1887

Abstract

The availability of the complete genome sequence of A3(2) has allowed the prediction of the Tat-exported proteins of this Gram-positive bacterium. To predict secreted proteins that potentially use the Tat pathway for their secretion, the TATscan program was developed. This program identified 129 putative Tat substrates. To test the validity of these predictions, nine signal sequences, including three which were not identified by existing prediction programs, were selected and fused to the structural gene in place of its native signal sequence. Xylanase C (XlnC) is a cofactorless enzyme which is secreted in an active form exclusively through the Tat-dependent pathway by . Among the nine chosen signal sequences, seven were shown to be Tat-dependent, one was Sec-dependent and one was probably not a signal sequence. The seven Tat-dependent signal sequences comprised two lipoprotein signal sequences and three sequences not predicted by previous programs. Pulse–chase experiments showed that the precursor-processing rate in the seven transformants was generally slower than wild-type XlnC, indicating that these signal peptides were not equivalent in secretion. This suggested that there might be some incompatibility between the signal peptide and the reporter protein fused to it. To test this possibility, the signal peptides were fused to a cofactorless chitosanase (SCO0677), a Tat-dependent protein validated in this work but structurally different from XlnC. With some fluctuations, similar results were obtained with this enzyme, indicating that the type of folding of the reporter protein had little effect on the Tat secretion process.

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2005-07-01
2019-10-20
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References

