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

R. A. McAdam, Torin Weisbrod, J. Martin, J. D. Scuderi, A. M. Brown, J. D. Cirillo, B. R. Bloom, W. R. Jacobs

Research output: Contribution to journalArticle

162 Citations (Scopus)

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 done. 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.

Original languageEnglish (US)
Pages (from-to)1004-1012
Number of pages9
JournalInfection and Immunity
Volume63
Issue number3
StatePublished - 1995
Externally publishedYes

Fingerprint

Mycobacterium bovis
Leucine
Mutagenesis
Methionine
Growth
Libraries
Clone Cells
Kanamycin Resistance
Mycobacterium smegmatis
Kanamycin
Insertional Mutagenesis
Cyanobacteria
Inbred C57BL Mouse
Open Reading Frames
Genes
Sulfates
Sequence Analysis
Carrier Proteins
Tuberculosis
Plasmids

ASJC Scopus subject areas

  • Immunology

Cite this

McAdam, R. A., Weisbrod, T., Martin, J., Scuderi, J. D., Brown, A. M., Cirillo, J. D., ... Jacobs, W. R. (1995). In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. Infection and Immunity, 63(3), 1004-1012.

In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. / McAdam, R. A.; Weisbrod, Torin; Martin, J.; Scuderi, J. D.; Brown, A. M.; Cirillo, J. D.; Bloom, B. R.; Jacobs, W. R.

In: Infection and Immunity, Vol. 63, No. 3, 1995, p. 1004-1012.

Research output: Contribution to journalArticle

McAdam, RA, Weisbrod, T, Martin, J, Scuderi, JD, Brown, AM, Cirillo, JD, Bloom, BR & Jacobs, WR 1995, 'In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis', Infection and Immunity, vol. 63, no. 3, pp. 1004-1012.
McAdam, R. A. ; Weisbrod, Torin ; Martin, J. ; Scuderi, J. D. ; Brown, A. M. ; Cirillo, J. D. ; Bloom, B. R. ; Jacobs, W. R. / In vivo growth characteristics of leucine and methionine auxotrophic mutants of Mycobacterium bovis BCG generated by transposon mutagenesis. In: Infection and Immunity. 1995 ; Vol. 63, No. 3. pp. 1004-1012.
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