A 3·9 kb DNA fragment containing the gene region of was cloned and its nucleotide sequence was determined. Nucleotide sequence analyses indicated that this region encodes three genes in the order (ribosomal protein L34), (the putative initiator protein) and (the β subunit of DNA polymerase III). The intergenic regions between the and genes were found to contain several putative DnaA boxes, 9 nt long DnaA protein recognition sequences. A DNA fragment containing the 3′ but not the 5′ flanking region of the gene when cloned in plasmids, which are otherwise non-replicative in mycobacteria, exhibited autonomous replication activity in but not in BCG and . The 5′ flanking region of , on the other hand, exhibited autonomous replication activity in BCG but not in and . The implications of these results for the understanding of the replication initiation process are discussed.


Article metrics loading...

Loading full text...

Full text loading...



  1. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. (1990). Basic local alignment search tool. J Mol Biol 215, 403-410.[CrossRef] [Google Scholar]
  2. Atlung, T., Lobner-Olesen, A. & Hansen, F. G. (1987). Overproduction of DnaA protein stimulates initiation of chromosome replication in Escherichia coli. Mol Gen Genet 206, 52-59. [Google Scholar]
  3. Bramhill, D. & Kornberg, A. (1988). A model for initiation at origins of DNA replication. Cell 54, 915-918.[CrossRef] [Google Scholar]
  4. Cole, S. T., Brosch, R., Parkhill, J. & 39 other authors (1998). Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Nature 393, 537–544.[CrossRef] [Google Scholar]
  5. Foley-Thomas, E. M., Whipple, D. L., Bermudez, L. E. & Barletta, R. G. (1995). Phage infection, transfection and transformation of Mycobacterium avium complex and Mycobacterium paratuberculosis. Microbiology 141, 1173-1181.[CrossRef] [Google Scholar]
  6. Fujita, M. Q., Yoshikawa, H. & Ogasawara, N. (1990). Structure of the dnaA region of Micrococcus luteus: conservation and variations among eubacteria. Gene 93, 73-78.[CrossRef] [Google Scholar]
  7. Goto, Y., Taniguchi, H., Udou, T., Mizuguchi, Y. & Tokunaga, T. (1991). Development of a new host vector system in mycobacteria. FEMS Microbiol Lett 83, 277-282.[CrossRef] [Google Scholar]
  8. Jacobs, W. R.Jr, Kalpana, G. V., Cirillo, J. D., Pascopella, L., Snapper, S. B., Udani, R., Jones, W., Barletta, R. G. & Bloom, B. R. (1991). Genetic systems for mycobacteria. Methods Enzymol 204, 537-555. [Google Scholar]
  9. Kornberg, A. & Baker, T. (1991).DNA Replication. New York: W. H. Freeman.
  10. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685.[CrossRef] [Google Scholar]
  11. Madiraju, M. V. V. S., Qin, M.-H. & Rajagopalan, M. (1999). Development of simple and efficient protocol for isolation of plasmids from mycobacteria using zirconia beads. Lett Appl Microbiol (in press).
  12. Moriya, S., Atlung, T., Hansen, F. G., Yoshikawa, H. & Ogasawara, N. (1992). Cloning of an autonomously replicating sequence (ars) from the Bacillus subtilis chromosome. Mol Microbiol 6, 309-315.[CrossRef] [Google Scholar]
  13. Qin, M.-H., Madiraju, M. V. V. S., Zachariah, S. & Rajagopalan, M. (1997). Characterization of the oriC region of Mycobacterium smegmatis. J Bacteriol 179, 6311-6317. [Google Scholar]
  14. Qin, M.-H., Madiraju, M. V. V. S. & Rajagopalan, M. (1999). Characterization of the functional replication origin of Mycobacterium tuberculosis. Gene 233, 121-130.[CrossRef] [Google Scholar]
  15. Rajagopalan, M., Qin, M.-H., Steingrube, V. A., Nash, D. R., Wallace, R. J.Jr & Madiraju, M. V. V. S. (1995a). Amplification and cloning of the Mycobacterium tuberculosis dnaA gene. Gene 163, 75-79.[CrossRef] [Google Scholar]
  16. Rajagopalan, M., Qin, M. H., Nash, D. R. & Madiraju, M. V. V. S. (1995b).Mycobacterium smegmatis dnaA region and autonomous replication activity. J Bacteriol 177, 6527-6535. [Google Scholar]
  17. Salazar, L., Fsihi, H., Rossi, E. D., Riccardi, G., Rios, C., Cole, S. T. & Takiff, H. E. (1996). Organization of the origins of replication of the chromosomes of Mycobacterium smegmatis, Mycobacterium leprae and Mycobacterium tuberculosis and isolation of a functional origin from M. smegmatis. Mol Microbiol 20, 283-293.[CrossRef] [Google Scholar]
  18. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989).Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  19. Skarstad, K. & Boye, E. (1994). The initiator protein DnaA: evolution, properties and function. Biochim Biophys Acta 1217, 111-130.[CrossRef] [Google Scholar]
  20. Skovgaard, O. & Hansen, F. G. (1987). Comparison of the dnaA nucleotide sequences of Escherichia coli, Salmonella typhimurium and Serratia marcescens. J Bacteriol 169, 3976-3981. [Google Scholar]
  21. Smith, D. W., Yee, T. W., Baird, C. & Krishnapillai, V. (1991). Pseudomonad replication origins: a paradigm for bacterial origins? Mol Microbiol 5, 2581-2587.[CrossRef] [Google Scholar]
  22. Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R.Jr (1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol 4, 1911-1919.[CrossRef] [Google Scholar]
  23. Stover, C. K., de la Cruz, V. F., Fuerst, T. R. & 11 other authors (1991). New use of BCG for recombinant vaccines. Nature 351, 456–460.[CrossRef] [Google Scholar]
  24. Studier, F. W., Rosenberg, A. H., Dunn, J. J. & Dubendorff, J. W. (1990). Use of T7 polymerase to direct expression of cloned genes. Methods Enzymol 185, 60-89. [Google Scholar]
  25. Wahl, S. M., Greenwell-Wild, T., Peng, G., Hale-Donze, H., Doherty, T. M., Mizel, D. & Orenstein, J. M. (1998).Mycobacterium avium complex augments macrophage HIV-1 production and increases CCR5 expression. Proc Natl Acad Sci USA 95, 12574-12579.[CrossRef] [Google Scholar]
  26. Wards, B. J. & Collins, D. M. (1996). Electroporation at elevated temperatures substantially improves transformation efficiency of slow-growing mycobacteria. FEMS Lett 145, 101-105.[CrossRef] [Google Scholar]
  27. Wheeler, P. R. & Ratledge, C. (1994). Metabolism of Mycobacterium tuberculosis. In Tuberculosis: Pathogenesis, Protection and Control, pp. 353-358. Edited by B. R. Bloom. Washington, DC: American Society for Microbiology.
  28. Yee, T. W. & Smith, D. W. (1990).Pseudomonas chromosomal replication origins: A bacterial class distinct from Escherichia coli-type origins. Proc Natl Acad Sci USA 87, 1278-1282.[CrossRef] [Google Scholar]
  29. Zakrzewska-Czerwinksa, J. & Schrempf, H. (1992). Characterization of an autonomously replicating region from the Streptomyces lividans chromosome. J Bacteriol 174, 2688-2693. [Google Scholar]
  30. Zyskind, J. W. & Smith, D. W. (1986). The bacterial origin of replication, oriC. Cell 46, 489-490.[CrossRef] [Google Scholar]

Data & Media loading...

Most cited this month Most Cited RSS feed

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