20. In the presence of zeocin during growth, pKM461 (Kan
R
) is
incompatible with the presence of pKM512 (Zeo
R
) and is lost,
resulting in cells that are senstive to kanamycin. In the following steps, the induction of Integrase and gp47 with Atc promotes excision of the integrating plasmid (attL x
attR ! attP) and loss of hygomycin resistance. Finally, plating
on 7H10 plates with 3% sucrose results in the selection of cells
cured of pKM512, resulting in zeocin sensitivity.
Acknowledgments
I would like to thank Kadamba Papavinasasundaram, Rujapak Sutiwisesak, Eun-Ik Koh, and Lisa Lojek for sharing their results of
oligo-mediated recombineering in M. tuberculosis. I would also like
to thank Christopher Sassetti for material support and helpful
suggestions.
References
1. Azad AK, Sirakova TD, Rogers LM et al
(1996) Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis
BCG produces a mutant that lacks mycosides.
Proc Natl Acad Sci U S A 93(10):4787–4792
2. Pavelka MS Jr (2008) Allelic exchange of
unmarked mutations in Mycobacterium tuberculosis. Methods Mol Biol 435:191–201.
https://doi.org/10.1007/978-1-59745-2328_14
3. Pavelka MS Jr, Jacobs WR Jr (1999) Comparison of the construction of unmarked deletion
mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guerin, and
Mycobacterium tuberculosis H37Rv by allelic
exchange. J Bacteriol 181(16):4780–4789
4. Pelicic V, Reyrat JM, Gicquel B (1996) Positive
selection of allelic exchange mutants in Mycobacterium bovis BCG. FEMS Microbiol Lett
144(2-3):161–166
5. Pelicic V, Reyrat JM, Gicquel B (1996) Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable
suicide vectors. Mol Microbiol 20(5):919–925
6. Reyrat JM, Berthet FX, 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(19):8768–8772
7. Bardarov S, Bardarov S Jr, Pavelka MS Jr et al
(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(Pt 10):3007–3017
8. Jain P, Hsu T, Arai M et al (2014) Specialized
transduction designed for precise highthroughput unmarked deletions in Mycobacterium tuberculosis. mBio 5(3):e01245–e01214.
https://doi.org/10.1128/mBio.01245-14
9. van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat
Methods 4(2):147–152
10. Marinelli LJ, Hatfull GF, Piuri M (2012)
Recombineering: a powerful tool for modification of bacteriophage genomes. Bacteriophage
2(1):5–14.
https://doi.org/10.4161/bact.
18778
11. van Kessel JC, Marinelli LJ, Hatfull GF (2008)
Recombineering mycobacteria and their
phages. Nat Rev Microbiol 6(11):851–857.
https://doi.org/10.1038/nrmicro2014
12. Marinelli LJ, Piuri M, Swigonova Z et al
(2008) BRED: a simple and powerful tool for
constructing mutant and recombinant bacteriophage genomes. PLoS One 3(12):e3957.
https://doi.org/10.1371/journal.pone.
0003957
13. Murphy KC, Papavinasasundaram K, Sassetti
CM (2015) Mycobacterial recombineering.
Methods Mol Biol 1285:177–199. https://
doi.org/10.1007/978-1-4939-2450-9_10
14. Murphy KC, Nelson SJ, Nambi S et al (2018)
ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes. mBio 9(6):
320
Kenan C. Murphy
R
) is
incompatible with the presence of pKM512 (Zeo
R
) and is lost,
resulting in cells that are senstive to kanamycin. In the following steps, the induction of Integrase and gp47 with Atc promotes excision of the integrating plasmid (attL x
attR ! attP) and loss of hygomycin resistance. Finally, plating
on 7H10 plates with 3% sucrose results in the selection of cells
cured of pKM512, resulting in zeocin sensitivity.
Acknowledgments
I would like to thank Kadamba Papavinasasundaram, Rujapak Sutiwisesak, Eun-Ik Koh, and Lisa Lojek for sharing their results of
oligo-mediated recombineering in M. tuberculosis. I would also like
to thank Christopher Sassetti for material support and helpful
suggestions.
References
1. Azad AK, Sirakova TD, Rogers LM et al
(1996) Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis
BCG produces a mutant that lacks mycosides.
Proc Natl Acad Sci U S A 93(10):4787–4792
2. Pavelka MS Jr (2008) Allelic exchange of
unmarked mutations in Mycobacterium tuberculosis. Methods Mol Biol 435:191–201.
https://doi.org/10.1007/978-1-59745-2328_14
3. Pavelka MS Jr, Jacobs WR Jr (1999) Comparison of the construction of unmarked deletion
mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guerin, and
Mycobacterium tuberculosis H37Rv by allelic
exchange. J Bacteriol 181(16):4780–4789
4. Pelicic V, Reyrat JM, Gicquel B (1996) Positive
selection of allelic exchange mutants in Mycobacterium bovis BCG. FEMS Microbiol Lett
144(2-3):161–166
5. Pelicic V, Reyrat JM, Gicquel B (1996) Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable
suicide vectors. Mol Microbiol 20(5):919–925
6. Reyrat JM, Berthet FX, 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(19):8768–8772
7. Bardarov S, Bardarov S Jr, Pavelka MS Jr et al
(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(Pt 10):3007–3017
8. Jain P, Hsu T, Arai M et al (2014) Specialized
transduction designed for precise highthroughput unmarked deletions in Mycobacterium tuberculosis. mBio 5(3):e01245–e01214.
https://doi.org/10.1128/mBio.01245-14
9. van Kessel JC, Hatfull GF (2007) Recombineering in Mycobacterium tuberculosis. Nat
Methods 4(2):147–152
10. Marinelli LJ, Hatfull GF, Piuri M (2012)
Recombineering: a powerful tool for modification of bacteriophage genomes. Bacteriophage
2(1):5–14.
https://doi.org/10.4161/bact.
18778
11. van Kessel JC, Marinelli LJ, Hatfull GF (2008)
Recombineering mycobacteria and their
phages. Nat Rev Microbiol 6(11):851–857.
https://doi.org/10.1038/nrmicro2014
12. Marinelli LJ, Piuri M, Swigonova Z et al
(2008) BRED: a simple and powerful tool for
constructing mutant and recombinant bacteriophage genomes. PLoS One 3(12):e3957.
https://doi.org/10.1371/journal.pone.
0003957
13. Murphy KC, Papavinasasundaram K, Sassetti
CM (2015) Mycobacterial recombineering.
Methods Mol Biol 1285:177–199. https://
doi.org/10.1007/978-1-4939-2450-9_10
14. Murphy KC, Nelson SJ, Nambi S et al (2018)
ORBIT: a new paradigm for genetic engineering of mycobacterial chromosomes. mBio 9(6):
320
Kenan C. Murphy