1887

Abstract

subspecies (MAH) is a pathogen that causes various non-tuberculous mycobacterial diseases in humans and animals worldwide. Among the genus, MAH is characterized by relatively slow growth. Here, we isolated a rapidly growing variant of the MAH 104 strain. The variant strain (named N104) exhibited an enhanced growth rate and higher motility compared to the parent MAH 104 strain (P104). Whole-genome sequencing analysis of N104 revealed the loss of the stop codon of due to a single nucleotide replacement, resulting in the substitution of the codon for tryptophan. Notably, exclusion of the stop codon ligated the open reading frames and caused the fusion of two adjacent proteins. A revertant parent strain, in which a mutation was introduced to restore the stop codon, revealed that elimination of the stop codon in was responsible for the N104 phenotype. Furthermore, we analysed the phenotypes of the parent and mutated strains by determining the functions of the and coding regions flanking the stop codon. The mutant strains, expected to express a fusion protein, exhibited increased resistance to antimicrobial drugs and exogenous copper toxicity compared to that of the parent strains. These findings suggest that the fusion of the - and -encoding regions in the mutant N104 strain could be related to the modified functions of these intrinsic proteins.

Funding
This study was supported by the:
  • Japan Agency for Medical Research and Development (Award JP20fk0108129)
    • Principle Award Recipient: ManabuAto
Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.001007
2020-12-23
2021-07-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/167/2/micro001007.html?itemId=/content/journal/micro/10.1099/mic.0.001007&mimeType=html&fmt=ahah

