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.
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References
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.
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.
Paget, E. and Davies, J. (1996) Apramycin resistance as a selective marker for gene transfer in mycobacteria. J Bacteriol, 178, 6357–6360.
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.
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.
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.
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.
Pashley, C.A. and Parish, T. (2003) Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett, 229, 211–215.
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.
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.
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.
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.
Qin, M., Taniguchi, H. and Mizuguchi, Y. (1994) Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol, 176, 419–425.
Radford, A.J. and Hodgson, A.L. (1991) Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector. Plasmid, 25, 149–153.
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.
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.
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.
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.
Marklund, B.I., Speert, D.P. and Stokes, R.W. (1995) Gene replacement through homologous recombination in Mycobacterium intracellulare. J Bacteriol, 177, 6100–6105.
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.
Talaat, A.M. and Trucksis, M. (2000) Transformation and transposition of the genome of Mycobacterium marinum. Am J Vet Res, 61, 125–128.
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.
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.
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.
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.
Labidi, A., Dauguet, C., Goh, K.S. and David, H.L. (1984) Plasmid profiles of Mycobacterium fortuitum complex isolates. Curr Microbiol, 11, 235–240.
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.
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.
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.
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.
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.
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.
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.
Bottger, E.C. (1994) Resistance to drugs targeting protein synthesis in mycobacteria. Trends Microbiol, 2, 416–421.
Hatfull, G.F. (1993) Genetic transformation of mycobacteria. Trends Microbiol, 1,310–314.
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.
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.
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.
Hammes, W., Schleifer, K.H. and Kandler, O. (1973) Mode of action of glycine on the biosynthesis of peptidoglycan. J Bacteriol, 116, 1029–1053.
Cruickshank, R. (1965) Medical Microbiology: A Guide to the Laboratory Diagnosis and Control of Infection, 11th ed., E & S Livingstone Limitated, London.
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 origen. Plasmid, 28, 267–271.
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.
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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
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DOI: https://doi.org/10.1007/978-1-59745-207-6_13
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