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Molecular taxonomic study of Mycobacterium yongonense : Mycobacterium yongonense 균주의 분자분류학적 연구

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의과대학 의과학과
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서울대학교 대학원
Mycobacterium yongonenseM. intracellulare INT-5 groupgenome sequencecomparative genome analysismulti-locus sequence typing (MLST)single nucleotide polymorphism (SNP)RNA polymerase β-subunit gene (rpoB)lateral gene transfer (LGT)DNA mismatch repair gene
학위논문 (박사)-- 서울대학교 대학원 : 의과학과, 2016. 8. 김범준.
Introduction: Members of Mycobacterium avium complex (MAC) are the most frequently isolated non-tuberculous mycobacteria (NTM). Traditionally, MAC complex includes two species of M. avium and M. intracellulare strains. Recently, using the partial three independent chronometer genes, hsp65, ITS1 and 16S rRNA, clinical isolated M. intracelllulare strains from Korean patients were separated into 5 groups (INT-1 to -5) due to their genetic diversities.
Methods: A novel strain 05-1390, which is related with M. intracellulare, especially INT-5 group, was isolated from a Korean pulmonary patient and proposed to a novel species (M. yongonense DSM 45126T) by biochemical and phylogenetic analyses. This strain is phylogenetically related to M. intracellulare, but has a distinct RNA polymerase β-subunit gene (rpoB) sequence that is identical to that of M. parascrofulaceum, suggesting the acquisition of the rpoB gene via a potential lateral gene transfer (LGT) event. To gain better insight into the possibility of LGT event mechanisms in the M. yongonense, the complete genome sequence of M. yongonense was determined by four types of sequencing methods. Also, to determine the exact taxonomic status of the M. yongonense and other INT-5 strains (MOTT-36Y and MOTT-H4Y), genome-based phylogenetic analysis and IS-elements identification were conducted. To this end, genome sequences of the two INT-5 strains were compared with M. intracellulare ATCC 13950T and M. yongonense DSM 45126T. Multilocus sequence typing (MLST) of 35 target genes and single nucleotide polymorphism (SNP) analyses were conducted to compare the relationship between the two INT-5 strains and M. yongonense DSM 45126T. And using the M. yongonense genome information, a novel IS-element was identified and applied to develop a novel diagnostic method based on a real-time PCR technique. To figure out the mechanism of LGT events in the genome of M. yongonense from M. parascrofulaceum, putative LGT regions in M. yongonense strains were identified by comparative genomic analysis and the regions were examined by SimPlot and BootScan analysis. Also, in the genome of M. yongonense strains, DNA mismatch repair genes (mutS4A and mutS4B) were identified, and examined the ability in recombination of DNA mismatch repair genes. To this end, recombinant M. smegmatis harboring DNA mismatch repair gene was constructed and partial rpoB sequences (684 bp) from rifampin resistant M. tuberculosis was introduced in the recombinant M. smegmatis. After that, the ability of resistance to rifampin and recombination frequencies of recombinant M. smegmatis were examined.
Results: A novel strain, 05-1390 was similar with M. intracellulare in biochemical traits. Phylogenetic analysis based on ITS1 and the hsp65 gene indicated that strain 05-1390 was closely related to M. intracellulare ATCC 13950T, but the rpoB gene sequence showed that it is closely related to M. parascrofulaceum ATCC BAA-614T. With these results, strain 05-1390 was proposed to a novel species as M. yongonense DSM 45126T. This strain have a circular chromosome (5,521,023 bp
GenBank accession no. of CP003347), a circular plasmid (122,976 bp
GenBank accession no. of JQ657805), and a linear plasmid (18,089 bp
GenBank accession no. of JQ657806). The genome based MLST and SNP analyses showed that the two INT-5 strains and M. yongonense DSM 45126T were closely related than other M. intracellulare strains. Also, a novel IS-elements, ISMyo2 (2,387 bp), belonging to the IS21 family were identified in the genome of M. yongonense DSM 45126T and two INT-5 strains. The developed real-time PCR diagnostic method targeting ISMyo2 could detect only M. yongonense strains including the two INT-5 strains. In the genome of M. yongonense DSM 45126T, two putative LGT regions with high sequence similarity with M. parascrofulaceum were identified. The first region is rpoBC operon (OEM_44170~44190) and the second region is containing ORFs from OEM_08030 to OEM_08590 (57 ORFs). SimPlot and BootScan analyses supported that these regions were laterally transferred from M. parascrofulaceum. Also, the recombinant M. smegmatis harboring DNA mismatch repair gene showed potential of resistance to rifampin after indroducing the partial rpoB sequences related with rifampin resistance.
Conclusions: The genome-based phylogenetic analysis showed that M. yongonense is taxonomically related with INT-5 strains (MOTT-36Y and MOTT-H4Y). So, previously described taxonomic status of the two INT-5 strains should be revised into M. yongonense strains. M. yongonense strains could be divided into two distinct genotypes. Type I genotype have the distinct rpoBC operon which was laterally transferred from M. parascrofulaceum, and Type II genotype have the similar rpoBC operon with M. intracellulare strains. The result of LGT events could be a criteria for differentiation between M. yongonense Type I and Type II genotypes, and the LGT events might be facilitated by introduction of DNA mismatch repair genes from the genus of Acidothermus species to M. yongonense strains.
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