The microbiome of buried soils demonstrates significant shifts in taxonomic structure and a general trend towards mineral horizons.

Burial mounds represent a challenge for microbiologists. Could ancient buried soils preserve microbiomes as they do archaeological artifacts? To investigate this question, we studied the soil microbiome under a burial mound dating from 2500 years ago in Western Kazakhstan. Two soil profile cuts were established: one under the burial mound and another adjacent to the mound surface steppe soil. Both soils represented the same dark chestnut soil type and had the same horizontal stratification (A, B, C horizons) with slight alterations. DNA samples isolated from all horizons were studied with molecular techniques including qPCR and high throughput sequencing of amplicon libraries of the 16S rRNA gene fragment. The taxonomic structure of the microbiome of the buried horizons demonstrated a deep divergence from ones of the surface, comparable to the variation between different soil types (representatives of the soil types were included in the survey). The cause of this divergence could be attributed to diagenetic processes characterized by the reduction of organic matter content and changes in its structure. Corresponding trends in the microbiome structure are obvious from the beta-diversity pattern: the A and B horizons of the buried soils form one cluster with the C horizons of both buried and surface soil. This trend could generally be designated as 'mineralization'. Statistically significant changes between the buried and surface soils microbiomes were detected in the number of phylogenetic clusters, the biology of which is in the line of diagenesis. The trend of 'mineralization' was also supported by PICRUSt2 functional prediction, demonstrating a higher occurrence of the processes of degradation in the buried microbiome. Our results show a profound shift in the buried microbiome relatively the "surface" microbiome, indicating the deep difference between the original and buried microbiomes.

[Proteomic Profile of the Bacterium Sinorhizobium meliloti Depends on Its Life Form and Host Plant 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 lipopolysaccha-rides and formation of a large conductance mechanosensitive channel, respectively, depend on a host plant species that confirms the results of proteomic analysis. Overall, our data show that the regulation of bacteroid development by the host plant has species-specific features.

[Nodulation competitiveness of nodule bacteria: Genetic control and adaptive significance].

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.

[Evolution of Root Nodule Bacteria: Reconstruction of the Speciation Processes Resulting from Genomic Rearrangements in a Symbiotic System].

The processes of speciation and macroevolution of root nodule bacteria (rhizobia), based on deep rearrangements of their genomes and occurring in the N2-fixing symbiotic system, are reconstructed. At the first stage of rhizobial evolution, transformation of free-living diazotrophs (related to Rhodopseudomonas) to symbiotic N2-fixers (Bradyrhizobium) occurred due to the acquisition of the fix gene system, which is responsible for providing nitrogenase with electrons and reducing equivalents, as well as for oxygen-dependent regulation of nitrogenase synthesis in planta, and then of the nod genes responsible for the synthesis of the lipo- chito-oligosaccharide Nod factors, which induce root nodule development. The subsequent rearrangements of bacterial genomes included: (1) increased volume of hereditary information supported by species, genera (pan-genome), and individual strains; (2) transition from the unitary genome to a multicomponent one; and (3) enhanced levels of bacterial genetic plasticity and horizontal gene transfer, resulting in formation of new genera, of which Mesorhizobium, Rhizobium, and Sinorhizobium are the largest, and of over 100 species. Rhizobial evolution caused by development and diversification of the Nod factor synthesizing systems may result in both increased host specificity range (transition of Bradyrhizobium from autotrophic to symbiotrophic carbon metabolism in interaction with a broad spectrum of legumes) and to its contraction (transition of Rhizobium and Sinorhizobium to "altruistic" interaction with legumes of the galegoid clade). Reconstruction of the evolutionary pathway from symbiotic N2-fixers to their free-living ancestors makes it possible to initiate the studies based on up-to-date genome screening technologies and aimed at the issues of genetic integration of organisms into supracpecies complexes, ratios of the macro- and microevolutionary mechanisms, and developmetn of cooperative adaptations based on altruistic relationship between the symbiotic partners.

[Effect of Long-Term Application of Agrotechnical Techniques and Crops on Soil Microbial Communities].

