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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.

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.

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.

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).

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).

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.

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.

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.

Instability of cryptic plasmids in Sinorhizobium meliloti laboratory strains SXM1, DM7-R, and P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation experiment (120 days) was studied. The isolated clones of strains SXM1 and DM7-R manifested stable inheritance of plasmids, whereas 12.7–14.0% of clones with changed plasmid profile were detected in a population of clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited significantly decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, compared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the natural vegetation period of alfalfa.

Nodulation, rhizospheral, and saprophytic types of competitiveness (NC, RC, and SC, respectively) were studied in the highly active strains CXM1-105 and CXM1-188 of the alfalfa rhizobium Sinorhizobium meliloti.The competitiveness was estimated with the use of markers of antibiotic resistance. It was found that the mutant strain T37, which was characterized by a drastically decreased NC, had higher SC and RC than the parental strain. The mutant T107 (with a moderately decreased NC) did not differ from the parental strain with respect to RC but had a higher SC. The mutant T27 (with the lowest NC) did not differ from the parental strain with respect to SC or RC. In the mutant Tb1, the NC and RC were decreased and the SC was the same as in the parental strain. In Tb7, the SC was decreased and RC was increased. In the mutant T795, all of the three types of competitiveness were decreased. The difference between the mutants studied and the parental strain with respect to NC and RC was confirmed using an indirect method (the ability to form effective symbiosis after mixed inoculation together with the an ineffective tester strain CXM1-48) and the X-Gluc staining method (using the S. melilotiRmM4gustester strain carrying the gene of β-glucuronidase). However, the decreased SC that the mutants exhibited when they were cultivated together with parental strains in a plant-growth substrate (vermiculite) was not observed in the case of their cocultivation in liquid media. The independent variation of different types of competitiveness indicate that rhizobia have several separate gene systems determining their survival in in plantaandex plantaecological niches.

The symbiotic plasmid (pSym1-32) of the highly effective Rhizobium leguminosarum bv. viceae 1-32 strain was identified after the conjugal transfer of replicons carrying Tn5-mob into the plasmidless Agrobacterium tumefaciens Gm1-9023 strain. Plasmid pSym1-32 was transferred into R. leguminosarum bv. viceae strains Y14 (showing low effectiveness of symbiosis with Vicia villosa) and Y57 (unable to fix nitrogen). Transconjugants formed Fix+ nodules on roots of V. villosa and had a highly enhanced nitrogen fixing ability, increased plant weight, and increased nitrogen accumulation compared to the recipient strains. Variation of transconjugants in symbiotic properties (accompanied by alterations in plasmid composition in some of the conjugants) was detected. Moreover, the donor strain R. leguminosarum bv. viceae 1-32 was shown to be more efficient in the competitiveness and acid tolerance than the recipient Y14 strain. Both these properties were transmitted upon transfer of pSym1-32 into the recipient. Thus, plasmid pSym1-32 was shown to carry genes involved in the control of the nitrogen fixing ability, symbiotic effectiveness, competitiveness, and acid tolerance in R. leguminosarum bv. viceae.