Chapter 12 : Gene Replacement Systems

MyBook is a cheap paperback edition of the original book and will be sold at uniform, low price.

Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase

Buy this Chapter
Digital (?) $15.00

Preview this chapter:
Zoom in

Gene Replacement Systems, Page 1 of 2

| /docserver/preview/fulltext/10.1128/9781555817657/9781555812959_Chap12-1.gif /docserver/preview/fulltext/10.1128/9781555817657/9781555812959_Chap12-2.gif


With the completion of the genome sequence, the focus of research has turned to functional characterization of genes, and, in particular, identification of virulence factors. The inability to identify double-crossover events was a major impediment in the study of pathogenicity. The first mutant of the complex made through allelic replacement was a BCG mutant, created using the gene as a target. A major factor in the small number of mutants obtained using suicide plasmids was the efficiency of transformation. This difficulty is avoided by using replicating plasmids, although there is the new problem of ensuring plasmid loss. The first counterselectable marker to be described in mycobacteria was streptomycin sensitivity. This system takes advantage of the fact that the S12 ribosomal protein is the target of streptomycin. A major drawback when using replicating plasmids was that it was necessary to isolate plasmid free cells. To induce efficient plasmid loss at will, a thermosensitive replicon was isolated. Marked mutants, in which an antibiotic resistance gene is used to interrupt the gene of interest, are the most straightforward to make. Even when the gene studied is essential, studying the phenotype of a conditional mutant can be useful because it can help to unravel the function of the gene. In conclusion, many genetic tools are now available for studying mycobacteria. It is possible to select directly for allelic exchange in a single-step strategy or to construct an unmarked mutation in a two-step strategy.

Citation: Stoker N, Sander P, Reyrat J. 2005. Gene Replacement Systems, p 183-190. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch12

Key Concept Ranking

Bacterial Genetics
Deletion Mutation
Periplasmic Space
Plasma Membrane
Periplasmic Space
Plasma Membrane
Mycobacterium tuberculosis
Highlighted Text: Show | Hide
Loading full text...

Full text loading...


Image of Figure 1
Figure 1

Two-step strategy for allelic replacement. Positive selection of allelic exchange mutants in a two-step selection strategy, using a counterselectable marker, is shown. CSM, counterselectable marker; SM, selectable marker; wt, wild-type allele; mut, mutated allele. Adapted from reference 54 with permission.

Citation: Stoker N, Sander P, Reyrat J. 2005. Gene Replacement Systems, p 183-190. In Cole S, Eisenach K, McMurray D, Jacobs, Jr. W (ed), Tuberculosis and the Tubercle Bacillus. ASM Press, Washington, DC. doi: 10.1128/9781555817657.ch12
Permissions and Reprints Request Permissions
Download as Powerpoint


1. Aldovini, A.,, R. N. Husson,, and R. A. Young. 1993. The uraA locus and homologous recombination in Mycobacterium bovis BCG. J. Bacteriol. 175:72827289.
2. Azad, A. K.,, T. D. Sirakova,, N. D. Fernandes,, and P. E. Kolattukudy. 1997. Gene knockout reveals a novel gene cluster for the synthesis of a class of cell wall lipids unique to pathogenic mycobacteria. J. Biol. Chem. 272:1674116745.
3. Azad, A. K.,, T. D. Sirakova,, L. M. Rogers,, and P. E. Kolattukudy. 1996. Targeted replacement of the mycocerosic acid synthase gene in Mycobacterium bovis BCG produces a mutant that lacks mycosides. Proc. Natl. Acad. Sci. USA 93:47874792.
4. Balasubramanian, V.,, M. S. Pavelka, Jr.,, S. S. Bardarov,, J. Martin,, T. R. Weisbrod,, R. A. McAdam,, B. R. Bloom,, and W. R. Jacobs, Jr. 1996. Allelic exchange in Mycobacterium tuberculosis with long linear recombination substrates. J. Bacteriol. 178:273279.
5. Bardarov, S.,, S. Bardarov, Jr.,, M. S. Pavelka, Jr.,, V. Sambandamurthy,, M. Larsen,, J. Tufariello,, J. Chan,, G. Hatfull,, and W. R. Jacobs, 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 148:30073017.
6. Bardarov, S.,, J. Kriakov,, C. Carriere,, S. Yu,, C. Vaamonde,, R. A. McAdam,, B. R. Bloom,, G. F. Hatfull,, and W. R. Jacobs, Jr. 1997. Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 94:1096110966.
7. Baulard, A.,, L. Kremer,, and C. Locht. 1996. Efficient homologous recombination in fast-growing and slow-growing mycobacteria. J. Bacteriol. 178:30913098.
8. Berthet, F. X.,, M. Lagranderie,, P. Gounon,, C. Laurent-Winter,, D. Ensergueix,, P. Chavarot,, F. Thouron,, E. Maranghi,, V. Pelicic,, D. Portnoi,, G. Marchal,, and B. Gicquel. 1998. Attenuation of virulence by disruption of the Mycobacterium tuberculosis erp gene. Science 282:759762.
9. Boeke, J. D.,, F. LaCroute,, and G. R. Fink. 1984. A positive selection for mutants lacking orotidine-5_-phosphate decarboxylase activity in yeast: 5-fluoroorotic acid resistance. Mol. Gen. Genet. 197:345346.
10. Camacho, L. R.,, D. Ensergueix,, E. Perez,, B. Gicquel,, and C. Guilhot. 1999. Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis. Mol. Microbiol. 34:257267.
11. Camus, J. C.,, M. J. Pryor,, C. Medigue,, and S. T. Cole. 2002. Re-annotation of the genome sequence of Mycobacterium tuberculosis H37Rv. Microbiology 148:29672973.
12. Casali, N.,, and S. Ehrt,. 2001. Plasmid vectors, p. 117. In T. Parish, and N. G. Stoker (ed.), Mycobacterium tuberculosis Protocols. Humana Press, Inc., Totowa, N.J..
13. Clark-Curtiss, J. E.,, and S. E. Haydel. 2003. Molecular genetics of Mycobacterium tuberculosis pathogenesis. Annu. Rev. Microbiol. 57:517549.
14. Cole, S. T.,, R. Brosch,, J. Parkhill,, T. Garnier,, C. Churcher,, D. Harris,, S. V. Gordon,, K. Eiglmeier,, S. Gas,, C. E. Barry, III, F. Tekaia, K. Badcock, D. Basham, D. Brown, T. Chillingworth, R. Connor, R. Davies, K. Devlin, T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby, K. Jagels, and B. G. Barrell. 1998. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393:537544.
15. Cox, J. S.,, B. Chen,, M. McNeil,, and W. R. Jacobs, Jr. 1999. Complex lipid determines tissue-specific replication of Mycobacterium tuberculosis in mice. Nature 402:7983.
16. Curcic, R.,, S. Dhandayuthapani,, and V. Deretic. 1994. Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis. Mol. Microbiol. 13:10571064.
17. Davis, E. O.,, P. J. Jenner,, P. C. Brooks,, M. J. Colston,, and S. G. Sedgwick. 1992. Protein splicing in the maturation of M. tuberculosis recA protein: a mechanism for tolerating a novel class of intervening sequence. Cell 71:201210.
18. Frischkorn, K.,, P. Sander,, M. Scholz,, K. Teschner,, T. Prammananan,, and E. C. Bottger. 1998. Investigation of mycobacterial recA function: protein introns in the RecA of pathogenic mycobacteria do not affect competency for homologous recombination. Mol. Microbiol. 29:12031214.
19. Glickman, M. S.,, and W. R. Jacobs, Jr. 2001. Microbial pathogenesis of Mycobacterium tuberculosis: dawn of a discipline. Cell 104:477485.
20. Guilhot, C.,, B. Gicquel,, and C. Martin. 1992. Temperaturesensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol. Lett. 77:181186.
21. Hinds, J.,, E. Mahenthiralingam,, K. E. Kempsell,, K. Duncan,, R. W. Stokes,, T. Parish,, and N. G. Stoker. 1999. Enhanced gene replacement in mycobacteria. Microbiology 145:519527.
