Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Jan;63(1):1–8. doi: 10.1128/iai.63.1.1-8.1995

Expression of recombinant exoenzyme S of Pseudomonas aeruginosa.

S M Kulich 1, D W Frank 1, J T Barbieri 1
PMCID: PMC172950  PMID: 7806344

Abstract

The structural gene for the 49-kDa form of exoenzyme S (exoS) isolated from Pseudomonas aeruginosa 388 was expressed in both Escherichia coli and P. aeruginosa PA103. Expression of exoS in E. coli under the transcriptional regulation of the T7 promoter yielded a soluble cytosolic protein with an apparent molecular mass of 49 kDa, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Expression of exoS in P. aeruginosa PA103 under the transcriptional regulation of the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS yielded a nitrilotriacetic acid-inducible extracellular protein with an apparent molecular mass of 49 kDa. Recombinant ExoS (rExoS) reacted with the anti-49-kDa form of exoenzyme S immunoglobulin G, existed as an aggregate as determined by gel filtration chromatography, and ADP-ribosylated soybean trypsin inhibitor at a specific activity that was similar (within twofold) to that of native exoenzyme S. Allelic exchange of exoS with a tetracycline gene cartridge yielded a strain of P. aeruginosa 388 that did not express detectable amounts of either ExoS in an immunoblot analysis using the anti-49-kDa form of exoenzyme S immunoglobulin G or ADP-ribosyltransferase activity under standard enzyme assay conditions. Expression of catalytically active rExoS in E. coli demonstrated that exoS was necessary and sufficient for the factor-activating exoenzyme S-dependent ADP-ribosyltransferase activity of exoenzyme S. Expression of nitrilotriacetic acid-inducible rExoS in P. aeruginosa PA103 demonstrated that the 0.9 kbp of Pseudomonas chromosomal DNA flanking the 5' end of exoS encoded a functional exoenzyme S promoter. Expression analysis and allelic exchange experiments suggest that the 49- and 53-kDa forms of exoenzyme S are encoded by separate genes.

Full Text

The Full Text of this article is available as a PDF (318.0 KB).