References

  1. James BW, Williams A, Marsh PD. The physiology and pathogenicity of Mycobacterium tuberculosis grown under controlled conditions in a defined medium. J Appl Microbiol 2000; 88:669–677 [View Article][PubMed]
    [Google Scholar]
  2. Klann AG, Belanger AE, Abanes-De Mello A, Lee JY, Hatfull GF. Characterization of the dnaG locus in Mycobacterium smegmatis reveals linkage of DNA replication and cell division. J Bacteriol 1998; 180:65–72 [View Article][PubMed]
    [Google Scholar]
  3. Runyon EH. Anonymous mycobacteria in pulmonary disease. Med Clin North Am 1959; 43:273–290 [View Article][PubMed]
    [Google Scholar]
  4. Kim C-J, Kim N-H, Song K-H, Choe PG, Kim ES et al. Differentiating rapid- and slow-growing mycobacteria by difference in time to growth detection in liquid media. Diagn Microbiol Infect Dis 2013; 75:73–76 [View Article][PubMed]
    [Google Scholar]
  5. Menéndez MDC, Rebollo MJ, Núñez MDC, Cox RA, García MJ. Analysis of the precursor rRNA fractions of rapidly growing mycobacteria: quantification by methods that include the use of a promoter (rrnA P1) as a novel standard. J Bacteriol 2005; 187:534–543 [View Article][PubMed]
    [Google Scholar]
  6. Beste DJV, Espasa M, Bonde B, Kierzek AM, Stewart GR et al. The genetic requirements for fast and slow growth in mycobacteria. PLoS One 2009; 4:e5349 [View Article][PubMed]
    [Google Scholar]
  7. Mariam DH, Mengistu Y, Hoffner SE, Andersson DI. Effect of rpoB mutations on fitness of Mycobacterium tuberculosis. Antimicrob Agents Chemother 2004; 48:1289–1294
    [Google Scholar]
  8. Lamichhane G, Raghunand TR, Morrison NE, Woolwine SC, Tyagi S et al. Deletion of a Mycobacterium tuberculosis proteasomal ATPase homologue gene produces a slow-growing strain that persists in host tissues. J Infect Dis 2006; 194:1233–1240 [View Article][PubMed]
    [Google Scholar]
  9. Malhotra V, Okon BP, Clark-Curtiss JE. Mycobacterium tuberculosis protein kinase K enables growth adaptation through translation control. J Bacteriol 2012; 194:4184–4196 [View Article][PubMed]
    [Google Scholar]
  10. Plocinska R, Purushotham G, Sarva K, Vadrevu IS, Pandeeti EVP et al. Septal localization of the Mycobacterium tuberculosis MtrB sensor kinase promotes MtrA regulon expression. J Biol Chem 2012; 287:23887–23899 [View Article][PubMed]
    [Google Scholar]
  11. Mathew R, Mukherjee R, Balachandar R, Chatterji D. Deletion of the rpoZ gene, encoding the omega subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis. Microbiology 2006; 152:1741–1750 [View Article][PubMed]
    [Google Scholar]
  12. Schorey JS, Sweet L. The mycobacterial glycopeptidolipids: structure, function, and their role in pathogenesis. Glycobiology 2008; 18:832–841 [View Article][PubMed]
    [Google Scholar]
  13. Martínez A, Torello S, Kolter R. Sliding motility in mycobacteria. J Bacteriol 1999; 181:7331–7338 [View Article][PubMed]
    [Google Scholar]
  14. Recht J, Martínez A, Torello S, Kolter R. Genetic analysis of sliding motility in Mycobacterium smegmatis . J Bacteriol 2000; 182:4348–4351 [View Article][PubMed]
    [Google Scholar]
  15. Elguezabal N, Bastida F, Sevilla IA, González N, Molina E et al. Estimation of Mycobacterium avium subsp. paratuberculosis growth parameters: strain characterization and comparison of methods. Appl Environ Microbiol 2011; 77:8615–8624 [View Article][PubMed]
    [Google Scholar]
  16. Yamada H, Yamaguchi M, Igarashi Y, Chikamatsu K, Aono A et al. Mycolicibacterium smegmatis, Basonym Mycobacterium smegmatis, Expresses Morphological Phenotypes Much More Similar to Escherichia coli Than Mycobacterium tuberculosis in Quantitative Structome Analysis and CryoTEM Examination. Front Microbiol 2018; 9:9 [View Article][PubMed]
    [Google Scholar]
  17. Yamada H, Bhatt A, Danev R, Fujiwara N, Maeda S et al. Non-acid-fastness in Mycobacterium tuberculosis ΔkasB mutant correlates with the cell envelope electron density. Tuberculosis 2012; 92:351–357 [View Article][PubMed]
    [Google Scholar]
  18. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M et al. Fiji: an open-source platform for biological-image analysis. Nat Methods 2012; 9:676–682 [View Article][PubMed]
    [Google Scholar]
  19. Wilson K. Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol 2001; Chapter 2:2.4.1–2.4.2 [View Article][PubMed]
    [Google Scholar]
  20. Nakanaga K, Ogura Y, Toyoda A, Yoshida M, Fukano H et al. Naturally occurring a loss of a giant plasmid from Mycobacterium ulcerans subsp. shinshuense makes it non-pathogenic. Sci Rep 2018; 8:1–12 [View Article]
    [Google Scholar]
  21. Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009; 25:1754–1760 [View Article][PubMed]
    [Google Scholar]
  22. McKenna A, Hanna M, Banks E, Sivachenko A, Cibulskis K et al. The genome analysis toolkit: a MapReduce framework for analyzing next-generation DNA sequencing data. Genome Res 2010; 20:1297–1303 [View Article][PubMed]
    [Google Scholar]
  23. DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011; 43:491–498 [View Article][PubMed]
    [Google Scholar]
