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Sinorhizobium meliloti are agriculturally valuable species of soil bacteria that form nitrogen-fixing symbiosis with alfalfa plants. Global climate changes lead to an increase of agricultural areas subjected to salinity. Current knowledge about about high-salt stress impact on soil saprophitic root nodulated microsymbionts of legumes is weakly studied and rhizobia gene pool responsible for salt tolerance are fragment and far from clear. An increase of bacteria nonspecific resistance (immune status) to unfavorable stress factors can occur through the induction of defense mechanisms like restrictionmodification systems and CRISPR/cas systems which are aimed to protect bacteria cells from bacteriophages widespread in soil microbiome. The aim of this research was to evaluate the role of the megaplasmid pSymA in the formation of ecological genome of S. meliloti, which is related to stress tolerance and to determine the location of elements of adaptive immune systems protecting root nodule bacteria against external foreign DNA. The analysis was done on 11 genes, products of which involved in response to ion stress and synthesis of osmoprotectors. It was found that 6 out of 11 genes were found in the genomes of all analyzed S. meliloti strains, while it was not a case for other 5 genes. It was found that, unlike chromosome, megaplasmid I of S. meliloti accumulated copies of 4 from 5 genes, except kdpA gene, which is represented by a single copy and localized on megaplasmid I in all so far studied strains. It was predicted that closest phylogenetic relatives of genes whose products are involved in response to ion stress as well in synthesis of osmoprotectors are homologous genes of closely related S. medicae species. The exception was for betI2, for which the closest phylogenetic relative was homologous gene of Klebsiella pneumonia, and another exception is kdpA gene introduced onto megaplasmid-I from actinobacteria. Regarding elements of immune systems it was revealed that nonsymbiotic plasmids of S. meliloti harbored incomplete elements of RMS-I, -II, and - III systems, while the 4 complete RMS-IV systems were detected on a single plasmid. It was found out that corresponding methylases had similarities with similar enzymes detected in nitrogen-fixing strains of Agrobacterium tumefaciens, Mezorhizobium sp., Bradyrhizobium sp. CRISPR sequences were not detected on megaplasmid-I, while they were on chromosome, megaplasmid-II and on cryptic plasmids. So, it was concluded that megaplasmid-I of S. meliloti are enriched in copies of genes related to osmotic stress tolerance, but it role in immune status of rhizobia is requested further elucidation. [ABSTRACT FROM AUTHOR]

Genomes of alfalfa root nodule bacteria, Sinorhizobium meliloti, symbionts of alfalfa are enriched in non-symbiotic (cryptic) plasmids, which gene pool is remained weakly studied. S. meliloti strains are significantly varied in number and size of these plasmids. The goal of the study was to assess the occurrence of phage-related sequences (PRS) on cryptic plasmids. Whole genome sequences of 12 S. meliloti strains native to Caucasian and Kazakhstan centers of alfalfa diversity (NCG and PAG, correspondingly) were studied and 20 cryptic plasmids, which sizes varied from 17.2 to 453.8 kb, were assembled. In total 55 PRS were identified on cryptic plasmids, and these sequences were represented by intact, questionable and incomplete sequences according to PHASTER. Significant differences in the occurrence of above-mentioned types of PRS on cryptic plasmids was detected between strains native to NCG and PAG (X² = 6.73, p = 0.03). The sizes of the desired PRS varied from 5.1 to 33 kb, and their number was from 1 to 11 per replicon in tested strains. It was revealed that PRS on plasmids of strains from NCG were predominantly related to Siphoviridae family (p < 0.05), while PRS homologous to phages of Siphoviridae and Podoviridae families prevailed with equal frequencies on plasmids of strains from PAG. For 40% of tested PRS the attL/attR sequences were detected and that is proving their site-specific integration type. ORFs of PRS as it was revealed are encoded integrases, fiber protein and tail shaft, and nearly all PRS are contained ORFs encoded transposases. Summarizing, S. meliloti strains native to origins of alfalfa diversity are enriched in cryptic plasmids, and the latest are attractive for soil bacteriophages, that is strongly evident the participation of small size plasmids in horizontal gene transfer process. [ABSTRACT FROM AUTHOR]

Nodule bacteria (rhizobia) forming nitrogen-fixing symbiosis with legumes have a multicomponent genome represented by chromosome and a number of plasmids of different sizes. Genes related to symbiotic activity of Sinorhizobium meliloti are located predominantly on two megaplasmids SMa and SMb (1.35 and 1.68 Mb, correspondingly). Genomes at about 80% of native rhizobia nodulating alfalfa contained cryptic plasmids with sizes varied from 7 up to 450 kb. Functional role of this class of plasmids remained unclear up to the postgenomic era. Using two high-throughput sequencing methods (MiSeq and nanopore), the complete genome sequences of three S. meliloti strains native to the Aral Sea area were done in this work. Two cryptic plasmids of the strains AK23 and AK89, and two cryptic plasmids of the strain AK555 were assembled and annotated by using bioinformatics approaches (Flye, Racon, Medaka, Pilon, Prokka). Whole genome sequence data for the strains AK21 and AK83 harboring per two cryptic plasmids are from GenBank. The objective of the study was a joint comparative analysis of the 8 cryptic plasmids of the 5 native strains of S. meliloti from one population adapted to saline environment. The average size of three and two plasmids was at about 225 and 400 kb, correspondingly, while sizes of other three plasmids was greatly different (from 31 kb up to 152 kb). Plasmids of similar size (209 kb) showed 99% identity in genetically distinct strains, besides that replicon of SMd (31 kb) of the AK555 showed 86% homology to half-replicon of pSINME02 of the AK83. All plasmids were screened for replication systems and repABC locus was detected in cases of the 6 plasmids, while some of these and other two plasmids contained additional locus, or single repA, repB or repC genes. No correlation between sizes and replication systems was detected for tested plasmids. The sequences of all plasmids were analyzed for ORFs and their affiliation with COGs groups. The number of ORFs on tested plasmid replicons was ranged from 34 up to 506, while in each particular case all detected ORFs were belonged to the next four functional groups: i) cellular processes and signaling, ii) information storage and processing, iii) metabolism and iv) poorly characterized. Proteins homologous to type IV restriction endonucleases responsible for protection of bacteria cells from invading foreign DNA, like phages, were identified on 209 kb plasmids. These proteins are relating to bacterial immune systems. Summarizing, cryptic plasmids of native S. meliloti strains adapted to salinized environment are varied greatly in sizes, replication systems and harbored ORFs relating to vitality and immune system of bacteria. Obtained data strongly evident that non-symbiotic plasmids of S. meliloti involving in horizontal gene transfer harbored ORFs essential for environmental fitness of rhizobia. [ABSTRACT FROM AUTHOR]