Selected References

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

  1. Bodey G. P., Bolivar R., Fainstein V., Jadeja L. Infections caused by Pseudomonas aeruginosa. Rev Infect Dis. 1983 Mar-Apr;5(2):279–313. doi: 10.1093/clinids/5.2.279. [DOI] [PubMed] [Google Scholar]
  2. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  3. Coburn J., Gill D. M. ADP-ribosylation of p21ras and related proteins by Pseudomonas aeruginosa exoenzyme S. Infect Immun. 1991 Nov;59(11):4259–4262. doi: 10.1128/iai.59.11.4259-4262.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Coburn J., Kane A. V., Feig L., Gill D. M. Pseudomonas aeruginosa exoenzyme S requires a eukaryotic protein for ADP-ribosyltransferase activity. J Biol Chem. 1991 Apr 5;266(10):6438–6446. [PubMed] [Google Scholar]
  5. Coburn J. Pseudomonas aeruginosa exoenzyme S. Curr Top Microbiol Immunol. 1992;175:133–143. doi: 10.1007/978-3-642-76966-5_7. [DOI] [PubMed] [Google Scholar]
  6. Coburn J., Wyatt R. T., Iglewski B. H., Gill D. M. Several GTP-binding proteins, including p21c-H-ras, are preferred substrates of Pseudomonas aeruginosa exoenzyme S. J Biol Chem. 1989 May 25;264(15):9004–9008. [PubMed] [Google Scholar]
  7. Drazin R., Kandel J., Collier R. J. Structure and activity of diphtheria toxin. II. Attack by trypsin at a specific site within the intact toxin molecule. J Biol Chem. 1971 Mar 10;246(5):1504–1510. [PubMed] [Google Scholar]
  8. Frank D. W., Iglewski B. H. Cloning and sequence analysis of a trans-regulatory locus required for exoenzyme S synthesis in Pseudomonas aeruginosa. J Bacteriol. 1991 Oct;173(20):6460–6468. doi: 10.1128/jb.173.20.6460-6468.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Frank D. W., Nair G., Schweizer H. P. Construction and characterization of chromosomal insertional mutations of the Pseudomonas aeruginosa exoenzyme S trans-regulatory locus. Infect Immun. 1994 Feb;62(2):554–563. doi: 10.1128/iai.62.2.554-563.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fu H., Coburn J., Collier R. J. The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2320–2324. doi: 10.1073/pnas.90.6.2320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Grimwood K., To M., Rabin H. R., Woods D. E. Inhibition of Pseudomonas aeruginosa exoenzyme expression by subinhibitory antibiotic concentrations. Antimicrob Agents Chemother. 1989 Jan;33(1):41–47. doi: 10.1128/aac.33.1.41. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ho S. N., Hunt H. D., Horton R. M., Pullen J. K., Pease L. R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene. 1989 Apr 15;77(1):51–59. doi: 10.1016/0378-1119(89)90358-2. [DOI] [PubMed] [Google Scholar]
  13. Iglewski B. H., Liu P. V., Kabat D. Mechanism of action of Pseudomonas aeruginosa exotoxin Aiadenosine diphosphate-ribosylation of mammalian elongation factor 2 in vitro and in vivo. Infect Immun. 1977 Jan;15(1):138–144. doi: 10.1128/iai.15.1.138-144.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iglewski B. H., Sadoff J., Bjorn M. J., Maxwell E. S. Pseudomonas aeruginosa exoenzyme S: an adenosine diphosphate ribosyltransferase distinct from toxin A. Proc Natl Acad Sci U S A. 1978 Jul;75(7):3211–3215. doi: 10.1073/pnas.75.7.3211. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kulich S. M., Frank D. W., Barbieri J. T. Purification and characterization of exoenzyme S from Pseudomonas aeruginosa 388. Infect Immun. 1993 Jan;61(1):307–313. doi: 10.1128/iai.61.1.307-313.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kulich S. M., Yahr T. L., Mende-Mueller L. M., Barbieri J. T., Frank D. W. Cloning the structural gene for the 49-kDa form of exoenzyme S (exoS) from Pseudomonas aeruginosa strain 388. J Biol Chem. 1994 Apr 8;269(14):10431–10437. [PubMed] [Google Scholar]
  17. 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]
  18. Liu P. V. Extracellular toxins of Pseudomonas aeruginosa. J Infect Dis. 1974 Nov;130 (Suppl)(0):S94–S99. doi: 10.1093/infdis/130.supplement.s94. [DOI] [PubMed] [Google Scholar]
  19. Lory S., Collier R. J. Expression of enzymic activity by exotoxin A from Pseudomonas aeruginosa. Infect Immun. 1980 May;28(2):494–501. doi: 10.1128/iai.28.2.494-501.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Mekalanos J. J., Collier R. J., Romig W. R. Enzymic activity of cholera toxin. II. Relationships to proteolytic processing, disulfide bond reduction, and subunit composition. J Biol Chem. 1979 Jul 10;254(13):5855–5861. [PubMed] [Google Scholar]
  21. Mekalanos J. J. Environmental signals controlling expression of virulence determinants in bacteria. J Bacteriol. 1992 Jan;174(1):1–7. doi: 10.1128/jb.174.1.1-7.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Nicas T. I., Frank D. W., Stenzel P., Lile J. D., Iglewski B. H. Role of exoenzyme S in chronic Pseudomonas aeruginosa lung infections. Eur J Clin Microbiol. 1985 Apr;4(2):175–179. doi: 10.1007/BF02013593. [DOI] [PubMed] [Google Scholar]
  23. Nicas T. I., Iglewski B. H. Contribution of exoenzyme S to the virulence of Pseudomonas aeruginosa. Antibiot Chemother (1971) 1985;36:40–48. doi: 10.1159/000410470. [DOI] [PubMed] [Google Scholar]
  24. Nicas T. I., Iglewski B. H. Isolation and characterization of transposon-induced mutants of Pseudomonas aeruginosa deficient in production of exoenzyme S. Infect Immun. 1984 Aug;45(2):470–474. doi: 10.1128/iai.45.2.470-474.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nicas T. I., Iglewski B. H. The contribution of exoproducts to virulence of Pseudomonas aeruginosa. Can J Microbiol. 1985 Apr;31(4):387–392. doi: 10.1139/m85-074. [DOI] [PubMed] [Google Scholar]
  26. Roilides E., Butler K. M., Husson R. N., Mueller B. U., Lewis L. L., Pizzo P. A. Pseudomonas infections in children with human immunodeficiency virus infection. Pediatr Infect Dis J. 1992 Jul;11(7):547–553. doi: 10.1097/00006454-199207000-00008. [DOI] [PubMed] [Google Scholar]
  27. Sanger F., Coulson A. R., Barrell B. G., Smith A. J., Roe B. A. Cloning in single-stranded bacteriophage as an aid to rapid DNA sequencing. J Mol Biol. 1980 Oct 25;143(2):161–178. doi: 10.1016/0022-2836(80)90196-5. [DOI] [PubMed] [Google Scholar]
  28. Schweizer H. P. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol Microbiol. 1992 May;6(9):1195–1204. doi: 10.1111/j.1365-2958.1992.tb01558.x. [DOI] [PubMed] [Google Scholar]
  29. Schweizer H. P. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene. 1991 Jan 2;97(1):109–121. doi: 10.1016/0378-1119(91)90016-5. [DOI] [PubMed] [Google Scholar]
  30. Sokol P. A., Dennis J. J., MacDougall P. C., Sexton M., Woods D. E. Cloning and expression of the Pseudomonas aeruginosa exoenzyme S toxin gene. Microb Pathog. 1990 Apr;8(4):243–257. doi: 10.1016/0882-4010(90)90051-q. [DOI] [PubMed] [Google Scholar]
  31. Studier F. W., Moffatt B. A. Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol. 1986 May 5;189(1):113–130. doi: 10.1016/0022-2836(86)90385-2. [DOI] [PubMed] [Google Scholar]
  32. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vasil M. L., Kabat D., Iglewski B. H. Structure-activity relationships of an exotoxin of Pseudomonas aeruginosa. Infect Immun. 1977 Apr;16(1):353–361. doi: 10.1128/iai.16.1.353-361.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Woods D. E., Iglewski B. H. Toxins of Pseudomonas aeruginosa: new perspectives. Rev Infect Dis. 1983 Sep-Oct;5 (Suppl 4):S715–S722. doi: 10.1093/clinids/5.supplement_4.s715. [DOI] [PubMed] [Google Scholar]
  35. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES