1887

Abstract

The genome sequences of two virulent strains of (H37Rv and CDC 1551) are now available. CDC 1551 is a recent clinical isolate and H37Rv is a commonly used lab strain which has been subject to passage. The two strains have been shown to display differing phenotypes both and . The proteome of the two strains grown in liquid culture were examined over time to determine whether there are any major differences between them at the protein level and the differences were compared to the genome data. Total cell lysates of the two strains were analysed by two-dimensional electrophoresis. Approximately 1750 protein spots were visualized by silver staining and the protein profiles of the two strains were found to be highly similar. Out of a total of 17 protein spot differences, seven were unique to CDC 1551 and three to H37Rv. Two further spots showed increased intensity in H37Rv, one spot showed differing vertical mobility between the strains and four showed differing spot intensities with time. Twelve of the spot differences were identified using mass spectrometry; however, no obvious association with phenotype could be deduced. When genome differences were analysed and related to the proteome differences, a mobility shift identified in the MoxR protein could be explained by a point mutation at the gene level. This proteome analysis reveals that, despite having been maintained under vastly different conditions, namely passage and transmission, these two strains have remained highly similar.

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/content/journal/micro/10.1099/00221287-146-12-3205
2000-12-01
2022-01-18
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References

  1. Adessi C., Miege C., Albrieux C., Rabilloud T. 1997; Two-dimensional electrophoresis of membrane proteins: a current challenge for immobilized pH gradients. . Electrophoresis 18:127–135 [CrossRef]
    [Google Scholar]
  2. 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]
  3. Berthet F.-X., Rasmussen P. B., Rosenkrands I., Andersen P., Gicquel B. 1998; A Mycobacterium tuberculosis operon encoding ESAT-6 and a novel low-molecular-mass culture filtrate antigen. Microbiology 144:3195–3203 [CrossRef]
    [Google Scholar]
  4. Betts J. C., Smith M. A. 2000; Proteomics. In Mycobacterium tuberculosis Protocols, Methods in Microbiology In press Edited by Parish T., Stoker N. G. Totowa, NJ: Humana;
    [Google Scholar]
  5. Bishai W. R., Dannenberg A. M. Jr., Parrish N., Ruiz R., Chen P., Zook B. C., Johnson W., Boles J. W., Pitt M. L. 1999; Virulence of Mycobacterium tuberculosis CDC 1551 and H37Rv in rabbits evaluated by Lurie’s pulmonary tubercle count method. Infect Immun 67:4931–4934
    [Google Scholar]
  6. Bjellqvist B., Pasquali Ch., Ravier F., Sanchez J.-Ch., Hochstrasser D. F. 1993; A nonlinear wide-range immobilized pH gradient for two-dimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14:1357–1365 [CrossRef]
    [Google Scholar]
  7. Bradford M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 [CrossRef]
    [Google Scholar]
  8. Brosch R., Philipp W. J., Stavropoulos E., Colston M. J., Cole S. T., Gordon S. V. 1999; Genomic analysis reveals variation between Mycobacterium tuberculosis H37Rv and the attenuated M. tuberculosis H37Ra strain. Infect Immun 67:5768–5774
    [Google Scholar]
  9. 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]
  10. Cordwell S. J., Nouwens A. S., Verrills N. M., Basseal D. J., Walsh B. J. 2000; Subproteomics based upon protein cellular location and relative solubilities in conjunction with composite two-dimensional electrophoresis gels. Electrophoresis 21:1094–1103 [CrossRef]
    [Google Scholar]
  11. Delcher A. L., Kasi S., Fleischmann R. D., Peterson J., White O., Salzberg S. L. 1999; Alignment of whole genomes. Nucleic Acids Res 27:2369–2376 [CrossRef]
    [Google Scholar]
  12. Garbe T. R., Hibler N. S., Deretic V. 1996; Response of Mycobacterium tuberculosis to reactive oxygen and nitrogen intermediates. Mol Med 2:134–142
    [Google Scholar]
  13. Garbe T. R., Hibler N. S., Deretic V. 1999; Response to reactive nitrogen intermediates in Mycobacterium tuberculosis: induction of the 16-kilodalton α-crystallin homolog by exposure to nitric oxide donors. Infect Immun 67:460–465
    [Google Scholar]
  14. Gordon S. V., Brosch R., Billault A., Garnier T., Eiglmeier K., Cole S. T. 1999; Identification of variable regions in the genomes of tubercle bacilli using bacterial artificial chromosome arrays. Mol Microbiol 32:643–655 [CrossRef]
