Skip to main content
NCBI home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Mar;63(3):1004–1012. doi: 10.1128/iai.63.3.1004-1012.1995

In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis.

R A McAdam 1, T R Weisbrod 1, J Martin 1, J D Scuderi 1, A M Brown 1, J D Cirillo 1, B R Bloom 1, W R Jacobs Jr 1
PMCID: PMC173102  PMID: 7868221

Abstract

Insertional mutagenesis in Mycobacterium bovis BCG, a member of the slow-growing M. tuberculosis complex, was accomplished with transposons engineered from the Mycobacterium smegmatis insertion element IS1096. Transposons were created by placing a kanamycin resistance gene in several different positions in IS1096, and the resulting transposons were electroporated into BCG on nonreplicating plasmids. These analyses demonstrated that only one of the two open reading frames was necessary for transposition. A library of insertions was generated. Southern analysis of 23 kanamycin-resistant clones revealed that the transposons had inserted directly, with no evidence of cointegrate formation, into different restriction fragments in each clone. Sequence analysis of nine of the clones revealed junctional direct 8-bp repeats with only a slight similarity in target sites. These results suggest that IS1096-derived transposons transposed into the BCG genome in a relatively random fashion. Three auxotrophs, two for leucine and one for methionine, were isolated from the library of transposon insertions in BCG. They were characterized by sequencing and found to be homologous to the leuD gene of Escherichia coli and a sulfate-binding protein of cyanobacteria, respectively. When inoculated intravenously into C57BL/6 mice, the leucine auxotrophs, in contrast to the parent BCG strain or the methionine auxotroph, showed an inability to grow in vivo and were cleared within 7 weeks from the lungs and spleen.

