Targeted Gene Knockout and Essentiality Testing by Homologous Recombination

  • Krishnamoorthy Gopinath
  • Digby F. Warner
  • Valerie MizrahiEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)


This chapter provides an updated experimental protocol for generating allelic exchange mutants of mycobacteria by two-step selection using the p2NIL/pGOAL system. The types of mutants that can be generated using this approach are targeted gene knockouts marked with a drug resistance gene, unmarked deletion mutants, or strains in which a point mutation/s has been introduced into the target gene. A method for assessing the essentiality of a gene for mycobacterial growth by means of allelic exchange is also described. This method, which utilizes a merodiploid strain carrying a second copy of the gene of interest on an integration vector, allows the exploration by means of complement switching of structure–function relationships in proteins that are essential for mycobacterial growth.

Key words

Allelic exchange Complement switching Homologous recombination Illegitimate recombination Phthiocerol dimycocerosate (PDIM) UV irradiation 



This work was financially supported by grants from South African Medical Research Council (to V.M.), the National Research Foundation (to V.M.), and the Howard Hughes Medical Institute (Senior International Research Scholar’s grant to V.M.).


  1. 1.
    Muttucumaru DN, Parish T (2004) The molecular biology of recombination in Mycobacteria: what do we know and how can we use it? Curr Issues Mol Biol 6:145–158PubMedGoogle Scholar
  2. 2.
    Lamrabet O, Drancourt M (2012) Genetic engineering of Mycobacterium tuberculosis: a review. Tuberculosis (Edinb) 92:365–376CrossRefGoogle Scholar
  3. 3.
    Machowski EE, Dawes S, Mizrahi V (2005) TB tools to tell the tale–molecular genetic methods for mycobacterial research. Int J Biochem Cell Biol 37:54–68CrossRefPubMedGoogle Scholar
  4. 4.
    Nebenzahl-Guimaraes H, Jacobson KR, Farhat MR, Murray MB (2014) Systematic review of allelic exchange experiments aimed at identifying mutations that confer drug resistance in Mycobacterium tuberculosis. J Antimicrob Chemother 69:331–342CrossRefPubMedGoogle Scholar
  5. 5.
    Bardarov S, Kriakov J, Carriere C, Yu S, Vaamonde C, McAdam RA, Bloom BR, Hatfull GF, Jacobs WR (1997) Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 94:10961–10966CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Bardarov S, Bardarov S, Pavelka MS, Sambandamurthy V, Larsen M, Tufariello J, Chan J, Hatfull G, Jacobs WR (2002) Specialized transduction: an efficient method for generating marked and unmarked targeted gene disruptions in Mycobacterium tuberculosis, M. bovis BCG and M. smegmatis. Microbiology 148:3007–3017CrossRefPubMedGoogle Scholar
  7. 7.
    Gordhan BG, Parish T (2001) Gene replacement using pretreated DNA. In: Parish T, Stoker NG (eds) Mycobacterium tuberculosis protocols, Methods in molecular medicine. Humana Press, NY, pp 77–92CrossRefGoogle Scholar
  8. 8.
    Kendall SL, Frita R (2009) Construction of targeted mycobacterial mutants by homologous recombination. In: Parish T, Brown AC (eds) Mycobacteria protocols, vol 465, 2nd edn, Methods in molecular biology. Humana Press, NY, pp 297–310CrossRefGoogle Scholar
  9. 9.
    Niederweis M (2009) Construction of unmarked deletion mutants in mycobacteria. In: Parish T, Brown AC (eds) Mycobacteria protocols, vol 465, 2nd edn, Methods in molecular biology. Humana Press, NY, pp 279–295Google Scholar
  10. 10.
    Parish T, Stoker NG (2000) Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146:1969–1975CrossRefPubMedGoogle Scholar
  11. 11.
    Ioerger TR, Feng Y, Ganesula K, Chen X, Dobos KM, Fortune S, Jacobs WR, Mizrahi V, Parish T, Rubin E (2010) Variation among genome sequences of H37Rv strains of Mycobacterium tuberculosis from multiple laboratories. J Bacteriol 192:3645–3653CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Cox JS, Chen B, McNeil M, Jacobs WR (1999) Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402:79–83CrossRefPubMedGoogle Scholar
  13. 13.
    Domenech P, Reed MB (2009) Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies. Microbiology 155:3532–3543CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Marrero J, Rhee KY, Schnappinger D, Pethe K, Ehrt S (2010) Gluconeogenic carbon flow of tricarboxylic acid cycle intermediates is critical for Mycobacterium tuberculosis to establish and maintain infection. Proc Natl Acad Sci U S A 107:9819–9824CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Pashley CA, Parish T (2003) Efficient switching of mycobacteriophage L5‐based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett 229:211–215CrossRefPubMedGoogle Scholar
  16. 16.
    Springer B, Sander P, Sedlacek L, Ellrott K, Bottger EC (2001) Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors. Int J Med Microbiol 290:669–675CrossRefPubMedGoogle Scholar
  17. 17.
