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Transposon Mutagenesis in Mycobacteria Using Conditionally Replicating Mycobacteriophages

  • Stoyan S. Bardarov
  • Svetoslav S. BardarovJr.
  • William R. JacobsJr.
Protocol
Part of the Methods in Molecular Medicine book series (MIMM, volume 54)

Abstract

Genetic analyses of pathogenic mycobacteria such as Mycobacterium tubeculosis and Mycobacterium bovis required improvement of existing methodologies for the generation of large representative libraries of mutants. Two basic methodologies have been used to generate mutant libraries in both fast- and slow-growing mycobacteria: chemical mutagenesis and transposon mutagenesis. Chemical mutagenesis has successfully been used to produce different auxotrophic mutants in the fast growing mycobacteria Mycobacterium phlei (1,2) and Mycobacterium smegmatis (3,4). A detailed chemical mutagenesis protocol for the generation of mutant libraries in the fast-growing mycobacteria can be found in the previous volume of this manual (5). Chemical mutagenesis is not the ideal method for producing large representative mutant libraries for the slow-growing mycobacteria because: (1) the mutation frequency is relatively low,(2) multiple mutations may occur in the same cells,(3) clumping of the mycobacteria makes the identification and purification of the mutant clones very difficult,and (4) no generalized transducing phage has been described for the slow-growing mycobacteria to allow transfer of the point mutations and construction of isogenic strains.

Keywords

Mobile Genetic Element Chemical Mutagenesis Phage Infection Wash Medium Smegmatis Mc2155 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

References

  1. 1.
    Konickova-Radochova M., et al. (1969) The use of ethyl methanesulfonate for the induction of mutants in Mycobacterium phlei PA. Folia Microbiol. (Praha). 14 470–474.CrossRefGoogle Scholar
  2. 2.
    Konickova-Radochova M., et al. (1974) Mutagenesis by N-methyl-N-nitroso-N′-nitroguanidine in synchronized cultures of Mycobacterium phlei. Folia Microbiol.(Praha). 19 16–23.CrossRefGoogle Scholar
  3. 3.
    Hinshelwood S. and Stoker N. G. (1992) Cloning of mycobacterial histidine synthesis genes by complementation of a Mycobacterium smegmatis auxotroph. Mol. Microbiol. 6 2887–2895.CrossRefPubMedGoogle Scholar
  4. 4.
    Holland K. T. and Ratledge C. (1971) A procedure for selecting and isolating specific auxotrophic mutants of Mycobacterium smegmatis. J. Gen. Microbiol. 66 115–118.PubMedGoogle Scholar
  5. 5.
    Brooks L. A. (1998) Chemical mutagenesis of mycobacteriar in Mycobacteria Protocols: Methods in Molecular Biology vol. 101 (Parish T. and Stoker N. G., eds.), Humana, Totowa, NJ, pp. 175–186.CrossRefGoogle Scholar
  6. 6.
    Kleckner N., Roth J., and Botstein D. (1977) Genetic engineering in vivo using translocatable drug-resistance elements. New methods in bacterial genetics. J. Mol. Biol. 116, 125–159.CrossRefPubMedGoogle Scholar
  7. 7.
    Kleckner N., Bender J., and Gottesman S. (1991) Uses of transposons with emphasis on Tn10. Methods Enzymol. 204, 139–180.CrossRefPubMedGoogle Scholar
  8. 8.
    Bardarov S., Kriakov J., Carriere C., Yu S., Vaamonde C., McAdam R.A., Bloom B. R., Hatfull G. F., and Jacobs W. R., Jr. (1997) Conditionally replicating mycobacteriophages: a system for transposon delivery to Mycobacterium tuberculosis. Proc. Natl. Acad. Sci. USA 94 10,961–10,966.CrossRefPubMedGoogle Scholar
  9. 9.
    McAdam R. A., Weisbrod T. R., Martin J., Scuderi J. D., Brown A. M., Cirillo J. D., Bloom B. R., and Jacobs W. R., Jr. (1995) In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. Infect. Immunol. 63 1004–1012.Google Scholar
  10. 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.PubMedGoogle Scholar
  11. 11.
    Fomukong N. G., et al. (1993) Transpositional activity of IS986 in Mycobacterium smegmatis. Gene 130, 99–1CrossRefPubMedGoogle Scholar
  12. 12.
    England P. M., Wall S., and McFadden J. (1991) IS900-promoted stable integration of a foreign gene into mycobacteria. Mol. Microbiol. 5 2047–2052.CrossRefPubMedGoogle Scholar
  13. 13.
    Berg C. M. and Berg D. E. (1996) Transposable element tools for microbial genetics in Escherichia coli and Salmonella: Cellular and Molecular Biology 2nd ed., vol. 2. (Niedhardt F. C., et al., eds.) ASM Press, Washington DC pp. 2588–2612.Google Scholar
  14. 14.
    De Bruijn F. J. and Rosenbach S. (1994) Transposon mutagenesis in Methods in General and Molecular Bacteriology vol. 1 (Gerhardt P., ed.) American Society of Microbiology, Washington DC pp. 387–405.Google Scholar
  15. 15.
    Green E. P., Tizard M. L., Moss M. T., Thompson J., Winterbourne D. J., McFadden J. J., and Hermon-Taylor J. (1989) Sequence and characteristics of IS900, an insertion element identified in a human Crohn′s disease isolate of Mycobacterium paratuberculosis. Nucleic Acids Res. 17, 9063–9073.CrossRefPubMedGoogle Scholar
  16. 16.
    Thierry D., Brisson-Noel A., Vincent-Levy-Frebault V., Nguyen S., Guesdon J. L., and Gicquel B. (1990) Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis}. J. Clin. Microbiol. 28, 2668–2673.PubMedGoogle Scholar
  17. 17.
    Thierry D., Cave M. D., Eisenach K. D., Crawford J. T., Bates J. H., Gicquel B., and Guesdon J. L. (1990) IS6110, an IS-like element of Mycobacterium tuberculosis complex. Nucleic Acids Res. 18, 188.CrossRefPubMedGoogle Scholar
  18. 18.
    McAdam R. A., Hermans P. W., van Soolingen D., Zainuddin Z. F., Catty D., van Embden J. D., and Dale J. W. (1990) Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family. Mol. Microbiol. 4, 1607–1613.CrossRefPubMedGoogle Scholar
  19. 19.
    McAdam R. A., Guilhot C., and Gicquel B. (1994) Transposition in Mycobacteria,in Tuberculosis: Pathogenesis,Protection and Control (Bloom B. R., ed.) American Society for Microbiology, Washington, DC,pp.Google Scholar
  20. 20.
    Dale J. W. (1995) Mobile genetic elements in mycobacteria. Eur. Respir. J. Suppl. 20, 633s–648s.PubMedGoogle Scholar
  21. 21.
    Eisenach K. D., Crawford J. T., andBates J. H. (1988) RepetitiveDNAsequences as probes for Mycobacterium tuberculosis. J. Clin. Microbiol. 26, 2240–2245.PubMedGoogle Scholar
  22. 22.
    Martin C., Timm J., Rauzier J., Gomez-Lus R., Davies J., and Gicquel B. (1990) Transposition of an antibiotic resistance element in mycobacteria. Nature 345, 739–743.CrossRefPubMedGoogle Scholar
  23. 23.
    Guilhot C., Gicquel B., Davies J., and Martin C. (1992) Isolation and analysis of IS6120, anew insertion sequence from Mycobacterium smegmatis.Mol. Microbiol. 6, 107–113.CrossRefPubMedGoogle Scholar
  24. 24.
    Cirillo J. D., Barletta R. G., Bloom B. R., and Jacobs W. R., Jr. (1991) A novel transposon trap for mycobacteria: isolation and characterization of IS1096. J. Bacteriol. 173, 7772–7780.PubMedGoogle Scholar
  25. 25.
    Cole S. T., Brosch R., Parkhill J., Garnier T., Churcher C., Harris D., et al. (1998) Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.CrossRefPubMedGoogle Scholar
  26. 26.
    Gordon S. V., Heym B., Parkhill J., Barrell B., and Cole S. T. (1999) New insertion sequences and a novel repeated sequence in the genome of Mycobacterium tuberculosis H37Rv. Microbiology 145, 881–892.CrossRefPubMedGoogle Scholar
  27. 27.
    Philipp W. J., Schwartz D. C., Telenti A., and Cole S. T. (1998) Mycobacterial genome structure. Electrophoresis 19, 573–576.CrossRefPubMedGoogle Scholar
  28. 28.
    van Soolingen D., de Haas P. E., Hermans P. W., and van Embden J. D. (1994) DNA fingerprinting of Mycobacterium tuberculosis. Methods Enzymol. 235, 196–205.CrossRefPubMedGoogle Scholar
  29. 29.
    van Soolingen D. and Hermans P. W. (1995) Epidemiology of tuberculosis by DNA fingerprinting. Eur. Respir. J. Suppl. 20, 649s–656s.PubMedGoogle Scholar
  30. 30.
    Perez E., Gavigan J. A., Otal I., Guilhot C., Pelicic V., Giquel B., and Martin C. (1998) Tn611 transposon mutagenesis in Mycobacterium smegmatis using a temperature-sensitive delivery system. Methods Mol. Biol. 101, 187–198.PubMedGoogle Scholar
  31. 31.
    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.CrossRefPubMedGoogle Scholar
  32. 32.
    Carriere C., Riska P. F., Zimhony O., Kriakov J., Bardarov S., Burns J., Chan J., and Jacobs W. R., Jr. (1997) Conditionally replicating luciferase reporter phages: improved sensitivity for rapid detection and assessment of drug susceptibility of Mycobacterium tuberculosis.35, 3232–32PubMedGoogle Scholar
  33. 34.
    Wards B. J. (1996) Evaluation of defined media suitable for isolation of auxotrophic mutants of mycobacteria. J. Basic Microbiol. 36, 355–362.CrossRefPubMedGoogle Scholar
  34. 35.
    Rado T. A. and Bates J. H. (1980) Mycobacteriophage structure and function: a review. Adv. Tuberc. Res. 20, 64–91.PubMedGoogle Scholar

Copyright information

© Humana Press Inc. 2001

Authors and Affiliations

  • Stoyan S. Bardarov
  • Svetoslav S. BardarovJr.
  • William R. JacobsJr.

There are no affiliations available

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