Abstract
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.
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References
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 6:145–158
Lamrabet O, Drancourt M (2012) Genetic engineering of Mycobacterium tuberculosis: a review. Tuberculosis (Edinb) 92:365–376
Machowski EE, Dawes S, Mizrahi V (2005) TB tools to tell the tale–molecular genetic methods for mycobacterial research. Int J Biochem Cell Biol 37:54–68
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 69:331–342
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 94:10961–10966
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 148:3007–3017
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 77–92
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 297–310
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 279–295
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 146:1969–1975
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 192:3645–3653
Cox JS, Chen B, McNeil M, Jacobs WR (1999) Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402:79–83
Domenech P, Reed MB (2009) Rapid and spontaneous loss of phthiocerol dimycocerosate (PDIM) from Mycobacterium tuberculosis grown in vitro: implications for virulence studies. Microbiology 155:3532–3543
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 107:9819–9824
Pashley CA, Parish T (2003) Efficient switching of mycobacteriophage L5‐based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol Lett 229:211–215
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 290:669–675
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 193:4487–4494
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 46:791–800
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 113:183–193
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 107:13093–13098
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 189:3655–3659
Gopinath K, Venclovas C, Ioerger TR, Sacchettini JC, McKinney JD, Mizrahi V, Warner DF (2013) A vitamin B12 transporter in Mycobacterium tuberculosis. Open Biol 3:120175
Sassetti CM, Boyd DH, Rubin EJ (2003) Genes required for mycobacterial growth defined by high density mutagenesis. Mol Microbiol 48:77–84
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 189:3721–3728
Parish T, Stoker NG (2000) glnE is an essential gene in Mycobacterium tuberculosis. J Bacteriol 182:5715–5720
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 191:985–995
Pena CE, Lee MH, Pedulla ML, Hatfull GF (1997) Characterization of the mycobacteriophage L5 attachment site, attP. J Mol Biol 266:76–92
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 135:941–955
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 71:6124–6131
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 6:91–96
Boshoff HI, Mizrahi V (2000) Expression of Mycobacterium smegmatis pyrazinamidase in Mycobacterium tuberculosis confers hypersensitivity to pyrazinamide and related amides. J Bacteriol 182:5479–5485
Smith AM, Klugman KP (1997) “Megaprimer” method of PCR-based mutagenesis: the concentration of megaprimer is a critical factor. Biotechniques 22:438–442
Hinds J, Mahenthiralingam E, Kempsell KE, Duncan K, Stokes RW, Parish T, Stoker NG (1999) Enhanced gene replacement in mycobacteria. Microbiology 145:519–527
Wards BJ, Collins DM (1996) Electroporation at elevated temperatures substantially improves transformation efficiency of slow‐growing mycobacteria. FEMS Microbiol Lett 145:101–105
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 190:3886–3895
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 66:3626–3634
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 337–353
Acknowledgments
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.).
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Gopinath, K., Warner, D.F., Mizrahi, V. (2015). Targeted Gene Knockout and Essentiality Testing by Homologous Recombination. In: Parish, T., Roberts, D. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 1285. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2450-9_8
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DOI: https://doi.org/10.1007/978-1-4939-2450-9_8
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