebs cycle, providing ATP for symbiotic N2 fixation, we suggest that a significant portion of this coenzyme utilized by bacteroids is provided by the plant cell supporting the energy-consuming nitrogenase reaction. Proteomic data analysis allow us to reveal the lability of enzymatic pathways which are involved in bacteroids in the production and catabolism of acetyl-CoA and which should be modified to obtain the Eff++ phenotype. This phenotype was developed also after inactivation of NoeB protein which is involved in the host-specific nodulation and is characterized by an elevated production in wild type S. meliloti bacteroids, suggesting a multifunctional role of noeB in the symbiosis operation.

Nodule bacteria are presented as models for developing the major problems of evolutionary genetics – analysis of the trade-off between macro- and microevolution, progressive and adaptive evolution, individual and cooperative adaptations.

It was shown that in the nodule bacteria of the vetch and goats’ rue, isolated in the North Caucasian region, the phylogenetic differences of biotypes for symbiotic genes are more pronounced than for the housekeeping genes.

R. leguminosarum bv. trifolii strains are characterized by narrow host specificity. We have identified a strain that forms nodules on several types of clover and studied the genetic organization of its symbiotic region.

The goat’s rue rhizobia (Neorhizobium galegae) represent a convenient model to study the evolution and speciation of symbiotic bacteria. This rhizobial species is composed of two biovars (bv. orientalis and bv. officinalis), which form N2-fixing nodules with certain species of goat’s rue (Galega orientalis and G. officinalis). The cross-inoculation between them results in the formation of nodules unable to fix nitrogen. On the basis of the data on the whole-genome sequencing, we studied the nucleotide polymorphism of 11 N. galegae strains isolated from the North Caucasus ecosystems, where G. orientalis has higher diversity than G. officinalis. The low level of differences in the polymorphism within the group of the sym genes in comparison with the nonsymbiotic genes can be associated with the active participation of host plants in the evolution of rhizobia. The intragenic polymorphism of bv. orientalis proved to be significantly higher than that of bv. officinalis. The level of polymorphism of nonsymbiotic genes was lower than that of the symbiotic genes, which are functionally more homogeneous. The divergence of the nitrogen fixation genes (nif/fix) is more pronounced than that of the nodule formation genes (nod) in the N. galegae biovars. These facts indicate the leading role of the host-specific nitrogen fixation in the evolution of the studied rizhobial species.

—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 lipopolysaccharides 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.

Comparative sequence analysis of symbiotic genes (nodA, nodC, nodD, nifH), which are elements of accessory component of the rhizobial genome, demonstrated that the strains of Rhizobium leguminosarum bv. viciae, isolated from the nodules of a relic legume, Vavilovia formosa, the closest relative of hypothetical common ancestor of the tribe Fabeae, represented a group separated from the strains of R. leguminosarum bv. viciae, isolated from other representatives of this tribe (Vicia, Lathyrus, Pisum, Lens). No isolation was observed relative to the genes representing the core component of the rhizobial genome (16S rDNA, ITS, glnII) or relative to host specificity of the rhizobia. The data obtained suggest that sequence divergence of symbiotic genes marks the initial stage of sympatric speciation, which can be classified as the isolation of the relic “vaviloviae” symbiotype, a possible evolutionary precursor of the “viciae” biotype.

Many bacteria belonging to the order Rhizobiales have fixNOQP genes which encode cytochrome oxidase with high affinity to oxygen required for oxidative phosphorylation in microaerophilic conditions. There is one copy of the identified fixNOQP operon in ancestral forms of rhizobia (Bradyrhizobium), as well as in their putative evolutionary predecessors (bacteria related to Rhodopseudomonas). At the same time, forms deeply specialized in symbiosis (Rhizobium leguminosarum, Sinorhizobium meliloti) have multiple (2–3) copies, some of them have a high similarity (>90%) to fixNOQP genes of Bradyrhizobium and Rhodopseudomonas, and others have only 30–50% similarity. Two divergent copies fixNOQP are detected in Tardiphaga, which is a representative of the Bradyrhizobiaceae family, lacking the ability to fix N2 (lack of nif genes encoding the synthesis of nitrogenase) and to induce the formation of nodules on legumes roots (lack of nod genes encoding the synthesis of signal Nod factors activating symbiosis development). The presence of Tardiphaga in nodule bacterial communities from a range of legumes, including Vavilovia formosa (relic representative of the tribe Fabeae, for which R. leguminosarum bv. viciae is the main microsymbiont), suggests that the ancestral gene duplication and subsequent divergence of fixNOQP operon in bacteria related to Tardiphaga opened the possibility of wide dissemination of functionally different copies of this cluster among symbiotically active forms of Rhizobiales. It is possible that the acquisition of fixNOQP genes determines adaptation of bacteria to microaerophilic niches not only in plants nodules but also in their environment (the rhizosphere, rhizoplane, internal portions of soil aggregates).

