1887

Abstract

Disruption of the gene of mc155, by standard gene replacement methods, revealed that there are two copies of this gene within a large duplication of the mc155 genome. AdhC and AdhC mutants were obtained when one or two copies, respectively, were disrupted by homologous recombination. Southern blot analysis of I restriction digests of the DNA from these mutants and from wild-type mc155, resolved by PFGE, showed that the duplication size may be at least ∼250 kb. The single and double knockout mutants were characterized and compared with the wild-type. A growth disadvantage and a different morphology were associated with the loss of expression of one or both of the copies, but both mutants were still acid-fast. Findings in this study indicate that the process of chromosomal duplication in is ongoing and remains a potent source of genome dynamics. Hence, the mc155 genome might be larger than previously thought.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-147-12-3281
2001-12-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/147/12/1473281a.html?itemId=/content/journal/micro/10.1099/00221287-147-12-3281&mimeType=html&fmt=ahah

References

  1. Anderson, R. P. & Roth, J. R. (1977). Tandem genetic duplications in phage and bacteria. Annu Rev Microbiol 31, 473-505.[CrossRef] [Google Scholar]
  2. Anderson, P. & Roth, J. (1981). Spontaneous tandem genetic duplications in Salmonella typhimurium arise by unequal recombination between rRNA (rrn) cistrons. Proc Natl Acad Sci USA 78, 3113-3117.[CrossRef] [Google Scholar]
  3. Baulard, A., Gurdyal, S., Brennan, B. & Brennan, P. J. (1999). The cell-wall core of Mycobacterium: structure, biogenesis and genetics. In Mycobacteria. Molecular Biology and Virulence , pp. 240-259. Edited by C. Ratledge & J. Dale. London:Blackwell Science.
  4. Besra, G. S., Morehouse, C. B., Rittner, C. M., Waechter, C. J. & Brennan, P. J. (1997). Biosynthesis of mycobacterial lipoarabinomannan. J Biol Chem 272, 18460-18466.[CrossRef] [Google Scholar]
  5. Bhatt, K., Banerjee, S. K. & Chakraborty, P. K. (2000). Evidence that phosphate specific transporter is amplified in a fluoroquinolone resistant Mycobacterium smegmatis. Eur J Biochem 267, 4028-4032.[CrossRef] [Google Scholar]
  6. Brosch, R., Gordon, S. V., Buchrieser, C., Pym, A. S., Garnier, T. & Cole, S. T. (2000). Comparative genomics uncovers large tandem chromosomal duplications in Mycobacterium bovis BCG Pasteur. Yeast 17, 111-123. [Google Scholar]
  7. Cole, S. T., Brosch, R., Parkhill, J. & 38 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef] [Google Scholar]
  8. De Bruyn, J., Johannes, A., Weckx, M. & Beumer-Jochmans, M. P. (1981a). Partial purification and characterization of an alcohol dehydrogenase of Mycobacterium tuberculosis var. bovis (BCG). J Gen Microbiol 124, 359-363. [Google Scholar]
  9. De Bruyn, J., Weckx, M. & Beumer-Jochmans, M. P. (1981b). Effect of zinc deficiency on Mycobacterium tuberculosis var. bovis (BCG). J Gen Microbiol 124, 353-357. [Google Scholar]
  10. Dye, C., Scheele, S., Dolin, P., Pathania, V. & Raviglione, M. C. (1999). Consensus statement. Global burden of tuberculosis: estimated incidence, prevalence, and mortality by country. WHO global surveillance and monitoring project. J Am Med Assoc 282, 677-686.[CrossRef] [Google Scholar]
  11. Edlund, T. & Normark, S. (1981). Recombination between short DNA homologies causes tandem duplication. Nature 292, 269-271.[CrossRef] [Google Scholar]
  12. Evans, M. & Dyson, P. (1993). Pulsed-field gel electrophoresis of Streptomyces lividans DNA. Trends Genet 9, 72.[CrossRef] [Google Scholar]
  13. Galamba, A., Soetaert, K., Buyssens, P., Monnaie, D., Jacobs, P. & Content, J. (2001). Molecular and biochemical characterisation of Mycobacterium smegmatis alcohol dehydrogenase C. FEMS Microbiol Lett 196, 51-56.[CrossRef] [Google Scholar]
  14. Haack, K. R. & Roth, J. R. (1995). Recombination between chromosomal IS200 elements supports frequent duplication formation in Salmonella typhimurium. Genetics 141, 1245-1252. [Google Scholar]
  15. Hauser, P. M. & Karamata, D. (1994). Characterization of the chromosomes of Bacillus subtilis merodiploid strains by quantitative DNA–DNA hybridization. Microbiology 140, 1605-1611.[CrossRef] [Google Scholar]
  16. Heath, J. D. (1992).Control of chromosomal rearrangements in Escherichia coli. PhD thesis, University of Texas, Health Science Center.
  17. Hinds, J., Mahenthiralingam, E., Kempsell, K. E., Duncan, K., Stokes, R. W., Parish, T. & Stoker, N. G. (1999). Enhanced gene replacement in mycobacteria. Microbiology 145, 519-527.[CrossRef] [Google Scholar]