22. Husson, R. N.,, B. E. James,, and R. A. Young. 1990. Gene replacement and expression of foreign DNA in mycobacteria. J. Bacteriol. 172:519524.
23. Jackson, M.,, D. C. Crick,, and P. J. Brennan. 2000. Phosphatidylinositol is an essential phospholipid of mycobacteria. J. Biol. Chem. 275:3009230099.
24. Jacobs, W. R., Jr., 2000. Mycobacterium tuberculosis: a once genetically intractable organism, p. 116. In G. F. Hatfull, and W. R. Jacobs, Jr. (ed.), Molecular Genetics of Mycobacteria. ASM Press, Washington, D.C..
25. Jacobs, W. R., Jr.,, G. V. Kalpana,, J. D. Cirillo,, L. Pascopella,, S. B. Snapper,, R. A. Udani,, W. Jones,, R. G. Barletta,, and B. R. Bloom. 1991. Genetic systems for mycobacteria. Methods Enzymol. 204:537555.
26. Jager, W.,, A. Schafer,, A. Puhler,, G. Labes,, and W. Wohlleben. 1992. Expression of the Bacillus subtilis sacB gene leads to sucrose sensitivity in the gram-positive bacterium Corynebacterium glutamicum but not in Streptomyces lividans. J. Bacteriol. 174:54625465.
27. Kalpana, G. V.,, B. R. Bloom,, and W. R. Jacobs, Jr. 1991. Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc. Natl. Acad. Sci. USA 88:54335437.
28. Knipfer, N.,, A. Seth,, and T. E. Shrader. 1997. Unmarked gene integration into the chromosome of Mycobacterium smegmatis via precise replacement of the pyrF gene. Plasmid 37:129140.
29. Kordulakova, J.,, M. Gilleron,, K. Mikusova,, G. Puzo,, P. J. Brennan,, B. Gicquel,, and M. Jackson. 2002. Definition of the first mannosylation step in phosphatidylinositol mannoside synthesis. PimA is essential for growth of mycobacteria. J. Biol. Chem. 277:3133531344.
30. Lamichhane, G.,, M. Zignol,, N. J. Blades,, D. E. Geiman,, A. Dougherty,, J. Grosset,, K. W. Broman,, and W. R. Bishai. 2003. A postgenomic method for predicting essential genes at subsaturation levels of mutagenesis: application to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 100:72137218.
31. Lederberg, J. 1951. Streptomycin resistance: a genetically recessive mutation. J. Bacteriol. 61:549550.
32. Lewis, J. A.,, and G. F. Hatfull. 2000. Identification and characterization of mycobacteriophage L5 excisionase. Mol. Microbiol. 35:350360.
33. Malaga, W.,, E. Perez,, and C. Guilhot. 2003. Production of unmarked mutations in mycobacteria using site-specific recombination. FEMS Microbiol. Lett. 219:261268.
34. Marklund, B. I.,, D. P. Speert,, and R. W. Stokes. 1995. Gene replacement through homologous recombination in Mycobacterium intracellulare. J. Bacteriol. 177:61006105.
35. McAdam, R. A.,, S. Quan,, D. A. Smith,, S. Bardarov,, J. C. Betts,, F. C. Cook,, E. U. Hooker,, A. P. Lewis,, P. Woollard,, M. J. Everett,, P. T. Lukey,, G. J. Bancroft,, W. R. Jacobs, Jr.,, and K. Duncan. 2002. Characterization of a Mycobacterium tuberculosis H37Rv transposon library reveals insertions in 351 ORFs and mutants with altered virulence. Microbiology 148:29752986.
36. Norman, E.,, O. A. Dellagostin,, J. McFadden,, and J. W. Dale. 1995. Gene replacement by homologous recombination in Mycobacterium bovis BCG. Mol. Microbiol. 16:755760.
37. Pan, F.,, M. Jackson,, Y. Ma,, and M. McNeil. 2001. Cell wall core galactofuran synthesis is essential for growth of mycobacteria. J. Bacteriol. 183:39913998.
38. Papavinasasundaram, K. G.,, M. J. Colston,, and E. O. Davis. 1998. Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function. Mol. Microbiol. 30:525534.
39. Parish, T.,, B. G. Gordhan,, R. A. McAdam,, K. Duncan,, V. Mizrahi,, and N. G. Stoker. 1999. Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology 145:34973503.
40. Parish, T.,, J. Lewis,, and N. G. Stoker. 2001. Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality. Tuberculosis 81:359364.
41. Parish, T.,, and N. G. Stoker. 2002. The common aromatic amino acid biosynthesis pathway is essential in Mycobacterium tuberculosis. Microbiology 148:30693077.
42. Parish, T.,, and N. G. Stoker. 1997. Development and use of a conditional antisense mutagenesis system in mycobacteria. FEMS Microbiol. Lett. 154:151157.
43. 43 Parish, T.,, and N. G. Stoker. 2000. glnE is an essential gene in Mycobacterium tuberculosis. J. Bacteriol. 182:57155720.
44. Parish, T.,, and N. G. Stoker. 2000. Use of a flexible cassette method to generate a double unmarked Mycobacterium tuberculosis tlyA plcABC mutant by gene replacement. Microbiology 146:19691975.
