An. Kh. Baymiev

Influence of the introduction of Caragana arborescens on the composition of its root-nodule bacteria

The genetic diversity and phylogeny of root module bacteria entering into symbiotic relations with Caragana arborescens introduced on the territory of the Republic of Bashkortostan (RB) were studied. Analysis of the 85 strains isolated from root-nodules showed that, according to the DNA polymorphism revealed by RAPD analysis, they can be divided into 12 groups. It was found that, both in natural habitats and on the territory of the RB, Caragana arborescens is primarily nodulated by Mesorhizobium bacteria whose 16S rRNA gene sequences differ to some degree from those of the bacteria described earlier by Chinese authors. Bacteria assigned to the genus Phyllobacterium based on their 16S rRNA gene sequences were also revealed in plants growing on the territory of the RB.

Polymorphism of lectin genes in Lathyrus plants

The carbohydrate-binding sequences of the lectin genes from spring vetchling Lathyrus vernus (L.) Bernh., marsh vetchling L. palustris (L.), and Gmelin’s vetchling L. gmelinii (Fitsch) (Fabaceae) were determined. Computer-aided analysis revealed substantial differences between nucleotide and predicted amino acid sequences of the lectin gene regions examined in each of the three vetchling species tested. In the phylogenetic trees based on sequence similarity of carbohydrate-biding regions of legume lectins, the sequences examined formed a compact cluster with the lectin genes of the plants belonging to the tribe Fabeae. In each plant, L. vernus, L. palustris, and L. gmelinii, three different lectin-encoding genes were detected. Most of the substitutions were identified within the gene sequence responsible for coding the carbohydrate-binding protein regions. This finding may explain different affinity of these lectins to different carbohydrates, and as a consequence, can affect the plant host specificity upon development of symbiosis with rhizobium bacteria.

Genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh.

The genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh. (Fabaceae) growing in various regions of the Republic of Bashkortostan were studied. RAPD analysis revealed a high degree of polymorphism of the DNA of the isolated strains giving evidence of the heterogeneity of the microorganisms in question. The study of the phylogeny of microsymbionts based on comparative analysis of the nucleotide sequences of 16S rRNA genes showed that the bacteria isolated from the plant nodules of L. vernus growing on the territory of Ufa and Beloretsk raions belonged to the species Rhizobium leguminosarum, whereas the microsymbionts of L. vernus growing on the territory of Tatyshly raion belonged to the species Rhizobium tropici,@ except for several strains of Rhizobium leguminosarum

Analysis of symbiotic genes of leguminous root nodule bacteria grown in the southern urals

Bacterial strains isolated from the nodules, tissues, and root surface of wild legumes growing in the Southern Urals related to the tribes Galegeae, Hedysareae, Genisteae, Trifolieae, and Loteae were examined for the presence in their genomes of symbiotic (sym) genes. It was found that the sym-genes are present in microorganisms isolated only from the nodules of the analyzed plants (sym+ strains). Phylogenetic analysis of sym+ strains on the basis of a comparative analysis of 16S rRNA gene sequences showed that sym+ strains belong to five families of nodule bacteria: Mesorhizobium, Bradyrhizobium, Sinorhizobium, Rhizobium, and Phyllobacterium. A study the phylogeny of the sym-genes showed that the nodule bacteria of leguminous plants of the Southern Urals at the genus level are mainly characterized by a parallel evolution of symbiotic genes and the 16S rRNA gene. Thus, cases of horizontal transfer of sym genes, which sometimes leads to the formation of certain types of atypical rhizobial strains of leguminous plants, are detected in nodule bacteria populations.

Genetic description of root nodule bacteria of Lathyrus species growing on the territory of the Republic of Bashkortostan

The genetic diversity and phylogeny of rhizobia isolated from nodules of nine wild-growing Lathyrus L. species (Fabaceae) growing in the Republic of Bashkortostan were studied. It is shown that, for the given plants, a large variety of heterogeneous strains of root nodule bacteria is characteristic. Nevertheless, it is revealed that the majority of them, in terms of phylogenetics, are closely related to Rhizobium leguminosarum. However, some plant species are found also: nodule bacteria, which were earlier considered unusual for Lathyrus. Thus, L. vernus L. Bernh. and L. sylvestris L. are found to have a root nodule bacteria close to R. tropici, L. palustris L. Agrobacterium sp., and L. gmelinii Fritsch, all bacteria isolated by us from root nodules by the sequence of genes of 16S rRNA, are closely related to Phyllobacterium myrsinacearum.