The role of different forms of natural selection in the evolution of genomes in root nodule bacteria (rhizobia) is analyzed for the first time. In these nitrogen-fixing symbionts of leguminous plants, two types of genome organization are revealed: (i) unitary type, where over 95% of genetic information is encoded by chromosomes (5.3–5.5 Mb in Azorhizobium, 7.0–7.8 Mb in Mesorhizobium, 7.3–10.1 Mb in Bradyrhizobium); (ii) multipartite type, where up to 50% of genetic information is allocated to plasmids or chromids which may exceed 2 Mb in size and usually control the symbiotic properties (pSyms) in fast-growing rhizobia (Rhizobium, Sinorhizobium, Neorhizobium). Emergence of fast-growing species with narrow host ranges are correlated to the extension of extrachromosomal parts of genomes, including the increase in pSyms sizes (in Sinorhizobium). An important role in this evolution is implemented by diversifying selection since the genomic diversity evolved in rhizobia owing to symbiotic interactions with highly divergent legumes. However, analysis of polymorphism in nod genes (encoding synthesis of lipo-chitooligosaccharide signaling Nod factors) suggests that the impacts of diversifying selection are restricted to the bacterial divergence for host specificity and do not influence the overall genome organization. Since the extension of rhizobia genome diversity results from the horizontal sym gene transfer occurring with low frequencies, we suggest that this extension is due to the frequency-dependent selection anchoring the rare genotypes in bacterial populations. It is implemented during the rhizobia competition for nodulation encoded by the functionally diverse cmp genes. Their location in different parts of bacterial genomes may be considered as an important factor of their adaptive diversification implemented in the host-associated microbial communities.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

The most recent data on the system of cmp (competitiveness) genes that determine the nodulation competitiveness of rhizobial strains, i.e., the ability to compete for nodule formation in leguminous plants, is analyzed. Three genetic approaches for the construction of economically valuable strains of rhizobia are proposed: the amplification of positive regulators of competitiveness, the inactivation of the negative regulators of this trait, and the introduction of efficient competitiveness factors into strains capable of active nitrogen fixation.

The structure of the plasmid locus containing the sym-genes (nod-, nif-, and fix-operons) was investigated in eight Rhizobium leguminosarum strains differing in their origin and host specificity, including five strains of the viciae biovar—symbionts of pea (3), forage beans (1), and Vavilovia (1)—as well as three strains of the biovar trifolii (clover symbionts). Strains of R. leguminosarum bv. viciae, which possess the nodX gene (controlling acetylation of the Nod factor, which is responsible for the ability of rhizobia to form symbioses with a broad spectrum of hosts, including the “Afghan” pea lines, homozygous by the allele sym2A), are characterized by a less compact location of the sym-genes than the strains lacking the nodX gene. The size of the symbiotic cluster in the strains possessing nodX was 94.5 ± 3.5 kb, with the share of the sym-genes of 36.5 ± 1.5%, while for the strains lacking nodX these values were 61.7 ± 3.7 kb and 56.3 ± 1.4%, respectively (significant difference at P0 < 0.01). Syntenic structures were revealed in the symbiotic regions of strains Vaf12, UPM1131, and TOM, as well as syntenic structures of non-symbiotic regions in strains Vaf12, TOM, and WSM1689. The correlation coefficients between the matrices of genetic distances in the analyzed strains for the nodABC, nifHDK, and fixABC operons were on average 0.993 ± 0.002, while their values for the plasmid sites located between the sym-genes were considerably less (0.706 ± 0.010). In these regions, 21 to 27% of the genes were involved in amino acid transport and metabolism, which was substantially higher than the average for the genome of R. leguminosarum bv. viciae (11–12%). These data suggest that the evolution of R. leguminosarum bv. viciae, defined by narrowing of the host specificity (associated with a loss of the nodX gene), was accompanied by reduction of the regions of plasmids located between the sym-genes, as well as by specialization of these areas to perform the functions related to symbiotic nitrogen fixation. The observed increase of density in the cluster of sym-genes may be associated with intensification of their horizontal transfer in the populations of rhizobia, which determines the speed of evolution of the symbiotic system.

The data on heavy metal (HM) accumulation and detoxification by plants and bacteria in plant–microbial systems (PMS) are reviewed. Bacteria are shown to be the labile component of the system, responsible for a considerable amelioration of HM stress impact on plants and for improved PMS adaptation to heavy metals. Simulation of plant–microbial interactions under conditions of soil contamination by HM revealed the protective role of bacterial migration from the rhizoplane to the rhizosphere.

Symbiogenic evolution, which involves transformations of bacteria into the cellular organelles, is represented as a model for reconstructing the early stages of genome evolution, including the origin of DNA genomes from RNA genomes and the emergence of template processes on the basis of self-replicating molecular complexes in the ancestral metabolic systems. The antiquity of RNA genomes is supported by an increased evolutionary stability of ribosomal protein synthesis (translation) with respect to the DNA-dependent template processes (replication, transcription, recombination, and reparation). This stability is demonstrated by analysis of the deeply reduced genomes of symbiotic bacteria and cellular organelles as well as the “minimal” genomes which are common to phylogenetically diverse organisms. Higher evolutionary conservation of template biosynthetic processes with respect to step processes determining the metabolism and development in cells does not support the hypothesis about emergence of genomes within the ancestral cellular metabolic systems which are thought to be of abiogenic origin, instead suggesting dualistic origin of life on Earth. We suppose that the genome-free organelles of some eukaryotes (mitosomes, many hydrogenosomes, and some plastids) represent the products of reversion of symbiotic bacteria into ancestral forms which implemented their basic cellular functions using the informational macromolecules of exogenic origin. In the framework of this hypothesis the eukaryotic cells functioning based on the massive transfer of gene products (RNAs, proteins) from cytosol to organelles may represent the analogs of ancestral biocenoses that possessed integral hereditary systems (metagenomes).

Using high throughput sequencing of the nodA gene, we studied the population dynamics of Rhizobium leguminosarum (bv. viciae, bv. trifolii) in rhizospheric and nodular subpopulations associated with the leguminous plants representing different cross-inoculation groups (Vicia sativa, Lathyrus pratensis of the vetch/vetchling/pea group and Trifolium hybridum of the clover group). The “rhizosphere → nodules” transitions result in either an increase or decrease in the frequencies of 10 of the 23 operational taxonomic units (OTUs) (which were identified with 95% similarity) depending on the symbiotic specificity and phylogenetic positions of OTUs. Statistical and bioinformatical analysis of the population structures suggest that the type of natural selection responsible for these changes may be diversifying at the whole-population level and frequency-dependent at the OTU-specific level, ensuring the divergent evolution of rhizobia interacting with different host species.

We propose the Evolutionary Moulding hypothesis that population diversities of partners in nitrogen-fixing rhizobium-legume symbiosis are matched, and tested it in nucleotide polymorphism of symbiotic genes encoding two components of the plant-bacteria signalling system. The first component is the rhizobialnodA acyltransferase involved in the fatty acid tail decoration of Nod factor (rhizobia signalling molecule). The second component is the plantNFR5receptor, putatively required for Nod-factor binding.We collected three wild growing legume species together with soil samples adjacent to the roots (soil pool) from one large 25-year fallow:Vicia sativa, Lathyrus pratensisandTrifolium hybridumnodulated by one of the twoRhizobium leguminosarumbiovars (viciaeandtrifolii). For each plant species we prepared three pools for DNA extraction: the plant pool (30 plant indiv.), the nodule pool (90 nodules) and the soil pool (30 samples).NFR5gene libraries from the plant pool andnodA gene libraries from nodule and soil pools were sequenced by Sanger technology and High-throughput pyrosequencing, respectively. Analysis of the data demonstrated concordance in population diversities of one symbiotic partner (rhizobia) the second partner (legume host), in line with the Evolutionary Moulding hypothesis. This effect was evinced by the following observations for each plant species: (1) significantly increased diversity in the nodulenodA popset (set of gene sequences derived from the nodule population) compared to the soil popset; (2) a monotonic relationship between the diversity in the plantNFR5gene popset and the nodule rhizobialnodA gene popset; and (3) higher topological similarity of theNFR5gene tree with thenodA gene tree of the nodule popset, than with thenodA gene tree of the soil popset. Both nonsynonymous diversity and Tajima’s D were increased in the nodule popsets compared to the soil popsets, consistent with relaxation of negative selection and/or admixture of balancing selection underlying the Evolutionary Moulding effect. We propose that the observed genetic concordance arises from the selection of particular characteristics of the nodulenodA genes by the host plant.

