1887

Abstract

The genome of H37Rv includes a homologue of the CRP/FNR (cAMP receptor protein/fumarate and nitrate reduction regulator) family of transcription regulators encoded by Rv3676. Sequencing of the orthologous gene from attenuated Bacille Calmette–Guérin (BCG) strains revealed point mutations that affect the putative DNA-binding and cNMP-binding domains of the encoded protein. These mutations are not present in the published sequences of the Rv3676 orthologues in , or . An reporter system was used to show that the Rv3676 protein binds to DNA sites for CRP, but this DNA binding was decreased or abolished with the Rv3676 protein counterparts from BCG strains. The DNA-binding ability of the Rv3676 protein was decreased by the introduction of base changes corresponding to the BCG point mutations. Conversely, the DNA binding of the BCG Rv3676 proteins from BCG strains was restored by removing the mutations. These data show that in this reporter system the point mutations present in the Rv3676 orthologue in BCG strains render its function defective (early strains) or abolished (late strains) and suggest that this protein might be naturally defective in BCG strains. This raises the possibility that a contributing factor to the attenuation of BCG strains may be an inability of this global regulator to control the expression of genes required for survival and persistence.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.27444-0
2005-02-01
2019-10-14
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/2/mic1510547.html?itemId=/content/journal/micro/10.1099/mic.0.27444-0&mimeType=html&fmt=ahah

References

  1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  2. Behr, M. A. & Small, P. M. ( 1997; ). Has BCG attenuated to impotence? Nature 389, 133–134.
    [Google Scholar]
  3. Behr, M. A. & Small, P. M. ( 1999; ). A historical and molecular phylogeny of BCG strains. Vaccine 17, 915–922.[CrossRef]
    [Google Scholar]
  4. Behr, M. A., Wilson, M. A., Gill, W. P., Salamon, H., Schoolnik, G. K., Rane, S. & Small, P. M. ( 1999; ). Comparative genomics of BCG vaccines by whole-genome DNA microarray. Science 284, 1520–1523.[CrossRef]
    [Google Scholar]
  5. Behr, M. A., Schroeder, B. G., Brinkman, J. N., Slayden, R. A. & Barry, C. E., III ( 2000; ). A point mutation in the mma3 gene is responsible for impaired methoxymycolic acid production in Mycobacterium bovis BCG strains obtained after 1927. J Bacteriol 182, 3394–3399.[CrossRef]
    [Google Scholar]
  6. Bell, A. & Busby, S. ( 1994; ). Location and orientation of an activating region in the Escherichia coli transcription factor, FNR. Mol Microbiol 11, 383–390.[CrossRef]
    [Google Scholar]
  7. Bell, A. I., Gaston, K. L., Cole, J. A. & Busby, S. J. ( 1989; ). Cloning of binding sequences for the Escherichia coli transcription activators, FNR and CRP: location of bases involved in discrimination between FNR and CRP. Nucleic Acids Res 17, 3865–3874.[CrossRef]
    [Google Scholar]
  8. Bell, A., Gaston, K., Williams, R., Chapman, K., Kolb, A., Buc, H., Minchin, S., Williams, J. & Busby, S. ( 1990; ). Mutations that alter the ability of the Escherichia coli cyclic AMP receptor protein to activate transcription. Nucleic Acids Res 18, 7243–7250.[CrossRef]
    [Google Scholar]
  9. Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. ( 2002; ). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol 43, 717–731.[CrossRef]
    [Google Scholar]
  10. Bloom, B. R. & Murray, C. J. L. ( 1992; ). Tuberculosis: commentary on a reemergent killer. Science 257, 1055–1064.[CrossRef]
    [Google Scholar]
  11. Botsford, J. L. & Harman, J. G. ( 1992; ). Cyclic AMP in prokaryotes. Microbiol Rev 56, 100–122.
    [Google Scholar]
  12. Brosch, R., Gordon, S. V., Billault, A., Garnier, T., Eiglmeier, K., Soravito, C., Barrell, B. G. & Cole, S. T. ( 1998; ). Use of a Mycobacterium tuberculosis H37Rv bacterial artificial chromosome library for genome mapping, sequencing, and comparative genomics. Infect Immun 66, 2221–2229.
    [Google Scholar]
  13. Busby, S. & Ebright, R. H. ( 1999; ). Transcription activation by catabolite activator protein (CAP). J Mol Biol 293, 199–213.[CrossRef]
    [Google Scholar]
  14. Busby, S. & Kolb, A. ( 1996; ). The CAP modulon. In Regulation of Gene Expression in Escherichia coli, pp. 255–279. Edited by E. C. C. Lin & A. S. Lynch. Georgetown, TX: R. G. Landes Company.
  15. Busby, S., Kotlarz, D. & Buc, H. ( 1983; ). Deletion mutagenesis of the Escherichia coli galactose operon promoter region. J Mol Biol 167, 259–274.[CrossRef]
    [Google Scholar]
  16. Calmette, A. ( 1927; ). La Vaccination Preventive Contre la Tuberculose. Paris: Masson et cie.
  17. Calmette, A. & Guérin, C. ( 1911; ). Recherches expérimentales sur la défense de l'organisme contre l'infection tuberculeuse. Ann Inst Pasteur 25, 625–641.
    [Google Scholar]
  18. Casadaban, M. J. & Cohen, S. N. ( 1980; ). Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138, 179–207.[CrossRef]
    [Google Scholar]
  19. Chou, P. Y. & Fasman, G. D. ( 1974; ). Conformational parameters for amino acids in helical, β-sheet, and random coil regions calculated from proteins. Biochemistry 13, 211–222.[CrossRef]
    [Google Scholar]
  20. Chou, P. Y. & Fasman, G. D. ( 1978; ). Empirical predictions of protein conformation. Annu Rev Biochem 47, 251–276.[CrossRef]
    [Google Scholar]
  21. 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; erratum, 396, 190.[CrossRef]
    [Google Scholar]
  22. Cole, S. T., Eiglmeier, K., Parkhill, J. & 41 other authors ( 2001; ). Massive gene decay in the leprosy bacillus. Nature 409, 1007–1011.[CrossRef]
    [Google Scholar]
  23. Comstock, G. W. ( 1988; ). Identification of an effective vaccine against tuberculosis. Am Rev Respir Dis 138, 479–480.[CrossRef]
    [Google Scholar]
  24. Fleischmann, R. D., Alland, D., Eisen, J. A. & 23 other authors ( 2002; ). Whole-genome comparison of Mycobacterium tuberculosis clinical and laboratory strains. J Bacteriol 184, 5479–5490.[CrossRef]
    [Google Scholar]
  25. Frimodt-Moller, J. ( 1939; ). Dissociation of Tubercle Bacilli. London: H. K. Lewis.
  26. Galtier, N., Gouy, M. & Gautier, C. ( 1996; ). seaview and phylo_win: two graphic tools for sequence alignment and molecular phylogeny. Comput Appl Biosci 12, 543–548.
    [Google Scholar]
  27. Garnier, T., Eiglmeier, K., Camus, J. C. & 19 other authors ( 2003; ). The complete genome sequence of Mycobacterium bovis. Proc Natl Acad Sci U S A 100, 7877–7882.[CrossRef]
    [Google Scholar]
  28. Glickman, M. S., Cox, J. S. & Jacobs, W. R., Jr ( 2000; ). A novel mycolic acid cyclopropane synthetase is required for cording, persistence, and virulence of Mycobacterium tuberculosis. Mol Cell 5, 717–727.[CrossRef]
    [Google Scholar]
  29. Green, J., Scott, C. & Guest, J. R. ( 2001; ). Functional versatility in the CRP-FNR superfamily of transcription factors: FNR and FLP. Adv Microbiol Physiol 44, 1–34.
    [Google Scholar]
  30. Guérin, C. & Rosenthal, S. R. ( 1957; ). The history of BCG: early history. In BCG Vaccination Against Tuberculosis, pp. 48–57. Edited by S. R. Rosenthal. London: Churchill.
  31. Jayaraman, P. S., Cole, J. A. & Busby, S. J. ( 1989; ). Mutational analysis of the nucleotide sequence at the FNR-dependent nirB promoter in Escherichia coli. Nucleic Acids Res 17, 135–145.[CrossRef]
    [Google Scholar]
  32. Kim, H.-J., Kim, T.-H., Kim, Y. & Lee, H.-S. ( 2004; ). Identification and characterization of glxR, a gene involved in regulation of glyoxylate bypass in Corynebacterium glutamicum. J Bacteriol 186, 3453–3460.[CrossRef]
    [Google Scholar]
  33. Kolb, A., Busby, S., Garges, S. & Adhya, S. ( 1993; ). Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem 62, 749–795.[CrossRef]
    [Google Scholar]
  34. Lewis, K. N., Liao, R., Guinn, K. M., Hickey, M. J., Smith, S., Behr, M. A. & Sherman, D. R. ( 2003; ). Deletion of RD1 from Mycobacterium tuberculosis mimics Bacille Calmette–Guérin attenuation. J Infect Dis 187, 117–123.[CrossRef]
    [Google Scholar]
  35. Lissenden, S., Mohan, S., Overton, T. & 9 other authors ( 2000; ). Identification of transcriptional activators that regulate gonococcal adaptation from aerobic to anaerobic or oxygen-limited growth. Mol Microbiol 37, 839–855.[CrossRef]
    [Google Scholar]
  36. Lodge, J., Fear, J., Busby, S., Gunasekaran, P. & Kamini, N. R. ( 1992; ). Broad host range plasmids carrying the Escherichia coli lactose and galactose operons. FEMS Microbiol Lett 95, 271–276.[CrossRef]
    [Google Scholar]
  37. Mahairas, G. G., Sabo, P. J., Hickey, M. J., Singh, D. C. & Stover, C. K. ( 1996; ). Molecular analysis of genetic differences between Mycobacterium bovis BCG and virulent M. bovis. J Bacteriol 178, 1274–1282.
    [Google Scholar]
  38. Mattow, J., Jungblut, P. R., Schaible, U. E., Mollenkopf, H.-J., Lamer, S., Zimny-Arndt, U., Hagens, K., Müller, E.-C. & Kaufmann, S. H. E. ( 2001; ). Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains. Electrophoresis 22, 2936–2946.[CrossRef]
    [Google Scholar]
  39. McKinney, J. D., Honer zu Bentrup, K., Munoz-Elias, E. J. & 9 other authors ( 2000; ). Persistence of Mycobacterium tuberculosis in macrophages and mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735–738.[CrossRef]
    [Google Scholar]
  40. Mukamolova, G. V., Turapov, O. A., Young, D. I., Kaprelyants, A. S., Kell, D. B. & Young, M. ( 2002; ). A family of autocrine growth factors in Mycobacterium tuberculosis. Mol Microbiol 46, 623–635.[CrossRef]
    [Google Scholar]
  41. Pym, A. S., Brodin, P., Brosch, R., Huerre, M. & Cole, S. T. ( 2002; ). Loss of RD1 contributed to the attenuation of the live tuberculosis vaccines Mycobacterium bovis BCG and Mycobacterium microti. Mol Microbiol 46, 709–717.[CrossRef]
    [Google Scholar]
  42. Spiro, S. & Guest, J. R. ( 1988; ). Inactivation of the FNR protein of Escherichia coli by targeted mutagenesis in the N-terminal region. Mol Microbiol 2, 701–707.[CrossRef]
    [Google Scholar]
  43. Spiro, S. & Guest, J. R. ( 1990; ). FNR and its role in oxygen-related gene expression in Escherichia coli. FEMS Microbiol Rev 75, 399–428.
    [Google Scholar]
  44. van Soolingen, D., Hermans, P. W. M., de Haas, P. E. W., Soll, D. R. & van Embden, J. D. A. ( 1991; ). Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 29, 2578–2586.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.27444-0
Loading
/content/journal/micro/10.1099/mic.0.27444-0
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