45. Pashley, C. A.,, T. Parish,, R. A. McAdam,, K. Duncan,, and N. G. Stoker. 2003. Gene replacement in mycobacteria by using incompatible plasmids. Appl. Environ. Microbiol. 69:517523.
46. Pavelka, M. S., Jr.,, and W. R. Jacobs, Jr. 1999. Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette- Guérin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J. Bacteriol. 181:47804789.
47. Pelicic, V.,, M. Jackson,, J. M. Reyrat,, W. R. Jacobs, Jr.,, B. Gicquel,, and C. Guilhot. 1997. Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 94:1095510960.
48. Pelicic, V.,, J. M. Reyrat,, and B. Gicquel. 1996. Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J. Bacteriol. 178:11971199.
49. Pelicic, V.,, J. M. Reyrat,, and B. Gicquel. 1996. Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol. Microbiol. 20:919925.
50. Pelicic, V.,, J. M. Reyrat,, and B. Gicquel. 1998. Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol. Microbiol. 28:413420.
51. Ramakrishnan, L.,, H. T. Tran,, N. A. Federspiel,, and S. Falkow. 1997. A crtB homolog essential for photochromogenicity in Mycobacterium marinum: isolation, characterization, and gene disruption via homologous recombination. J. Bacteriol. 179:58625865.
52. Raynaud, C.,, K. G. Papavinasasundaram,, R. A. Speight,, B. Springer,, P. Sander,, E. C. Bottger,, M. J. Colston,, and P. Draper. 2002. The functions of OmpATb, a pore-forming protein of Mycobacterium tuberculosis. Mol. Microbiol. 46:191201.
53. Reyrat, J. M.,, F. X. Berthet,, and B. Gicquel. 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. USA 92:87688772.
54. Reyrat, J. M.,, V. Pelicic,, B. Gicquel,, and R. Rappuoli. 1998. Counterselectable markers: untapped tools for bacterial genetics and pathogenesis. Infect. Immun. 66:40114017.
55. Sander, P.,, A. Meier,, and E. C. Bottger. 1995. rpsL_: a dominant selectable marker for gene replacement in mycobacteria. Mol. Microbiol. 16:9911000.
56. Sander, P.,, K. G. Papavinasasundaram,, T. Dick,, E. Stavropoulos,, K. Ellrott,, B. Springer,, M. J. Colston,, and E. C. Bottger. 2001. Mycobacterium bovis BCG recA deletion mutant shows increased susceptibility to DNA-damaging agents but wildtype survival in a mouse infection model. Infect. Immun. 69:35623568.
57. Sander, P.,, T. Prammananan,, and E. C. Bottger. 1996. Introducing mutations into a chromosomal rRNA gene using a genetically modified eubacterial host with a single rRNA operon. Mol. Microbiol. 22:841848.
58. Sassetti, C. M.,, D. H. Boyd,, and E. J. Rubin. 2001. Comprehensive identification of conditionally essential genes in mycobacteria. Proc. Natl. Acad. Sci. USA 98:1271212717.
59. Sassetti, C. M.,, D. H. Boyd,, and E. J. Rubin. 2003. Genes required for mycobacterial growth defined by high density mutagenesis. Mol. Microbiol. 48:7784.
60. Sassetti, C. M.,, and E. J. Rubin. 2003. Genetic requirements for mycobacterial survival during infection. Proc. Natl. Acad. Sci. USA 100:1298912994.
61. Smith, I. 2003. Mycobacterium tuberculosis pathogenesis and molecular determinants of virulence. Clin. Microbiol. Rev. 16:463496.
62. Springer, B.,, S. Master,, P. Sander,, T. Zahrt,, M. McFalone,, J. Song,, K. G. Papavinasasundaram,, M. J. Colston,, E. Boettger,, and V. Deretic. 2001. Silencing of oxidative stress response in Mycobacterium tuberculosis: expression patterns of ahpC in virulent and avirulent strains and effect of ahpC inactivation. Infect. Immun. 69:59675973.
63. Springer, B.,, P. Sander,, L. Sedlacek,, K. Ellrott,, and E. C. Bottger. 2001. Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors. Int. J. Med. Microbiol. 290:669675.
64. Stewart, G. R.,, V. A. Snewin,, G. Walzl,, T. Hussell,, P. Tormay,, P. O’Gaora,, M. Goyal,, J. Betts,, I. N. Brown,, and D. B. Young. 2001. Overexpression of heatshock proteins reduces survival of Mycobacterium tuberculosis in the chronic phase of infection. Nat. Med. 7:732737.
65. Stewart, G. R.,, L. Wernisch,, R. Stabler,, J. A. Mangan,, J. Hinds,, K. G. Laing,, D. B. Young,, and P. D. Butcher. 2002. Dissection of the heat-shock response in Mycobacterium tuberculosis using mutants and microarrays. Microbiology 148:31293138.
66. Stibitz, S. 1994. Use of conditionally counterselectable suicide vectors for allelic exchange. Methods Enzymol. 235:458465.

This is a required field
Please enter a valid email address
Please check the format of the address you have entered.
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error