Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1987 Jun;169(6):2691–2696. doi: 10.1128/jb.169.6.2691-2696.1987

Cloning and characterization of elastase genes from Pseudomonas aeruginosa.

P A Schad, R A Bever, T I Nicas, F Leduc, L F Hanne, B H Iglewski
PMCID: PMC212161  PMID: 3034864

Abstract

A gene bank was constructed from Pseudomonas aeruginosa PAO1 and used to complement three P. aeruginosa elastase-deficient strains. One clone, pRF1, contained a gene which restored elastase production in two P. aeruginosa isolates deficient in elastase production (PA-E15 and PAO-E105). This gene also encoded production of elastase antigen and activity in Escherichia coli and is the structural gene for Pseudomonas elastase. A second clone, pHN13, contained a 20-kilobase (kb) EcoRI insert which was not related to the 8-kb EcoRI insert of pRF1 as determined by restriction analysis and DNA hybridization. A 2.2-kb SalI-HindIII fragment from pHN3 was subcloned into pUC18, forming pRB1822-1. Plasmid pRB1822-1 restored normal elastolytic activity to PAO-E64, a mutant for elastase activity. Clones derived from pHN13 failed to elicit elastase antigen or enzymatic activity in E. coli.

Full text

PDF
2691

Images in this article

2694Image
on p.2694

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. Bjorn M. J., Sokol P. A., Iglewski B. H. Influence of iron on yields of extracellular products in Pseudomonas aeruginosa cultures. J Bacteriol. 1979 Apr;138(1):193–200. doi: 10.1128/jb.138.1.193-200.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Döring G., Obernesser H. J., Botzenhart K. Extrazelluläre Toxine von Pseudomonas aeruginosa. II. Einwirkung zweier gereinigter Proteasen auf die menschlichen Immunoglobuline IgG, IgA und sekretorisches IgA. Zentralbl Bakteriol A. 1981 Mar;249(1):89–98. [PubMed] [Google Scholar]
  5. Fecycz I. T., Campbell J. N. Mechanisms of activation and secretion of a cell-associated precursor of an exocellular protease of Pseudomonas aeruginosa 34362A. Eur J Biochem. 1985 Jan 2;146(1):35–42. doi: 10.1111/j.1432-1033.1985.tb08616.x. [DOI] [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. Finn C. W., Jr, Silver R. P., Habig W. H., Hardegree M. C., Zon G., Garon C. F. The structural gene for tetanus neurotoxin is on a plasmid. Science. 1984 May 25;224(4651):881–884. doi: 10.1126/science.6326263. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Goldberg J. B., Ohman D. E. Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol. 1984 Jun;158(3):1115–1121. doi: 10.1128/jb.158.3.1115-1121.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hedstrom R. C., Funk C. R., Kaper J. B., Pavlovskis O. R., Galloway D. R. Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa. Infect Immun. 1986 Jan;51(1):37–42. doi: 10.1128/iai.51.1.37-42.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Hindahl M. S., Iglewski B. H. Outer membrane proteins from Legionella pneumophila serogroups and other Legionella species. Infect Immun. 1986 Jan;51(1):94–101. doi: 10.1128/iai.51.1.94-101.1986. [DOI] [PMC free article] [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. Homma J. Y., Tomiyama T., Sano H., Hirao Y., Saku K. Passive hemagglutination reaction test using formalinized sheep erythrocytes treated with tannic acid and coated with proteast or elastase of Pseudomonas aeruginosa. Jpn J Exp Med. 1975 Oct;45(5):361–365. [PubMed] [Google Scholar]
  15. Howe T. R., Wretlind B., Iglewski B. H. Comparison of two methods of genetic exchange in determination of the genetic locus of the structural gene for Pseudomonas aeruginosa elastase. J Bacteriol. 1983 Oct;156(1):58–61. doi: 10.1128/jb.156.1.58-61.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Janda J. M., Atang-Nomo S., Bottone E. J., Desmond E. P. Correlation of proteolytic activity of Pseudomonas aeruginosa with site of isolation. J Clin Microbiol. 1980 Oct;12(4):626–628. doi: 10.1128/jcm.12.4.626-628.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Jensen S. E., Fecycz I. T., Campbell J. N. Nutritional factors controlling exocellular protease production by Pseudomonas aeruginosa. J Bacteriol. 1980 Nov;144(2):844–847. doi: 10.1128/jb.144.2.844-847.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jensen S. E., Fecycz I. T., Stemke G. W., Campbell J. N. Demonstration of a cell-associated, inactive precursor of an exocellular protease produced by Pseudomonas aeruginosa. Can J Microbiol. 1980 Jan;26(1):87–93. doi: 10.1139/m80-013. [DOI] [PubMed] [Google Scholar]
  19. Jensen S. E., Phillippe L., Teng Tseng J., Stemke G. W., Campbell J. N. Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa. Can J Microbiol. 1980 Jan;26(1):77–86. doi: 10.1139/m80-012. [DOI] [PubMed] [Google Scholar]
  20. Kado C. I., Liu S. T. Rapid procedure for detection and isolation of large and small plasmids. J Bacteriol. 1981 Mar;145(3):1365–1373. doi: 10.1128/jb.145.3.1365-1373.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kawaharajo K., Homma J. Y. Effects of elastase, protease and common antigen (OEP) from Pseudomonas aeruginosa on protection against burns in mice. Jpn J Exp Med. 1977 Dec;47(6):495–500. [PubMed] [Google Scholar]
  22. Kreger A. S., Griffin O. K. Physicochemical fractionation of extracellular cornea-damaging proteases of Pseudomonas aeruginosa. Infect Immun. 1974 May;9(5):828–834. doi: 10.1128/iai.9.5.828-834.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. MORIHARA K., TSUZUKI H., OKA T., INOUE H., EBATA M. PSEUDOMONAS AERUGINOSA ELASTASE. ISOLATION, CRYSTALLIZATION, AND PRELIMINARY CHARACTERIZATION. J Biol Chem. 1965 Aug;240:3295–3304. [PubMed] [Google Scholar]
  27. Morihara K., Tsuzuki H., Oda K. Protease and elastase of Pseudomonas aeruginosa: inactivation of human plasma alpha 1-proteinase inhibitor. Infect Immun. 1979 Apr;24(1):188–193. doi: 10.1128/iai.24.1.188-193.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Morihara K., Tsuzuki H. Substrate specificity of elastolytic and nonelastolytic proteinases from Pseudomonas aeruginosa. Arch Biochem Biophys. 1966 Apr;114(1):158–165. doi: 10.1016/0003-9861(66)90317-1. [DOI] [PubMed] [Google Scholar]
  29. Mull J. D., Callahan W. S. The role of the elastase of Pseudomonas aeruginosa in experimental infection. Exp Mol Pathol. 1965 Dec;4(6):567–575. doi: 10.1016/0014-4800(65)90037-7. [DOI] [PubMed] [Google Scholar]
  30. Nicas T. I., Iglewski B. H. Production of elastase and other exoproducts by environmental isolates of Pseudomonas aeruginosa. J Clin Microbiol. 1986 May;23(5):967–969. doi: 10.1128/jcm.23.5.967-969.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nishino N., Powers J. C. Pseudomonas aeruginosa elastase. Development of a new substrate, inhibitors, and an affinity ligand. J Biol Chem. 1980 Apr 25;255(8):3482–3486. [PubMed] [Google Scholar]
  32. Ohman D. E., Cryz S. J., Iglewski B. H. Isolation and characterization of Pseudomonas aeruginosa PAO mutant that produces altered elastase. J Bacteriol. 1980 Jun;142(3):836–842. doi: 10.1128/jb.142.3.836-842.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  34. Schultz D. R., Miller K. D. Elastase of Pseudomonas aeruginosa: inactivation of complement components and complement-derived chemotactic and phagocytic factors. Infect Immun. 1974 Jul;10(1):128–135. doi: 10.1128/iai.10.1.128-135.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  36. VOGEL H. J., BONNER D. M. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem. 1956 Jan;218(1):97–106. [PubMed] [Google Scholar]
  37. Weiss A. A., Falkow S. Transposon insertion and subsequent donor formation promoted by Tn501 in Bordetella pertussis. J Bacteriol. 1983 Jan;153(1):304–309. doi: 10.1128/jb.153.1.304-309.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Wretlind B., Pavlovskis O. R. Pseudomonas aeruginosa elastase and its role in pseudomonas infections. Rev Infect Dis. 1983 Nov-Dec;5 (Suppl 5):S998–1004. doi: 10.1093/clinids/5.supplement_5.s998. [DOI] [PubMed] [Google Scholar]
  39. Wretlind B., Wadström T. Purification and properties of a protease with elastase activity from Pseudomonas aeruginosa. J Gen Microbiol. 1977 Dec;103(2):319–327. doi: 10.1099/00221287-103-2-319. [DOI] [PubMed] [Google Scholar]
  40. 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]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES