ANALYSIS OF CRISPR CASSETTE ELEMENTS IN NATIVE ISOLATES OF SINORHIZOBIUM MELILOTI ISOLATED IN THE CENTRAL ASIAN ORIGIN OF PLANT DIVERSITY

Abstract

CRISPR-Cas adaptive immunity systems of prokaryotes have been identified in 42% of bacteria and 85% of archaea [1] and are actively used in genetic engineering. However, in nitrogen-fixing nodule bacteria required for agriculture, these systems are very poorly characterized. Therefore, in this study, we evaluated the diversity of CRISPR-Cas systems in the genomes of 39 isolates of alfalfa nodule bacteria Sinorhizobium meliloti recovered from soil samples from Kazakhstan and Turkmenistan, which belong to the Central Asian origion of plants diversity. The diversity of detected CRISPR elements was assessed by changes in the size of the PCR amplified DNA fragments. As a result, the presence of 7 species-specific CRISPR loci (CR1- CR7) was shown for each of 39 isolates, of which three (CR4, CR5 and CR6) were selected for further analysis. It was shown that structural changes in the analyzed CRISPR loci sequences were significantly 2-fold more frequent in strains from Turkmenistan than in strains from Kazakhstan (?2=4.07, P less than 0.05). The most frequent changes in the size of the amplified sequences were detected at the CR6 locus (frequency 0.39), and 7.5-fold less frequent at the CR4 locus. Analysis of the nucleotide sequences of the loci for which changes in the size of amplified fragments were detected showed that at the CR4 locus one spacer was replaced with another; at the CR6 locus a deletion of the spacer and one direct repeat were involved. No changes were detected in the sequences of the CR-5 locus. The results suggest that strains in which changes in cassette compositions have occurred may have significant differences in resistance to lytic phages. The possibility of directionally modulating the spacers sequence in the CRISPR loci of nodule bacteria used in bioproducts may undoubtedly be of interest for agrobiotechnology.