Using the example of N2-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve: (i) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; (ii) the loss of genes encoding for functions that are required for autonomous performance but interfere with symbiotic functions (negative regulators of symbiosis). Therefore, the construction of effective rhizobia strains should involve improvement of sym genes activities (for instance, nif, fix, and dct genes encoding for nitrogenase synthesis or for the energy supply of N2 fixation), as well as the inactivation of negative regulators of symbiosis identified in our lab (eff genes encoding for the transport of sugars and the production of polysaccharides and storage compounds, as well as for oxidative-reductive processes).

Vavilovia formosa is a relict leguminous plant growing in hard-to-reach habitats in the rocky highlands of the Caucasus and Middle East, and it is considered as the putative closest living relative of the last common ancestor (LCA) of the Fabeae tribe. Symbionts of Vavilovia belonging to Rhizobium leguminosarum bv. viciae compose a discrete group that differs from the other strains, especially in the nucleotide sequences of the symbiotically specialised (sym) genes. Comparison of the genomes of Vavilovia strains with the reference group composed of R. leguminosarum bv. viciae strains isolated from Pisum and Vicia demonstrated that the vavilovia strains have a set of genomic features, probably indicating the important stages of microevolution of the symbiotic system. Specifically, symbionts of Vavilovia (considered as an ancestral group) demonstrated a scattered arrangement of sym genes (&gt, 90 kb cluster on pSym), with the location of nodT gene outside of the other nod operons, the presence of nodX and fixW, and the absence of chromosomal fixNOPQ copies. In contrast, the reference (derived) group harboured sym genes as a compact cluster (&lt, 60 kb) on a single pSym, lacking nodX and fixW, with nodT between nodN and nodO, and possessing chromosomal fixNOPQ copies. The TOM strain, obtained from nodules of the primitive &ldquo, Afghan&rdquo, peas, occupied an intermediate position because it has the chromosomal fixNOPQ copy, while the other features, the most important of which is presence of nodX and fixW, were similar to the Vavilovia strains. We suggest that genome evolution from the ancestral to the derived R. leguminosarum bv. viciae groups follows the &ldquo, gain-and-loss of sym genes&rdquo, and the &ldquo, compaction of sym cluster&rdquo, strategies, which are common for the macro-evolutionary and micro-evolutionary processes. The revealed genomic features are in concordance with a relict status of the vavilovia strains, indicating that V. formosa coexists with ancestral microsymbionts, which are presumably close to the LCA of R. leguminosarum bv. viciae.

In the N2-fixing symbionts of legumes (rhizobia), the evolution of (altruistic) characters that are useful for a host occurs in the populations colonizing the subscellular compartments of nodules (infection threads, symbiosomes). These compartments appear as a result of the coevolution of partners, which is associated with the complication of the trophic and regulatory interactions determining the ecological efficiency of symbiosis. Their analysis allows us to study the correlation of the mechanisms of the adaptive and progressive evolution of symbiosis, which is still unclear for free-living organisms.

The mathematical model is constructed which describes the impacts of chaotically changing environment on the frequencies and productivity of partners in the legume-rhizobia symbiosis. The most sensitive for external impacts are the adaptively prospective bacteria strains which are specific with respect to hosts and are capable for the intensive evolution towards an improved symbiotic efficiency. An increased stability of these strains in symbiotic system may be an important factor of its evolution for the improved efficiency of partners’ interaction.

