Skip to main content
SpringerLink
Log in
Book cover

Mycobacteria Protocols pp 203–215Cite as

Electroporation of Mycobacteria

  • Protocol
  • First Online:

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

Abstract

High-efficiency transformation is a major limitation in the study of mycobacteria. The genus Mycobacterium can be difficult to transform; this is mainly caused by the thick and waxy cell wall but is compounded by the fact that most molecular techniques have been developed for distantly related species such as Escherichia coli and Bacillus subtilis. In spite of these obstacles, mycobacterial plasmids have been identified, and DNA transformation of many mycobacterial species has now been described. The most successful method for introducing DNA into mycobacteria is electroporation. Many parameters contribute to successful transformation; these include the species/strain, the nature of the transforming DNA, the selectable marker used, the growth medium, and the conditions for the electroporation pulse. Optimized methods for the transformation of both slow-grower and fast-grower are detailed here. Transformation efficiencies for different mycobacterial species and with various selectable markers are reported.

This is a preview of subscription content, log in via an institution.

Protocol
USD   49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD   129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD   169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD   169.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Learn about institutional subscriptions

Springer Nature is developing a new tool to find and evaluate Protocols. Learn more

References

  1. Jacobs, W.R. Jr., Kalpana, G.V., Cirillo, J.D., Pascopella, L., Snapper, S.B., Udani, R.A., Jones, W., Barletta, R.G. and Bloom, B.R. (1991) Genetic systems for mycobacteria. Methods Enzymol, 204, 537–555.

    Article  PubMed  CAS  Google Scholar 

  2. 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, 5433–5437.

    Article  PubMed  CAS  Google Scholar 

  3. Paget, E. and Davies, J. (1996) Apramycin resistance as a selective marker for gene transfer in mycobacteria. J Bacteriol, 178, 6357–6360.

    PubMed  CAS  Google Scholar 

  4. Parish, T., Gordhan, B.G., McAdam, R.A., Duncan, K., Mizrahi, V. and Stoker, N.G. (1999) Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology, 145, 3497–3503.

    PubMed  CAS  Google Scholar 

  5. Ranes, M.G., Rauzier, J., Lagranderie, M., Gheorghiu, M. and Gicquel, B. (1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a “mini” mycobacterium-Escherichia coli shuttle vector. J Bacteriol, 172, 2793–2797.

    PubMed  CAS  Google Scholar 

  6. Yuan, Y., Crane, D.D., Simpson, R.M., Zhu, Y.Q., Hickey, M.J., Sherman, D.R. and Barry, C.E. III. (1998) The 16-kDa alpha-crystallin (Acr) protein of Mycobacterium tuberculosis is required for growth in macrophages. Proc Natl Acad Sci U S A, 95, 9578–9583.

    Article  PubMed  CAS  Google Scholar 

  7. Consaul, S.A. and Pavelka, M.S. Jr. (2004) Use of a novel allele of the Escherichia coli aacC4 aminoglycoside resistance gene as a genetic marker in mycobacteria. FEMS Microbiol Lett, 234, 297–301.

    Article  PubMed  CAS  Google Scholar 

  8. Pashley, C.A. and Parish, T. (2003) Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett, 229, 211–215.

    Article  PubMed  CAS  Google Scholar 

  9. Dellagostin, O.A., Wall, S., Norman, E., O'Shaughnessy, T., Dale, J.W. and McFadden, J. (1993) Construction and use of integrative vectors to express foreign genes in mycobacteria. Mol Microbiol, 10, 983–993.

    Article  PubMed  CAS  Google Scholar 

  10. Garbe, T.R., Barathi, J., Barnini, S., Zhang, Y., Abou-Zeid, C., Tang, D., Mukherjee, R. and Young, D.B. (1994) Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology, 140, 133–138.

    Article  PubMed  CAS  Google Scholar 

  11. Goto, Y., Taniguchi, H., Udou, T., Mizuguchi, Y. and Tokunaga, T. (1991) Development of a new host vector system in mycobacteria. FEMS Microbiol Lett, 67, 277–282.