    Williams A, Guthlein C, Beresford N, Bottger EC, Springer B, Davis EO (2011) UvrD2 is essential in Mycobacterium tuberculosis, but its helicase activity is not required. J Bacteriol 193:4487–4494CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Davis EO, Springer B, Gopaul KK, Papavinasasundaram KG, Sander P, Bottger EC (2002) DNA damage induction of recA in Mycobacterium tuberculosis independently of RecA and LexA. Mol Microbiol 46:791–800CrossRefPubMedGoogle Scholar
  19. 19.
    Boshoff HI, Reed MB, Barry CE 3rd, Mizrahi V (2003) DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113:183–193CrossRefPubMedGoogle Scholar
  20. 20.
    Warner DF, Ndwandwe DE, Abrahams GL, Kana BD, Machowski EE, Venclovas C, Mizrahi V (2010) Essential roles for imuA′- and imuB-encoded accessory factors in DnaE2-dependent mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 107:13093–13098CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Warner DF, Savvi S, Mizrahi V, Dawes SS (2007) A riboswitch regulates expression of the coenzyme B12-independent methionine synthase in Mycobacterium tuberculosis: implications for differential methionine synthase function in strains H37Rv and CDC1551. J Bacteriol 189:3655–3659CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Gopinath K, Venclovas C, Ioerger TR, Sacchettini JC, McKinney JD, Mizrahi V, Warner DF (2013) A vitamin B12 transporter in Mycobacterium tuberculosis. Open Biol 3:120175CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84CrossRefPubMedGoogle Scholar
  24. 24.
    Parish T, Roberts G, Laval F, Schaeffer M, Daffé M, Duncan K (2007) Functional complementation of the essential gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but not Escherichia coli fabG. J Bacteriol 189:3721–3728CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Parish T, Stoker NG (2000) glnE is an essential gene in Mycobacterium tuberculosis. J Bacteriol 182:5715–5720CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mowa MB, Warner DF, Kaplan G, Kana BD, Mizrahi V (2009) Function and regulation of class I ribonucleotide reductase-encoding genes in mycobacteria. J Bacteriol 191:985–995CrossRefPubMedGoogle Scholar
  27. 27.
    Pena CE, Lee MH, Pedulla ML, Hatfull GF (1997) Characterization of the mycobacteriophage L5 attachment site, attP. J Mol Biol 266:76–92CrossRefPubMedGoogle Scholar
  28. 28.
    Lydiate DJ, Ashby AM, Henderson DJ, Kieser HM, Hopwood DA (1989) Physical and genetic characterization of chromosomal copies of the Streptomyces coelicolor mini-circle. J Gen Microbiol 135:941–955Google Scholar
  29. 29.
    Dawes SS, Warner DF, Tsenova L, Timm J, McKinney JD, Kaplan G, Rubin H, Mizrahi V (2003) Ribonucleotide reduction in Mycobacterium tuberculosis: function and expression of genes encoding class Ib and class II ribonucleotide reductases. Infect Immun 71:6124–6131CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    O’Gaora P, Barnini S, Hayward C, Filley E, Rook G, Young D, Thole J (1997) Mycobacteria as immunogens. Development of expression vectors for use in multiple mycobacterial species. Med Princ Pract 6:91–96Google Scholar
  31. 31.
    Boshoff HI, Mizrahi V (2000) Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides. J Bacteriol 182:5479–5485CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Smith AM, Klugman KP (1997) “Megaprimer” method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor. Biotechniques 22:438–442PubMedGoogle Scholar
  33. 33.
    Hinds J, Mahenthiralingam E, Kempsell KE, Duncan K, Stokes RW, Parish T, Stoker NG (1999) Enhanced gene replacement in mycobacteria. Microbiology 145:519–527CrossRefPubMedGoogle Scholar
  34. 34.
    Wards BJ, Collins DM (1996) Electroporation at elevated temperatures substantially improves transformation efficiency of slow‐growing mycobacteria. FEMS Microbiol Lett 145:101–105CrossRefPubMedGoogle Scholar
  35. 35.
    Savvi S, Warner DF, Kana BD, McKinney JD, Mizrahi V, Dawes SS (2008) Functional characterization of a vitamin B12-dependent methylmalonyl pathway in Mycobacterium tuberculosis: implications for propionate metabolism during growth on fatty acids. J Bacteriol 190:3886–3895CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Mahenthiralingam E, Marklund BI, Brooks LA, Smith DA, Bancroft GJ, Stokes RW (1998) Site-directed mutagenesis of the 19-kilodalton lipoprotein antigen reveals no essential role for the protein in the growth and virulence of Mycobacterium intracellulare. Infect Immun 66:3626–3634PubMedPubMedCentralGoogle Scholar
  37. 37.
    Brown AC (2009) Gene switching and essentiality testing. In: Parish T, Brown AC (eds) Mycobacteria protocols, vol 465, 2nd edn, Methods in molecular biology. Humana Press, NY, pp 337–353CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Krishnamoorthy Gopinath
    • 1
  • Digby F. Warner
    • 1
  • Valerie Mizrahi
    • 1
    Email author
  1. 1.MRC/NHLS/UCT Molecular Mycobacteriology Research Unit and DST/NRF Centre of Excellence for Biomedical TB Research, Institute of Infectious Disease and Molecular Medicine and Department of Clinical Laboratory Sciences, Faculty of Health SciencesUniversity of Cape TownCape TownSouth Africa

Personalised recommendations