The review summarizes the results of studies on the comigration of tubercular bacteria and bean plants to new habitats, which is often accompanied by a decrease in the symbiosis efficiency due to a loss of the diversity of genes responsible for the interaction. This migration may lead to a rise in new symbionts as a result of gene transfers from initial symbionts to local bacteria. It was demonstrated that typically new symbionts lack an ability for N2 fixation but are highly competitive, blocking the inoculation of bean cultures by industrial strains. The design of coadapted systems of recognition and signal interaction of partners is a perspective approach to ensure competitive advantages of efficient rhizobia strains introduced into agrocenoses, together with host plants, over inactive local strains.

Factor analysis has been conducted for the data on the interaction between the genes of the root nodule bacteria (rhizobia), which influence the efficiency of symbiosis with leguminous plants, including dctA (encoding succinate permease), dctBD (activating the dctA gene due to binding its enhancer in the presence of succinate), rpoN (activating the promoters of dctA and nitrogenase genes nifHDK), and nifA (activating the nitrogenase genes due to binding their enhancers). The analysis of the alfalfa rhizobia (Sinorhizobium meliloti) recombinants that contain additional copies of these genes suggested the antagonistic (epistatic) interaction between nifA and rpoN. It may be associated either with the competition for C compounds imported into the nodules between the energy production and nitrogen assimilation processes or with the competition for redox potentials between the oxidative phosphorylation and nitrogen fixation processes. Since the phenotypic effects of the studied genes depend on the activity of nitrogen export into the aerial parts of plants, we suppose that its accumulation in bacteroids impairs the activation of the nifHDK genes by the NifA protein due to its interaction with the GlnB protein (the nitrogen metabolism regulator) or with the FixLJ and ActSR proteins (the redox potential regulators).

In N2-fixing symbionts of leguminous plants (rhizobia) evolution of the host-beneficial (“altruistic”) traits occurs in populations colonizing the subcellular compartments in nodules (infection threads, symbiosomes). These compartments are developed as a result of partners’ coevolution related to complications of trophic and regulatory interactions elevating the ecological efficiency of symbiosis. Their analysis enables us to study correlations between genetic mechanisms of adaptive and progressive symbiosis evolution which remain obscure in free-living organisms

Using the example of nodular legume-rhizobia symbiosis (LRS), we discuss the evolution in plant micro-symbionts of mutualistic traits that are apparently host-beneficial and therefore the products of inter-species evolution. These traits include: in planta activation of N2 fixation machinery; exporting the products of nitrogenase reaction into the plant cells/tissues; and the terminal differentiation of bacteria into non-reproductive N2-fixing bacteroids. It seems probable that such adaptive traits evolved by natural selection within the populations of endosymbiotic bacteria that colonize the extra- and intra-cellular compartments provided by the hosts (i.e., infection threads and symbiosomes). This evolution would occur under the impacts of group (inter-deme, kin) selection pressures induced by the partners’ metabolic and regulatory feedbacks that ensure the high activity of symbiotic N2 fixation. These important feedbacks include: progressive allocation of C compounds into N2-fixing nodules; maintenance of micro-aerobic intracellular environments that are indispensable for intensive N2 fixation; and stringent control by the host over bacterial reproduction in planta. A computational simulation of the associated co-evolutionary processes reveals the trade-off between inter-species and individual species components of progressive and adaptive LRS evolution. This is expressed as a correlated increase of ecological efficiency, functional integrity and genotypic specificity of mutualistic symbiosis. Thus, the evolution of rhizobia in symbiosis may be represented by a progressive multi-level scenario based on increasing the dependency of bacteria on the host-provided nutrients accompanied by increasing complexity of the bacterial genomes and of the symbiosis-encoding gene networks.

Comparative study of Rhizobium leguminosarum populations formed under the conditions of the Srednii Island (White Sea) demonstrated the introduced clover rhizobia (R. l. bv. trifolii) to be more variable than the aboriginal vetch/vetchling rhizobia (R. l. bv. viceae) in the chromosomal IGS locus, while being less variable in the plasmid-located symbiotic genes nodD and nifH. The analysis of these genes revealed the most pronounced differences between the clover and vetch/vetchling rhizobia populations. These differences, together with the results of ERIC-fingerprinting, indicated that the evolution of the clover rhizobia was mainly linked with the adaptation to local soil environment, and the evolution of the vetch/vetchling rhizobia, to the adaptation to various species of the host plants. High panmixia of R. leguminosarum population suggests its evolution to be based on the combinatory variability associated with the transfer of Sym-plasmids between R. l. bv. trifolii and R. l. bv. viceae, as well as with genomic rearrangements in the resulting recombinants.

