생명과학회지 (Journal of Life Science)

  • 제23권2호
  • /
  • Pages.290-298
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  • 2013
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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DOI QR코드

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Mycobacteria에 적용 가능한 genetic tool로서의 새로운 vector system 개발

Development of New Vector Systems as Genetic Tools Applicable to Mycobacteria

  • 정지아 (부산대학교 미생물학과) ;
  • 이하나 (부산대학교 미생물학과) ;
  • 고인정 (카이스트부설 한국과학영재학교) ;
  • 오정일 (부산대학교 미생물학과)
  • Jeong, Ji-A (Department of Microbiology, Pusan National University) ;
  • Lee, Ha-Na (Department of Microbiology, Pusan National University) ;
  • Ko, In-Jeong (Korea Science Academy of KAIST) ;
  • Oh, Jeong-Il (Department of Microbiology, Pusan National University)
  • 투고 : 2013.01.23
  • 심사 : 2013.02.15
  • 발행 : 2013.02.28
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초록

Mycobacterium 속은 Mycobacterium tuberculosis, Mycobacterium leprae, Mycobacterium bovis와 같은 동물과 인체에 병원성을 나타내는 세균 종을 다수 포함하고 있다. 이들의 숙주에서의 생존과 병원성에 관한 유전학적 정보를 확보하는 것은 매우 중요하지만, 효과적인 유전학적 도구가 부족하였기 때문에 이들에 관한 연구가 미비하였다. 따라서 mycobacteria의 연구를 위한 분자생물학적 실험 도구로서 다양한 기능성 vector들이 고안되었고, 이러한 기능성 vector의 개발은 실질적으로 mycobacteria에서의 연구 효과를 증진시켰다. 본 연구에서는 Mycobacterium smegmatis에 적용 가능하고 기존에 제시되었던 mycobacteria 연구에 있어서의 한계점을 극복하기 위한 노력의 일환으로, 기능성 vector인 temperature-sensitive replication origin (TSRO)과 counterselectable marker로 levansucrase를 암호화하는 sacB 유전자를 포함하는 suicide vector pKOTs, chromosomal DNA로 site-specific recombination을 통해 삽입되는 lacZ transcriptional fusion vector pMV306lacZ, 그리고 TSRO를 가지는 minitransposon vector pTnMod-OKmTs를 개발하였다. 이 vector들은 실질적으로 M. smegmatis에서 효과적으로 작동하는 것이 확인되었으며 목적으로 하는 실험 결과 도출 가능성 또한 보여주었다. 따라서 이들 vector는 앞으로의 mycobacteria에 대한 효과적인 연구 기반이 될 것으로 기대된다.

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.

키워드

참고문헌

  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. https://doi.org/10.1073/pnas.93.10.4787
  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. https://doi.org/10.1099/mic.0.2007/012419-0
  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. https://doi.org/10.1016/0378-1119(89)90162-5
  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. https://doi.org/10.1128/JB.184.18.5001-5010.2002
  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. https://doi.org/10.1016/j.imbio.2011.07.008
  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. https://doi.org/10.1111/j.1365-2958.1993.tb01131.x
  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. https://doi.org/10.1016/0378-1119(95)00597-X
  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. https://doi.org/10.1111/j.1574-6968.1998.tb13200.x
  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. https://doi.org/10.1038/327532a0
  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. https://doi.org/10.1073/pnas.88.8.3111
  21. McFadden, J. 1996. Recombination in mycobacteria. Mol Microbiol 21, 205-211. https://doi.org/10.1046/j.1365-2958.1996.6271345.x
  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. https://doi.org/10.1128/JB.00286-10
  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. https://doi.org/10.1128/AEM.69.9.5039-5044.2003
  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. https://doi.org/10.1128/AEM.69.1.517-523.2003
  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. https://doi.org/10.1073/pnas.93.26.15411
  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. https://doi.org/10.1073/pnas.94.20.10955
  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. https://doi.org/10.1111/j.1365-2958.1996.tb02533.x
  32. Pelicic, V., Reyrat, J. M. and Gicquel, B. 1998. Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol Microbiol 28, 413-420. https://doi.org/10.1046/j.1365-2958.1998.00807.x
  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. https://doi.org/10.1073/pnas.121172498
  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. https://doi.org/10.1073/pnas.85.18.6987
  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. https://doi.org/10.1111/j.1365-2958.1990.tb02040.x
  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. https://doi.org/10.1038/351456a0

피인용 문헌

  1. Regulation of the ahpC Gene Encoding Alkyl Hydroperoxide Reductase in Mycobacterium smegmatis vol.9, pp.11, 2014, https://doi.org/10.1371/journal.pone.0111680
  2. under Respiration-Inhibitory Conditions vol.200, pp.14, 2018, https://doi.org/10.1128/JB.00152-18