Ins and Outs of Mycobacterial Plasmids

  • Farahnaz Movahedzadeh
  • Wilbert Bitter
Part of the Methods in Molecular Biology book series (MIMB, volume 465)


The importance of plasmids for molecular research cannot be underestimated. These double-stranded DNA units that replicate independently of the chromosomal DNA are as valuable to bacterial geneticists as a carpenter’s hammer. Fortunately, today the mycobacterial research community has a number of these genetic tools at its disposal, and the development of these tools has greatly accelerated the study of mycobacterial pathogens. However, working with mycobacterial cloning plasmids is still not always as straightforward as working with Escherichia coli plasmids, and therefore a number of precautions and potential pitfalls will be discussed in this chapter.


antibiotic resistance marker integrating vector Mycobacterium natural plasmid shuttle vector 


  1. 1.
    Jucker, M. T. & Falkinham, J. O. III. (1990). Epidemiology of infection by nontuberculous mycobacteria IX. Evidence for two DNA homology groups among small plasmids in Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. Am. Rev. Respir. Dis. 142, 858–62.PubMedGoogle Scholar
  2. 2.
    Kirby, C., Waring, A., Griffin, T. J., Falkinham, J. O. III, Grindley, N. D. & Derbyshire, K. M. (2002). Cryptic plasmids of Mycobacterium avium: Tn552 to the rescue. Mol. Microbiol. 43, 173–86.Google Scholar
  3. 3.
    Zainuddin, Z. F. & Dale, J. W. (1990). Does Mycobacterium tuberculosis have plasmids? Tubercle 71, 43–9.PubMedCrossRefGoogle Scholar
  4. 4.
    Le Dantec, C., Winter, N., Gicquel, B., Vincent, V. & Picardeau, M. (2001). Genomic sequence and transcriptional analysis of a 23-kilobase mycobacterial linear plasmid: evidence for horizontal transfer and identification of plasmid maintenance systems. J. Bacteriol. 183, 2157–64.PubMedCrossRefGoogle Scholar
  5. 5.
    Stinear, T. P., Pryor, M. J., Porter, J. L. & Cole, S. T. (2005). Functional analysis and annotation of the virulence plasmid pMUM001 from Mycobacterium ulcerans. Microbiology 151, 683–92.PubMedCrossRefGoogle Scholar
  6. 6.
    Labidi, A., David, H. L. & Roulland-Dussoix, D. (1985). Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000. Ann. Inst. Pasteur. Microbiol. 136B, 209–15.PubMedCrossRefGoogle Scholar
  7. 7.
    Pashley, C. & Stoker, N. G. Plasmids in mycobacteria. In: Hatfull, G. F. and Jacobs, W.R. (eds.), Molecular Genetics of Mycobacteria, ASM Press, 2000: 55–68.Google Scholar
  8. 8.
    Crawford, J. T. & Bates, J. H. (1986). Analysis of plasmids in Mycobacterium avium-intracellulare isolates from persons with acquired immunodeficiency syndrome. Am. Rev. Respir. Dis. 134, 659–61.PubMedGoogle Scholar
  9. 9.
    Gangadharam, P. R., Perumal, V. K., Crawford, J. T. & Bates, J. H. (1988). Association of plasmids and virulence of Mycobacterium avium complex. Am. Rev. Respir. Dis. 137, 212–4.PubMedCrossRefGoogle Scholar
  10. 10.
    Stinear, T. P., Mve-Obiang, A., Small, P. L., Frigui, W., Pryor, M. J., Brosch, R., Jenkin, G. A., Johnson, P. D., Davies, J. K., Lee, R. E., Adusumilli, S., Garnier, T., Haydock, S. F., Leadlay, P. F. & Cole, S. T. (2004). Giant plasmid-encoded polyketide synthases produce the macrolide toxin of Mycobacterium ulcerans. Proc. Natl. Acad. Sci. U. S. A. 101, 1345–9.PubMedCrossRefGoogle Scholar
  11. 11.
    George, K. M., Chatterjee, D., Gunawardana, G., Welty, D., Hayman, J., Lee, R. & Small, P. L. (1999). Mycolactone: a polyketide toxin from Mycobacterium ulcerans required for virulence. Science 283, 854–7.PubMedCrossRefGoogle Scholar
  12. 12.
    Ranger, B. S., Mahrous, E. A., Mosi, L., Adusumilli, S., Lee, R. E., Colorni, A., Rhodes, M. & Small, P. L. (2006). Globally distributed mycobacterial fish pathogens produce a novel plasmid-encoded toxic macrolide, mycolactone F. Infect. Immun. 74(11), 6037–45.Google Scholar
  13. 13.
    Harth, G., Maslesa-Galic, S. & Horwitz, M. A. (2004). A two-plasmid system for stable, selective-pressure-independent expression of multiple extracellular proteins in mycobacteria. Microbiology 150, 2143–51.PubMedCrossRefGoogle Scholar
  14. 14.
    Wang, J., Parsons, L. M. & Derbyshire, K. M. (2003). Unconventional conjugal DNA transfer in mycobacteria. Nat. Genet. 34, 80–4.PubMedCrossRefGoogle Scholar