Based on the knowledge on the structural and functional organization, ecological potential, and evolution of symbiotic complexes, we suggest to formulate the subject, aims, and methodology of symbiogenetics as a science studying the genetic control of interspecies interactions. It is based on the view on the superspecies system of variation and heredity (symbiogenome) controlling the development of novel properties lacking in the unitary organisms and radically extending their adaptive potentials. Investigation of symbiogenomes represents the first step toward genetic analysis of microbiomes and metagenomes, which are superspecies hereditary systems responsible for developing the multicomponent complexes of biocenotic type, such as rumen microflora, endophytic and rhizospheric communities, soil microbial consortia. The approaches of symbiogenetics can be used for developing biotechnologies of integration of plants or animals with beneficial microbes ensuring host nutrition and development as well as resistance to biotic and abiotic stresses.

The methodology is suggested for analyzing the variability and heredity in super-organism genetic systems of different complexity ranging from two-component plant-microbe and animal-microbe symbiosis to the endosymbiotic and soil microbial communities. The approaches based on symbiogenetics and metagenomics may be used for development of highlyproductive ecologically sustainable agrocenoses based on the substitution of agrochemicals (mineral fertilizers, pesticides) by the microbial preparations. A possibility is emphasized to use the natural analogs of agrocenoses for development of models to implement the directed construction and improvement of productivity in the sustainable agricultural systems.

Polymorphism analysis was performed in Rhizobium leguminosarum bv. viceae populations isolated from geographically distant regions of Ukraine and Middle Asia. Examination of cultural, biochemical, and symbiotic traits revealed interpopulation differences, which were attributed to the difference in conditions between natural ecosystems and agrocenoses. Vetch has high species diversity and is not cultivated in Middle Asia, and the corresponding rhizobial population displayed higher genetic diversity and higher polymorphism of adaptive traits ensuring saprophytic development in soil and the rhizosphere, including melanin synthesis (35%) and active exopolysaccharide production (90%). Strains of the Ukrainian population had a lower exopolysaccharide production (10%), did not produce melanin, had higher herbicide resistance, and utilized glucose and succinate (main components of plant root exudation) as carbon sources. Strains capable of efficient symbiosis with Vicia villosa Roth. had a higher frequency in the Middle Asian than in the Ukrainian population, especially among strains isolated from soil (80 and 35%, respectively). In addition, strains of the Middle Asian population better competed for nodulation. It was assumed that the formation of rhizobial populations in vetch cultivation regions (Ukraine) is aimed at adaptation to ectosymbiotic (rhizospheric) interactions with plants and anthropogenic stress factors, while strains of the vetch original center (Middle Asia) are mostly adapted to the endosymbiotic interaction and to natural edaphic stress factors.

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.

Legumes develop different mutually beneficial microbial-root symbioses such as arbuscular mysorrhiza (AM), rhizobium-legume symbiosis (RLS) and epiphytic or endophytic associations with plant growth-promoting bacteria (PGPB) which are distinguished in level of integration of the partners. Evidences of the role of AM as ancestral form of symbiosis which might be a source of the legume pre-adaptation to form some RLS are demonstrated. The RLS is supposed to evolve for a few times in ancient legumes in parallel ways based on the universal organization and regulatory mechanisms of the plant genetic material. Associations of plant roots with PGPB probably are the vestige of the early stages of evolution in morphologically differentiated RLS. Also, it is quite possible that 'first' rhizobia have originated from bacterial endosymbionts of AM fungi; then AM fungi might operate as effective vectors for introducing bacteria into the plants. Thus, the legume root symbioses may be considered as a single 'evolutionary plant-microbial continuum'. The acquired knowledge about evolution of plantmicrobe symbioses would contribute to the creation of new commercial varieties of plants with the use of both bio-engineered methods and traditional plant breeding. An original conception of legume breeding to improve their symbiotic effectiveness is proposed.

