| Objective The CRISPR system is a special structure widely distributed in bacteria and archaea that can provide adaptive immunity to foreign genetic elements.Pseudomonas aeruginosa is an intractable pathogen of antibiotics with a large genome and wide genotypic diversity,and is a common opportunistic pathogen in hospitals.In this study,we compared the CRISPR/cas content and diversity in P.aeruginosa genomes and clinical isolates including CRISPR repeats,spacers,leader sequences,and cas gene sequences,that provide more insights to the evolution,diversity,conservation of CRISPR system and information of resistance to strains.Methods We analyzed the CRISPR system of Pseudomonas aeruginosa from two aspects,including the bioinformatics analysis of the CRISPR system in the genome of the strain and the study of the CRISPR array and cas1 gene of the clinical isolates.We downloaded the complete genome of 95 strains of Pseudomonas aeruginosa and collected CRISPRs information.The alignment analysis of repetitive sequences and cas genes was performed using Clustal W in DNAstar,and the phylogenetic tree was completed with MEGA7.The RNA secondary structure was predicted by RNAfold.The spacer sequence was BLASTed in the Gen Bank database to find the homologous sequence.The sequence between the cas gene and the upstream of the first repeat sequence was selected as the search target for the leader sequence,using Clustal W to find conserved regions by repeated analysis;108strains of Pseudomonas aeruginosa were collected in laboratory departments of Qingdao major hospitals with drug resistance information,CRISPR array and cas1 gene fragments were amplified by PCR and analyzed the spacer sequences.Results Of 95 strains of Pseudomonas aeruginosa,we identified 130 CRISPR loci in 58 strains,of which 47 have a complete CRISPR system structurally,30 are I-F type,and 7are I-C type,6 are I-E type,the remaining can not be classified.I-F type system and I-C type or I-E type can exist in the same strain at the same time.18 repeats were found in130 CRISPR loci.The repeats were conserved and basically formed a conserved dumbbell-like secondary structure.Different types of CRISPR systems have different cas,cas1 and cas3 exist in all CRISPR-cas systems of this study.Cas1 and cas3 are more conservative and can be used as a basis for typing.The length and number of spacers in different loci are different.The target genes of 2132 spacers in this study are mainly derived from bacterial genome sequences.Only 32 genes are matched with plasmid sequences,and 526 are matched with phage sequences;The PCR results of the CRISPR array and cas1 gene of the bacteria showed that among 108 strains of clinical bacteria,36.11%(39/108)could detect the cas1 gene,37.96%(41/108)could detect CRISPR array gene,cas1 gene and CRISPR array gene can be detected at the same time(30.60%).The repeat sequence of these CRISPR sites is very conservative,but the locus size is different.The analysis of the correlation between antibiotic resistance and the CRISPR system found that its presence can reduce the resistance rate of P.aeruginosa to imipenem.Conclusions ?The CRISPR system in the Pseudomonas aeruginosa genome is mainly type I-F,and I-E and I-C CRISPR systems are also found;?cas1 and cas3 genes are highly conserved in the same type of CRISPR system;?Exogenous genes that are homologous to the spacer sequence are mainly phage,only a few are plasmids;?The carrying rate of antibiotic resistance-associated genes in the P.aeruginosa genome is high,and the presence of the CRISPR locus and the complete system can reduce the resistance against imipenem... |
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