1887

Abstract

Proteins secreted by may play a key role in virulence and may also constitute antigens that elicit the host immune response. However, the protein export machinery has not been characterized. A library of H37Rv genomic DNA fragments ligated into a signal sequence selection vector that contained a leaderless β-lactamase gene and an upstream Tac promoter was constructed. Transformation of with the DNA library and selection on plates containing 50-100 μg ampicillin ml resulted in the identification of 15 Amp clones out of a total of 14000 transformants. Twelve of the β-lactamase gene fusions conferred high levels of Amp (up to 1 mg ampicillin ml); insert sizes ranged from 350 to 3000 bp. Of ten inserts that were completely sequenced, two were identified as fragments of the genes for antigens 85A and 85C, which are the major secreted proteins of this pathogen. Seven of the remaining inserts were ≥97% identical to hypothetical ORFs in the genome, one of which encoded a protein with 35% identity to a low-affinity penicillin-binding protein (PBP) from . Four of the seven hypothetical ORFs encoded putative exported proteins with one or more membrane interaction elements, including lipoprotein attachment sites and type I and II transmembrane (TM) segments. All of the inserts encoded typical signal sequences, with the exception of a possible type II membrane protein. It is concluded that expression of β-lactamase gene fusions in provides a useful system for the identification and analysis of signal-sequence-encoding genes.

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1998-06-01
2024-12-03
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References

  1. Andersen, P. (1994); Effective vaccination of mice against Mycobacterium tuberculosis infection with a soluble mixture of secreted mycobacterial proteins.. Infect Immun 62,:2536–2544
    [Google Scholar]
  2. Belisle, J. T., Vissa, V. D., Sievert, T., Takayama, K., Brennan, P. J., Besra, G. S. (1997); Role of the major antigen of Mycobacterium tuberculosis in cell wall biogenesis.. Science 276,:1420–1422
    [Google Scholar]
  3. Bolhuis, A., Sorokin, A., Azevedo, V., Ehrlich, S. D., Braun, P. G., de long, A., Venema, G., Bron, S., van Dijl, J. M. (1996); Bacillus subtilis can modulate its capacity and specificity for protein secretion through temporally controlled expression of the sipS gene for signal peptidase I.. Mole Microbiol 22,:605–618
    [Google Scholar]
  4. Brennan, P. J., Draper, P. (1994) Ultrastructure of Mycobacterium tuberculosis.. In Tuberculosis: Pathogenesis, Protection, and Control, Washington, DC:: American Society for Microbiology,;271–284
    [Google Scholar]
  5. Broome-Smith, J. K., Spratt, B. G. (1986); A vector for the construction of translational fusions to TEM β-lactamase and the analysis of protein export signals and membrane protein topology.. Gene 49,:341–349
    [Google Scholar]
  6. Broome-Smith, J. K., Tadayyon, M., Zhang, Y. (1990); 0-Lactamase as a probe of membrane protein assembly and protein export.. Mol Microbiol 4,:1637–1644
    [Google Scholar]
  7. Content, J., de la Cavellerie, A., de Wit, L., Vincent-Levy-Fr6bault, V., Ooms, J., de Bruyn, J. (1991); The genes coding for the antigen 85 complexes of Mycobacterium tuberculosis and Mycobacterium bovis BCG are members of a gene family: cloning, sequence determination, and genomic organization of the gene coding for antigen 85-C of M. tuberculosis.. Infect Immun 59,:3205–3212
    [Google Scholar]
  8. Cywes, C., Hoppe, H. C., Daffe, M., Ehlers, M. R. W. (1997); Nonopsonic binding of Mycobacterium tuberculosis to complement receptor type 3 is mediated by capsular polysaccharides and is strain dependent.. Infect Immun 65,:4258–1266
