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BACTERIOPHAGES OF SINORHIZOBIUM MELILOTI NATIVE TO ALFALFA ORIGIN OF DIVERSITY AT THE CAUCASUS
Abstract
Sinorhizobium meliloti species are saprophytic soil bacteria able to fix atmospheric nitrogen in symbiosis with host-plants from alfalfa cross inoculated group. These biotechnologically important species of rhizobia are commonly present in various soil environments, while little is known about their phages. The main objective of the research was to isolate and characterize phages (rhizobiophages) of S. meliloti from soil samples collected at the NE Caucasian origin of alfalfa diversity (NEC). Under our particular focus of the research was to find template phages in genomes of S. meliloti isolated from the NEC. Soil samples used for bacteriophage and rhizobia strains isolation were taken under wild grown alfalfa plants. Final collection of phages was tested for morphological characteristics of phage plaque formed on the strain S. meliloti L5-30 lawn and for lytic activity on the collection of S. meliloti isolates from the NEC. All isolated rhizobiophages were different according to morphological types of phage plaques. The four lytic rhizobiophages and the four genetically unrelated S. meliloti strains resistant to isolated phages were identified. All the four lytic rhizobiophages were DNA-containing viruses. Genome of S. meliloti strain S35 from NEC which is one of the four strains resistant to all tested phages was sequenced (Illumina, nanopore) and annotated (Prokka). The one intact and two incomplete prophages were identified on the chromosome and on each of the two megaplasmids of the strain S35 by PHASTER web server. On the chromosome and on the megaplasmid-I were detected sequences similar to the intact Sinorhizobium phage phiLM21 (NC_029046) and incomplete Escherichia phage slur01 (NC_028831), respectively. These both prophage sequences are related to Siphoviridae family. On the megaplasmid-II a sequence similar to incomplete Enterobacteria phage phi92-like (NC_023693) from Myoviridae family was identified. The sequence of the intact Sinorhizobium phage phiLM21-like is mosaic, since 20% of ORFs are homologous to phage phiLM21 and 13% of ORFs are related to sequences of other phages from the same Siphoviridae family, while other 12.2% and 6.7% of ORFs are related to Myoviridae and Podoviridae families, correspondingly. The rest 47.8% of ORFs are encoding hypothetical bacterial related proteins. The intact phiLM21-like phage did not have the sequence encoding terminase TerL used as a phylogenetic marker in comparative genomics of phages. At the same time one of the ORFs of this phage encodes chitinase-like protein with 84% nucleotide homology to phage sequence encoding chitinase-like protein (lytic enzyme; BLASTn) abundant among bacteria and plants was identified. Summarizing, our findings showed that rhizobiophages isolated from soils are active against rhizobia from the same soil sources, that there are a high recombination activity occurred between phages, as well as phages are actively participate in intracellular recombination processes and in horizontal transfer of bacterial genes.
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