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. 1991 Sep;173(17):5290–5297. doi: 10.1128/jb.173.17.5290-5297.1991

Pseudomonas aeruginosa alkaline protease: evidence for secretion genes and study of secretion mechanism.

J Guzzo 1, J M Pages 1, F Duong 1, A Lazdunski 1, M Murgier 1
PMCID: PMC208238  PMID: 1832151

Abstract

A 6.5-kb DNA fragment carrying the functions required for specific secretion of the extracellular alkaline protease produced by Pseudomonas aeruginosa was cloned. The whole 6.5-kb DNA fragment was transcribed in one direction and probably carried three genes involved in secretion. The expression in trans of these genes, together with the apr gene, in Escherichia coli allowed synthesis and secretion of the alkaline protease, which was extensively investigated by performing pulse-chase experiments under various conditions. We demonstrated the absence of a precursor form, as well as the independence of alkaline protease translocation from SecA. The absence of secretion genes impaired alkaline protease secretion; the protein then remained intracellular and was partially degraded.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Chang A. C., Cohen S. N. Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol. 1978 Jun;134(3):1141–1156. doi: 10.1128/jb.134.3.1141-1156.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi. Proteases B and C are synthesized and secreted as zymogens without a signal peptide. J Biol Chem. 1989 May 25;264(15):9083–9089. [PubMed] [Google Scholar]
  4. Douglas C. M., Guidi-Rontani C., Collier R. J. Exotoxin A of Pseudomonas aeruginosa: active, cloned toxin is secreted into the periplasmic space of Escherichia coli. J Bacteriol. 1987 Nov;169(11):4962–4966. doi: 10.1128/jb.169.11.4962-4966.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Fellay R., Frey J., Krisch H. Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene. 1987;52(2-3):147–154. doi: 10.1016/0378-1119(87)90041-2. [DOI] [PubMed] [Google Scholar]
  6. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  7. Gray G. L., Smith D. H., Baldridge J. S., Harkins R. N., Vasil M. L., Chen E. Y., Heyneker H. L. Cloning, nucleotide sequence, and expression in Escherichia coli of the exotoxin A structural gene of Pseudomonas aeruginosa. Proc Natl Acad Sci U S A. 1984 May;81(9):2645–2649. doi: 10.1073/pnas.81.9.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gray L., Baker K., Kenny B., Mackman N., Haigh R., Holland I. B. A novel C-terminal signal sequence targets Escherichia coli haemolysin directly to the medium. J Cell Sci Suppl. 1989;11:45–57. doi: 10.1242/jcs.1989.supplement_11.4. [DOI] [PubMed] [Google Scholar]
  9. Guzzo J., Duong F., Wandersman C., Murgier M., Lazdunski A. The secretion genes of Pseudomonas aeruginosa alkaline protease are functionally related to those of Erwinia chrysanthemi proteases and Escherichia coli alpha-haemolysin. Mol Microbiol. 1991 Feb;5(2):447–453. doi: 10.1111/j.1365-2958.1991.tb02128.x. [DOI] [PubMed] [Google Scholar]
  10. Guzzo J., Murgier M., Filloux A., Lazdunski A. Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli. J Bacteriol. 1990 Feb;172(2):942–948. doi: 10.1128/jb.172.2.942-948.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Halegoua S., Inouye M. Translocation and assembly of outer membrance proteins of Escherichia coli. Selective accumulation of precursors and novel assembly intermediates caused by phenethyl alcohol. J Mol Biol. 1979 May 5;130(1):39–61. doi: 10.1016/0022-2836(79)90551-5. [DOI] [PubMed] [Google Scholar]
  12. Holmes D. S., Quigley M. A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem. 1981 Jun;114(1):193–197. doi: 10.1016/0003-2697(81)90473-5. [DOI] [PubMed] [Google Scholar]