The development of symbioses ensures formation of the super-organism systems for heredity (symbiogenomes) which represent the products of joint adaptations of partners towards an unfavorable environment. Using the examples of symbioses which enable plants and microorganisms to cooperatively overcome the limitations in the major biogenic elements (C, N, P) or impacts of the biotic and abiotic stresses we demonstrate that symbiosis involves not only the de novo formation (epigenesis) by plant of the ecological niches for hosting the microsymbionts, but also the reorganizations of relevant genetic systems in accordance to the partners’ genotypes and environmental conditions. A possibility to address the ongoing processes in terms of epigenetics is evident when the microsymbionts occurring in the novel niches are included into the host reproduction cycle ensuring a stable maintenance of novel adaptation in the next generations suggesting that the newly formed symbiogenome have acquired the properties of a system for inheritance of the newly acquired adaptive traits.

Simulation of evolution in the legume-rhizobial symbiosis allowed us to suggest the mathematical criterion (index) for the functional integrity of symbiosis (FIS). Calculation of FIS index is based on analysis of the covariance matrix of partners’ frequencies influenced by fluctuations of the systemic parameters. The positive correlations of FIS with ecological efficiency of symbiosis (its impact on the seed productivity of plants) and a negative correlation with the partners’ diversity indices were demonstrated. The revealed dependences allow us to explain the increasing of structural-functional integrity in mutualistic symbiosis byoperation of the natural selection in favor of an improved ecological efficiency of partners’ interactions.

Interactions were studied between oat (Avena sativa) and two bacterial species, Bacillus subtilis and Pantoea agglomerans, in soils contaminated with heavy metals (HM), cadmium (50 mg/kg), and lead (200 mg/kg). Exposure to HM resulted in decreased (by 30–50%) length, mass, and ratio of shoot to root dimensions. Inoculation with bacteria lead to restoration and further enhancement of plant productivity, raising it above the level achieved via inoculation of oat in uncontaminated soils. It also reduced HM accumulation by plants. Pure cultures of P. agglomerans accumulate HM more intensively than those of B. subtilis (adsorbing activity was studied for both cells and extracellular metabolites). After the introduction of bacteria, lead, and cadmium content in soil decreased four- to fivefold and two- to threefold, respectively. Protection from HM is attributable to reorganizations in the populations of root-associated bacteria: cell number increases in the rhizoplane while decreasing in the rhizosphere.

Inactivation of genes involved in the dicarbyxylic acid transport in alfalfa rhizobia, Sinorhizobium meliloti (structural gene of succinate permease dctA and its transcriptional regulators dctBD, nifA, ntrA) resulted in the full or partial loss of Nsub2/sub-fixing activity while amplifications of these genes - in its improvement. It lead to the marked increases of N and C accumulation in alfalfa while its shoot mass was increased by a much lesser degree due to the incomplete N translocation from the roots. Factorial analysis suggested that dctABD amplification was important for improving the symbiotic efficiency in all trials while the effects of dctA, nifA and ntrA amplifications depend on the plant genotypes and growth conditions.

The model is suggested for evolution of Nsub2/sub-fixing legume-rhizobia symbiosis implemented under the conditions of multi-strain bacteria competition for inoculation of symbiotic habitats (rhizosphere, nodules). Competitiveness of each strain is characterized by the power coefficients which reflect the operation of frequency-dependent selection in the rhizobia population. When polymorphic bacteria populations are interacting with the dimorphic plant population, the selective pressures in favor of hostspecific symbionts (forming Nsub2/sub-fixing nodules only with one of the available plant genotypes) are higher than the pressures in favor of non-host-specific symbionts (forming these nodules with both plant genotypes). The highest mutualism efficiency is reached under an intermediate level of plant population diversity.

The molecular mechanism of the agronomically important nutritional and defensive plantmicrobe symbioses are reviewed. These symbioses are based on the signaling interactions which result in the development of novel tissue/cellular structures and of extended metabolic capacities in the partners which improve greatly the adaptive potential of plants due to an increased tolerance to biotic or abiotic stresses. The molecular, genetic and ecological knowledge on plant-microbe interactions provide a strategy for a sustainable crop production based on substituting the agrochemicals (mineral fertilizers, pesticides) by the microbial inoculants. An improvement of plantmicrobe symbioses should involve the coordinated partners' modifications resulted in complementary combinations of their genotypes. The research presented in this paper is supported by grants from Russian Foundation of Basic Research (04-04-48457, 06-04-48800, 06-04-89000NWO); NWO Centre of Excellence: 047. 018. 001.

