Development of New Vector Systems as Genetic Tools Applicable to Mycobacteria

Mycobacteria에 적용 가능한 genetic tool로서의 새로운 vector system 개발

  • Received : 2013.01.23
  • Accepted : 2013.02.15
  • Published : 2013.02.28


The genus Mycobacterium includes crucial animal and human pathogens such as Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium bovis. Although it is important to understand the genetic basis for their virulence and persistence in host, genetic analysis in mycobacteria was hampered by a lack of sufficient genetic tools. Therefore, many functional vectors as molecular genetic tools have been designed for understanding mycobacterial biology, and the application of these tools to mycobacteria has accelerated the study of mechanisms involved in virulence and gene expression. To overcome the pre-existing problems in genetic manipulation of mycobacteria, this paper reports new vector systems as effective genetic tools in Mycobacterium smegmatis. Three vectors were developed; pKOTs is a suicide vector for mutagenesis containing a temperature-sensitive replication origin (TSRO) and the sacB gene encoding levansucrase as a counterselectable marker. pMV306lacZ is an integrative lacZ transcriptional fusion vector that can be inserted into chromosomal DNA by site-specific recombination. pTnMod-OKmTs is a minitransposon vector harboring the TSRO that can be used in random mutagenesis. It was demonstrated in this study that these vectors effectively worked in M. smegmatis. The vector systems reported here are expected to successfully applicable to future research of mycobacterial molecular genetics.


Integration vector;minitransposon;mutagenesis;mycobacteria;temperature-sensitive replication origin