Preparation of fluorescent labeled nodule bacteria strains of wild legumes for their detection in vivo and in vitro

A series of expression vectors containing TurboGFP and TurboRFP genes of fluorescent proteins under the control of the T5 phage constitutive promoter was created for a vital staining of nodule bacteria. These vectors were either obtained using the broad host range pBBRI replicon for labeling of strains, where a marker gene was expressed from a transformed plasmid, or they were prepared using the pRL765 gfp plasmid for labeling of strains via the introduction of genes of fluorescent proteins into the bacterial chromosome. Transformation was shown to be the most convenient method of transfer of constructions into cells of nodule bacteria, as there exists the possibility of spontaneous plasmid mobilization and, consequently, its transition from cells of labeled strains into other soil bacteria if the mob locus is present in vectors needed for conjugation. Fluorescent labeled strains of Rhizobium sp., Mesorhizobium sp., Ensifer (Sinorhizobium) sp., Bradyrhizobium sp., Phyllobacterium sp., and Agrobacterium sp. were prepared using the obtained vector constructions. The suitability of the obtained strains for both in vivo and in vitro experiments was demonstrated.

Inverse PCR-based site-directed mutagenesis of nucleotide sequences coding for carbohydrate-binding fragments of legume lectins

A new method of site-directed mutagenesis was developed to allow manipulation with extended plasmid-cloned gene fragments irrespective of their position and the presence of restriction sites. The method was used to obtain chimeric constructs encoding a Pisum sativum lectin with the native carbohydrate-binding region replaced by its counterpart from other legumes. The method can be used in plasmid construction to clone a coding gene fragment under the control of a promoter in a certain reading frame.

Genetic markers for search of rhizobia based on symbiotic genes

The possible application of rhizobial symbiotic genes as markers for the search and primary identification of rhizobia from temperate-zone legumes was studied. It was shown that conservative sym genes nifH and nifD could be used as markers for rapid search of rhizobia among the analyzed isolates, while more variable genes nifK and nodC could be used for their primary identification. Efficiency of the proposed method was shown in analysis of bacterial isolates obtained from Onobrychis arenaria and Astragalus cicer root nodules.

Carbohydrate-binding sequences of the lectin genes in leguminous plants from the Galegeae and Hedysareae Tribes

The carbohydrate-binding sequences (CBS) of the lectin genes from legume plants of the genera Astragalus Lam., Oxytropis DC., and Hedysarum L. were determined. Computer-assisted analysis of nucleotide and predicted amino acid sequences of the lectin gene fragments examined revealed a high homology level between their CBS. At the same time, the CBS of Astragalus and Oxytropis were considerably different from the lectin gene CBS in the earlier examined representatives of the tribe Galegeae, Caragana frutex and C. arborescens. This fact probably points to the differences in the carbohydrate-binding specificity of the proteins examined, which can eventually affect their functional activity.

Phylogenetic analysis of symbiotic genes of nodule bacteria in plants of the genus Lathyrus (L.) (Fabaceae)

We performed a comparative analysis of the symbiotic genes nifH, nifD, and nodA of microsymbionts of wild plants of the Lathyrus L. (Fabaceae) genus. According to 16S rRNA gene sequences, the microsymbionts belonged to Rhizobium leguminosarum bv. viceae, Rhizobium tropici, Agrobacterium sp., and Phyllobacterium sp. It was shown that all the studied genes from the analyzed strains were highly homologous to the similar genes of R. leguminosarum bv. viceae. We propose that these symbiotic genes were transferred into R. tropici, Agrobacterium sp., and Phyllobacterium sp. via horizontal transfer of genes from R. leguminosarum bv. viceae strains, which resulted in the appearance of recombinant strains able to form symbiotic relationships with plants of the Lathyrus L. genus. Such relationships previously seemed unlikely for these plants.