  18. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  19. Lee, R. E., Brennan, P. J. & Besra, G. S. (1996).Mycobacterium tuberculosis cell envelope. Curr Top Microbiol Immunol 215, 1-27. [Google Scholar]
  20. Levy-Frebault, V. V., Thorel, M. F., Varnerot, A. & Gicquel, B. (1989). DNA polymorphism in Mycobacterium paratuberculosis, ‘wood pigeon mycobacteria’, and related mycobacteria analyzed by field inversion gel electrophoresis. J Clin Microbiol 27, 2823-2826. [Google Scholar]
  21. Lupski, J. R., Roth, J. R. & Weinstock, G. M. (1996). Chromosomal duplications in bacteria, fruit flies, and humans. Am J Hum Genet 58, 21-27. [Google Scholar]
  22. McFadden, J. (1996). Recombination in mycobacteria. Mol Microbiol 21, 205-211.[CrossRef] [Google Scholar]
  23. Menard, R., Sansonetti, P. J. & Parsot, C. (1993). Nonpolar mutagenesis of the ipa genes defines IpaB, IpaC, and IpaD as effectors of Shigella flexneri entry into epithelial cells. J Bacteriol 175, 5899-5906. [Google Scholar]
  24. Merkel, J., Feldblyum, T., Young, B., Kriakov, J., Jacobs, W. R.Jr & Fleishmann, R. D. (2001). Sequencing and assembly of the Mycobacterium smegmatis genome. In Keystone Symposium B1: Molecular and Cellular Aspects of Tuberculosis Research in the Post Genome Era , pp. 62. Edited by G. Kaplan, S. T. Cole & B. Brennan. Taos,New Mexico.
  25. Mikusova, K., Mikus, M., Besra, G. S., Hancock, I. & Brennan, P. J. (1996). Biosynthesis of the linkage region of the mycobacterial cell wall. J Biol Chem 271, 7820-7828.[CrossRef] [Google Scholar]
  26. Papavinasasundaram, K. G., Colston, M. J. & Davis, E. O. (1998). Construction and complementation of a recA deletion mutant of Mycobacterium smegmatis reveals that the intein in Mycobacterium tuberculosis recA does not affect RecA function. Mol Microbiol 30, 525-534.[CrossRef] [Google Scholar]
  27. 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, 1969-1975. [Google Scholar]
  28. Parish, T., Gordhan, B. G., McAdam, R. A., Duncan, K., Mizrahi, V. & Stoker, N. G. (1999). Production of mutants in amino acid biosynthesis genes of Mycobacterium tuberculosis by homologous recombination. Microbiology 145, 3497-3503. [Google Scholar]
  29. Pavelka, M. S.Jr & Jacobs, W. R.Jr (1999). Comparison of the construction of unmarked deletion mutations in Mycobacterium smegmatis, Mycobacterium bovis bacillus Calmette-Guerin, and Mycobacterium tuberculosis H37Rv by allelic exchange. J Bacteriol 181, 4780-4789. [Google Scholar]
  30. Pelicic, V., Reyrat, J. M. & Gicquel, B. (1996a). Expression of the Bacillus subtilis sacB gene confers sucrose sensitivity on mycobacteria. J Bacteriol 178, 1197-1199. [Google Scholar]
  31. Pelicic, V., Reyrat, J. M. & Gicquel, B. (1996b). Generation of unmarked directed mutations in mycobacteria using sucrose counter-selectable suicide vectors. Mol Microbiol 20, 919-925.[CrossRef] [Google Scholar]
  32. Philipp, W. J., Poulet, S., Eiglmeier, K. & 7 other authors (1996). An integrated map of the genome of the tubercle bacillus, Mycobacterium tuberculosis H37Rv, and comparison with Mycobacterium leprae. Proc Natl Acad Sci USA 93, 3132–3137.[CrossRef] [Google Scholar]
  33. Ray, T., Weaden, J. & Dyson, P. (1992). Tris-dependent site-specific cleavage of Streptomyces lividans DNA. FEMS Microbiol Lett 96, 247-252.[CrossRef] [Google Scholar]
  34. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  35. Spector, T. (1978). Refinement of the Coomassie blue method of protein quantitation. A simple and linear spectrophotometric assay for less than or equal to 0·5 to 5·0 microgram of protein. Anal Biochem 86, 142-146.[CrossRef] [Google Scholar]
  36. Stelandre, M., Bosseloir, Y., De Bruyn, J., Maes, P. & Content, J. (1992). Cloning and sequence analysis of the gene encoding an NADP-dependent alcohol dehydrogenase in Mycobacterium bovis BCG. Gene 121, 79-86.[CrossRef] [Google Scholar]
  37. Tekaia, F., Gordon, S. V., Garnier, T., Brosch, R., Barrell, B. G. & Cole, S. T. (1999). Analysis of the proteome of Mycobacterium tuberculosis in silico. Tuber Lung Dis 79, 329-342.[CrossRef] [Google Scholar]
  38. Wilkin, J. M., Soetaert, K., Stelandre, M. & 7 other authors (1999). Overexpression, purification and characterization of Mycobacterium bovis BCG alcohol dehydrogenase. Eur J Biochem 262, 299–307.[CrossRef] [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-147-12-3281
Loading
/content/journal/micro/10.1099/00221287-147-12-3281
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error