  15. 15.
    Gormley, E. P. & Davies, J. (1991). Transfer of plasmid RSF1010 by conjugation from Escherichia colito Streptomyces lividans and Mycobacterium smegmatis. J. Bacteriol. 173, 6705–8.PubMedGoogle Scholar
  16. 16.
    Stolt, P. & Stoker, N. G. (1996). Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000. Microbiology 142(Pt 10), 2795–802.PubMedCrossRefGoogle Scholar
  17. 17.
    Bachrach, G., Colston, M. J., Bercovier, H., Bar-Nir, D., Anderson, C. & Papavinasasundaram, K. G. (2000). A new single-copy mycobacterial plasmid, pMF1, from Mycobacterium fortuitum which is compatible with the pAL5000 replicon. Microbiology 146(Pt 2), 297–303.PubMedGoogle Scholar
  18. 18.
    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. & et al. (1991). New use of BCG for recombinant vaccines. Nature 351, 456–60.PubMedCrossRefGoogle Scholar
  19. 19.
    Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R., Jr. (1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol. Microbiol. 4, 1911–9.PubMedCrossRefGoogle Scholar
  20. 20.
    Al-Zarouni, M. & Dale, J. W. (2002). Expression of foreign genes in Mycobacterium bovisBCG strains using different promoters reveals instability of the hsp60 promoter for expression of foreign genes in Mycobacterium bovisBCG strains. Tuberculosis(Edinb) 82, 283–91.CrossRefGoogle Scholar
  21. 21.
    Parish, T., Roberts, G., Laval, F., Schaeffer, M., Daffe, M. & Duncan, K. (2007). Functional Complementation of the Essential Gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but Not Escherichia coli fabG. J. Bacteriol. 189, 3721–8.PubMedCrossRefGoogle Scholar
  22. 22.
    Parish, T., Liu, J., Nikaido, H. & Stoker, N. G. (1997). A Mycobacterium smegmatis mutant with a defective inositol monophosphate phosphatase gene homolog has altered cell envelope permeability. J. Bacteriol. 179, 7827–33.PubMedGoogle Scholar
  23. 23.
    Carroll, P., Muttucumaru, D. G. & Parish, T. (2005). Use of a tetracycline-inducible system for conditional expression in Mycobacterium tuberculosis and Mycobacterium smegmatis. Appl. Environ. Microbiol. 71, 3077–84.PubMedCrossRefGoogle Scholar
  24. 24.
    Ehrt, S., Guo, X. V., Hickey, C. M., Ryou, M., Monteleone, M., Riley, L. W. & Schnappinger, D. (2005). Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 33, e21.PubMedCrossRefGoogle Scholar
  25. 25.
    Blokpoel, M. C., Murphy, H. N., O'Toole, R., Wiles, S., Runn, E. S., Stewart, G. R., Young, D. B. & Robertson, B. D. (2005). Tetracycline-inducible gene regulation in mycobacteria. Nucleic Acids Res. 33, e22.PubMedCrossRefGoogle Scholar
  26. 26.
    Das Gupta, S. K., Bashyam, M. D. & Tyagi, A. K. (1993). Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector. J. Bacteriol. 175, 5186–92.PubMedGoogle Scholar
  27. 27.
    Timm, J., Lim, E. M. & Gicquel, B. (1994). Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions tolacZ: the pJEM series. J. Bacteriol. 176, 6749–53.PubMedGoogle Scholar
  28. 28.
    Valdivia, R. H., Hromockyj, A. E., Monack, D., Ramakrishnan, L. & Falkow, S. (1996). Applications for green fluorescent protein (GFP) in the study of host-pathogen interactions. Gene 173, 47–52.PubMedCrossRefGoogle Scholar
  29. 29.
    Kenney, T. J. & Churchward, G. (1996). Genetic analysis of the Mycobacterium smegmatis rpsL promoter. J. Bacteriol. 178, 3564–71.PubMedGoogle Scholar
  30. 30.
    Guilhot, C., Gicquel, B. & Martin, C. (1992). Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol. Lett. 77, 181–6.PubMedCrossRefGoogle Scholar
  31. 31.
    Guilhot, C., Otal, I., Van Rompaey, I., Martin, C. & Gicquel, B. (1994). Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J. Bacteriol. 176, 535–9.Google Scholar
  32. 32.
    Pelicic, V., Reyrat, J. M. & Gicquel, B. (1996). Generation of unmarked directed mutations in mycobacteria, using sucrose counter-selectable suicide vectors. Mol. Microbiol. 20, 919–25.PubMedCrossRefGoogle Scholar
  33. 33.
    Pelicic, V., Jackson, M., Reyrat, J. M., Jacobs, W. R. Jr., Gicquel, B. & Guilhot, C. (1997). Efficient allelic exchange and transposon mutagenesis in Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A. 94, 10955–60.PubMedCrossRefGoogle Scholar
  34. 34.