    Article  PubMed  CAS  Google Scholar 

  12. Matsuo, K., Yamaguchi, R., Yamazaki, A., Tasaka, H., Terasaka, K., Totsuka, M., Kobayashi, K., Yukitake, H. and Yamada, T. (1990) Establishment of a foreign antigen secretion system in mycobacteria. Infect Immun, 58, 4049–4054.

    PubMed  CAS  Google Scholar 

  13. Qin, M., Taniguchi, H. and Mizuguchi, Y. (1994) Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol, 176, 419–425.

    PubMed  CAS  Google Scholar 

  14. Radford, A.J. and Hodgson, A.L. (1991) Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector. Plasmid, 25, 149–153.

    Article  PubMed  CAS  Google Scholar 

  15. Snapper, S.B., Lugosi, L., Jekkel, A., Melton, R.E., Kieser, T., Bloom, B.R. and Jacobs, W.R., Jr. (1988) Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A, 85, 6987–6991.

    Article  PubMed  CAS  Google Scholar 

  16. Wards, B.J. and Collins, D.M. (1996) Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol Lett, 145, 101–105.

    Article  PubMed  CAS  Google Scholar 

  17. Hermans, J., Martin, C., Huijberts, G.N., Goosen, T. and de Bont, J.A. (1991) Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215. Mol Microbiol, 5, 1561–1566.

    Article  PubMed  CAS  Google Scholar 

  18. Houssaini-Iraqui, M., Lazraq, M.H., Clavel-Seres, S., Rastogi, N. and David, H.L. (1992) Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis. FEMS Microbiol Lett, 69, 239–244.

    Article  PubMed  CAS  Google Scholar 

  19. Marklund, B.I., Speert, D.P. and Stokes, R.W. (1995) Gene replacement through homologous recombination in Mycobacterium intracellulare. J Bacteriol, 177, 6100–6105.

    PubMed  CAS  Google Scholar 

  20. Hermans, J., Suy, I.M.L. and De Bont, J.A.M. (1993) Transformation of Gram-positive microorganisms with the Gram-negative broad-host-range cosmid vector pJRD215. FEMS Microbiol Lett, 108, 201–204.

    Article  CAS  Google Scholar 

  21. Talaat, A.M. and Trucksis, M. (2000) Transformation and transposition of the genome of Mycobacterium marinum. Am J Vet Res, 61, 125–128.

    Article  PubMed  CAS  Google Scholar 

  22. Beggs, M.L., Crawford, J.T. and Eisenach, K.D. (1995) Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7. J Bacteriol, 177, 4836–4840.

    PubMed  CAS  Google Scholar 

  23. Foley-Thomas, E.M., Whipple, D.L., Bermudez, L.E. and Barletta, R.G. (1995) Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis. Microbiology, 141, 1173–1181.

    Article  PubMed  CAS  Google Scholar 

  24. Lee, S.H., Cheung, M., Irani, V., Carroll, J.D., Inamine, J.M., Howe, W.R. and Maslow, J.N. (2002) Optimization of electroporation conditions for Mycobacterium avium. Tuberculosis (Edinb), 82, 167–174.

    Article  Google Scholar 

  25. Snapper, S.B., Melton, R.E., Mustafa, S., Kieser, T. and Jacobs, W.R., Jr. (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol, 4, 1911–1919.

    Article  PubMed  CAS  Google Scholar 

  26. Labidi, A., Dauguet, C., Goh, K.S. and David, H.L. (1984) Plasmid profiles of Mycobacterium fortuitum complex isolates. Curr Microbiol, 11, 235–240.

    Article  CAS  Google Scholar 

  27. Stover, C.K., de la Cruz, V.F., Fuerst, T.R., Burlein, J.E., Benson, L.A., Bennett, L.T., Bansal, G.P., Young, J.F., Lee, M.H., Hatfull, G.F. et al. (1991) New use of BCG for recombinant vaccines. Nature, 351, 456–460.