  1. Bendtsen, J. D., Nielsen, H., von Heijne, G. & Brunak, S. ( 2004; ). Improved prediction of signal peptides: SignalP 3.0. J Mol Biol 340, 783–795.[CrossRef]
    [Google Scholar]
  2. Bentley, S. D., Chater, K. F., Cerdeno-Tarraga, A. M. & 40 other authors ( 2002; ). Complete genome sequence of the model actinomycete Streptomyces coelicolor A3(2). Nature 417, 141–147.[CrossRef]
    [Google Scholar]
  3. Berks, B. C. ( 1996; ). A common export pathway for proteins binding complex redox cofactors? Mol Microbiol 22, 393–404.[CrossRef]
    [Google Scholar]
  4. Berks, B. C., Sargent, F. & Palmer, T. ( 2000; ). The Tat protein export pathway. Mol Microbiol 35, 260–274.[CrossRef]
    [Google Scholar]
  5. Blaudeck, N., Sprenger, G. A., Freudl, R. & Wiegert, T. ( 2001; ). Specificity of signal peptide recognition in tat-dependent bacterial protein translocation. J Bacteriol 183, 604–610.[CrossRef]
    [Google Scholar]
  6. Bogsch, E., Brink, S. & Robinson, C. ( 1997; ). Pathway specificity for a ΔpH-dependent precursor thylakoid lumen protein is governed by a ‘Sec-avoidance’ motif in the transfer peptide and a ‘Sec-incompatible’ mature protein. EMBO J 16, 3851–3859.[CrossRef]
    [Google Scholar]
  7. Cristobal, S., de Gier, J. W., Nielsen, H. & von Heijne, G. ( 1999; ). Competition between Sec- and TAT-dependent protein translocation in Escherichia coli. EMBO J 18, 2982–2990.[CrossRef]
    [Google Scholar]
  8. Dilks, K., Rose, R. W., Hartmann, E. & Pohlschröder, M. ( 2003; ). Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey. J Bacteriol 185, 1478–1483.[CrossRef]
    [Google Scholar]
  9. Ding, Z. & Christie, P. J. ( 2003; ). Agrobacterium tumefaciens twin-arginine-dependent translocation is important for virulence, flagellation, and chemotaxis but not type IV secretion. J Bacteriol 185, 760–771.[CrossRef]
    [Google Scholar]
  10. Faury, D., Saidane, S., Li, H. & Morosoli, R. ( 2004; ). Secretion of active xylanase C from Streptomyces lividans is exclusively mediated by the Tat protein export system. Biochim Biophys Acta 1699, 155–162.[CrossRef]
    [Google Scholar]
  11. Fukamizo, T., Honda, Y., Goto, S., Boucher, I. & Brzezinski, R. ( 1995; ). Reaction mechanism of chitosanase from Streptomyces sp. N174. Biochem J 311, 377–383.
    [Google Scholar]
  12. Fukamizo, T., Juffer, A. H., Vogel, H. J., Honda, Y., Tremblay, H., Boucher, I., Neugebauer, W. A. & Brzezinski, R. ( 2000; ). Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase. J Biol Chem 275, 25633–25640.[CrossRef]
    [Google Scholar]
  13. Gebler, J., Gilkes, N. R., Claeyssens, M. & 7 other authors ( 1992; ). Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases. J Biol Chem 267, 12559–12561.
    [Google Scholar]
  14. Halbig, D., Wiegert, T., Blaudeck, N., Freudl, R. & Sprenger, G. A. ( 1999; ). The efficient export of NADP-containing glucose-fructose oxidoreductase to the periplasm of Zymomonas mobilis depends both on an intact twin-arginine motif in the signal peptide and on the generation of a structural export signal induced by cofactor binding. Eur J Biochem 263, 543–551.[CrossRef]
    [Google Scholar]
  15. Hurtubise, Y., Shareck, F., Kluepfel, D. & Morosoli, R. ( 1995; ). A cellulase/xylanase-negative mutant of Streptomyces lividans 1326 defective in cellobiose and xylobiose uptake is mutated in a gene encoding a protein homologous to ATP-binding proteins. Mol Microbiol 17, 367–377.[CrossRef]
    [Google Scholar]
  16. Hutcheon, G. W. & Bolhuis, A. ( 2003; ). The archaeal twin-arginine translocation pathway. Biochem Soc Trans 31, 686–689.
    [Google Scholar]
  17. Jongbloed, J. D. H., Martin, U., Antelmann, H., Hecker, M., Tjalsma, H., Venema, G., Bron, S., van Dijl, J. M. & Müller, J. ( 2000; ). TatC is a specificity determinant for protein secretion via the twin-arginine translocation pathway. J Biol Chem 275, 41350–41357.[CrossRef]
    [Google Scholar]
  18. Jongbloed, J. D. H., Antelmann, H., Hecker, M. & 7 other authors ( 2002; ). Selective contribution of the twin-arginine translocation pathway to protein secretion in Bacillus subtilis. J Biol Chem 277, 44068–44078.[CrossRef]
    [Google Scholar]
  19. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. ( 2000; ). Practical Streptomyces Genetics. Norwich: John Innes Centre.
  20. Kluepfel, D., Vats-Metha, S., Aumont, F., Shareck, F. & Morosoli, R. ( 1990; ). Purification and characterization of a new xylanase (xylanase B) produced by Streptomyces lividans 66. Biochem J 267, 45–50.
    [Google Scholar]
  21. Kluepfel, D., Daigneault, N., Morosoli, R. & Shareck, F. ( 1992; ). Purification of a new xylanase (xylanase C) produced by Streptomyces lividans. Appl Microbiol Biotechnol 36, 626–631.
    [Google Scholar]
  22. Kyte, J. & Doolittle, R. F. ( 1982; ). A simple method for displaying the hydropathic character of a protein. J Mol Biol 157, 105–132.[CrossRef]
    [Google Scholar]
  23. Lever, M. ( 1972; ). A new reaction for colorimetric determination of carbohydrates. Anal Biochem 47, 273–279.[CrossRef]
    [Google Scholar]
  24. Marcotte, E. M., Monzingo, A. F., Ernst, S. R., Brzezinski, R. & Robertus, J. D. ( 1996; ). X-ray structure of an anti-fungal chitosanase from Streptomyces N174. Nat Struct Biol 3, 155–162.[CrossRef]
    [Google Scholar]
  25. Mori, H. & Ito, K. ( 2001; ). The Sec protein-translocation pathway. Trends Microbiol 9, 494–500.[CrossRef]
    [Google Scholar]
  26. Morosoli, R., Bertrand, J. L., Mondou, F., Shareck, F. & Kluepfel, D. ( 1986; ). Purification and properties of a xylanase from Streptomyces lividans. Biochem J 239, 587–592.
    [Google Scholar]
  27. Ochsner, U. A., Snyder, A., Vasil, A. I. & Vasil, M. L. ( 2002; ). Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis. Proc Natl Acad Sci U S A 99, 8312–8317.[CrossRef]
    [Google Scholar]
  28. Pagé, N., Kluepfel, D., Shareck, F. & Morosoli, R. ( 1996; ). Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans. Appl Environ Microbiol 62, 109–114.
    [Google Scholar]
  29. Schaerlaekens, K., Schierová, M., Lammertyn, E., Geukens, N., Anné, J. & van Mellaert, L. ( 2001; ). Twin-arginine translocation pathway in Streptomyces lividans. J Bacteriol 183, 6727–6732.[CrossRef]
    [Google Scholar]
  30. Schaerlaekens, K., van Mellaert, L., Lammertyn, E., Geukens, N. & Anné, J. ( 2004; ). The importance of the Tat-dependent protein secretion pathway in Streptomyces as revealed by phenotypic changes in tat deletion mutants and genome analysis. Microbiology 150, 21–31.[CrossRef]
    [Google Scholar]
  31. Sutcliffe, I. C. & Russell, R. R. B. ( 1995; ). Lipoproteins of Gram-positive bacteria. J Bacteriol 177, 1123–1128.
    [Google Scholar]
  32. Törrönen, A. & Rouvinen, J. ( 1995; ). Structural comparison of two major endo-1,4-xylanases from Trichoderma reesei. Biochemistry 34, 847–856.[CrossRef]
    [Google Scholar]
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vol. , part 7, pp. 2189-2198

A complete set of 129 candiodate signal sequences is presented in an Excel spreadsheet.



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