1887

Abstract

The propeptide of elastase functions both as an intramolecular chaperone required for the folding of the enzyme and as an inhibitor that prevents activity of the enzyme before its secretion into the extracellular medium. Since expression of the gene, which encodes elastase, in did not result in extracellular elastase activity, it has been suggested that the enzyme is not recognized by the Xcp secretion machinery of the heterologous host. Here, it is demonstrated that the proenzyme is normally processed in and that it is indeed not actively secreted by the Xcp machinery. Nevertheless, substantial amounts of the enzyme were detected in the extracellular medium. Co-immunoprecipitations revealed that the extracellular enzyme was associated with the propeptide, which explains the lack of enzymic activity. Since the propeptide–enzyme complex in apparently does not dissociate spontaneously, it is concluded that a host-specific factor is required to induce this event. Mutants were selected which showed extracellular elastase activity. Two mutations, located within the gene, were further characterized. These mutations, resulting in the substitution of Ala and Thr at positions −15 and −153, respectively, of the propeptide (where position +1 is defined as the first residue of the mature enzyme) destabilized the propeptide–enzyme complex. It is concluded that Ala-15 and Thr-153 are required for the inhibitor function, but not for the chaperone function of the propeptide.

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2000-10-01
2019-11-22
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References

  1. Braun, P. & Tommassen, J. ( 1998; ). Function of bacterial propeptides. Trends Microbiol 6, 6-8.[CrossRef]
    [Google Scholar]
  2. Braun, P., Tommassen, J. & Filloux, A. ( 1996; ). Role of the propeptide in folding and secretion of elastase of Pseudomonas aeruginosa. Mol Microbiol 19, 297-306.[CrossRef]
    [Google Scholar]
  3. Braun, P., de Groot, A., Bitter, W. & Tommassen, J. ( 1998; ). Secretion of elastinolytic enzymes with their propeptides by Pseudomonas aeruginosa. J Bacteriol 180, 3467-3469.
    [Google Scholar]
  4. Chowdhury, K. ( 1991; ). One step ‘miniprep’ method for isolation of plasmid DNA. Nucleic Acids Res 19, 2792.[CrossRef]
    [Google Scholar]
  5. Figurski, D. H. & Helinski, D. R. ( 1979; ). Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A 76, 1648-1652.[CrossRef]
    [Google Scholar]
  6. Filloux, A., Michel, G. & Bally, M. ( 1998; ). GSP-dependent protein secretion in Gram-negative bacteria: the Xcp system of Pseudomonas aeruginosa. FEMS Microbiol Rev 22, 177-198.[CrossRef]
    [Google Scholar]
  7. Folders, J., Tommassen, J., van Loon, L. C. & Bitter, W. ( 2000; ). Identification of a chitin-binding protein secreted by Pseudomonas aeruginosa. J Bacteriol 182, 1257-1263.[CrossRef]
    [Google Scholar]
  8. Frenken, L. G. J., Bos, J. W., Visser, C., Müller, W., Tommassen, J. & Verrips, C. T. ( 1993; ). An accessory gene, lipB, required for the production of active Pseudomonas glumae lipase. Mol Microbiol 9, 579-589.[CrossRef]
    [Google Scholar]
  9. Fürste, J. P., Pansegrau, W., Frank, R., Blöcker, H., Scholz, P., Bagdasarian, M. & Lanka, E. ( 1986; ). Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene 48, 119-131.[CrossRef]
    [Google Scholar]
  10. Geels, F. P. & Schippers, B. ( 1983; ). Reduction of yield depressions in high frequency potato cropping soil after seed tuber treatment with antagonistic fluorescent Pseudomonas spp. Phytopathol Z 108, 207-214.[CrossRef]
    [Google Scholar]
  11. van der Goot, F. G., Hardie, K. R., Parker, M. W. & Buckley, J. T. ( 1994; ). The C-terminal peptide produced upon proteolytic activation of the cytolytic toxin aerolysin is not involved in channel formation. J Biol Chem 269, 30496-30501.