Effects of long-term application ofvarious fertilizers and crops on soil microbiomes an a long-term field experiment were investigated using the library of the 16S rRNA gene sequences obtained by high-throughput sequencing of the total DNA. The communities exhibited high diversity, with 655 microbial genera belonging to 34 phyla detected (31 bacterial and 3 archaeal ones). For analysis of the effect of the studied factors on community structure, a linear model was developed in order to simplify interpretation of the data of high-throughput sequencing and to obtain biologically important information. Liming was shown to modulate the effect of mineral fertilizers on the structure of microbial populations. The differences in the structure and alpha-diversity of microbial communities were shown to depend more on the crops and liming, rather than on the fertilizers applied. Interaction between the crop factor and liming expressed as an ambiguous effect of liming on the microbiome in the presence of different plants was reliably demonstrated. Thus, in the case of barley and clover, liming resulted in increased taxonomic diversity of the community, while in the case of potato and flax it had an opposite effect.

[Symbiogenesis as a Model for Reconstructing the Early Stages of Genome Evolution].

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 ofgenomes 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).

[Evolution of nitrogen-fixing symbioses based on the migration of bacteria from mycorrhizal fungi and soil into the plant tissues].

The hypothesis is suggested on the emergence of N2-fixing plant symbionts from soil diazotrophs and from the satellites of Glomeromycota fungi forming arbuscular mycorrhizae (AM). This universal form of plant-microbe symbiosis possibly appeared from integration of ancestral land plants (rhyniophytes, psylophytes) and microbial consortia composed of AM-fungi assimilating soil phosphates and bacteria fixing atmospheric CO2 and/or N2. Releasing of these bacteria from AM-fungal hyphae into the plant tissues elicited the selection of genotypes capable of the fungi-independent multiplication in planta, as well as the fixation in bacterial genomes of the genes for synthesis of chitin-like signal factors stimulating the development of symbiotic structures. An early stage of this evolution might been represented by formation of N2-fixing syncyanoses, the late stage - by formation of nodular symbioses of dicots from Eurosid I clade with rhizobia (α- and ß-proteobacteria) and with actinobacteria Frankia. Emergence of these symbioses was possibly based on the migration of soil and endophytic bacteria into the storage organs (modified stems or lateral roots), where the optimal conditions were established not only for N2 fixation but also for the evolution of bacteria towards an increased symbiotic activity. This evolution resulted in the emergence of primary rhizobia (Bradyrhizobium, Burkholderia) which acted as the donors of sym-genes for a broad spectrum of microbes transformed into the secondary rhizobia (Rhizobium, Sinorhizobium). The succeeding evolution of nodular symbioses was directed at an increased efficiency of symbiotrophic nitrogen nutrition in host plants following two scenarios: (i) "expensive", based on the increase of N2- fixing activity via transformation of bacteria into non-reproducible bacteroids; (ii) "economic", based on acquiring the determinate nodule structure and ureide nitrogen assimilation.

[The Principle of Genome Complementarity in the Enhancement of Plant Adaptive Capacities].

In the present work, the potential for the enhancement of the adaptive capacity of microbe-plant systems (MPSs) through the integration of the symbiosis partners' genomes is considered on the example of different types of symbiotic relationships. The accumulated data on the genetic control of interactions for both the plant and microbe, which are discussed in the paper with respect to signaling genes, suggest that it is the complementarity of genetic determinants that underlies the successful formation of MPSs. A eukaryotic genome with limited information content, which is stable throughout a generation, is complemented by a virtually unlimited prokaryotic metagenome. The microsymbiont's ability to adapt to different living conditions is based on the restructuring of the accessory genome by different mechanisms, which are likely to be activated under the influence of plants, although the details of such a regulation remain unknown. Features of the genetic control of the interaction, particularly its universal character for different symbionts, allow us to formulate a principle of genome-complementarity with respect to interacting organisms and consider it an important factor, an adaptation that enhances the abilities of M PSs for their sustainable development in natural ecosystems and for high plant productivity in agrocenoses.

[Evolution of Host-Beneficial Traits in Nitrogen-Fixing Bacteria: Modeling and Construction of Systems for Interspecies Altruism].