    [Google Scholar]
  15. Henzel W. J., Billeci T. M., Stults J. T., Wong S. C. 1993; Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci USA 90:5011–5015 [CrossRef]
    [Google Scholar]
  16. Herbert B. R., Molloy M. P., Gooley A. A., Walsh B. J., Bryson W. G., Williams K. L. 1998; Improved protein solubility in two-dimensional electrophoresis using tributyl phosphine as reducing agent. Electrophoresis 19:845–851 [CrossRef]
    [Google Scholar]
  17. Humphery-Smith I., Cordwell S. J., Blackstock W. P. 1997; Proteome research: complementarity and limitations with respect to the RNA and DNA-worlds. Electrophoresis 18:1217–1242 [CrossRef]
    [Google Scholar]
  18. Jacobs W., Brennan P., Curlin G.10 other authors 1996; Comparative sequencing. Science 274:17–18 [CrossRef]
    [Google Scholar]
  19. James P., Quadroni M., Carafoli E., Gonnet G. 1993; Protein identification by mass profile fingerprinting. Biochem Biophys Res Commun 195:58–64 [CrossRef]
    [Google Scholar]
  20. Jensen O. N., Podtelejnikov A., Mann M. 1996; Delayed extraction improves specificity in database searches by matrix-assisted laser desorption/ionisation peptide maps. Rapid Commun Mass Spectrom 10:1371–1378 [CrossRef]
    [Google Scholar]
  21. Jensen O. N., Wilm M., Shevchenko A., Mann M. 1999; Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels. Methods Mol Biol 112:513–530
    [Google Scholar]
  22. Jungblut P. R., Schaible U. E., Mollenkopf H.7 other authors 1999; Comparative proteome analysis of Mycobacterium tuberculosis and Mycobacterium bovis BCG strains: towards functional genomics of microbial pathogens. Mol Microbiol 33:1103–1117
    [Google Scholar]
  23. Lee B. Y., Horwitz M. A. 1995; Identification of macrophage and stress-induced proteins of Mycobacterium tuberculosis. J Clin Invest 96:245–249 [CrossRef]
    [Google Scholar]
  24. Manca C., Tsenova L., Barry C. E. III, Bergtold A., Freeman S., Haslett P. A., Musser J. M., Freedman V. H., Kaplan G. 1999; Mycobacterium tuberculosis CDC 1551 induces a more vigorous host response in vivo and in vitro, but is not more virulent than other clinical isolates. J Immunol 162:6740–6746
    [Google Scholar]
  25. Mann M., Hojrup P., Roepstorff P. 1993; Use of mass spectrometric molecular weight information to identify proteins in sequence databases. Biol Mass Spectrom 22:338–345 [CrossRef]
    [Google Scholar]
  26. Mollenkopf H. J., Jungblut P. R., Raupach B., Mattow J., Lamer S., Zimny-Arndt U., Schaible U. E., Kaufmann S. H. 1999; A dynamic two-dimensional polyacrylamide gel electrophoresis database: the mycobacterial proteome via Internet. Electrophoresis 20:2172–2180 [CrossRef]
    [Google Scholar]
  27. Molloy M. P., Herbert B. R., Walsh B. J., Tyler M. I., Traini M., Sanchez J. C., Hochstrasser D. F., Williams K. L., Gooley A. A. 1998; Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844 [CrossRef]
    [Google Scholar]
  28. Mustafa A. S., Amoudy H. A., Wiker H. G., Abal A. T., Ravn P., Oftung F., Anderson P. 1998; Comparison of antigen specific T cell responses of tuberculosis patients using complex or single antigens of Mycobacterium tuberculosis. Scand J Immunol 48:535–543 [CrossRef]
    [Google Scholar]
  29. O’Farrell P. H. 1975; High resolution two-dimensional electrophoresis of proteins. . J Biol Chem 250:4007–4021
    [Google Scholar]
  30. Pappin D., Hojrup P., Bleasby A. J. 1993; Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 3:327–332 [CrossRef]
    [Google Scholar]
  31. Rabilloud T., Adessi C., Giraudel A., Lunardi J. 1997; Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307–316 [CrossRef]
    [Google Scholar]
  32. Ravn P., Demissie A., Eguale T.11 other authors 1999; Human T cell responses to the ESAT-6 antigen secreted from. Mycobacterium tuberculosis. J Infect Dis 179:637–645 [CrossRef]
    [Google Scholar]
  33. Rosenkrands I., Weldingh K., Jacobsen S., Hansen C. V., Florio W., Gianetri I., Andersen P. 2000; Mapping and identification of Mycobacterium tuberculosis proteins by two-dimensional gel electrophoresis, microsequencing and immunodetection. Electrophoresis 21:935–948 [CrossRef]
    [Google Scholar]
  34. Santoni V., Rabilloud T., Doumas P., Rouquie D., Mansion M., Kieffer S., Garin J., Rossignol M. 1999; Towards the recovery of hydrophobic proteins on two-dimensional electrophoresis gels. . Electrophoresis 20:705–711 [CrossRef]
    [Google Scholar]
  35. Shevchenko A., Wilm M., Vorm O., Mann M. 1996a; Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem 68:850–858 [CrossRef]
    [Google Scholar]
  36. Shevchenko A., Jensen O. N., Podtelejnikov A. V.7 other authors 1996b; Linking genome and proteome by mass spectrometry: large-scale identification of yeast proteins from two dimensional gels. Proc Natl Acad Sci USA 93:14440–14445 [CrossRef]
    [Google Scholar]
  37. Skjøt R. L. V., Oettinger T., Rosenkrands I., Ravn P., Brock I., Jacobsen S., Andersen P. 2000; Comparative evaluation of low-molecular-mass proteins from Mycobacterium tuberculosis identifies members of the ESAT-6 family as immunodominant T-cell antigens. Infect Immun 68:214–220 [CrossRef]
    [Google Scholar]
  38. Sonnenberg M. G., Belisle J. T. 1997; Definition of Mycobacterium tuberculosis culture filtrate proteins by two-dimensional polyacrylamide gel electrophoresis, N-terminal amino acid sequencing, and electrospray mass spectrometry. Infect Immun 65:4515–4524
    [Google Scholar]
  39. Sorensen A. L., Nagai S., Houen G., Andersen P., Andersen A. B. 1995; Purification and characterization of a low-molecular-mass T-cell antigen secreted by Mycobacterium tuberculosis. Infect Immun 63:1710–1717
    [Google Scholar]
  40. 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]
  41. Ulrichs T., Munk M. E., Mollenkopf H., Behr-Perst S., Colangeli R., Gennaro M. L., Kaufmann S. H. 1998; Differential T cell responses to Mycobacterium tuberculosis ESAT-6 in tuberculosis patients and healthy donors. Eur J Immunol 28:3949–3958 [CrossRef]
    [Google Scholar]
  42. Urquhart B. L., Atsalos T. E., Roach D., Basseal D. J., Bjellqvist B., Britton W. L., Humphery-Smith I. 1997; ‘Proteomic contigs’ of Mycobacterium tuberculosis and Mycobacterium bovis (BCG) using novel immobilised pH gradients. . Electrophoresis 18:1384–1392 [CrossRef]
    [Google Scholar]
  43. Urquhart B. L., Cordwell S. J., Humphery-Smith I. 1998; Comparison of predicted and observed properties of proteins encoded in the genome of Mycobacterium tuberculosis H37Rv. Biochem Biophys Res Commun 253:70–79 [CrossRef]
    [Google Scholar]
  44. Valway S. E., Sanchez M. P., Shinnick T. F., Orme I., Agerton T., Hoy D., Jones J. S., Westmoreland H., Onorato I. M. 1998; An outbreak involving extensive transmission of a virulent strain of Mycobacterium tuberculosis. N Engl J Med 338:633–639 [CrossRef]
    [Google Scholar]
  45. Weldingh K., Rosenkrands I., Jacobsen S., Rasmussen P. B., Elhay M. J., Andersen P. 1998; Two-dimensional electrophoresis for analysis of Mycobacterium tuberculosis culture filtrate and purification and characterization of six novel proteins. . Infect Immun 66:3492–3500
    [Google Scholar]
  46. Wilkins M. R., Gasteiger E., Sanchez J. C., Bairoch A., Hochstrasser D. F. 1998; Two-dimensional gel electrophoresis for proteome projects: the effects of protein hydrophobicity and copy number. Electrophoresis 19:1501–1505 [CrossRef]
    [Google Scholar]
  47. Wilm M., Mann M. 1994; Electrospray and taylor-cone theory: Dole’s beam of macromolecules at last?. Int J Mass Spectrom Ion Proc 136:167–180 [CrossRef]
    [Google Scholar]
  48. Wilm M., Mann M. 1996; Analytical properties of the nanoelectrospray ion source. Anal Chem 68:1–8
    [Google Scholar]
  49. Wilm M., Shevchenko A., Houthaeve T., Breit S., Schweigerer L., Fotsis T., Mann M. 1996; Femtomole sequencing of proteins from polyacrylamide gels by nanoelectrospray mass spectrometry. . Nature 379:466–469 [CrossRef]
    [Google Scholar]
  50. Wong D. K., Lee B. Y., Horwitz M. A., Gibson B. W. 1999; Identification of Fur, aconitase, and other proteins expressed by Mycobacterium tuberculosis under conditions of low and high concentrations of iron by combined two-dimensional gel electrophoresis and mass spectrometry. Infect Immun 67:327–336
    [Google Scholar]
  51. Yates J. R. III, Speicher S., Griffin P. R., Hunkapiller T. 1993; Peptide mass maps: a highly informative approach to protein identification. Anal Biochem 214:397–408 [CrossRef]
    [Google Scholar]
  52. Yuan Y., Crane D. D., Barry C. E. III 1996; Stationary phase-associated protein expression in Mycobacterium tuberculosis: function of the mycobacterial α-crystallin homolog. J Bacteriol 178:4484–4492
    [Google Scholar]
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