  24. Cingolani P, Platts A, Wang LL, Coon M, Nguyen T et al. A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. Fly 2012; 6:80–92 [View Article][PubMed]
    [Google Scholar]
  25. Tanizawa Y, Fujisawa T, Kaminuma E, Nakamura Y, Arita M. DFAST and DAGA: web-based integrated genome annotation tools and resources. Biosci Microbiota Food Health 2016; 35:173–184 [View Article][PubMed]
    [Google Scholar]
  26. Darling AE, Mau B, Perna NT. progressiveMauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS One 2010; 5:e11147 [View Article][PubMed]
    [Google Scholar]
  27. Daugelat S, Kowall J, Mattow J, Bumann D, Winter R et al. The RD1 proteins of Mycobacterium tuberculosis: expression in Mycobacterium smegmatis and biochemical characterization. Microbes Infect 2003; 5:1082–1095 [View Article][PubMed]
    [Google Scholar]
  28. Ouchi Y, Mukai T, Koide K, Yamaguchi T, Park J-H et al. WQ-3810: a new fluoroquinolone with a high potential against fluoroquinolone-resistant Mycobacterium tuberculosis . Tuberculosis 2020; 120:101891 [View Article][PubMed]
    [Google Scholar]
  29. van Kessel JC, Hatfull GF. Recombineering in Mycobacterium tuberculosis . Nat Methods 2007; 4:147–152 [View Article][PubMed]
    [Google Scholar]
  30. Miyamoto Y, Mukai T, Takeshita F, Nakata N, Maeda Y et al. Aggregation of mycobacteria caused by disruption of fibronectin-attachment protein-encoding gene. FEMS Microbiol Lett 2004; 236:227–234 [View Article][PubMed]
    [Google Scholar]
  31. Wolschendorf F, Ackart D, Shrestha TB, Hascall-Dove L, Nolan S et al. Copper resistance is essential for virulence of Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 2011; 108:1621–1626 [View Article][PubMed]
    [Google Scholar]
  32. Philalay JS, Palermo CO, Hauge KA, Rustad TR, Cangelosi GA. Genes required for intrinsic multidrug resistance in Mycobacterium avium. Antimicrob Agents Chemother 2004; 48:3412–3418 [View Article][PubMed]
    [Google Scholar]
  33. Nisa S, Hazen TH, Assatourian L, Nougayrède J-P, Rasko DA et al. In vitro evolution of an archetypal enteropathogenic Escherichia coli strain. J Bacteriol 2013; 195:4476–4483 [View Article][PubMed]
    [Google Scholar]
  34. Cangelosi GA, Palermo CO, Laurent J-P, Hamlin AM, Brabant WH. Colony morphotypes on Congo red agar segregate along species and drug susceptibility lines in the Mycobacterium avium-intracellulare complex. Microbiology 1999; 145:1317–1324 [View Article][PubMed]
    [Google Scholar]
  35. Recht J, Kolter R. Glycopeptidolipid acetylation affects sliding motility and biofilm formation in Mycobacterium smegmatis . J Bacteriol 2001; 183:5718–5724 [View Article][PubMed]
    [Google Scholar]
  36. Fujiwara N, Ohara N, Ogawa M, Maeda S, Naka T et al. Glycopeptidolipid of Mycobacterium smegmatis J15cs affects morphology and survival in host cells. PLoS One 2015; 10:e0126813–11 [View Article][PubMed]
    [Google Scholar]
  37. Song H, Sandie R, Wang Y, Andrade-Navarro MA, Niederweis M. Identification of outer membrane proteins of Mycobacterium tuberculosis . Tuberculosis 2008; 88:526–544 [View Article][PubMed]
    [Google Scholar]
  38. Wagner D, Maser J, Lai B, Cai Z, Barry CE et al. Elemental analysis of Mycobacterium avium, Mycobacterium tuberculosis and Mycobacterium smegmatis-containing phagosomes indicates pathogen-induced microenvironments within the host cell's endosomal system. J Immunol 2005; 174:1491–1500 [View Article][PubMed]
    [Google Scholar]
  39. Fan Y, Evans CR, Barber KW, Banerjee K, Weiss KJ et al. Heterogeneity of stop codon readthrough in single bacterial cells and implications for population fitness. Mol Cell 2017; 67:826–836 [View Article][PubMed]
    [Google Scholar]
  40. Firth AE, Brierley I. Non-canonical translation in RNA viruses. J Gen Virol 2012; 93:1385–1409 [View Article][PubMed]
    [Google Scholar]
  41. Sawyer EB, Grabowska AD, Cortes T. Translational regulation in mycobacteria and its implications for pathogenicity. Nucleic Acids Res 2018; 46:6950–6961 [View Article][PubMed]
    [Google Scholar]
  42. Bidnenko V, Nicolas P, Grylak-Mielnicka A, Delumeau O, Auger S et al. Termination factor Rho: from the control of pervasive transcription to cell fate determination in Bacillus subtilis. PLoS Genet 2017; 13:e1006909 [View Article][PubMed]
    [Google Scholar]
  43. LM F, Fu-Liu CS. The gene expression data of Mycobacterium tuberculosis based on affymetrix gene chips provide insight into regulatory and hypothetical genes. BMC Microbiol 2007; 7:1–11
    [Google Scholar]
  44. Goodsmith N, Guo XV, Vandal OH, Vaubourgeix J, Wang R et al. Disruption of an M. tuberculosis membrane protein causes a magnesium-dependent cell division defect and failure to persist in mice. PLoS Pathog 2015; 11:e1004645–23 [View Article]
    [Google Scholar]
  45. Bardarov S, Bardarov S, Pavelka MS, Sambandamurthy V, Larsen M et al. Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology [View Article][PubMed]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.001007
Loading
/content/journal/micro/10.1099/mic.0.001007
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF
This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error