    [Google Scholar]
  9. Da Silva Tatley, F. M., Steyn, L. M. (1993); Characterization of a replicon of the moderately promiscuous plasmid, pGSH5000, with features of both the mini-replicon of pCUl and the ori-2 of F.. Mol Microbiol 7,:805–823
    [Google Scholar]
  10. Das Gupta, S. K., Bashyam, M. D., Tyagi, A. K. (1993); Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector.. J Bacteriol 175,:5186–5192
    [Google Scholar]
  11. Dobos, K. M., Khoo, K.-H., Swiderek, K. M., Brennan, P. J., Belisle, J. T. (1996); Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis.. J Bacteriol 178,:2498–2506
    [Google Scholar]
  12. Dreyfus, G., Williams, A. W., Kawagishi, I., MacNab, R. M. (1993); Genetic and biochemical analysis of Salmonella typbi- murium Flil, a flagellar protein related to the catalytic subunit of the F0F1 ATPase and to virulence proteins of mammalian and plant pathogens.. J Bacteriol 175,:3131–3138
    [Google Scholar]
  13. Fenton, M. J., Vermeulen, M. W. (1996); Immunopathology of tuberculosis: roles of macrophages and monocytes.. Infect Immu 64,:683–690
    [Google Scholar]
  14. Herrmann, J. L., O'Gaora, P., Gallagher, A., Thole, J. E. R., Young, D. B. (1996); Bacterial glycoproteins: a link between glycosylation and proteolytic cleavage of a 19 kDa antigen from Mycobacterium tuberculosis.. EMBO J 15,:3547–3554
    [Google Scholar]
  15. Hewinson, R. G., Russell, W. P. (1993); Processing and secretion by Escherichia coli of a recombinant form of the immunogenic protein MPB70 of Mycobacterium bovis.. J Gen Microbiol 139,:1253–1259
    [Google Scholar]
  16. Hewinson, R. G., Harris, D. P., Whelan, A., Russell, W. P. (1996a); Secretion of the mycobacterial 19-kilodalton protein by Escherichia coli, a novel method for the purification of recombinant mycobacterial antigens.. Clin Diagn Lab Immunol 3,23–29
    [Google Scholar]
  17. Hewinson, R. G., Michell, S. L., Russell, W. P., McAdam, R. A., Jacobs, W. R. Jr (1996b); Molecular characterization of MPT83: a seroreactive antigen of Mycobacterium tuberculosis with homology to MPT70.. Scand J Immunol 43,:49CM199
    [Google Scholar]
  18. Hoppe, H. C., de Wet, B. J. M., Cywes, C., Daffé, M., Ehlers, M. R. W. (1997); Identification of phosphatidylinositol mannoside as a mycobacterial adhesin mediating both direct and opsonic binding to nonphagocytic mammalian cells.. Infect Immun 65,:3896–3905
    [Google Scholar]
  19. Horwitz, M. A., Lee, B.-W. E., Dillon, B. J., Harth, G. (1995); Protective immunity against tuberculosis induced by vaccination with major extracellular proteins of Mycobacterium tuberculosis.. Proc Natl Acad Sci USA 92,:1530–1534
    [Google Scholar]
  20. Izard, J. W., Kendall, D. A. (1994); Signal peptides: exquisitely designed transport promoters.. Mol Microbiol 13,:765–773
    [Google Scholar]
  21. Jackson, M., Portnoi, D., Catheline, D., Dumail, L., Rauzier, J., Legrand, P., Gicquel, B. (1997); Mycobacterium tuberculosis Des protein: an immunodominant target for the humoral response of tuberculous patients.. Infect Immun 65,:2883–2889
    [Google Scholar]
  22. Kuo, C.-C., Takahashi, N., Swanson, A. F., Ozeki, Y., Hakomori, S.-l. (1996); An N-linked high-mannose type oligosaccharide, expressed at the major outer membrane protein of Chlamydia trachomatis, mediates attachment and infectivity of the microorganism to HeLa cells.. J Clin Invest 98,:2813–2818
    [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
    [Google Scholar]
