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Construction of Targeted Mycobacterial Mutants by Homologous Recombination

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Mycobacteria Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 465))

Abstract

The ability to select genes to knock out of mycobacterial genomes has greatly improved our understanding of mycobacteria. This chapter describes a method for doing this. The gene (including a 1-kb flanking region) is cloned into a pNIL series vector and disrupted by deletion or insertion of a cassette. A selection of marker genes obtained from the pGOAL series of vectors are inserted into the pNIL vector to create a suicide delivery system. This delivery vector is introduced into mycobacteria where the disrupted version of the gene replaces the wild-type version by a two-step homologous recombination process. The method involves selecting for a single crossover event followed by selection of double crossovers. Single crossovers have incorporated plasmid marker genes and are sucroseS, kanamycinR and blue on media containing X-gal. Double crossovers have lost plasmid markers and are sucroseR, kanamycinS and white on media containing X-gal.

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References

  1. Shin, S. J., Wu, C. W., Steinberg, H., and Talaat, A. M. (2006) Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants. Infect Immun 74, 3825–33.

    Article  PubMed  CAS  Google Scholar 

  2. Mehta, P. K., Pandey, A. K., Subbian, S., El-Etr, S. H., Cirillo, S. L., Samrakandi, M. M., and Cirillo, J. D. (2006) Identification of Mycobacterium marinum macrophage infection mutants. Microb Pathog 40, 139–51.

    Article  PubMed  CAS  Google Scholar 

  3. Ruley, K. M., Ansede, J. H., Pritchett, C. L., Talaat, A. M., Reimschuessel, R., and Trucksis, M. (2004) Identification of Mycobacterium marinum virulence genes using signature-tagged mutagenesis and the goldfish model of mycobacterial pathogenesis. FEMS Microbiol Lett 232, 75–81.

    Article  PubMed  CAS  Google Scholar 

  4. Philalay, J. S., Palermo, C. O., Hauge, K. A., Rustad, T. R., and Cangelosi, G. A. (2004) Genes required for intrinsic multidrug resistance in Mycobacterium avium. Antimicrob Agents Chemother 48, 3412–8.

    Article  PubMed  CAS  Google Scholar 

  5. Sassetti, C. M., and Rubin, E. J. (2003) Genetic requirements for mycobacterial survival during infection. Proc Natl Acad Sci U S A 100, 12989–94.

    Article  PubMed  CAS  Google Scholar 

  6. McAdam, R. A., Quan, S., Smith, D. A., Bardarov, S., Betts, J. C., Cook, F. C., Hooker, E. U., Lewis, A. P., Woollard, P., Everett, M. J., Lukey, P. T., Bancroft, G. J., Jacobs W. R. Jr., and Duncan, K. (2002) Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence. Microbiology 148, 2975–86.

    PubMed  CAS  Google Scholar 

  7. Li, Y., Miltner, E., Wu, M., Petrofsky, M., and Bermudez, L. E. (2005) A Mycobacterium avium PPE gene is associated with the ability of the bacterium to grow in macrophages and virulence in mice. Cell Microbiol 7, 539–48.

    Article  PubMed  CAS  Google Scholar 

  8. Chen, J. M., German, G. J., Alexander, D. C., Ren, H., Tan, T., and Liu, J. (2006) Roles of Lsr2 in colony morphology and biofilm formation of Mycobacterium smegmatis. J Bacteriol 188, 633–41.

    Article  PubMed  CAS  Google Scholar 

  9. Movahedzadeh, F., Smith, D. A., Norman, R. A., Dinadayala, P., Murray-Rust, J., Russell, D. G., Kendall, S. L., Rison, S. C., McAlister, M. S., Bancroft, G. J., McDonald, N. Q., Daffe, M., Av-Gay, Y., and Stoker, N. G. (2004) The Mycobacterium tuberculosis ino1 gene is essential for growth and virulence. Mol Microbiol 51, 1003–14.

    Article  PubMed  CAS  Google Scholar 

  10. Parish, T., Schaeffer, M., Roberts, G., and Duncan, K. (2005) HemZ is essential for heme biosynthesis in Mycobacterium tuberculosis. Tuberculosis (Edinb) 85, 197–204.

    Article  CAS  Google Scholar 

  11. Parish, T., and Stoker, N. G. (2000) glnE is an essential gene in Mycobacterium tuberculosis. J Bacteriol 182, 5715–20.

    Article  PubMed  CAS  Google Scholar 

  12. Hernandez Pando, R., Aguilar, L. D., Infante, E., Cataldi, A., Bigi, F., Martin, C., and Gicquel, B. (2006) The use of mutant mycobacteria as new vaccines to prevent tuberculosis. Tuberculosis 86, 203–10.

    Article  PubMed  CAS  Google Scholar 

  13. Perez, E., Samper, S., Bordas, Y., Guilhot, C., Gicquel, B., and Martin, C. (2001) An essential role for phoP in Mycobacterium tuberculosis virulence. Mol Microbiol 41, 179–87.

    Article  PubMed  CAS  Google Scholar 

  14. Smith, D. A., Parish, T., Stoker, N. G., and Bancroft, G. J. (2001) Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates. Infect Immun 69, 1142–50.

    Article  PubMed  CAS  Google Scholar 

  15. Vilcheze, C., Wang, F., Arai, M., Hazbon, M. H., Colangeli, R., Kremer, L., Weisbrod, T. R., Alland, D., Sacchettini, J. C., and Jacobs, W. R. Jr. (2006) Transfer of a point mutation in Mycobacterium tuberculosis inhA resolves the target of isoniazid. Nat Med 12, 1027–9.

    Article  PubMed  CAS  Google Scholar 

  16. Brooks, L. A. (1998) Chemical mutagenesis of mycobacteria. Methods Mol Biol 101, 175–86.

    PubMed  CAS  Google Scholar 

  17. Guilhot, C., Otal, I., Van Rompaey, I., Martin, C., and Gicquel, B. (1994) Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176, 535–9.

    Google Scholar 

  18. Martin, C., Timm, J., Rauzier, J., Gomez-Lus, R., Davies, J., and Gicquel, B. (1990) Transposition of an antibiotic resistance element in mycobacteria. Nature 345, 739–43.

    Article  PubMed  CAS  Google Scholar 

  19. McAdam, R. A., Weisbrod, T. R., Martin, J., Scuderi, J. D., Brown, A. M., Cirillo, J. D., Bloom, B. R., and Jacobs, W. R., Jr. (1995) In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. Infect Immun 63, 1004–12.

    PubMed  CAS  Google Scholar 

  20. Rengarajan, J., Bloom, B. R., and Rubin, E. J. (2005) Genome-wide requirements for Mycobacterium tuberculosis adaptation and survival in macrophages. Proc Natl Acad Sci U S A 102, 8327–32.

    Article  PubMed  CAS  Google Scholar 

  21. Husson, R. N., James, B. E., and Young, R. A. (1990) Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol 172, 519–24.

    PubMed  CAS  Google Scholar 

  22. Kalpana, G. V., Bloom, B. R., and Jacobs, W. R., Jr. (1991) Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc Natl Acad Sci U S A 88, 5433–7.

    Article  PubMed  CAS  Google Scholar 

  23. Machowski, E. E., Dawes, S., and Mizrahi, V. (2005) TB tools to tell the tale-molecular genetic methods for mycobacterial research. Int J Biochem Cell Biol 37, 54–68.

    Article  PubMed  CAS  Google Scholar 

  24. Reyrat, J. M., Berthet, F. X., and Gicquel, B. (1995) The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guerin. Proc Natl Acad Sci U S A 92, 8768–72.

    Article  PubMed  CAS  Google Scholar 

  25. Balasubramanian, V., Pavelka, M. S., Jr., Bardarov, S. S., Martin, J., Weisbrod, T. R., McAdam, R. A., Bloom, B. R., and Jacobs, W. R., Jr. (1996) Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J Bacteriol 178, 273–9.

    PubMed  CAS  Google Scholar 

  26. Parish, T., and Stoker, N. G. (2000) Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146 (Pt 8), 1969–75.

    PubMed  CAS  Google Scholar 

  27. Cox, M. M. (1999) Recombinational DNA repair in bacteria and the RecA protein. Prog Nucleic Acid Res Mol Biol 63, 311–66.

    Article  PubMed  CAS  Google Scholar 

  28. Muttucumaru, D. G., and 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, 145–57.

    PubMed  CAS  Google Scholar 

  29. Hinds, J., Mahenthiralingam, E., Kempsell, K. E., Duncan, K., Stokes, R. W., Parish, T., and Stoker, N. G. (1999) Enhanced gene replacement in mycobacteria. Microbiology 145, 519–27.

    Article  PubMed  CAS  Google Scholar 

  30. Pelicic, V., Reyrat, J. M., and Gicquel, B. (1996) Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol Microbiol 20, 919–25.

    Article  PubMed  CAS  Google Scholar 

  31. Pashley, C. A., Parish, T., McAdam, R. A., Duncan, K., and Stoker, N. G. (2003) Gene replacement in mycobacteria by using incompatible plasmids. Appl Environ Microbiol 69, 517–23.

    Article  PubMed  CAS  Google Scholar 

  32. Pelicic, V., Reyrat, J. M., and Gicquel, B. (1998) Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol Microbiol 28, 413–20.

    Article  PubMed  CAS  Google Scholar 

  33. Parish, T., Smith, D. A., Kendall, S., Casali, N., Bancroft, G. J., and Stoker, N. G. (2003) Deletion of two-component regulatory systems increases the virulence of Mycobacterium tuberculosis. Infect Immun 71, 1134–40.

    Article  PubMed  CAS  Google Scholar 

  34. Parish, T., Smith, D. A., Roberts, G., Betts, J., and Stoker, N. G. (2003) The senX3-regX3 two-component regulatory system of Mycobacterium tuberculosis is required for virulence. Microbiology 149, 1423–35.

    Article  PubMed  CAS  Google Scholar 

  35. Parish, T., Turner, J., and Stoker, N. G. (2001) amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis. BMC Microbiol 1, 19.

    Article  PubMed  CAS  Google Scholar 

  36. Chauhan, A., Madiraju, M. V., Fol, M., Lofton, H., Maloney, E., Reynolds, R., and Rajagopalan, M. (2006) Mycobacterium tuberculosis cells growing in macrophages are filamentous and deficient in FtsZ rings. J Bacteriol 188, 1856–65.

    Article  PubMed  CAS  Google Scholar 

  37. Dawes, S. S., Warner, D. F., Tsenova, L., Timm, J., McKinney, J. D., Kaplan, G., Rubin, H., and Mizrahi, V. (2003) Ribonucleotide reduction in Mycobacterium tuberculosis: function and expression of genes encoding class Ib and class II ribonucleotide reductases. Infect Immun 71, 6124–31.

    Article  PubMed  CAS  Google Scholar 

  38. Matsoso, L. G., Kana, B. D., Crellin, P. K., Lea-Smith, D. J., Pelosi, A., Powell, D., Dawes, S. S., Rubin, H., Coppel, R. L., and Mizrahi, V. (2005) Function of the cytochrome bc1-aa3 branch of the respiratory network in mycobacteria and network adaptation occurring in response to its disruption. J Bacteriol 187, 6300–8.

    Article  PubMed  CAS  Google Scholar 

  39. Curry, J. M., Whalan, R., Hunt, D. M., Gohil, K., Strom, M., Rickman, L., Colston, M. J., Smerdon, S. J., and Buxton, R. S. (2005) An ABC transporter containing a forkhead-associated domain interacts with a serine-threonine protein kinase and is required for growth of Mycobacterium tuberculosis in mice. Infect Immun 73, 4471–7.

    Article  PubMed  CAS  Google Scholar 

  40. Onwueme, K. C., Ferreras, J. A., Buglino, J., Lima, C. D., and Quadri, L. E. (2004) Mycobacterial polyketide-associated proteins are acyltransferases: proof of principle with Mycobacterium tuberculosis PapA5. Proc Natl Acad Sci USA 101, 4608–13.

    Article  PubMed  CAS  Google Scholar 

  41. Shimono, N., Morici, L., Casali, N., Cantrell, S., Sidders, B., Ehrt, S., and Riley, L. W. (2003) Hypervirulent mutant of Mycobacterium tuberculosis resulting from disruption of the mce1 operon. Proc Natl Acad Sci U S A 100, 15918–23.

    Article  PubMed  CAS  Google Scholar 

  42. Boshoff, H. I., Reed, M. B., Barry, C. E., 3rd, and Mizrahi, V. (2003) DnaE2 polymerase contributes to in vivo survival and the emergence of drug resistance in Mycobacterium tuberculosis. Cell 113, 183–93.

    Article  PubMed  CAS  Google Scholar 

  43. Primm, T. P., Andersen, S. J., Mizrahi, V., Avarbock, D., Rubin, H., and Barry, C. E. III (2000) The stringent response of Mycobacterium tuberculosis is required for long-term survival. J Bacteriol 182, 4889–98.

    Google Scholar 

  44. Hu, Y., Movahedzadeh, F., Stoker, N. G., and Coates, A. R. (2006) Deletion of the Mycobacterium tuberculosis alpha-crystallin-like hspX gene causes increased bacterial growth in vivo. Infect Immun 74, 861–8.

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Pathology and Infectious Diseases, The Royal Veterinary College, Royal College Street, London, NW1 OTU, UK

    Sharon L. Kendall PhD

  2. Department of Pathology and Infectious Diseases, The Royal Veterinary College, London, UK

    Rosangela Frita BSc

Authors
  1. Sharon L. Kendall PhD
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  2. Rosangela Frita BSc
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Corresponding author

Correspondence to Sharon L. Kendall PhD .

Editor information

Editors and Affiliations

  1. Queen Mary's School of Med. & Dentistry, Inst. Cell & Molecular Science, Barts and the London, Newark Street 4, London, E1 2AT, United Kingdom

    Tanya Parish

  2. Queen Mary's School of Med. & Dentistry, Inst. Cell & Molecular Science, Barts and the London, Newark Street 4, London, E1 2AT, United Kingdom

    Amanda Claire Brown

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Kendall, S.L., Frita, R. (2009). Construction of Targeted Mycobacterial Mutants by Homologous Recombination. In: Parish, T., Brown, A. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 465. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-207-6_20

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  • DOI: https://doi.org/10.1007/978-1-59745-207-6_20

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-889-8

  • Online ISBN: 978-1-59745-207-6

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