Abstract
Site-specific recombinases such as the Saccharomyces cerevisiae Flp and the P1 phage Cre proteins have been increasingly used for the construction of unmarked deletions in bacteria. Both systems consist of an antibiotic resistance gene flanked by recognition sites in direct orientation and a curable plasmid for temporary expression of the respective recombinase gene. In this chapter, we describe strategies and methods of how to use sequence-specific recombination mediated by Flp and Cre to construct mutants of Mycobacterium smegmatis, Mycobacterium bovis BCG, and Mycobacterium tuberculosis.
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References
Pelicic, V., Jackson, M., Reyrat, J. M., Jacobs, W. R. Jr., Gicquel, B., and Guilhot, C. (1997) Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc Natl Acad Sci U S A 94, 10955–10960.
Bardarov, S., Bardarov S. Jr., Pavelka M. S. Jr., Sambandamurthy, V., Larsen, M., Tufariello, J., Chan, J., Hatfull, G., and Jacobs W. R. Jr. (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.
Machowski, E. E., Dawes, S., and Mizrahi, V. (2005) TB tools to tell the tale—molecular genetic methods for mycobacterial research. Int J Biochem Cell Biol 37, 54–68.
Stephan, J., Stemmer, V., and Niederweis, M. (2004) Consecutive gene deletions in Mycobacterium smegmatis using the yeast FLP recombinase. Gene 343, 181–190.
Kana, B. D., and Mizrahi, V. (2004) Molecular genetics of Mycobacterium tuberculosis in relation to the discovery of novel drugs and vaccines. Tuberculosis 84, 63–75.
Merlin, C., McAteer, S., and Masters, M. (2002) Tools for characterization of Escherichia coli genes of unknown function. J Bacteriol 184, 4573–4581.
Hasan, N., Koob, M., and Szybalski, W. (1994) Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori- plasmids and their in vivo chromosomal integration and retrieval. Gene 150, 51–56.
Tsuda, M. (1998) Use of a transposon-encoded site-specific resolution system for construction of large and defined deletion mutations in bacterial chromosome. Gene 207, 33–41.
Denome, S. A., Elf, P. K., Henderson, T. A., Nelson, D. E., and Young, K. D. (1999) Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis. J Bacteriol 181, 3981–3993.
Malaga, W., Perez, E., and Guilhot, C. (2003) Production of unmarked mutations in mycobacteria using site-specific recombination. FEMS Microbiol Lett 219, 261–268.
Stephan, J., Bender, J., Wolschendorf, F., Hoffmann, C., Roth, E., Mailander, C., Engelhardt, H., and Niederweis, M. (2005) The growth rate of Mycobacterium smegmatis depends on sufficient porin-mediated influx of nutrients. Mol Microbiol 58, 714–730.
Sander, P., Meier, A., and Boettger, E. C. (1995) rpsL+: a dominant selectable marker for gene replacement in mycobacteria. Mol Microbiol 16, 991–1000.
Guilhot, C., Gicquel, B. and Martin, C. (1992) Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol Lett 98, 181–186.
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This work was supported by grant AI063432 from the National Institutes of Health.
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Song, H., Wolschendorf, F., Niederweis, M. (2009). Construction of Unmarked Deletion Mutants in 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_19
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DOI: https://doi.org/10.1007/978-1-59745-207-6_19
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