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A Comprehensive Map of Mycobacterium tuberculosis Complex Regions of Difference.

ABSTRACT: Mycobacterium tuberculosis complex (MTBC) species are classic examples of genetically monomorphic microorganisms due to their low genetic variability. Whole-genome sequencing made it possible to describe both the main species within the complex and M. tuberculosis lineages and sublineages. This differentiation is based on single nucleotide polymorphisms (SNPs) and large sequence polymorphisms in the so-called regions of difference (RDs). Although a number of studies have been performed to elucidate RD localizations, their distribution among MTBC species, and their role in the bacterial life cycle, there are some inconsistencies and ambiguities in the localization of RDs in different members of the complex. To address this issue, we conducted a thorough search for all possible deletions in the WGS data collection comprising 721 samples representing the full MTBC diversity. Discovered deletions were compared with a list of all previously described RDs. As with the SNP-based analysis, we confirmed the specificities of 79 regions at the species, lineage, or sublineage level, 17 of which are described for the first time. We also present RDscan (https://github.com/dbespiatykh/RDscan), an open-source workflow, which detects deletions from short-read sequencing data and correlates the results with high-specificity RDs, curated in this study. Testing of the workflow on a collection comprising ∼7,000 samples showed a high specificity of the found RDs. This study provides novel details that can contribute to a better understanding of the species differentiation within the MTBC and can help to determine how individual clusters evolve within various MTBC species. IMPORTANCE Reductive genome evolution is one of the most important and intriguing adaptation strategies of different living organisms to their environment. Mycobacterium offers several notorious examples of either naturally reduced (Mycobacterium leprae) or laboratory-reduced (Mycobacterium bovis BCG) genomes. Mycobacterium tuberculosis complex has its phylogeny unambiguously framed by large sequence polymorphisms that present unidirectional unique event changes. In the present study, we curated all known regions of difference and analyzed both Mycobacterium tuberculosis and animal-adapted MTBC species. For 79 loci, we have shown a relationship with phylogenetic units, which can serve as a marker for diagnosing or studying biological effects. Moreover, intersections were found for some loci, which may indicate the nonrandomness of these processes and the involvement of these regions in the adaptation of bacteria to external conditions.

SUBMITTER: Bespiatykh D

PROVIDER: S-EPMC8386458 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundPseudomonas aeruginosa is a common bacterium which is recognized for its association with hospital-acquired infections and its advanced antibiotic resistance mechanisms. Tuberculosis, one of the major causes of mortality, is initiated by the deposition of Mycobacterium tuberculosis. Accessory sequences shared by a subset of strains of a species play an important role in a species' evolution, antibiotic resistance and infectious potential.ResultsHere, with a multiple sequence aligner, we segmented 25 P. aeruginosa genomes and 28 M. tuberculosis genomes into core blocks (include sequences shared by all the input genomes) and dispensable blocks (include sequences shared by a subset of the input genomes), respectively. For each input genome, we then constructed a scaffold consisting of its core and dispensable blocks sorted by blocks' locations on the chromosomes. Consecutive dispensable blocks on these scaffold formed instable regions. After a comprehensive study of these instable regions, three characteristics of instable regions are summarized: instable regions were short, site specific and varied in different strains. Three DNA elements (directed repeats (DRs), transposons and integrons) were then studied to see whether these DNA elements are associated with the variation of instable regions. A pipeline was developed to search for DR pairs on the flank of every instable sequence. 27 DR pairs in P. aeruginosa strains and 6 pairs in M. tuberculosis strains were found to exist in the instable regions. On the average, 14% and 12% of instable regions in P. aeruginosa strains covered transposase genes and integrase genes, respectively. In M. tuberculosis strains, an average of 43% and 8% of instable regions contain transposase genes and integrase genes, respectively.ConclusionsInstable regions were short, site specific and varied in different strains for both P. aeruginosa and M. tuberculosis. Our experimental results showed that DRs, transposons and integrons may be associated with variation of instable regions.

Project description: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). 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.NGS 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.Although 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.

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A Comprehensive Map of Mycobacterium tuberculosis Complex Regions of Difference.

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