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. 1995 Nov;177(21):6100–6105. doi: 10.1128/jb.177.21.6100-6105.1995

Gene replacement through homologous recombination in Mycobacterium intracellulare.

B I Marklund 1, D P Speert 1, R W Stokes 1
PMCID: PMC177448  PMID: 7592373

Abstract

Mycobacterium intracellulare is a slow-growing pathogenic mycobacterium closely related to Mycobacterium avium. In contrast to Mycobacterium tuberculosis and Mycobacterium bovis BCG, M. intracellulare has received little attention as a model species for studies of mycobacterial molecular biology and genetics. This study shows that M. intracellulare 1403 (ATCC 35761) can be transformed by electroporation with high frequencies (up to 10(6) transformants per microgram of DNA), using plasmids pYT937 and pMH94 as replicative and integrative vectors, respectively. We also describe an experimental system that we used to study DNA recombination in M. intracellulare. First, an integrative plasmid was introduced into M. intracellulare 1403. A nonreplicative, nonintegrative plasmid having homology with the integrated plasmid was then introduced, and the resultant recombinants were analyzed to distinguish between events of homologous and illegitimate recombination. No illegitimate recombination occurred; in all recombinants, a single crossover between homologous regions of the two plasmids was noted. During subsequent growth of a recombinant clone, a spontaneous deletion occurred that resulted in a gene replacement on the chromosome of M. intracellulare 1403. The ability to construct site-specific mutations in M. intracellulare will provide novel insights into the biology of slow-growing mycobacteria.

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Selected References

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  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. Barletta R. G., Kim D. D., Snapper S. B., Bloom B. R., Jacobs W. R., Jr Identification of expression signals of the mycobacteriophages Bxb1, L1 and TM4 using the Escherichia-Mycobacterium shuttle plasmids pYUB75 and pYUB76 designed to create translational fusions to the lacZ gene. J Gen Microbiol. 1992 Jan;138(1):23–30. doi: 10.1099/00221287-138-1-23. [DOI] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Crawford J. T., Cave M. D., Bates J. H. Evidence for plasmid-mediated restriction-modification in Mycobacterium avium intracellulare. J Gen Microbiol. 1981 Dec;127(2):333–338. doi: 10.1099/00221287-127-2-333. [DOI] [PubMed] [Google Scholar]
  5. Curcic R., Dhandayuthapani S., Deretic V. 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. 1994 Sep;13(6):1057–1064. doi: 10.1111/j.1365-2958.1994.tb00496.x. [DOI] [PubMed] [Google Scholar]
  6. Davis E. O., Sedgwick S. G., Colston M. J. Novel structure of the recA locus of Mycobacterium tuberculosis implies processing of the gene product. J Bacteriol. 1991 Sep;173(18):5653–5662. doi: 10.1128/jb.173.18.5653-5662.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Davis E. O., Thangaraj H. S., Brooks P. C., Colston M. J. Evidence of selection for protein introns in the recAs of pathogenic mycobacteria. EMBO J. 1994 Feb 1;13(3):699–703. doi: 10.1002/j.1460-2075.1994.tb06309.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Goto Y., Taniguchi H., Udou T., Mizuguchi Y., Tokunaga T. Development of a new host vector system in mycobacteria. FEMS Microbiol Lett. 1991 Oct 15;67(3):277–282. doi: 10.1016/0378-1097(91)90489-w. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. 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]
  11. Husson R. N., James B. E., Young R. A. Gene replacement and expression of foreign DNA in mycobacteria. J Bacteriol. 1990 Feb;172(2):519–524. doi: 10.1128/jb.172.2.519-524.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Inderlied C. B., Kemper C. A., Bermudez L. E. The Mycobacterium avium complex. Clin Microbiol Rev. 1993 Jul;6(3):266–310. doi: 10.1128/cmr.6.3.266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jacobs W. R., Jr, Barletta R. G., Udani R., Chan J., Kalkut G., Sosne G., Kieser T., Sarkis G. J., Hatfull G. F., Bloom B. R. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science. 1993 May 7;260(5109):819–822. doi: 10.1126/science.8484123. [DOI] [PubMed] [Google Scholar]
  14. Ji B., Lounis N., Truffot-Pernot C., Grosset J. Effectiveness of various antimicrobial agents against Mycobacterium avium complex in the beige mouse model. Antimicrob Agents Chemother. 1994 Nov;38(11):2521–2529. doi: 10.1128/aac.38.11.2521. [DOI] [PMC free article] [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. Kunze Z. M., Wall S., Appelberg R., Silva M. T., Portaels F., McFadden J. J. IS901, a new member of a widespread class of atypical insertion sequences, is associated with pathogenicity in Mycobacterium avium. Mol Microbiol. 1991 Sep;5(9):2265–2272. doi: 10.1111/j.1365-2958.1991.tb02157.x. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Lee M. H., Pascopella L., Jacobs W. R., Jr, Hatfull G. F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. Proc Natl Acad Sci U S A. 1991 Apr 15;88(8):3111–3115. doi: 10.1073/pnas.88.8.3111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Meissner P. S., Falkinham J. O., 3rd Plasmid DNA profiles as epidemiological markers for clinical and environmental isolates of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum. J Infect Dis. 1986 Feb;153(2):325–331. doi: 10.1093/infdis/153.2.325. [DOI] [PubMed] [Google Scholar]
  20. Norman E., Dellagostin O. A., McFadden J., Dale J. W. Gene replacement by homologous recombination in Mycobacterium bovis BCG. Mol Microbiol. 1995 May;16(4):755–760. doi: 10.1111/j.1365-2958.1995.tb02436.x. [DOI] [PubMed] [Google Scholar]
  21. Oka A., Sugisaki H., Takanami M. Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol. 1981 Apr 5;147(2):217–226. doi: 10.1016/0022-2836(81)90438-1. [DOI] [PubMed] [Google Scholar]
  22. Radford A. J., Hodgson A. L. Construction and characterization of a Mycobacterium-Escherichia coli shuttle vector. Plasmid. 1991 Mar;25(2):149–153. doi: 10.1016/0147-619x(91)90029-v. [DOI] [PubMed] [Google Scholar]
  23. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R., Jr Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci U S A. 1988 Sep;85(18):6987–6991. doi: 10.1073/pnas.85.18.6987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Stahl D. A., Urbance J. W. The division between fast- and slow-growing species corresponds to natural relationships among the mycobacteria. J Bacteriol. 1990 Jan;172(1):116–124. doi: 10.1128/jb.172.1.116-124.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  26. Wohlleben W., Arnold W., Bissonnette L., Pelletier A., Tanguay A., Roy P. H., Gamboa G. C., Barry G. F., Aubert E., Davies J. On the evolution of Tn21-like multiresistance transposons: sequence analysis of the gene (aacC1) for gentamicin acetyltransferase-3-I(AAC(3)-I), another member of the Tn21-based expression cassette. Mol Gen Genet. 1989 Jun;217(2-3):202–208. doi: 10.1007/BF02464882. [DOI] [PubMed] [Google Scholar]
  27. Young L. S. Mycobacterium avium complex infection. J Infect Dis. 1988 May;157(5):863–867. doi: 10.1093/infdis/157.5.863. [DOI] [PubMed] [Google Scholar]

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