Full Text

The Full Text of this article is available as a PDF (380.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aldovini A., Husson R. N., Young R. A. The uraA locus and homologous recombination in Mycobacterium bovis BCG. J Bacteriol. 1993 Nov;175(22):7282–7289. doi: 10.1128/jb.175.22.7282-7289.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bloom B. R., Murray C. J. Tuberculosis: commentary on a reemergent killer. Science. 1992 Aug 21;257(5073):1055–1064. doi: 10.1126/science.257.5073.1055. [DOI] [PubMed] [Google Scholar]
  3. Christie G. E., Farnham P. J., Platt T. Synthetic sites for transcription termination and a functional comparison with tryptophan operon termination sites in vitro. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4180–4184. doi: 10.1073/pnas.78.7.4180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cirillo J. D., Barletta R. G., Bloom B. R., Jacobs W. R., Jr A novel transposon trap for mycobacteria: isolation and characterization of IS1096. J Bacteriol. 1991 Dec;173(24):7772–7780. doi: 10.1128/jb.173.24.7772-7780.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donnelly-Wu M. K., Jacobs W. R., Jr, Hatfull G. F. Superinfection immunity of mycobacteriophage L5: applications for genetic transformation of mycobacteria. Mol Microbiol. 1993 Feb;7(3):407–417. doi: 10.1111/j.1365-2958.1993.tb01132.x. [DOI] [PubMed] [Google Scholar]
  6. Fields P. I., Swanson R. V., Haidaris C. G., Heffron F. Mutants of Salmonella typhimurium that cannot survive within the macrophage are avirulent. Proc Natl Acad Sci U S A. 1986 Jul;83(14):5189–5193. doi: 10.1073/pnas.83.14.5189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Garbe T. R., Barathi J., Barnini S., Zhang Y., Abou-Zeid C., Tang D., Mukherjee R., Young D. B. Transformation of mycobacterial species using hygromycin resistance as selectable marker. Microbiology. 1994 Jan;140(Pt 1):133–138. doi: 10.1099/13500872-140-1-133. [DOI] [PubMed] [Google Scholar]
  8. Gormley E. P., Davies J. Transfer of plasmid RSF1010 by conjugation from Escherichia coli to Streptomyces lividans and Mycobacterium smegmatis. J Bacteriol. 1991 Nov;173(21):6705–6708. doi: 10.1128/jb.173.21.6705-6708.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Guilhot C., Gicquel B., Martín C. Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol Lett. 1992 Nov 1;77(1-3):181–186. doi: 10.1016/0378-1097(92)90152-e. [DOI] [PubMed] [Google Scholar]
  10. Guilhot C., Otal I., Van Rompaey I., Martìn C., Gicquel B. Efficient transposition in mycobacteria: construction of Mycobacterium smegmatis insertional mutant libraries. J Bacteriol. 1994 Jan;176(2):535–539. doi: 10.1128/jb.176.2.535-539.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Hatfull G. F. Genetic transformation of mycobacteria. Trends Microbiol. 1993 Nov;1(8):310–314. doi: 10.1016/0966-842x(93)90008-f. [DOI] [PubMed] [Google Scholar]
  13. Jacobs W. R., Jr, Kalpana G. V., Cirillo J. D., Pascopella L., Snapper S. B., Udani R. A., Jones W., Barletta R. G., Bloom B. R. Genetic systems for mycobacteria. Methods Enzymol. 1991;204:537–555. doi: 10.1016/0076-6879(91)04027-l. [DOI] [PubMed] [Google Scholar]
  14. Jacobs W. R., Jr, Tuckman M., Bloom B. R. Introduction of foreign DNA into mycobacteria using a shuttle phasmid. Nature. 1987 Jun 11;327(6122):532–535. doi: 10.1038/327532a0. [DOI] [PubMed] [Google Scholar]
  15. Kalpana G. V., Bloom B. R., Jacobs W. R., Jr Insertional mutagenesis and illegitimate recombination in mycobacteria. Proc Natl Acad Sci U S A. 1991 Jun 15;88(12):5433–5437. doi: 10.1073/pnas.88.12.5433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kleckner N., Bender J., Gottesman S. Uses of transposons with emphasis on Tn10. Methods Enzymol. 1991;204:139–180. doi: 10.1016/0076-6879(91)04009-d. [DOI] [PubMed] [Google Scholar]
  17. Kung S. S., Chen J., Chow W. Y. Molecular and genetic characterization of an Alcaligenes eutrophus insertion element. J Bacteriol. 1992 Dec;174(24):8023–8029. doi: 10.1128/jb.174.24.8023-8029.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Labidi A., Mardis E., Roe B. A., Wallace R. J., Jr Cloning and DNA sequence of the Mycobacterium fortuitum var fortuitum plasmid pAL5000. Plasmid. 1992 Mar;27(2):130–140. doi: 10.1016/0147-619x(92)90013-z. [DOI] [PubMed] [Google Scholar]
  19. Leung K. Y., Finlay B. B. Intracellular replication is essential for the virulence of Salmonella typhimurium. Proc Natl Acad Sci U S A. 1991 Dec 15;88(24):11470–11474. doi: 10.1073/pnas.88.24.11470. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mahan M. J., Slauch J. M., Mekalanos J. J. Selection of bacterial virulence genes that are specifically induced in host tissues. Science. 1993 Jan 29;259(5095):686–688. doi: 10.1126/science.8430319. [DOI] [PubMed] [Google Scholar]
  21. Manoil C., Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. doi: 10.1073/pnas.82.23.8129. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Marquis H., Bouwer H. G., Hinrichs D. J., Portnoy D. A. Intracytoplasmic growth and virulence of Listeria monocytogenes auxotrophic mutants. Infect Immun. 1993 Sep;61(9):3756–3760. doi: 10.1128/iai.61.9.3756-3760.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Martin C., Timm J., Rauzier J., Gomez-Lus R., Davies J., Gicquel B. Transposition of an antibiotic resistance element in mycobacteria. Nature. 1990 Jun 21;345(6277):739–743. doi: 10.1038/345739a0. [DOI] [PubMed] [Google Scholar]
  24. McDonough K. A., Kress Y., Bloom B. R. Pathogenesis of tuberculosis: interaction of Mycobacterium tuberculosis with macrophages. Infect Immun. 1993 Jul;61(7):2763–2773. doi: 10.1128/iai.61.7.2763-2773.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. PIEZ K. A., EAGLE H. The free amino acid pool of cultured human cells. J Biol Chem. 1958 Mar;231(1):533–545. [PubMed] [Google Scholar]
  26. Pflugrath J. W., Quiocho F. A. The 2 A resolution structure of the sulfate-binding protein involved in active transport in Salmonella typhimurium. J Mol Biol. 1988 Mar 5;200(1):163–180. doi: 10.1016/0022-2836(88)90341-5. [DOI] [PubMed] [Google Scholar]
  27. Rauzier J., Moniz-Pereira J., Gicquel-Sanzey B. Complete nucleotide sequence of pAL5000, a plasmid from Mycobacterium fortuitum. Gene. 1988 Nov 30;71(2):315–321. doi: 10.1016/0378-1119(88)90048-0. [DOI] [PubMed] [Google Scholar]
  28. Raviglione M. C., Sudre P., Rieder H. L., Spinaci S., Kochi A. Secular trends of tuberculosis in western Europe. Bull World Health Organ. 1993;71(3-4):297–306. [PMC free article] [PubMed] [Google Scholar]
  29. Slauch J. M., Silhavy T. J. Genetic fusions as experimental tools. Methods Enzymol. 1991;204:213–248. doi: 10.1016/0076-6879(91)04011-c. [DOI] [PubMed] [Google Scholar]
  30. Somers J. M., Amzallag A., Middleton R. B. Genetic fine structure of the leucine operon of Escherichia coli K-12. J Bacteriol. 1973 Mar;113(3):1268–1272. doi: 10.1128/jb.113.3.1268-1272.1973. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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. New use of BCG for recombinant vaccines. Nature. 1991 Jun 6;351(6326):456–460. doi: 10.1038/351456a0. [DOI] [PubMed] [Google Scholar]
  32. Sudre P., ten Dam G., Kochi A. Tuberculosis: a global overview of the situation today. Bull World Health Organ. 1992;70(2):149–159. [PMC free article] [PubMed] [Google Scholar]
  33. Vertès A. A., Inui M., Kobayashi M., Kurusu Y., Yukawa H. Isolation and characterization of IS31831, a transposable element from Corynebacterium glutamicum. Mol Microbiol. 1994 Feb;11(4):739–746. doi: 10.1111/j.1365-2958.1994.tb00351.x. [DOI] [PubMed] [Google Scholar]
  34. Villar C. A., Benitez J. Functional analysis of pAL5000 plasmid in Mycobacterium fortuitum. Plasmid. 1992 Sep;28(2):166–169. doi: 10.1016/0147-619x(92)90047-e. [DOI] [PubMed] [Google Scholar]
  35. Young D. B., Cole S. T. Leprosy, tuberculosis, and the new genetics. J Bacteriol. 1993 Jan;175(1):1–6. doi: 10.1128/jb.175.1.1-6.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. van Soolingen D., Hermans P. W., de Haas P. E., Soll D. R., van Embden J. D. Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol. 1991 Nov;29(11):2578–2586. doi: 10.1128/jcm.29.11.2578-2586.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. van der Zee A., Agterberg C., van Agterveld M., Peeters M., Mooi F. R. Characterization of IS1001, an insertion sequence element of Bordetella parapertussis. J Bacteriol. 1993 Jan;175(1):141–147. doi: 10.1128/jb.175.1.141-147.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES