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Electroporation of Mycobacteria

  • Renan Goude
  • David M. Roberts
  • Tanya ParishEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1285)

Abstract

High-efficiency transformation of DNA is integral to the study of mycobacteria, allowing genetic manipulation. Electroporation is the most widely used method for introducing DNA into mycobacterial strains. Many parameters contribute to high-efficiency transformation; these include the species per strain, the transforming DNA, the selectable marker, the growth medium additives, and the conditions of electroporation. In this chapter we provide an optimized method for the transformation of representative slow- and fast-growing species of mycobacteria—Mycobacterium tuberculosis and M. smegmatis, respectively.

Key words

Transforming DNA Selectable marker Electrocompetent cells Transformants 

References

  1. 1.
    Jacobs WR Jr, Kalpana GV, Cirillo JD, Pascopella L, Snapper SB, Udani RA, Jones W, Barletta RG, Bloom BR (1991) Genetic systems for mycobacteria. Methods Enzymol 204:537–555CrossRefPubMedGoogle Scholar
  2. 2.
    Kalpana GV, Bloom BR, Jacobs WR Jr (1991) Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc Natl Acad Sci U S A 88(12):5433–5437CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Paget E, Davies J (1996) Apramycin resistance as a selective marker for gene transfer in mycobacteria. J Bacteriol 178(21):6357–6360CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Consaul SA, Pavelka MS 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(2):297–301CrossRefPubMedGoogle Scholar
  5. 5.
    Parish T, Gordhan BG, McAdam RA, Duncan K, Mizrahi V, Stoker NG (1999) Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology 145(Pt 12):3497–3503CrossRefPubMedGoogle Scholar
  6. 6.
    Ranes MG, Rauzier J, Lagranderie M, Gheorghiu M, Gicquel B (1990) Functional analysis of pAL5000, a plasmid from Mycobacterium fortuitum: construction of a “mini” mycobacterium-Escherichia coli shuttle vector. J Bacteriol 172(5):2793–2797CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Yuan Y, Crane DD, Simpson RM, Zhu YQ, Hickey MJ, Sherman DR, Barry CE 3rd (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(16):9578–9583CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Dellagostin OA, Wall S, Norman E, O’Shaughnessy T, Dale JW, McFadden J (1993) Construction and use of integrative vectors to express foreign genes in mycobacteria. Mol Microbiol 10(5):983–993CrossRefPubMedGoogle Scholar
  9. 9.
    Goto Y, Taniguchi H, Udou T, Mizuguchi Y, Tokunaga T (1991) Development of a new host vector system in mycobacteria. FEMS Microbiol Lett 67(3):277–282CrossRefPubMedGoogle Scholar
  10. 10.
    Matsuo K, Yamaguchi R, Yamazaki A, Tasaka H, Terasaka K, Totsuka M, Kobayashi K, Yukitake H, Yamada T (1990) Establishment of a foreign antigen secretion system in mycobacteria. Infect Immun 58(12):4049–4054PubMedPubMedCentralGoogle Scholar
  11. 11.
    Qin M, Taniguchi H, Mizuguchi Y (1994) Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol 176(2):419–425CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Radford AJ, Hodgson AL (1991) Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector. Plasmid 25(2):149–153CrossRefPubMedGoogle Scholar
  13. 13.
    Snapper SB, Lugosi L, Jekkel A, Melton RE, Kieser T, Bloom BR, Jacobs WR Jr (1988) Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A 85(18):6987–6991CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Garbe TR, Barathi J, Barnini S, Zhang Y, Abou-Zeid C, Tang D, Mukherjee R, Young DB (1994) Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology 140:133–138CrossRefPubMedGoogle Scholar
  15. 15.
    Wards BJ, Collins DM (1996) Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Microbiol Lett 145(1):101–105CrossRefPubMedGoogle Scholar
  16. 16.
    Hermans J, Martin C, Huijberts GN, Goosen T, de Bont JA (1991) Transformation of Mycobacterium aurum and Mycobacterium smegmatis with the broad host-range gram-negative cosmid vector pJRD215. Mol Microbiol 5(6):1561–1566CrossRefPubMedGoogle Scholar
  17. 17.
    Houssaini-Iraqui M, Lazraq MH, Clavel-Seres S, Rastogi N, David HL (1992) Cloning and expression of Mycobacterium aurum carotenogenesis genes in Mycobacterium smegmatis. FEMS Microbiol Lett 69(3):239–244CrossRefPubMedGoogle Scholar
  18. 18.
    Marklund BI, Speert DP, Stokes RW (1995) Gene replacement through homologous recombination in Mycobacterium intracellulare. J Bacteriol 177(21):6100–6105CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hermans J, Suy IML, De Bont JAM (1993) Transformation of Gram-positive microorganisms with the Gram-negative broad-host-range cosmid vector pJRD215. FEMS Microbiol Lett 108:201–204CrossRefGoogle Scholar
  20. 20.
    Talaat AM, Trucksis M (2000) Transformation and transposition of the genome of Mycobacterium marinum. Am J Vet Res 61(2):125–128CrossRefPubMedGoogle Scholar
  21. 21.
    Beggs ML, Crawford JT, Eisenach KD (1995) Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7. J Bacteriol 177(17):4836–4840CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Foley-Thomas EM, Whipple DL, Bermudez LE, Barletta RG (1995) Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis. Microbiology 141:1173–1181CrossRefPubMedGoogle Scholar
  23. 23.
    Lee SH, Cheung M, Irani V, Carroll JD, Inamine JM, Howe WR, Maslow JN (2002) Optimization of electroporation conditions for Mycobacterium avium. Tuberculosis (Edinb) 82(4–5):167–174CrossRefGoogle Scholar
  24. 24.
    Snapper SB, Melton RE, Mustafa S, Kieser T, Jacobs WR Jr (1990) Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4(11):1911–1919CrossRefPubMedGoogle Scholar
  25. 25.
    Labidi A, Dauguet C, Goh KS, David HL (1984) Plasmid profiles of Mycobacterium fortuitum complex isolates. Curr Microbiol 11:235–240CrossRefGoogle Scholar
  26. 26.
    Stover CK, de la Cruz VF, Fuerst TR, Burlein JE, Benson LA, Bennett LT, Bansal GP, Young JF, Lee MH, Hatfull GF et al (1991) New use of BCG for recombinant vaccines. Nature 351(6326):456–460CrossRefPubMedGoogle Scholar
  27. 27.
    Bachrach G, Colston MJ, Bercovier H, Bar-Nir D, Anderson C, Papavinasasundaram KG (2000) A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146(Pt 2):297–303CrossRefPubMedGoogle Scholar
  28. 28.
    Gavigan JA, Ainsa JA, Perez E, Otal I, Martin C (1997) Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum. J Bacteriol 179(13):4115–4122CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Lee MH, Pascopella L, Jacobs WR Jr, Hatfull GF (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(8):3111–3115CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Anes E, Portugal I, Moniz-Pereira J (1992) Insertion into the Mycobacterium smegmatis genome of the aph gene through lysogenization with the temperate mycobacteriophage Ms6. FEMS Microbiol Lett 74:21–25CrossRefPubMedGoogle Scholar
  31. 31.
    England PM, Mazodier P, Mediola MV, Gicquel B, Smokvina T, Thompson CJ, Davies J (1991) Site-specific integration of the Streptomyces plasmid pSAM2 in Mycobacterium smegmatis. Mol Microbiol 5:2499–2502CrossRefGoogle Scholar
  32. 32.
    Bottger EC (1994) Resistance to drugs targeting protein synthesis in mycobacteria. Trends Microbiol 2(10):416–421CrossRefPubMedGoogle Scholar
  33. 33.
    Hatfull GF (1993) Genetic transformation of mycobacteria. Trends Microbiol 1(8):310–314CrossRefPubMedGoogle Scholar
  34. 34.
    Kana BD, Mizrahi V (2004) Molecular genetics of Mycobacterium tuberculosis in relation to the discovery of novel drugs and vaccines. Tuberculosis (Edinb) 84(1–2):63–75CrossRefGoogle Scholar
  35. 35.
    Aldovini A, Husson RN, Young RA (1993) The uraA locus and homologous recombination in Mycobacterium bovis BCG. J Bacteriol 175(22):7282–7289CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Husson RN, James BE, Young RA (1990) Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol 172(2):519–524CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Hermans J, Boschloo JG, de Bont JAM (1990) Transformation of Mycobacterium aurum by electroporation: the use of glycine, lysozyme and isonicotinic acid hydrazide in enhancing transformation efficiency. FEMS Microbiol Lett 72:221–224CrossRefGoogle Scholar
  38. 38.
    Hammes W, Schleifer KH, Kandler O (1973) Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol 116(2):1029–1053PubMedPubMedCentralGoogle Scholar
  39. 39.
    Cruickshank R (1965) Medical microbiology: a guide to the laboratory diagnosis and control of infection, 11th edn. E & S Livingstone Limited, LondonGoogle Scholar
  40. 40.
    David M, Lubinsky-Mink S, Ben-Zvi A, Ulitzur S, Kuhn J, Suissa M (1992) A stable Escherichia coli-Mycobacterium smegmatis plasmid shuttle vector containing the mycobacteriophage D29 origin. Plasmid 28(3):267–271CrossRefPubMedGoogle Scholar
  41. 41.
    Gormley EP, Davies J (1991) Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol 173(21):6705–6708CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Pashley CA, Parish T (2003) Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett 229(2):211–215CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.University of Rennes, Campus scientifique de BeaulieuRennesFrance
  2. 2.TB Discovery Research GroupInfectious Disease Research InstituteSeattleUSA

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