    Article  PubMed  CAS  Google Scholar 

  28. Bachrach, G., Colston, M.J., Bercovier, H., Bar-Nir, D., Anderson, C. and Papavinasasundaram, K.G. (2000) A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology, 146(Pt 2), 297–303.

    PubMed  CAS  Google Scholar 

  29. Gavigan, J.A., Ainsa, J.A., Perez, E., Otal, I. and Martin, C. (1997) Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum. J Bacteriol, 179, 4115–4122.

    PubMed  CAS  Google Scholar 

  30. Lee, M.H., Pascopella, L., Jacobs, W.R. Jr. and Hatfull, G.F. (1991) Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guerin. Proc Natl Acad Sci U S A, 88, 3111–3115.

    Article  PubMed  CAS  Google Scholar 

  31. Anes, E., Portugal, I. and Moniz-Pereira, J. (1992) Insertion into the Mycobacterium smegmatis genome of theaph gene through lysogenization with the temperate mycobacteriophage Ms6. FEMS Microbiol Lett, 74, 21–25.

    Article  PubMed  CAS  Google Scholar 

  32. Martin, C., Mazodier, P., Mediola, M.V., Gicquel, B., Smokvina, T., Thompson, C.J. and Davies, J. (1991) Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol Microbiol, 5, 2499–2502.

    Article  PubMed  CAS  Google Scholar 

  33. England, P.M., Mazodier, P., Mediola, M.V., Gicquel, B., Smokvina, T., Thompson, C.J. and Davies, J. (1991) Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol Microbiol, 5, 2499–2502.

    Article  Google Scholar 

  34. Bottger, E.C. (1994) Resistance to drugs targeting protein synthesis in mycobacteria. Trends Microbiol, 2, 416–421.

    Article  PubMed  CAS  Google Scholar 

  35. Hatfull, G.F. (1993) Genetic transformation of mycobacteria. Trends Microbiol, 1,310–314.

    Article  PubMed  CAS  Google Scholar 

  36. Aldovini, A., Husson, R.N. and Young, R.A. (1993) The uraA locus and homologous recombination in Mycobacterium bovis BCG. J Bacteriol, 175, 7282–7289.

    PubMed  CAS  Google Scholar 

  37. Hermans, J., Boschloo, J.G. and de Bont, J.A.M. (1990) Transformation of Mycobacterium aurum by electroporation: the use of glycine, lysozyme and isonicotinic acid hydrazide in enhancing transformation efficiency. FEMS Microbiol Lett, 72, 221–224.

    Article  CAS  Google Scholar 

  38. Husson, R.N., James, B.E. and Young, R.A. (1990) Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol, 172, 519–524.

    PubMed  CAS  Google Scholar 

  39. Hammes, W., Schleifer, K.H. and Kandler, O. (1973) Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol, 116, 1029–1053.

    PubMed  CAS  Google Scholar 

  40. Cruickshank, R. (1965) Medical Microbiology: A Guide to the Laboratory Diagnosis and Control of Infection, 11th ed., E & S Livingstone Limitated, London.

    Google Scholar 

  41. David, M., Lubinsky-Mink, S., Ben-Zvi, A., Ulitzur, S., Kuhn, J. and Suissa, M. (1992) A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin. Plasmid, 28, 267–271.

    Article  PubMed  CAS  Google Scholar 

  42. Gormley, E.P. and Davies, J. (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol, 173, 6705–6708.

    PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Institute of Cell and Molecular Science, Barts and the London, Queen Mary’s School of Medicine and Dentistry, 4 Newark Street, Whitechapel, London, E1 2AT, UK

    Renan Goude PhD

  2. Centre for Infectious Disease, Institute of Cell and Molecular Science,Barts and The London, Queen Mary’s School of Medicine and Dentistry, London, UK

    Tanya Parish PhD

Authors
  1. Renan Goude PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tanya Parish PhD
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Renan Goude 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

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Goude, R., Parish, T. (2009). Electroporation of Mycobacteria. 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_13

Download citation

  • DOI: https://doi.org/10.1007/978-1-59745-207-6_13

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-58829-889-8

  • Online ISBN: 978-1-59745-207-6

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation