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. 1990 Feb;172(2):942–948. doi: 10.1128/jb.172.2.942-948.1990

Cloning of the Pseudomonas aeruginosa alkaline protease gene and secretion of the protease into the medium by Escherichia coli.

J Guzzo 1, M Murgier 1, A Filloux 1, A Lazdunski 1
PMCID: PMC208522  PMID: 2153662

Abstract

Pseudomonas virulence is thought to depend on multiple characteristics, including the production of an extracellular alkaline protease. We report the isolation, from a PAO1 DNA genomic bank, of a cosmid carrying the structural gene coding for alkaline protease. By in vivo mutagenesis using transposon Tn1735, which functions as a transposable promoter, the expression of an 8.8-kilobase DNA fragment under control the tac promoter was obtained. When expressed in Escherichia coli, active alkaline protease was synthesized and secreted to the extracellular medium in the absence of cell lysis.

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Selected References

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

  1. Bally M., Wretlind B., Lazdunski A. Protein secretion in Pseudomonas aeruginosa: molecular cloning and characterization of the xcp-1 gene. J Bacteriol. 1989 Aug;171(8):4342–4348. doi: 10.1128/jb.171.8.4342-4348.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bever R. A., Iglewski B. H. Molecular characterization and nucleotide sequence of the Pseudomonas aeruginosa elastase structural gene. J Bacteriol. 1988 Sep;170(9):4309–4314. doi: 10.1128/jb.170.9.4309-4314.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Chen S. T., Jordan E. M., Wilson R. B., Draper R. K., Clowes R. C. Transcription and expression of the exotoxin A gene of Pseudomonas aeruginosa. J Gen Microbiol. 1987 Nov;133(11):3081–3091. doi: 10.1099/00221287-133-11-3081. [DOI] [PubMed] [Google Scholar]
  5. 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]
  6. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Filloux A., Bally M., Murgier M., Wretlind B., Lazdunski A. Cloning of xcp genes located at the 55 min region of the chromosome and involved in protein secretion in Pseudomonas aeruginosa. Mol Microbiol. 1989 Feb;3(2):261–265. doi: 10.1111/j.1365-2958.1989.tb01816.x. [DOI] [PubMed] [Google Scholar]
  8. Filloux A., Bally M., Soscia C., Murgier M., Lazdunski A. Phosphate regulation in Pseudomonas aeruginosa: cloning of the alkaline phosphatase gene and identification of phoB- and phoR-like genes. Mol Gen Genet. 1988 Jun;212(3):510–513. doi: 10.1007/BF00330857. [DOI] [PubMed] [Google Scholar]
  9. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Holloway B. W., Krishnapillai V., Morgan A. F. Chromosomal genetics of Pseudomonas. Microbiol Rev. 1979 Mar;43(1):73–102. doi: 10.1128/mr.43.1.73-102.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Horvat R. T., Parmely M. J. Pseudomonas aeruginosa alkaline protease degrades human gamma interferon and inhibits its bioactivity. Infect Immun. 1988 Nov;56(11):2925–2932. doi: 10.1128/iai.56.11.2925-2932.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Kessler E., Safrin M. Synthesis, processing, and transport of Pseudomonas aeruginosa elastase. J Bacteriol. 1988 Nov;170(11):5241–5247. doi: 10.1128/jb.170.11.5241-5247.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Lory S., Strom M. S., Johnson K. Expression and secretion of the cloned Pseudomonas aeruginosa exotoxin A by Escherichia coli. J Bacteriol. 1988 Feb;170(2):714–719. doi: 10.1128/jb.170.2.714-719.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lory S., Tai P. C. Characterization of the phospholipase C gene of Pseudomonas aeruginosa cloned in Escherichia coli. Gene. 1983 Apr;22(1):95–101. doi: 10.1016/0378-1119(83)90068-9. [DOI] [PubMed] [Google Scholar]
  20. 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]
  21. Lüthi E., Mercenier A., Haas D. The arcABC operon required for fermentative growth of Pseudomonas aeruginosa on arginine: Tn5-751-assisted cloning and localization of structural genes. J Gen Microbiol. 1986 Oct;132(10):2667–2675. doi: 10.1099/00221287-132-10-2667. [DOI] [PubMed] [Google Scholar]
  22. MORIHARA K. PRODUCTION OF ELASTASE AND PROTEINASE BY PSEUDOMONAS AERUGINOSA. J Bacteriol. 1964 Sep;88:745–757. doi: 10.1128/jb.88.3.745-757.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ohman D. E., Sadoff J. C., Iglewski B. H. Toxin A-deficient mutants of Pseudomonas aeruginosa PA103: isolation and characterization. Infect Immun. 1980 Jun;28(3):899–908. doi: 10.1128/iai.28.3.899-908.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Ubben D., Schmitt R. A transposable promoter and transposable promoter probes derived from Tn1721. Gene. 1987;53(1):127–134. doi: 10.1016/0378-1119(87)90100-4. [DOI] [PubMed] [Google Scholar]
  25. Ubben D., Schmitt R. Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis. Gene. 1986;41(2-3):145–152. doi: 10.1016/0378-1119(86)90093-4. [DOI] [PubMed] [Google Scholar]
  26. Vasil M. L., Berka R. M., Gray G. L., Nakai H. Cloning of a phosphate-regulated hemolysin gene (phospholipase C) from Pseudomonas aeruginosa. J Bacteriol. 1982 Oct;152(1):431–440. doi: 10.1128/jb.152.1.431-440.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Watson J. M., Holloway B. W. Suppressor mutations in Pseudomonas aeruginosa. J Bacteriol. 1976 Mar;125(3):780–786. doi: 10.1128/jb.125.3.780-786.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Wretlind B., Hedén L., Sjöberg L., Wadström T. Production of enzymes and toxins by hospital strains of Pseudomonas aeruginosa in relation to serotype and phage-typing pattern. J Med Microbiol. 1973 Feb;6(1):91–100. doi: 10.1099/00222615-6-1-91. [DOI] [PubMed] [Google Scholar]
  29. 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]
  30. 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]

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