  13. Howe T. R., Iglewski B. H. Isolation and characterization of alkaline protease-deficient mutants of Pseudomonas aeruginosa in vitro and in a mouse eye model. Infect Immun. 1984 Mar;43(3):1058–1063. doi: 10.1128/iai.43.3.1058-1063.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klauser T., Pohlner J., Meyer T. F. Extracellular transport of cholera toxin B subunit using Neisseria IgA protease beta-domain: conformation-dependent outer membrane translocation. EMBO J. 1990 Jun;9(6):1991–1999. doi: 10.1002/j.1460-2075.1990.tb08327.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lazdunski A., Guzzo J., Filloux A., Bally M., Murgier M. Secretion of extracellular proteins by Pseudomonas aeruginosa. Biochimie. 1990 Feb-Mar;72(2-3):147–156. doi: 10.1016/0300-9084(90)90140-c. [DOI] [PubMed] [Google Scholar]
  16. Lederberg E. M., Cohen S. N. Transformation of Salmonella typhimurium by plasmid deoxyribonucleic acid. J Bacteriol. 1974 Sep;119(3):1072–1074. doi: 10.1128/jb.119.3.1072-1074.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lugtenberg B., Meijers J., Peters R., van der Hoek P., van Alphen L. Electrophoretic resolution of the "major outer membrane protein" of Escherichia coli K12 into four bands. FEBS Lett. 1975 Oct 15;58(1):254–258. doi: 10.1016/0014-5793(75)80272-9. [DOI] [PubMed] [Google Scholar]
  18. Létoffé S., Delepelaire P., Wandersman C. Protease secretion by Erwinia chrysanthemi: the specific secretion functions are analogous to those of Escherichia coli alpha-haemolysin. EMBO J. 1990 May;9(5):1375–1382. doi: 10.1002/j.1460-2075.1990.tb08252.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Oliver D. B., Beckwith J. E. coli mutant pleiotropically defective in the export of secreted proteins. Cell. 1981 Sep;25(3):765–772. doi: 10.1016/0092-8674(81)90184-7. [DOI] [PubMed] [Google Scholar]
  20. Oropeza-Wekerle R. L., Müller E., Kern P., Meyermann R., Goebel W. Synthesis, inactivation, and localization of extracellular and intracellular Escherichia coli hemolysins. J Bacteriol. 1989 May;171(5):2783–2788. doi: 10.1128/jb.171.5.2783-2788.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Pages J. M., Bolla J. M. Assembly of the OmpF porin of Escherichia coli B. Immunological and kinetic studies of the integration pathway. Eur J Biochem. 1988 Oct 1;176(3):655–660. doi: 10.1111/j.1432-1033.1988.tb14327.x. [DOI] [PubMed] [Google Scholar]
  22. Saier M. H., Jr, Werner P. K., Müller M. Insertion of proteins into bacterial membranes: mechanism, characteristics, and comparisons with the eucaryotic process. Microbiol Rev. 1989 Sep;53(3):333–366. doi: 10.1128/mr.53.3.333-366.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Varenne S., Buc J., Lloubes R., Lazdunski C. Translation is a non-uniform process. Effect of tRNA availability on the rate of elongation of nascent polypeptide chains. J Mol Biol. 1984 Dec 15;180(3):549–576. doi: 10.1016/0022-2836(84)90027-5. [DOI] [PubMed] [Google Scholar]
  24. Wandersman C. Secretion, processing and activation of bacterial extracellular proteases. Mol Microbiol. 1989 Dec;3(12):1825–1831. doi: 10.1111/j.1365-2958.1989.tb00169.x. [DOI] [PubMed] [Google Scholar]
  25. Wretlind B., Pavlovskis O. R. Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins. J Bacteriol. 1984 Jun;158(3):801–808. doi: 10.1128/jb.158.3.801-808.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  27. d'Enfert C., Ryter A., Pugsley A. P. Cloning and expression in Escherichia coli of the Klebsiella pneumoniae genes for production, surface localization and secretion of the lipoprotein pullulanase. EMBO J. 1987 Nov;6(11):3531–3538. doi: 10.1002/j.1460-2075.1987.tb02679.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

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