The literature and our own data on N2-fixing bacteria forming symbioses with plants and providing convenient models to study the evolution of interspecies (microsymbionts --> hosts) altruism are considered in the review. It is presented as a deeply reorganized intraspecies altruism implemented in the clonal population of rhizobia (bacteroids --> undifferentiated bacteria) under the control of kin selection induced by plant hosts. The analysis of this model suggests that it is possible to engineer practically valuable rhizobial strains in which high N2-fixing activity is combined with decreased survival outside of plants.

[Bacterial Genome Evolution in Superspecies Systems: an Approach to the Reconstruction of Symbiogenesis Processes].

Bacteria form a broad spectrum of symbioses with eukaryotes. This permits reconstruction of the symbiogenesis processes providing the transformation of free-living microorganisms into cellular organelles. In ecologically (conditionally) obligate symbioses, an increase in the size and complexity of the bacterial genome structure was observed. This was associated with segregation of the regions controlling symbiosis into gene clusters, islands, and plasmids. In genetically (strictly) obligate symbioses, a reduction of "nonsymbiotic" regions of microbial genome occurs, which could begin from genes encoding metabolic and regulatory functions. It is extended towards genes encoding template processes. Conditionally obligate symbioses are characterised by the activation of horizontal gene transfer between various forms of microsymbionts, while for strictly obligate intracellular symbioses an activation of endo-symbiotic gene transfer between microsymbionts and their hosts was detected. The latter is responsible for bacterial transition from the functional (based on gene cross-regulation) to structural (based on recombination) genetic integration with hosts, which later could be followed by the complete assimilation of microbial genomes. In α-proteobacteria this evolutionary pathway could result in the formation of cellular organelles that are deficient in their own genomes but capable of preserving proteomic and cytological traits as a result of the gene-product import synthesized in cytosol (hydrogenosomes and mitosomes). The symbiogenic evolution of cyanobacteria could result in the loss of the plasmids generated from them, while the host maintains a significant part of their genome in nuclear chromosomes.

[Characteristics of Natural Selection in Populations of Nodule Bacteria (Rhizobium leguminosarum) Interacting With Different Host Plants].

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.

[Influence of salt stress on the genetically polymorphic system of Sinorhizobium meliloti-Medicago truncatula].

The impacts of salt stress (75 mM NaC1) on the ecological efficiency of the genetically polymorphic Sinorhizobium meliloti-Medicago truncatula system were studied. Its impact on a symbiotic system results in an increase of the partners' variability for symbiotic traits and of the symbiosis integrity as indicated by: a) the specificity of the partners' interactions--the nonadditive inputs of their genotypes into the variation of symbiotic parameters; and b) the correlative links between these parameters. The structure of the nodDI locus and the content correlates to the efficiency of the symbiosis between S. meliloti and M. truncatula genotypes under stress conditions more sufficiently than in the absence of stress. Correlations between the symbiotic efficiency of rhizobia strains and their growth rate outside symbiosis are expressed under stress conditions, not in the absence of stress. Under salt stress symbiotic effectiveness was decreased for M. truncatula line F83005.5, which was salt sensitive for mineral nutrition. The inhibition of symbiotic activity for this line is linked with decreased nodule formation, whereas for Jemalong 6 and DZA315.16 lines it is associated with repressed N2-fixation. It was demonstrated for the first time that salt stress impairs the M. truncatula habitus (the mass : height ratio increased 2- to 6-fold), which in the salt-resistant cultivar Jemalong 6 is normalized as the result of rhizobia inoculation.

[Super-species genetic systems].

Genetic integration of diverse organisms results in generation of three types of the super-species systems of heredity: metagenome (set of genetic factors of the microbial community which occupies a certain ecological niche), symbiogenome (functionally integrated system of the partners' symbiotic genes) and hologenome (entire hereditary system of a symbiotically originated organism). The integrity of metagenome is based on the cross-regulation and horizontal transfer of genes in co-evolving organisms which in the soil microbial communities are accompanied by maintenance of the stable extracellular DNA pool. Formation of symbiogenome is related to the highly specific partners' signaling interactions which are responsible for development of the joint metabolic pathways based on the specialized cellular and tissue structures. Transitions of symbiogenome into hologenome are due to the endosymbiotic gene transfer from microsymbionts to their hosts. In symbiotic bacteria, these transitions are coupled with establishments of multi-component, reduced and rudimentary genomes revealed for the ecologically obligatory symbionts, genetically obligatory symbionts, and cellular organelles, respectively. Their evolution is related to the stringency of transmission of microsymbionts by hosts increased from pseudo-vertical (via environment) to the trans-embryonic (via embryos and the surrounding tissues) and trans-ovarian transmission (via germ cells) which are culminated in the cytoplasmic inheritance of cellular organelles. We suggest the hypothesis about generation of endophytic plant symbiogenome on the basis of soil metagenome subjected to the control of host by its involvement into the quorum sensing auto-regulation of microbial community.

[Population structure of the clover rhizobia Rhizobium leguminosarum bv. trifolii upon transition from soil into the nodular niche].

High-throughput sequencing of the amplicon gene library revealed variations in the population structure of clover rhizobia (Rhizobium leguminosarum bv. trifolii) upon transition from soil into the root nodules of the host plant (Trifolium-hybridum). Analysis of rhizobial-diversity using the nodA gene revealed 3258 and 1449 nucleotide sequences (allelic-variants) for the soil and root nodule population, respectively. They were combined into 29 operational taxonomic units (OTU) according to the 97% identity level; 24 OTU were. foundin the soil population, 12 were present in the root nodulepopulation, and 7 were common. The predominant OTE13 (77.4 and 91.5% of the soil and root nodule populations, respectively) contained 155 and -200 variants of the soil and root nodule populations, respectively, with the nucleotide diversity increasing significantly upon the "soil-->root" transition. The "moving window" approach was used to reveal the sites of the nodA gene in which polymorphism, including that associated with increased frequency of non-synonymous substitution frequency, increased sharply upon transition from soil into root nodiles. PCR analysis of the IGS genotypes of individual strains revealed insignificant changes in rhizobial diversity upon transition from soil into root nodules. These results indicate that acceleration of rhizobial evolutioin in the course of symbiosis may be associated with development of highly polymorphic virulent:subpopulations subjected to directional selection in the "plant-soil" system.

[Construction of high-effective symbiotic bacteria: evolutionary models and genetic approaches].

Using the example of N2-fixing legume-rhizobial symbiosis, we demonstrated that the origin and evolution of bacteria symbiotic for plants involve the following: 1) the formation of novel sym gene systems based on reorganizations of the bacterial genomes and on the gene transfer from the distant organisms; 2) 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).

[Comigration of root nodule bacteria and bean plants to new habitats: coevolution mechanisms and practical importance (review)].

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 of interactions between alfalfa nodule bacteria (Sinorhizobium meliloti) genes that regulate symbiotic nitrogen fixation].

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 ofthese 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).

[Co-evolution of partners and the integrity of symbiotic systems].

Symbioses are very suitable models for studying the integrity of biosystems which characterizes their structural/functional organization enabling the partners to respond adequately to the environmental changes. Analysis of different forms of plant-microbe and animal-microbe symbiosis suggests that a qualitative increase of its integrity occurs under the facultative and ecologically obligatory interactions and is culminated under the genetically obligatory interactions. By use of mathematical models, we demonstrate that the functional integrity of N2-fixing legume-rhizobia symbiosis (concordance of changes in partners' genotypic frequencies induced by environmental fluctuations) correlates to its ecological efficiency which increases under force of natural selection. It results in the tight partners' regulatory feedbacks leading to their genetic integration manifested in the establishment of "symbiogenome". The genetic integrity of symbiosis determines its high evolutionary potential based on: a) epigenetic inheritance of symbiotic traits by hosts which may occur in the form of vertical transmission of either microsymbionts themselves or genes obtained from them; b) interspecies altruism interactions related to the positive partners' feedbacks which determine the ecological efficiency of mutualistic interactions. Realization of this potential results in the deep genetic integration of initially independent partners including their fusions into the novel integral organisms.

[Comparison of the adaptive potential for Rhizobium leguminosarum bv. viceae nodule bacterial populations isolated in natural ecosystems and agrocenoses].

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.