致病性分枝杆菌仍然是人类健康的重要威胁。相对于传统模式原核生物, 在分枝杆菌中进行基因敲除一直是一个难题。文章介绍了分枝杆菌中用于基因敲除的转运载体和序列特异性重组系统的研究进展, 并进一步介绍了新近发展的分枝杆菌重组工程在基因敲除中的应用。最后, 对分枝杆菌基因敲除系统的发展历程进行了回顾, 并对未来的发展方向进行了展望。
Pathogenic mycobacteria were and remain a heavy burden to public health. Unfortunately, genetic manipulation including knockout technologies of Mycobacterium is difficult compared with other traditional model organisms. To overcome this obstacle, achievements in Mycobacterium knockout technologies were summarized, including deliv-ery vector, sequence-specific recombination system, as well as the recently developed recombinogenic engineering and its application. The future for this tool innovation is also addressed.
[1] WHO. Worl Health Organization Report 2010, Global tuberculosis control. 2010.
[2] Machowski EE, Dawes S, Mizrahi V. TB tools to tell the tale-molecular genetic methods for mycobacterial research. Int J Biochem Cell Biol, 2005, 37(1): 54-68.
[3] Muttucumaru DG, Parish T. The molecular biology of recombination in Mycobacteria: what do we know and how can we use it? Curr Issues Mol Biol, 2004, 6(2): 145-157.
[4] Bardarov S, Kriakov J, Carriere C, Yu SW, Vaamonde C, McAdam RA, Bloom BR, Hatfull GF, Jacobs WR Jr. Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc Natl Acad Sci USA, 1997, 94(20): 10961-10966.
[5] Guilhot C, Gicquel B, Martín C. Temperature-sensitive mutants of the mycobacterium plasmid pAL5000. FEMS Microbiol Lett, 1992, 77(1-3): 181-186.
[6] Pelicic V, Reyrat JM, Gicquel B. Generation of unmarked directed mutations in mycobacteria, using sucrose counter- selectable suicide vectors. Mol Microbiol, 1996, 20(5): 919-925.
[7] Pelicic V, Reyrat JM, Gicquel B. Positive selection of allelic exchange mutants in Mycobacterium bovis BCG. FEMS Microbiol Lett, 1996, 144(2-3): 161-166.
[8] Pelicic V, Reyrat JM, Gicquel B. Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol Microbiol, 1998, 28(3): 413-420.
[9] Reyrat JM, Berthet FX, Gicquel B. The urease locus of Mycobacterium tuberculosis and its utilization for the demonstration of allelic exchange in Mycobacterium bovis bacillus Calmette-Guérin. Proc Natl Acad Sci USA, 1995, 92(19): 8768-8772.
[10] Parish T, Stoker NG. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology, 2000, 146(Pt 8): 1969-1975.
[11] Parish T, Gordhan BG, McAdam RA, Duncan K, Mizrahi V, Stoker NG. Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology, 1999, 145(Pt 12): 3497-3503.
[12] Balhana R, Stoker NG, Sikder MH, Chauviac FX, Kendall SL. Rapid construction of mycobacterial mutagenesis vectors using ligation-independent cloning. J Microbiol Methods, 2010, 83(1): 34-41.
[13] Hasan N, Koob M, Szybalski W. Escherichia coli genome targeting, I. Cre-lox-mediated in vitro generation of ori plasmids and their in vivo chromosomal integration and retrieval. Gene, 1994, 150(1): 51-56.
[14] Tsuda M. Use of a transposon-encoded site-specific resolution system for construction of large and defined deletion mutations in bacterial chromosome. Gene, 1998, 207(1): 33-41.
[15] Merlin C, McAteer S, Masters M. Tools for characterization of Escherichia coli genes of unknown function. J Bacteriol, 2002, 184(16): 4573-4581.
[16] Reed RR. Transposon-mediated site-specific recombination: a defined in vitro system. Cell, 1981, 25(3): 713-719.
[17] Stephan J, Stemmer V, Niederweis M. Consecutive gene deletions in Mycobacterium smegmatis using the yeast FLP recombinase. Gene, 2004, 343(1): 181-190.
[18] Song HH, Niederweis M. Functional expression of the Flp recombinase in Mycobacterium bovis BCG. Gene, 2007, 399(2): 112-119.
[19] Malaga W, Perez E, Guilhot C. Production of unmarked mutations in mycobacteria using site-specific recombination. FEMS Microbiol Lett, 2003, 219(2): 261-268.
[20] Bloor AE, Cranenburgh RM. An efficient method of selectable marker gene excision by Xer recombination for gene replacement in bacterial chromosomes. Appl Environ Microbiol, 2006, 72(4): 2520-2525.
[21] Hendrickson H, Lawrence JG. Mutational bias suggests that replication termination occurs near the dif site, not at Ter sites. Mol M