[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.

[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.

[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).

[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).

[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.

[Symbiosis between the nodule bacterium Sinorhizobium meliloti and alfalfa (Medicago sativa) under salinization conditions].

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

[Identification of Sinorhizobium meliloti genes influencing synthesis of surface polysaccharides and competitiveness].

A new approach to isolating mutants with altered composition of capsular polysaccharides (CPS) and lipopolysaccharides (LPS) in nodule bacteria of alfalfa Sinorhizobium meliloti based on analysis of their respiratory activity was proposed. Random Tn5-mob mutants of symbiotically effective strain SKhM1-105 were tested for slime-production ability and coloration on diagnostic media containing the indicator of reducing equivalents, triphenyltetrasolium chloride (TTC), the inhibitor of respiratory activity, 2-methyl-4-chlorphenoxybutyric acid and Congo Red, the stain for LPS and exopolysaccharides (EPS). Electrophoretic analysis (SDS-PAGE) of polysaccharides in seven mutants, markedly differing from the parental strain with respect to their growth on diagnostic media demonstrated that (1) the production of CPS was either decreased (in mutants T64 and T835) or blocked (in T71, T94, T124, T134, and T170); (2) the R form of LPS had changed mobility (in T134); (3) the S form of LPS contained only one component instead of two (T71). In symbiosis with alfalfa Medicago sativa, all mutants exhibited a decreased competitive ability for adsorption on roots of host alfalfa plant, compared to the parental strain. At early stages of symbiosis, mutants had a tendency toward a decrease in the number of nodules, in comparison with the parental strain (a statistically significant decrease was observed in mutants T71 and T64). In mutants T64, T71, and T134, nodulation competitiveness was lower than in the parental strain. Analysis of Tn5-mob tagged sequences of the mutants revealed homologies with the genes encoding methionine synthase, luciferase-like monooxygenase, UDP-glucuronic acid epimerase, sensor hystidine kinase, acetoacetyl-CoA-reductase, oligopeptide uptake ABC transporter, and a transcription activator.

[Analysis of various types of competition in Tn5-mutants of alfalfa rhizobium bacteria (Sinorhizobium meliloti)]. / Analiz razlichnykh tipov konkurentosposobnosti u Tn5-muntantov kluben'kovykh bakterii liutserny (Sinorhizobium meliloti).

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. meliloti RmM4gus tester strain carrying the gene of beta-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 planta and ex planta ecological niches.