  1. Azad, A. K., Sirakova, T. D., Rogers, L. M. and Kolattukudy, P. E. 1996. Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides. Proc Natl Acad Sci USA 93, 4787-4792.
  2. Balasubramanian, V., Pavelka, M. S., Jr., Bardarov, S. S., Martin, J., Weisbrod, T. R., McAdam, R. A., Bloom, B. R. and Jacobs, W. R., Jr. 1996. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J Bacteriol 178, 273-279.
  3. 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.
  4. Brown, A. K., Bhatt, A., Singh, A., Saparia, E., Evans, A. F. and Besra, G. S. 2007. Identification of the dehydratase component of the mycobacterial mycolic acid-synthesizing fatty acid synthase-II complex. Microbiology 153, 4166-4173.
  5. De Lorenzo, V., Herrero, M., Jakubzik, U. and Timmis, K. N. 1990. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol 172, 6568-6572.
  6. Dennis, J. J. and Zylstra, G. J. 1998. Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial genomes. Appl Environ Microbiol 64, 2710-2715.
  7. Fellay, R., Krisch, H. M., Prentki, P. and Frey, J. 1989. Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria. Gene 76, 215-226.
  8. Feng, Z., Caceres, N. E., Sarath, G. and Barletta, R. G. 2002. Mycobacterium smegmatis L-alanine dehydrogenase (Ald) is required for proficient utilization of alanine as a sole nitrogen source and sustained anaerobic growth. J Bacteriol 184, 5001-5010.
  9. Guilhot, C., Gicquel, B. and Martin, C. 1992. Temperaturesensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol Lett 77, 181-186.
  10. Guilhot, C., Otal, I., Van Rompaey, I., Martin, C. and Gicquel, B. 1994. Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol 176, 535-539.
  11. Gupta, A., Kaul, A., Tsolaki, A. G., Kishore, U. and Bhakta, S. 2012. Mycobacterium tuberculosis: immune evasion, latency and reactivation. Immunobiology 217, 363-374.
  12. Hatfull, G. F. and Sarkis, G. J. 1993. DNA sequence, structure and gene expression of mycobacteriophage L5: a phage system for mycobacterial genetics. Mol Microbiol 7, 395-405.
  13. Howard, N. S., Gomez, J. E., Ko, C. and Bishai, W. R. 1995. Color selection with a hygromycin-resistance-based Escherichia coli-mycobacterial shuttle vector. Gene 166, 181-182.
  14. 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.
  15. Hutter, B. and Dick, T. 1998. Increased alanine dehydrogenase activity during dormancy in Mycobacterium smegmatis. FEMS Microbiol Lett 167, 7-11.
  16. Jacobs, W. R., Jr., Tuckman, M. and Bloom, B. R. 1987. Introduction of foreign DNA into mycobacteria using a shuttle phasmid. Nature 327, 532-535.
  17. Jessee, J. 1986. New subcloning efficiency. Competent cells:>1${\times}$106 transformants/${\mu}g$. Focus 8:9.
  18. Kong, D. and Kunimoto, D. Y. 1995. Secretion of human interleukin 2 by recombinant Mycobacterium bovis BCG. Infect Immun 63, 799-803.
  19. Lee, M. H. and Hatfull, G. F. 1993. Mycobacteriophage L5 integrase-mediated site-specific integration in vitro. J Bacteriol 175, 6836-6841.
  20. 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 USA 88, 3111-3115.
  21. McFadden, J. 1996. Recombination in mycobacteria. Mol Microbiol 21, 205-211.
  22. Miller, J. H. 1972. Experiments in Molecular Genetics, Cold Spring Harbor, NY, USA
  23. Oh, J. I., Park, S. J., Shin, S. J., Ko, I. J., Han, S. J., Park, S. W., Song, T. and Kim, Y. M. 2010. Identification of transand cis-control elements involved in regulation of the carbon monoxide dehydrogenase genes in Mycobacterium sp. strain JC1 DSM 3803. J Bacteriol 192, 3925-3933.
  24. Otero, J., Jacobs, W. R., Jr. and Glickman, M. S. 2003. Efficient allelic exchange and transposon mutagenesis in Mycobacterium avium by specialized transduction. Appl Environ Microbiol 69, 5039-5044.
  25. Parish, T., Stoker, N. G. 1999. Mycobacteria protocols, Humana Press, NJ, USA.
  26. Parish, T. and Stoker, N. G. 2000. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146, 1969-1975.
  27. Pashley, C. A., Parish, T., McAdam, R. A., Duncan, K. and Stoker, N. G. 2003. Gene replacement in mycobacteria by using incompatible plasmids. Appl Environ Microbiol 69, 517-523.
  28. Pedulla, M. L., Lee, M. H., Lever, D. C. and Hatfull, G. F. 1996. A novel host factor for integration of mycobacteriophage L5. Proc Natl Acad Sci USA 93, 15411-15416.
  29. 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 USA 94, 10955-10960.
  30. Pelicic, V., Reyrat, J. M. and Gicquel, B. 1996. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 178, 1197-1199.
  31. Pelicic, V., Reyrat, J. M. and Gicquel, B. 1996. Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol Microbiol 20, 919-925.
  32. Pelicic, V., Reyrat, J. M. and Gicquel, B. 1998. Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol Microbiol 28, 413-420.
  33. Sambrook, J. and Russell, D. W. 2001. Molecular Cloning: A Laboratory Manual. 3rd ed., Cold Spring Harbor, NY, USA.
  34. Sherman, D. R., Voskuil, M., Schnappinger, D., Liao, R., Harrell, M. I. and Schoolnik, G. K. 2001. Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding ${\alpha}$-crystallin. Proc Natl Acad Sci USA 98, 7534-7539.
  35. 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 USA 85, 6987-6991.
  36. 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.
  37. 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. , Snapper, S. B., Barletta, R. G. Jocobs, W. R, Jr. and Bloom, B. R. 1991. New use of BCG for recombinant vaccines. Nature 351, 456-460.

Cited by

  1. Regulation of the ahpC Gene Encoding Alkyl Hydroperoxide Reductase in Mycobacterium smegmatis vol.9, pp.11, 2014,
  2. under Respiration-Inhibitory Conditions vol.200, pp.14, 2018,


Supported by : 부산대학교