Genome-Wide Transposon Mutagenesis in Mycobacterium tuberculosis and Mycobacterium smegmatis

  • Gaurav Majumdar
  • Rendani Mbau
  • Vinayak Singh
  • Digby F. WarnerEmail author
  • Marte Singsås Dragset
  • Raju MukherjeeEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1498)


TnSeq, or transposon (Tn) insertion sequencing, is a powerful method for identifying the essential—as well as conditionally essential—regions in a genome, both coding and noncoding. The advent of accessible massively parallel DNA sequencing technologies in particular has resulted in the increased use of TnSeq-based approaches to elucidate various aspects of bacterial physiology and metabolism. Moreover, the availability of detailed protocols has enabled even nonspecialist laboratories to adapt and develop TnSeq approaches to address specific research questions. In this chapter, we describe a recently modified experimental protocol used in our laboratory for TnSeq in the major human pathogen, Mycobacterium tuberculosis, as well as the related non-pathogenic mycobacterium, M. smegmatis. The method, which was developed in close consultation with pioneers in the field of mycobacterial genetics, includes the steps involved in preparing a phage stock, generating a mutant library, selection of the library under a specific experimental condition, isolation of genomic DNA from the pooled population of mutants, amplification of the sites of Tn insertion and, finally, determining the essential genomic regions by next-generation sequencing.

Key words

Transposon mutagenesis TnSeq analysis MycoMarT7 



This work is funded by grants from the South African Medical Research Council (to DFW), the National Research Foundation (to DFW), and the Research Council of Norway through its Centres of Excellence funding scheme, project number 223255 (to MSD). RM is supported by the Howard Hughes Medical Institute through a Senior International Research Scholars grant to Valerie Mizrahi. GM is supported by a grant from the South African Medical Research Council (to DFW) with funds from National Treasury under the Economic Competitiveness and Support Package (MRC-RFA-UFSP-01-2013/CCAMP).


  1. 1.
    Cole ST, Brosch R, Parkhill J, Garnier T, Churcher C, Harris D, Gordon SV, Eiglmeier K, Gas S, Barry CE III, Tekaia F, Badcock K, Basham D, Brown D, Chillingworth T, Connor R, Davies R, Devlin K, Feltwell T, Gentles S, Hamlin N, Holroyd S, Hornsby T, Jagels K, Krogh A, McLean J, Moule S, Murphy L, Oliver K, Osborne J, Quail MA, Rajandream MA, Rogers J, Rutter S, Seeger K, Skelton J, Squares R, Squares S, Sulston JE, Taylor K, Whitehead S, Barrell BG (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537–544CrossRefPubMedGoogle Scholar
  2. 2.
    Lew JM, Kapopoulou A, Jones LM, Cole ST (2011) TubercuList-10 years after. Tuberculosis (Edinb) 91:1–7CrossRefGoogle Scholar
  3. 3.
    Ates LS, Ummels R, Commandeur S, van de Weerd R, Sparrius M, Weerdenburg E, Alber M, Kalscheuer R, Piersma SR, Abdallah AM, Abd El Ghany M, Abdel-Haleem AM, Pain A, Jimenez CR, Bitter W, Houben EN (2015) Essential role of the ESX-5 secretion system in outer membrane permeability of pathogenic mycobacteria. PLoS Genet 11:e1005190CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    van Opijnen T, Camilli A (2013) Transposon insertion sequencing: a new tool for systems-level analysis of microorganisms. Nat Rev Microbiol 11:435–442CrossRefPubMedGoogle Scholar
  5. 5.
    Chao MC, Abel S, Davis BM, Waldor MK (2016) The design and analysis of transposon insertion sequencing experiments. Nat Rev Microbiol 14:119–128CrossRefPubMedGoogle Scholar
  6. 6.
    Zhang YJ, Ioerger TR, Huttenhower C, Long JE, Sassetti CM, Sacchettini JC, Rubin EJ (2012) Global assessment of genomic regions required for growth in Mycobacterium tuberculosis. PLoS Pathog 8:e1002946CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    DeJesus MA, Ambadipudi C, Baker R, Sassetti C, Ioerger TR (2015) TRANSIT–a software tool for Himar1 TnSeq analysis. PLoS Comput Biol 11:e1004401CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Pritchard JR, Chao MC, Abel S, Davis BM, Baranowski C, Zhang YJ, Rubin EJ, Waldor MK (2014) ARTIST: high-resolution genome-wide assessment of fitness using transposoninsertion sequencing. PLoS Genet 10:e1004782CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    DeJesus MA, Ioerger TR (2013) A Hidden Markov Model for identifying essential and growth-defect regions in bacterial genomes from transposon insertion sequencing data. BMC Bioinformatics 14:303CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Nambi S, Long JE, Mishra BB, Baker R, Murphy KC, Olive AJ, Nguyen HP, Shaffer SA, Sassetti CM (2015) The oxidative stress network of Mycobacterium tuberculosis reveals coordination between radical detoxification systems. Cell Host Microbe 17:829–837CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Kieser KJ, Baranowski C, Chao MC, Long JE, Sassetti CM, Waldor MK, Sacchettini JC, Ioerger TR, Rubin EJ (2015) Peptidoglycan synthesis in Mycobacterium tuberculosis is organized into networks with varying drug susceptibility. Proc Natl Acad Sci U S A 112:13087–13092CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Griffin JE, Gawronski JD, Dejesus MA, Ioerger TR, Akerley BJ, Sassetti CM (2011) High-resolution phenotypic profiling defines genes essential for mycobacterial growth and cholesterol catabolism. PLoS Pathog 7:e1002251CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84CrossRefPubMedGoogle Scholar
  14. 14.
    Long JE, DeJesus M, Ward D, Baker RE, Ioerger T, Sassetti CM (2015) Identifying essential genes in Mycobacterium tuberculosis by global phenotypic profiling. Methods Mol Biol 1279:79–95CrossRefPubMedGoogle Scholar
  15. 15.
    DeJesus MA, Zhang YJ, Sassetti CM, Rubin EJ, Sacchettini JC, Ioerger TR (2013) Bayesian analysis of gene essentiality based on sequencing of transposon insertion libraries. Bioinformatics 29:695–703CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  • Gaurav Majumdar
    • 1
  • Rendani Mbau
    • 1
  • Vinayak Singh
    • 1
  • Digby F. Warner
    • 1
    • 2
    Email author
  • Marte Singsås Dragset
    • 3
  • Raju Mukherjee
    • 1
    • 4
    Email author
  1. 1.MRC/NHLS/UCT Molecular Mycobacteriology Research Unit, DST/NRF Centre of Excellence for Biomedical TB Research, Department of Pathology, Faculty of Health SciencesUniversity of Cape TownCape TownSouth Africa
  2. 2.Institute of Infectious Disease and Molecular MedicineUniversity of Cape TownCape TownSouth Africa
  3. 3.Centre of Molecular Inflammation Research, Department of Cancer Research and Molecular MedicineNorwegian University of Science and TechnologyTrondheimNorway
  4. 4.Division of BiologyIndian Institute of Science Education and ResearchTirupatiIndia

Personalised recommendations