  24. Lim, E. M., Rauzier, J., Timm, J., Torrca, G., Murray, A., Gicquel, B., Portnoi, D. (1995); Identification of Mycobacterium tuberculosis DNA sequences encoding exported proteins by using phoA gene fusions.. J Bacteriol 177,:59–65
    [Google Scholar]
  25. Liu, J., Barry, C. E., III, Besra, G. S., Nikaido, H. (1996); Mycolic acid structure determines the fluidity of the mycobacterial cell wall.. J Biol Chem 271,:29545–29551
    [Google Scholar]
  26. Mdluli, K. E., Treit, J. D., Kerr, V. J., Nano, F. E. (1995); New vectors for the in vitro generation of alkaline phosphatase fusions to proteins encoded by G + C-rich DNA.. Gene 155,:133–134
    [Google Scholar]
  27. Orme, I. M., Andersen, P., Boom, W. H. (1993); T cell response to Mycobacterium tuberculosis.. J Infect Dis 167,:1481–1497
    [Google Scholar]
  28. Paradkar, A. S., Aidoo, K. A., Wong, A., Jensen, S. E. (1996); Molecular analysis of a β-lactam resistance gene encoded within the cephamycin gene cluster of Streptomyces clavuligerus.. J Bacteriol 178,:6266–6274
    [Google Scholar]
  29. Perez-Martinez, G., Kok, J., Venema, G., van Dijl, J. M., Smith, H., Bron, S. (1992); Protein export elements from Lactococcus lactis.. Mole Gene Genet 234,:401–411
    [Google Scholar]
  30. Prinz, W. A., Beckwith, J. (1994); Gene fusion analysis of membrane protein topology: a direct comparison of alkaline phosphatase and β-lactamase fusions.. J Bacteriol 176,:6410–6413
    [Google Scholar]
  31. Pugsley, A. P. (1993); The complete general secretory pathway in Gram-negative bacteria.. Microbiol Rev 57,:50–108
    [Google Scholar]
  32. Reeves, P. J., Douglas, P., Salmond, G. P. C. (1994); Beta-lactamase topology probe analysis of the OutO NMePhe peptidase, and six other Out protein components of the Erwinia carotovora general secretion pathway apparatus.. Mol Microbiol 12,:445–457
    [Google Scholar]
  33. Roggenkamp, R., Dargatz, H., Hollenberg, C. (1985); Precursor of β-lactamase is enzymatically inactive.. J Biol Chem 260,:1508–1512
    [Google Scholar]
  34. Salmond, G. P. C., Reeves, P. J. (1993); Membrane traffic wardens and protein secretion in Gram-negative bacteria.. Trends Bioche Sci 18,:7–12
    [Google Scholar]
  35. 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]
  36. Silhavy, T. J. (1997); Death by lethal injection.. Science 278,:1085–1086
    [Google Scholar]
  37. Smith, H., Bron, S., van Ee, J., Venema, G. (1987); Construction and use of signal sequence selection vectors in Escherichia coli and Bacillus subtilis.. J Bacteriol 169,:3321–3328
    [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. Timm, J., Perilli, M. G., Duez, C. (1994); Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum β-lactamase genes cloned from a natural isolate and a high-level β-lactamase producer.. Mol Microbiol 12,:491–504
    [Google Scholar]
  40. Vosloo, W., Tippoo, P., Hughes, J. E., Harriman, N., Emms, D. W., Beatty, M., Zappe, H., Steyn, L. M. (1997); Characterisation of a lipoprotein in Mycobacterium bovis (BCG) with sequence similarity to the secreted protein MPB70.. Gene 188,:123–128
    [Google Scholar]
  41. Wiker, H. G., Harboe, M., Nagai, S. (1991); A localization index for distinction between extracellular and intracellular antigens of Mycobacterium tuberculosis.. J Gen Microbiol 137,:875–884
    [Google Scholar]
  42. Williams, N. (1998); Genome of TB culprit deciphered.. Science 279,:25
    [Google Scholar]
  43. Young, D. B., Kaufmann, S. H. E., Hermans, P. W. M., Thole, J. E. R. (1992); Mycobacterial protein antigens: a compilation.. Mol Microbiol 6,:133–145
    [Google Scholar]
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