The mathematical model for evolution of legume-rhizobia mutualism based on the partners' positive feedbacks resulted from their metabolic (C-N) exchange is presented. Negative FDS in rhizobia population, combined with the partners' positive feedbacks ensure anchoring or even domination of the mutants which either acquired the mutualistic traits or changed the specificity in their expression with different host genotypes. The created model allows us to consider the mutualistic symbiosis as of a finely balanced population system in which the equilibrium may be shifted in favor of beneficial microbial genotypes due to natural selection for an improved symbiotic efficiency implemented in plant population. Research is supported by RFBR grant 06-04-48800a.

Using the examples of diverse interactions among prokaryotes and eukaryotes, the relationships between molecular and population mechanisms of evolution of symbiotic bacteria are addressed. Their circulation in host-environment systems activates microevolutionary factors that direct combinative or reductive genome evolution in facultative, ecologically obligatory, and genetically obligatory symbioses. It is shown on the example of symbiosis of rhizobia with legumes, that due to intensive systemic intra-genome rearrangements and horizontal gene transfer, two types of gene systems evolve in these bacteria: (1) controlling the pathogenesis-like processes of host recognition and penetration and (2) responsible for mutualistic interactions that are related to nitrogen fixation and its transfer to the host. The evolution of gene systems of type 1 is directed by individual (Darwinian, frequency-dependent) selection, which is responsible for gene-for-gene interactions between the partners. In the evolution of the type 2 systems, group (interdeme, kin) selection plays the key role, being responsible for the development of bacterial traits beneficial for the host. It is shown that evolution of mutualism can be described in terms of biological altruism, whose regularities are common for intraspecific and interspecific relationships. Macroevolutionary rearrangements of bacterial genomes result from the structural changes in their populations, wherein various selection modes are combined with stochastic processes (genetic drift, population waves) induced in the symbiotic systems.

The purpose of the course "Genetic mechanisms of individual and cooperative adaptation" (12 semester 18 hours) - is to provide students with a broad view in population mechanisms of the different types of adaptation. The problem of the course consists in benchmark analysis of the ways and mechanisms of the origin of these adaptations, as well as in their possible macroevolution consequence. The individual adaptation is considered on model of stressful influence on populations. Cooperative adaptation is considered basically on model of symbiosis, whose formation associates with origin of new traits, which greatly increase evolution potential of biological species. The course develops the knowledge obtained from prerequisite courses, such as "General genetics" "Symbiogenetics", "General ecology" and "Theory of evolution".

The method of membrane filters was used to study the population dynamics of bacteria belonging to the genera Arthrobacter, Flavobacterium, and Klebsiella in barley (Hordeum vulgare) rhizosphere under conditions of cadmium stress (5–15 mg Cd/g soil). Mathematical simulation allowed us to demonstrate that the phytoprotective effect is implemented via the following succession of events: the bacteria synthesize phytohormones (IAA and ethylene) → root excretory activity increases → the number of the bacteria in the rhizoplane grows → the flux of bacteria migrating from the rhizoplane to the rhizosphere increases → the number of bacteria binding cadmium ions in the rhizosphere grows → the amount of free ions entering the plant decreases. Among the bacteria studied, K. mobilis 880 displayed the highest migration and immobilization activity and the best survival rate under conditions of cadmium stress. Consequently, K. mobilis 880 is recommended for use in biopreparations for stimulating plant growth under conditions of heavy metal pollution.

The method for mathematical simulation is suggested to analyze the balanced polymorphism in rhizobia population generated due to the interplay of Darwinian and frequency-dependent selection. Analysis of the model suggested that this polymorphism is determined not only by the selection pressures but also by the capacities of ecological niches occupied by bacteria in the «plant-soil» system. The model may be used for analyzing the selective processes in various symbiotic systems and for predicting the consequences of releasing of genetically modified plant symbionts into environment.