    Papavinasasundaram, K. G., Colston, M. J. & Davis, E. O. (1998). Construction and complementation of a recAdeletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function. Mol. Microbiol. 30, 525–34.PubMedCrossRefGoogle Scholar
  35. 35.
    Pedulla, M. L. & Hatfull, G. F. (1998). Characterization of the mIHFgene of Mycobacterium smegmatis. J. Bacteriol. 180, 5473–7.PubMedGoogle Scholar
  36. 36.
    Pavelka, M. S. Jr. & Jacobs, W. R. Jr. (1999). Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovisbacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J. Bacteriol. 181, 4780–9.PubMedGoogle Scholar
  37. 37.
    Parish, T. & 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(Pt 8), 1969–75.PubMedGoogle Scholar
  38. 38.
    Sander, P., Meier, A. & Bottger, E. C. (1995). rpsL+: a dominant selectable marker for gene replacement in mycobacteria. Mol. Microbiol. 16, 991–1000.PubMedCrossRefGoogle Scholar
  39. 39.
    Bardarov, S., Kriakov, J., Carriere, C., Yu, S., Vaamonde, C., McAdam, R. A., Bloom, B. R., Hatfull, G. F. & Jacobs, W. R. Jr. (1997). Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. U. S. A. 94, 10961–6.PubMedCrossRefGoogle Scholar
  40. 40.
    Rubin, E. J., Akerley, B. J., Novik, V. N., Lampe, D. J., Husson, R. N. & Mekalanos, J. J. (1999). In vivo transposition of mariner-based elements in enteric bacteria and mycobacteria. Proc. Natl. Acad. Sci. U. S. A. 96, 1645–50.PubMedCrossRefGoogle Scholar
  41. 41.
    Snapper, S. B., Lugosi, L., Jekkel, A., Melton, R. E., Kieser, T., Bloom, B. R. & Jacobs, W. R. Jr. (1988). Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc. Natl. Acad. Sci. U. S. A. 85, 6987–91.PubMedCrossRefGoogle Scholar
  42. 42.
    Mahenthiralingam, E., Marklund, B. I., Brooks, L. A., Smith, D. A., Bancroft, G. J. & Stokes, R. W. (1998). Site-directed mutagenesis of the 19-kilodalton lipoprotein antigen reveals no essential role for the protein in the growth and virulence of Mycobacterium intracellulare. Infect. Immun. 66, 3626–34.PubMedGoogle Scholar
  43. 43.
    Murry, J., Sassetti, C. M., Moreira, J., Lane, J. & Rubin, E. J. (2005). A new site-specific integration system for mycobacteria. Tuberculosis (Edinb) 85, 317–23.CrossRefGoogle Scholar
  44. 44.
    Springer, B., Sander, P., Sedlacek, L., Ellrott, K. & Bottger, E. C. (2001). Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors. Int. J. Med. Microbiol. 290, 669–75.PubMedCrossRefGoogle Scholar
  45. 45.
    Vultos, T. D., Mederle, I., Abadie, V., Pimentel, M., Moniz-Pereira, J., Gicquel, B., Reyrat, J. M. & Winter, N. (2006). Modification of the mycobacteriophage Ms6 attP core allows the integration of multiple vectors into different tRNAala T-loops in slow- and fast-growing mycobacteria. BMC Mol. Biol. 7, 47.PubMedCrossRefGoogle Scholar
  46. 46.
    Saviola, B. & Bishai, W. R. (2004). Method to integrate multiple plasmids into the mycobacterial chromosome. Nucleic Acids Res. 32, e11.PubMedCrossRefGoogle Scholar
  47. 47.
    Pashley, C. A. & Parish, T. (2003). Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis. FEMS Microbiol. Lett. 229, 211–5.PubMedCrossRefGoogle Scholar
  48. 48.
    Lewis, J. A. & Hatfull, G. F. (2000). Identification and characterization of mycobacteriophage L5 excisionase. Mol. Microbiol. 35, 350–60.PubMedCrossRefGoogle Scholar
  49. 49.
    Parish, T. & Stoker, N. G. Electroporation of mycobacteria. In Nickoloff, J. A. (ed.), Methods in Molecular Biology: Electroporation Protocols for Microorganisms, Vol. 47. Humana Press, 1995: 237–52.Google Scholar
  50. 50.
    Consaul, S. A. & Pavelka, M. S. Jr. (2004). Use of a novel allele of the Escherichia coli aacC4 aminoglycoside resistance gene as a genetic marker in mycobacteria. FEMS Microbiol. Lett. 234, 297–301.PubMedCrossRefGoogle Scholar
  51. 51.
    Goto, Y., Taniguchi, H., Udou, T., Mizuguchi, Y. & Tokunaga, T. (1991). Development of a new host vector system in mycobacteria. FEMS Microbiol. Lett. 67, 277–82.PubMedCrossRefGoogle Scholar
  52. 52.
    Beggs, M. L., Crawford, J. T. & Eisenach, K. D. (1995). Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7. J. Bacteriol. 177, 4836–40.PubMedGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Institute for Tuberculosis Research, College of Pharmacy, Rm 412, University of Illinois at ChicagoChicagoUSA
  2. 2.Medical MicrobiologyVU University Medical CentreAmsterdamThe Netherlands

Personalised recommendations