Abstract
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.
Highlights
clustered regularly interspaced short palindromic repeats (CRISPR) 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
Variations in the CRISPR and CRISPR-flanking regions of negative spoligotype (Additional file 1: Table S1) MTBC isolates from patients A, B, and C were observed in the visualization of Next Generation Sequencing (NGS) reads mapped on reference H37Rv NC_000962.3 (Fig. 1) and annotated alignments of DRplus (DR plus flanking sequences) subsequences to the DRplus reference H37Rv subsequence (Fig. 2)
Evidence presented in this paper showed that all deleted-Direct Repeat region (DR) strains lost at least their cas1 and cas2 genes (Fig. 2, Table 2), but previously Beijing was considered the only natural instance known of CRISPR-containing bacteria to be without a cas1 gene [27]
Summary
CRISPR 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). Variation in the structure of CRISPR and CRISPRflanking regions of these negative spoligotype MTBC isolates was observed when NGS reads were mapped on reference whole genome sequence H37Rv NC_000962.3. This yielded visualization of coverage (Fig. 1). After substitution of manual-PCR-derived DR-spanning sequences for the corresponding map-derived regions, further details of structural variation were revealed by alignments of annotated subsequences containing CRISPR plus CRISPR-flanking regions to the annotated reference H37Rv sequence (Fig. 2)
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