Характеристика CRISPR/CAS-системы штамма Pseudomonas aeruginosa DSM 50071 на основе биоинформационного анализа её структур

Abstract

An algorithm for bioinformatic search and analysis of the structures of CRISPR/Cas systems of bacteria and screening of phages and plasmids through spacer sequences of CRISPR cassettes in the genome of the Pseudomonas aeruginosa strain DSM 50071 is presented.Using several search algorithms in the CRISPR/Cas system of the studied strain, the presence of three CRISPR loci and a group of Cas genes characteristic of Type-I Subtype-I-F was determined.Analysis of the spacer composition of CRISPR cassettes showed the presence of 31 to 43 spacers and a universal consensus repeat in all cassettes.Screening of the spacer sequences of the CRISPR cassettes of the studied strain showed their correspondence to the protospacers of phages and plasmids of bacteria of the families Pseudomonadaceae and Enterobacteriaceae. A complete characterization of bacteriophages to which this strain is resistant is given with their accession number in NCBI. A complete identification of spacers to protospacers of phages specific for bacteria of the Pseudomonadaceae family, most often isolated from the lungs of patients with bronchiectasis, pneumonia, as well as from hospitals and reservoirs, has been established.Full correspondence between spacers and protospacers of bacterial plasmids with pan-resistance and causing the development of respiratory failure and pneumonia was revealed. Correspondence of a segment of one spacer with protospacers of several bacterial phages of the same family was noted. This may indicate that the bacterium “expediently” acquires new spacers from DNA regions that are conserved for phages of bacteria of the same family.Genes that have phage protospacers in their structure have been identified.It has been established that these genes are responsible for the synthesis of enzymes that regulate the processes of virus reproduction.Therefore, activation of the CRISPR/Cas system in the genome of this strain will allow the restriction endonuclease to introduce breaks into unmethylated DNA, which will lead to disruption of the synthesis of this enzyme, and, consequently, disruption of bacteriophage replication.Correspondences of spacer sequences with protospacers of plasmids included in the structure of genes responsible for the synthesis of conjugative transfer enzymes were revealed.These results suggested that activation of the CRISPR/Cas system of this strain would disrupt the processes replication of bacteriophage and conjugation.The proposed algorithm made it possible to obtain information about the structure of the CRISPR/Cas system of the P. aeruginosa DSM 50071 strain, about its resistance to certain phages and plasmids. In the future, this will serve as the basis for creating approaches for targeted therapy of infectious diseases.