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The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were "hot spots" of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration "hot spots", not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial–phage coevolution. [ABSTRACT FROM AUTHOR]

This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage–microbe interactions with nitrogen-fixing symbiotic microorganisms. [ABSTRACT FROM AUTHOR]

Medicago lupulina L. possesses a complex of valuable economic and biological traits: high adaptive ability to climatic and edaphic growing conditions, good nutrition and digestibility of green mass. The aim of this work was to study the chloroplast genome, which is associated with plant defense responses of M. lupulina L. var. vulgaris Koch, cultivar-population VIK32, line MlS-1, which is highly responsive to mycorrhization and forms a highly effective symbiosis with salt-sensitive strain Sinorhizobium meliloti L6-AK89. The studied complete chloroplast genome is a double-stranded circular DNA molecule of 118340 bp in length with an average GC composition of 34.18%. Short inverted repeats (IRa and IRb), presumably involved in chloroplast replication processes, are 1.8 times shorter in M. lupulina VIK32 than in M. lupulinaNC_042847. Two extended regions 12501 and 5001 bp in length, presumably introduced due to HGT, and the 7 regions containing short sequences showed similarity to sequences of 260 viruses (Identity 78–96%, E-value 5 × 10–33–4 × 10–3) were identified. In addition, a number of deletions, insertions, and SNPs are significantly differentiate the chloroplast genomes at the interspecies level, this suggestion requires further investigation. The cpDNA of the M. lupulina VIK32 was shown to be phylogenetically closely related to cpDNA of M. lupulinaNC_042847 and M. minima, but it was the most divergent from the putative common Medicago-Melilotus-Glycine ancestral cpDNA sequence. [ABSTRACT FROM AUTHOR]

Soil Sinorhizobium phage AP-16-3, a strain phylogenetically close to Rhizobium phage 16-3, was isolated in a mountainous region of Dagestan, belonging to the origin of cultivated plants in the Caucasus, according to Vavilov N.I. The genome of phage AP-16-3 is 61 kbp in size and contains 62 ORFs, of which 42 ORFs have homologues in the genome of Rhizobium phage 16-3, which was studied in the 1960s–1980s. A search for Rhizobium phage 16-3-related sequences was performed in the genomes of modern strains of root nodule bacteria belonging to different species, genera, and families. A total of 43 prophages of interest were identified out of 437 prophages found in the genomes of 42 strains, of which 31 belonged to Sinorhizobium meliloti species. However, almost all of the mentioned prophages contained single ORFs, and only two prophages contained 51 and 39 ORFs homologous to phages related to 16-3. These prophages were detected in S. meliloti NV1.1.1 and Rh. leguminosarum OyaliB strains belonging to different genera; however, the similarity level of these two prophages did not exceed 14.7%. Analysis of the orphan genes in these prophages showed that they encoded predominantly virion structural elements, but also enzymes and an extensive group of hypothetical proteins belonging to the L, S, and E regions of viral genes of phage 16-3. The data obtained indicate that temperate phages related to 16-3 had high infectivity against nodule bacteria and participated in intragenomic recombination events involving other phages, and in horizontal gene transfer between rhizobia of different genera. According to the data obtained, it is assumed that the repetitive lysogenic cycle of temperate bacteriophages promotes the dissolution of the phage genetic material in the host bacterial genome, and radical updating of phage and host bacterial genomes takes place. [ABSTRACT FROM AUTHOR]

This paper presents a deep analysis of the accessory genome of an economically promising strain of Ensifer (Sinorhizobium) meliloti, L6-AK89, obtained as a result of next-generation high-throughput sequencing (MiSeq, MinIon). Strain L6-AK89 is a StrR mutant of the native strain CIAM1775, a symbiont of Medicago lupulina that adapted to a saline and arid habitat in NW Kazakhstan. CIAM1775 is an effective inoculant of M. lupulina cv. Mira (fodder type standard), cultivated on moderately acid soils in the NW agricultural region of Russia. Strain L6-AK89 makes it possible to obtain the expected high (>150%) increases in dry mass of the same plant variety in plant tests. The L6-AK89 genome has an increased proportion of sequences related to the accessory elements relative to reference strain Rm1021, 7.4% versus 4.8%. A set of 53 nod/noe/nol/nif/fdx/fix genes and 32 genes involved in stress tolerance together with 16S rRNA and recA–atpD–glnII–gyrB–dnaJ were evaluated. The high symbiotic efficiency of L6-АК89 with hop clover is most likely due to unique features of its genome, in combination with structural differences in its nod and stress-related genes, as well as unique clusters of quorum-sensing genes and osmoprotector synthesis. [ABSTRACT FROM AUTHOR]

Analysis of the structural polymorphism of eight genes in Sinorhizobium meliloti (nodA, nodB, nodC, and nodH, as well as betA, betB, betC, and betB2) involved in virulence control and salt tolerance, respectively, was carried out in native populations from two geographically distant areas of alfalfa diversity. These areas are located in the North Caucasian gene center of cultivated plants (NCG) and in the modern center of introgressive hybridization of alfalfa located next to the Aral Sea area (PAG) subjected to salinization. RFLP types (alleles) of the nod and bet genes, similar to those in the reference strain Rm1021 (A-type) and different from them (divergent, or D-type alleles) were revealed. The combinations for A- and D-type alleles of the aforementioned genes (analysis of the linkage disequilibrium, LD) were studied in both populations. It was shown that D-type alleles of the nod genes were two times more frequent in the NCG population, while D-type alleles of the bet genes were predominantly identified in the PAG population. At the same time, different combinations of D-type alleles of both the nod and bet genes prevailed in populations. For instance, in the case of the glycine betaine metabolism pathway, these were the betC and betB2 genes in NCG population and betB and betA genes in PAG population. The state of linkage disequilibrium was shown for 60.7% of combinations of alleles of the nod and bet genes in the S. meliloti strains from NCG and more than twice less in strains from the PAG population. It was concluded that clonal lines prevailed in NCG, while the PAG population of S. meliloti had a panmictic structure with revealed single clonal lines. [ABSTRACT FROM AUTHOR]