期刊论文详细信息
BMC Genomics
Structure and variation of CRISPR and CRISPR-flanking regions in deleted-direct repeat region Mycobacterium tuberculosis complex strains
Research Article
Daniel Chemtob1  Paul Jeffrey Freidlin2  Hasia Kaidar-Shwartz2  Drora Goldblatt2  Israel Nissan2  Anna Luria3  Zeev Dveyrin4  Efrat Rorman4  Steven Robert Head5  Lana Schaffer5 
[1] Department of Tuberculosis and AIDS, Ministry of Health, Jerusalem, Israel;National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel;National Mycobacterium Reference Center, National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel;current address: Ben-Gurion University of the Negev, Beer Sheva, Israel;National Public Health Laboratory Tel Aviv, Ministry of Health, Tel Aviv, Israel;Scripps Research Institute, San Diego, CA, USA;
关键词: Mycobacterium tuberculosis;    Next Generation Sequencing NGS;    CRISPR-Cas;    Spacers;    cas1;    IS6110;    Deleted-direct repeat region deleted-DR;    Region of difference deletion RD deletion;    Spoligotype;    MIRU-VNTR;   
DOI  :  10.1186/s12864-017-3560-6
 received in 2016-10-10, accepted in 2017-02-07,  发布年份 2017
来源: Springer
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【 摘 要 】

BackgroundCRISPR and CRISPR-flanking genomic regions are important for molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) strains, and potentially for adaptive immunity to phage and plasmid DNA, and endogenous roles in the bacterium. Genotyping in the Israel National Mycobacterium Reference Center Tel-Aviv of over 1500 MTBC strains from 2008–2013 showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions (deleted-DR/CRISPR regions). Two isolates of each of three negative spoligotype MTBC (a total of 6 isolates) were subjected to Next Generation Sequencing (NGS). As positive controls, NGS was performed for three intact-DR isolates belonging to T3_Eth, the largest multiple-drug-resistant (MDR)-containing African-origin cluster in Israel. Other controls consisted of NGS reads and complete whole genome sequences from GenBank for 20 intact-DR MTBC and for 1 deleted-DR MTBC strain recognized as CAS by its defining RD deletion.ResultsNGS reads from negative spoligotype MTBC mapped to reference H37Rv NC_000962.3 suggested that the DR/CRISPR regions were completely deleted except for retention of the middle IS6110 mobile element. Clonally specific deletion of CRISPR-flanking genes also was observed, including deletion of at least cas2 and cas1 genes. Genomic RD deletions defined lineages corresponding to the major spoligotype families Beijing, EAI, and Haarlem, consistent with 24 loci MIRU-VNTR profiles. Analysis of NGS reads, and analysis of contigs obtained by manual PCR confirmed that all 43 gold standard DR/CRISPR spacers were missing in the deleted-DR genomes.ConclusionsAlthough many negative spoligotype strains are recorded as spoligotype-international-type (SIT) 2669 in the SITVIT international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least 4 different 24 loci MIRU-VNTR and RD deletion families. We report for the first time negative spoligotype-associated total loss of CRISPR region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least CRISPR-associated genes cas2 and cas1. Since cas1 deleted E.coli shows increased sensitivity to DNA damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-DR strains and Beijing family strains as both lack the cas1 gene.

【 授权许可】

CC BY   
© The Author(s). 2017

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