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Genome Sequences

Draft Genome Sequences of 12 Mycolicibacterium smegmatis Strains Resistant to Imidazo[1,2-b][1,2,4,5]Tetrazines

Aleksey A. Vatlin, Kirill V. Shur, Valery N. Danilenko, Dmitry A. Maslov
Frank J. Stewart, Editor
Aleksey A. Vatlin
aVavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
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Kirill V. Shur
aVavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
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Valery N. Danilenko
aVavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
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Dmitry A. Maslov
aVavilov Institute of General Genetics, Russian Academy of Sciences, Moscow, Russia
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  • ORCID record for Dmitry A. Maslov
Frank J. Stewart
Georgia Institute of Technology
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DOI: 10.1128/MRA.00263-19
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ABSTRACT

Here, we report 12 draft genome sequences of mutant Mycolicibacterium smegmatis strains resistant to imidazo[1,2-b][1,2,4,5]tetrazines, which are antituberculosis drug candidates. We have identified 7 different mutations in the MSMEG_1380 gene, which encodes the AcrR/TetR_N transcriptional repressor, which may activate efflux-mediated resistance.

ANNOUNCEMENT

We have previously described a number of substituted imidazo[1,2-b][1,2,4,5]tetrazines that showed promising MICs against both drug-susceptible and drug-resistant Mycobacterium tuberculosis strains. We used Mycolicibacterium smegmatis (formerly Mycobacterium smegmatis) as the model organism to study drug resistance mechanisms of mycobacteria to these compounds. We were able to obtain M. smegmatis mc2 155 spontaneous mutants that were resistant to 4 different imidazo[1,2-b][1,2,4,5]tetrazines at 3.5 to 4× MIC (1; D. A. Maslov, A. V. Korotina, K. V. Shur, A. A. Vatlin, O. B. Bekker, S. G. Tolshchina, R. I. Ishmetova, N. K. Ignatenko, G. L. Rusinov, V. N. Charushin, and V. N. Danilenko, unpublished data). We conducted whole-genome sequencing of the mutant strains and of wild-type M. smegmatis mc2 155 cultured in our laboratory. Here, we report the de novo assembly of a total of 13 M. smegmatis genomes, namely, 12 imidazo[1,2-b][1,2,4,5]tetrazine-resistant mutants and the original wild-type strain.

M. smegmatis strains were cultured in Middlebrook 7H9 medium with addition of oleic acid-albumin-dextrose-catalase (OADC; HiMedia, India) at 37°C for 16 to 40 h. Genomic DNA was isolated and purified by phenol-chloroform/isoamyl alcohol extraction after enzymatic cell lysis, as described in Belisle et al. (2). An NEBNext Ultra II DNA library prep kit for Illumina (New England Biolabs, USA) was used to prepare paired-end libraries. Quality control of the libraries was performed using gel electrophoresis and a Bioanalyzer 2100 instrument. The raw sequencing data were obtained using a HiSeq 2500 instrument (Illumina, USA) in rapid run mode with a HiSeq Rapid Sequencing by Synthesis (SBS) kit v. 2 (2 × 100 bp; Illumina, USA).

The read quality was checked with FastQC (v. 0.11.7) (3), which showed deviations in G+C content in the first 7 to 9 bases in most of the read archives and excessive adapter content in some of them, while Trimmomatic (v. 0.36) (4) was used for quality and adapter trimming (with the options HEADCROP:9 MINLEN:36 ILLUMINACLIP:TruSeq3-PE-2.fa:2:30:10 ILLUMINACLIP:TruSeq2-PE.fa:2:30:10 ILLUMINACLIP:NexteraPE-PE.fa:2:30:10 where applicable according to FastQC results). The reads were assembled to initial draft genomes by SPAdes (v. 3.13) with default settings (5). QUAST (v. 5.0.2) was used to assess the assembly metrics (6). The NCBI Prokaryotic Genome Annotation Pipeline was used for annotation. The resulting draft genome sequence metrics are shown in Table 1.

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TABLE 1

Sequencing data for 13 M. smegmatis genomes

As a result of a comparative bioinformatics analysis of M. smegmatis atR strain genomes, we were able to identify 7 different mutations in 11 strains (4 insertions, 2 deletions, and 1 single nucleotide substitution) in the MSMEG_1380 gene encoding the AcrR/TetR_N transcriptional regulator, which is involved in the regulation of gene expression of MmpS5/MmpL5 transmembrane transporters. It has been previously reported that overexpression of the mmpS5-mmpL5 operon modulates resistance to the derivatives of thiacetazone in Mycobacterium abscessus (7) and cross-resistance to bedaquiline and clofazimine in M. tuberculosis (8).

Data availability.The whole-genome shotgun (WGS) assemblies have been deposited in NCBI GenBank; the versions described in this paper are the first versions. The read archives have been deposited in NCBI SRA. The WGS (GenBank) and SRA accession numbers are listed in Table 1. All of the data are part of BioProject identifier (ID) PRJNA454808, except those for M. smegmatis mc2 155 (BioProject ID PRJNA523130).

ACKNOWLEDGMENT

This work was supported by the Russian Science Foundation (grant 17-75-20060 to D. A. Maslov).

FOOTNOTES

    • Received 5 March 2019.
    • Accepted 27 March 2019.
    • Published 18 April 2019.
  • Copyright © 2019 Vatlin et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.

REFERENCES

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Draft Genome Sequences of 12 Mycolicibacterium smegmatis Strains Resistant to Imidazo[1,2-b][1,2,4,5]Tetrazines
Aleksey A. Vatlin, Kirill V. Shur, Valery N. Danilenko, Dmitry A. Maslov
Microbiology Resource Announcements Apr 2019, 8 (16) e00263-19; DOI: 10.1128/MRA.00263-19

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Draft Genome Sequences of 12 Mycolicibacterium smegmatis Strains Resistant to Imidazo[1,2-b][1,2,4,5]Tetrazines
Aleksey A. Vatlin, Kirill V. Shur, Valery N. Danilenko, Dmitry A. Maslov
Microbiology Resource Announcements Apr 2019, 8 (16) e00263-19; DOI: 10.1128/MRA.00263-19
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