1887

Abstract

A 9·2 kb cryptic plasmid, pMF1, was isolated from strain 110 and its restriction map constructed. A 42 kb dIII fragment of pMF1 was found to support replication in mycobacteria and this fragment was cloned and sequenced to characterize the replication elements of the plasmid. Computer analysis identified a putative Rep protein (362 amino acids) with high homology to the putative Rep protein of the plasmid pCLP and limited homology, mostly in the N-terminal region, to the Rep proteins of pLR7, pJAZ38 and pMSC262. A region containing a putative site was located upstream of the gene; this region displayed high homology at the nucleotide level with the predicted of pCLP and pJAZ38. A plasmid carrying the 42 kb dIII fragment and a kanamycin resistance marker, designated pBP4, was maintained as a single-copy plasmid in and was stably inherited in the absence of antibiotic selection. Plasmid pBP4 was incompatible with the pJAZ38 replicon but was compatible with the widely used pAL5000 replicon, indicating that among the mycobacterial vectors now available there are two incompatibility groups. Significantly, the plasmid was able to replicate in the pathogen , making it a useful tool for gene expression studies. To provide a choice of restriction sites and easy manipulation, a 21 kb fragment containing the minimal replication region was cloned to make the mycobacterial shuttle vector pBP10, which showed similar stability to pBP4.

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2000-02-01
2024-04-25
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References

  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. Beggs M. L., Crawford J. T., Eisenach K. D. 1995; Isolation and sequencing of the replication region of Mycobacterium avium plasmid pLR7. J Bacteriol 177:4836–4840
    [Google Scholar]
  3. Bouet J. Y., Funnell B. E. 1999; P1 ParA interacts with the P1 partition complex at parS and an ATP-ADP switch controls ParA activities. EMBO J 18:1415–1424 [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. Crawford J. T., Bates J. H. 1984; Restriction endonuclease mapping and cloning of Mycobacterium intracellulare plasmid pLR7. Gene 27:331–333 [CrossRef]
    [Google Scholar]
  6. Davis M. A., Radnedge L., Martin K. A., Hayes F., Youngren B., Austin S. J. 1996; The P1 ParA protein and its ATPase activity play a direct role in the segregation of plasmid copies to daughter cells. Mol Microbiol 21:1029–1036 [CrossRef]
    [Google Scholar]
  7. Dodd H. M., Bennett P. M. 1987; The R46 site-specific recombination system is a homologue of the Tn3 and γδ (Tn1000) cointegrate resolution system. J Gen Microbiol 133:2031–2039
    [Google Scholar]
  8. Dominy C. N., Deane S. M., Rawlings D. E. 1997; A geographically widespread plasmid from Thiobacillus ferrooxidans has genes for ferredoxin-, FNR-, prismane- and NADH-oxidoreductase-like proteins which are also located on the chromosome. Microbiology 143:3123–3136 [CrossRef]
    [Google Scholar]
  9. Gavigan J. A., Ainsa J. A., Perez E., Otal I., Martin C. 1997; Isolation by genetic labeling of a new mycobacterial plasmid, pJAZ38, from Mycobacterium fortuitum. J Bacteriol 179:4115–4122
    [Google Scholar]
  10. Hatfull G. F. 1993; Genetic transformation of mycobacteria. Trends Microbiol 1:310–314 [CrossRef]
    [Google Scholar]
  11. Labidi A., Dauguet C., Goh K. S., David H. L. 1984; Plasmid profiles of Mycobacterium fortuitum complex isolates. Curr Microbiol 11:235–240 [CrossRef]
    [Google Scholar]
  12. Labidi A., David H. L., Roulland-Dussoix D. 1985; Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000. Ann Inst Pasteur Microbiol 136:209–215
    [Google Scholar]
  13. Lydiate D. J., Ashby A. M., Henderson D. J., Kieser H. M., Hopwood D. A. 1989; Physical and genetic characterization of chromosomal copies of the Streptomyces coelicolor mini-circle. J Gen Microbiol 135:941–955
    [Google Scholar]
  14. Meissner P. S., Falkinham J. O. III 1984; Plasmid-encoded mercuric reductase in Mycobacterium scrofulaceum. J Bacteriol 157:669–672
    [Google Scholar]
  15. Papavinasasundaram K. G., Movahedzadeh F., Keer J. T., Stoker N. G., Colston M. J., Davis E. O. 1997; Mycobacterial recA is cotranscribed with a potential regulatory gene called recX. Mol Microbiol 24:141–153 [CrossRef]
    [Google Scholar]
  16. 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]
  17. Pelicic V., Reyrat J.-M., Gicquel B. 1998; Genetic advances for studying Mycobacterium tuberculosis pathogenicity. Mol Microbiol 28:413–420 [CrossRef]
    [Google Scholar]
  18. Picardeau M., Vincent V. 1997; Characterization of large linear plasmids in mycobacteria. J Bacteriol 179:2753–2756
    [Google Scholar]
  19. Picardeau M., Vincent V. 1998; Mycobacterial linear plasmids have an invertron-like structure related to other linear replicons in actinomycetes. Microbiology 144:1981–1988 [CrossRef]
    [Google Scholar]
  20. Picardeau M., Le Dantec C., Vincent V. 2000; Analysis of the internal replication region of a mycobacterial linear plasmid. Microbiology 146:305–313
    [Google Scholar]
  21. Qin M., Taniguchi H., Mizuguchi Y. 1994; Analysis of the replication region of a mycobacterial plasmid, pMSC262. J Bacteriol 176:419–425
    [Google Scholar]
  22. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  23. Snapper S. B., Lugosi L., Jekkel A., Melton R. E., Kieser T., Bloom B. R., Jacobs W. R. Jr 1988; Lysogeny and transformation in mycobacteria: stable expression of foreign genes. Proc Natl Acad Sci USA 85:6987–6991 [CrossRef]
    [Google Scholar]
  24. 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]
  25. Stolt P., Stoker N. G. 1996a; Functional definition of regions necessary for replication and incompatibility in the Mycobacterium fortuitum plasmid pAL5000. Microbiology 142:2795–2802 [CrossRef]
    [Google Scholar]
  26. Stolt P., Stoker N. G. 1996b; Protein–DNA interactions in the ori region of the Mycobacterium fortuitum plasmid pAL5000. J Bacteriol 178:6693–6700
    [Google Scholar]
  27. Stolt P., Stoker N. G. 1997; Mutational analysis of the regulatory region of the Mycobacterium plasmid pAL5000. Nucleic Acids Res 25:3840–3846 [CrossRef]
    [Google Scholar]
  28. 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]
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