    [Google Scholar]
  12. de Groot, A., Filloux, A. & Tommassen, J. ( 1991; ). Conservation of xcp genes, involved in the two-step protein secretion process, in different Pseudomonas species and other gram-negative bacteria. Mol Gen Genet 229, 278-284.[CrossRef]
    [Google Scholar]
  13. de Groot, A., Krijger, J.-J., Filloux, A. & Tommassen, J. ( 1996; ). Characterization of type II protein secretion (xcp) genes in the plant growth-stimulating Pseudomonas putida, strain WCS358. Mol Gen Genet 250, 491-504.
    [Google Scholar]
  14. de Groot, A., Gerritse, G., Tommassen, J., Lazdunski, A. & Filloux, A. ( 1999; ). Molecular organization of the xcp gene cluster in Pseudomonas putida: absence of an xcpX (gspK) homologue. Gene 226, 35-40.[CrossRef]
    [Google Scholar]
  15. Guzzo, J., Murgier, M., Filloux, A. & Lazdunski, A. ( 1990; ). Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli. J Bacteriol 172, 942-948.
    [Google Scholar]
  16. Howard, S. P. & Buckley, J. T. ( 1985; ). Protein export by a gram-negative bacterium: production of aerolysin by Aeromonas hydrophila. J Bacteriol 161, 1118-1124.
    [Google Scholar]
  17. Kessler, E. & Safrin, M. ( 1988; ). Synthesis, processing, and transport of Pseudomonas aeruginosa elastase. J Bacteriol 170, 5241-5247.
    [Google Scholar]
  18. Kessler, E. & Safrin, M. ( 1994; ). The propeptide of Pseudomonas aeruginosa elastase acts as an elastase inhibitor. J Biol Chem 269, 22726-22731.
    [Google Scholar]
  19. Kessler, E., Safrin, M., Gustin, J. K & Ohman, D. E. ( 1998; ). Elastase and the LasA protease of Pseudomonas aeruginosa are secreted with their propeptides. J Biol Chem 273, 30225-30231.[CrossRef]
    [Google Scholar]
  20. Kobayashi, T. & Inouye, M. ( 1992; ). Functional analysis of the intramolecular chaperone. Mutational hot spots in the subtilisin pro-peptide and a second-site suppressor mutation within the subtilisin molecule. J Mol Biol 226, 931-933.[CrossRef]
    [Google Scholar]
  21. Lugtenberg, B., Peters, R., Bernheimer, H. & Berendsen, W. ( 1976; ). Influence of cultural conditions and mutations on the composition of the outer membrane proteins of Escherichia coli. Mol Gen Genet 147, 251-262.[CrossRef]
    [Google Scholar]
  22. McIver, K., Kessler, E. & Ohman, D. E. ( 1991; ). Substitution of active-site His-223 in Pseudomonas aeruginosa elastase and expression of mutated lasB alleles in Escherichia coli show evidence for autoproteolytic processing of proelastase. J Bacteriol 173, 7781-7789.
    [Google Scholar]
  23. McIver, K. S., Kessler, E., Olson, J. C. & Ohman, D. E. ( 1995; ). The elastase propeptide functions as an intramolecular chaperone required for elastase activity and secretion in Pseudomonas aeruginosa. Mol Microbiol 18, 877-889.[CrossRef]
    [Google Scholar]
  24. Michel, G., Ball, G., Goldberg, J. B. & Lazdunski, A. ( 2000; ). Alteration of the lipopolysaccharide structure affects the functioning of the Xcp secretory system in Pseudomonas aeruginosa. J Bacteriol 182, 696-703.[CrossRef]
    [Google Scholar]
  25. Norrander, J., Kempe, T. & Messing, J. ( 1983; ). Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene 26, 101-106.[CrossRef]
    [Google Scholar]
  26. Pugsley, A. P. ( 1993; ). The complete general secretory pathway in Gram-negative bacteria. Microbiol Rev 57, 50-108.
    [Google Scholar]
  27. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  28. Sellman, B. R. & Tweten, R. K. ( 1997; ). The propeptide of Clostridium septicum alpha toxin functions as an intramolecular chaperone and is a potent inhibitor of alpha toxin-dependent cytolysis. Mol Microbiol 25, 429-440.[CrossRef]
    [Google Scholar]
  29. Wretlind, B. & Pavlovskis, O. R. ( 1984; ). Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol 158, 801-808.
    [Google Scholar]
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