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. 1984 Aug;159(2):683–687. doi: 10.1128/jb.159.2.683-687.1984

Cloning and expression of a gene segment encoding the enzymatic moiety of Pseudomonas aeruginosa exotoxin A.

M A Mozola, R B Wilson, E M Jordan, R K Draper, R C Clowes
PMCID: PMC215698  PMID: 6086583

Abstract

Using the broad-host-range plasmid vector pRO1614, we cloned a segment of the gene from Pseudomonas aeruginosa PA103 encoding the enzymatically active part of the exotoxin A protein. Expression of the cloned gene segment has been achieved both in Escherichia coli and in a nontoxigenic P. aeruginosa host, as assayed by the production of exotoxin A-related antigen and by the ability of the gene product to ADP-ribosylate elongation factor 2. Western blot hybridization analysis revealed a series of polypeptides antigenically related to exotoxin A, the largest of which had a molecular weight of ca. 50,000.

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Selected References

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  1. Bjorn M. J., Vasil M. L., Sadoff J. C., Iglewski B. H. Incidence of exotoxin production by Pseudomonas species. Infect Immun. 1977 Apr;16(1):362–366. doi: 10.1128/iai.16.1.362-366.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. Brosius J., Cate R. L., Perlmutter A. P. Precise location of two promoters for the beta-lactamase gene of pBR322. S1 mapping of ribonucleic acid isolated from Escherichia coli or synthesized in vitro. J Biol Chem. 1982 Aug 10;257(15):9205–9210. [PubMed] [Google Scholar]
  4. 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]
  5. Chiang S. J., Clowes R. C. Intramolecular transposition and inversion in plasmid R6K. J Bacteriol. 1980 May;142(2):668–682. doi: 10.1128/jb.142.2.668-682.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chung D. W., Collier R. J. Enzymatically active peptide from the adenosine diphosphate-ribosylating toxin of Pseudomonas aeruginosa. Infect Immun. 1977 Jun;16(3):832–841. doi: 10.1128/iai.16.3.832-841.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cohen S. N., Chang A. C., Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. doi: 10.1073/pnas.69.8.2110. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cross A. S., Sadoff J. C., Iglewski B. H., Sokol P. A. Evidence for the role of toxin A in the pathogenesis of infection with Pseudomonas aeruginosa in humans. J Infect Dis. 1980 Oct;142(4):538–546. doi: 10.1093/infdis/142.4.538. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Haas D., Holloway B. W. Chromosome mobilization by the R plasmid R68.45: a tool in Pseudomonas genetics. Mol Gen Genet. 1978 Jan 17;158(3):229–237. doi: 10.1007/BF00267194. [DOI] [PubMed] [Google Scholar]
  11. Haas D., Holloway B. W. R factor variants with enhanced sex factor activity in Pseudomonas aeruginosa. Mol Gen Genet. 1976 Mar 30;144(3):243–251. doi: 10.1007/BF00341722. [DOI] [PubMed] [Google Scholar]
  12. Hanne L. F., Howe T. R., Iglewski B. H. Locus of the Pseudomonas aeruginosa toxin A gene. J Bacteriol. 1983 Apr;154(1):383–386. doi: 10.1128/jb.154.1.383-386.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Holmans P. L., Clowes R. C. Transposition of a duplicate antibiotic resistance gene and generation of deletions in plasmid R6K. J Bacteriol. 1979 Feb;137(2):977–989. doi: 10.1128/jb.137.2.977-989.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Iglewski B. H., Kabat D. NAD-dependent inhibition of protein synthesis by Pseudomonas aeruginosa toxin,. Proc Natl Acad Sci U S A. 1975 Jun;72(6):2284–2288. doi: 10.1073/pnas.72.6.2284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Iglewski B. H., Sadoff J. C. Toxin inhibitors of protein synthesis: production, purification, and assay of Pseudomonas aeruginosa toxin A. Methods Enzymol. 1979;60:780–793. doi: 10.1016/s0076-6879(79)60071-x. [DOI] [PubMed] [Google Scholar]
  16. Iglewski W. J., Lee H. Purification and properties of an altered form of elongation factor 2 from mutant cells resistant to intoxication by diphtheria toxin. Eur J Biochem. 1983 Aug 1;134(2):237–240. doi: 10.1111/j.1432-1033.1983.tb07556.x. [DOI] [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. The roles of various fractions of Pseudomonas aeruginosa in its pathogenesis. 3. Identity of the lethal toxins produced in vitro and in vivo. J Infect Dis. 1966 Oct;116(4):481–489. doi: 10.1093/infdis/116.4.481. [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. Markwell M. A. A new solid-state reagent to iodinate proteins. I. Conditions for the efficient labeling of antiserum. Anal Biochem. 1982 Sep 15;125(2):427–432. doi: 10.1016/0003-2697(82)90025-2. [DOI] [PubMed] [Google Scholar]
  21. Mee B. J., Lee B. T. A map order for his I, one of the genetic regions controlling histidine biosynthesis in Pseudomonas aeruginosa, using the transducing phage F116. Genetics. 1969 Jul;62(3):687–696. [PMC free article] [PubMed] [Google Scholar]
  22. Meyer T. F., Mlawer N., So M. Pilus expression in Neisseria gonorrhoeae involves chromosomal rearrangement. Cell. 1982 Aug;30(1):45–52. doi: 10.1016/0092-8674(82)90010-1. [DOI] [PubMed] [Google Scholar]
  23. Olsen R. H., DeBusscher G., McCombie W. R. Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol. 1982 Apr;150(1):60–69. doi: 10.1128/jb.150.1.60-69.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Olsnes S., Pihl A. Chimeric toxins. Pharmacol Ther. 1981;15(3):355–381. doi: 10.1016/0163-7258(81)90050-4. [DOI] [PubMed] [Google Scholar]
  25. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  26. Peden K. W. Revised sequence of the tetracycline-resistance gene of pBR322. Gene. 1983 May-Jun;22(2-3):277–280. doi: 10.1016/0378-1119(83)90112-9. [DOI] [PubMed] [Google Scholar]
  27. Pollack M., Taylor N. S., Callahan L. T., 3rd Exotoxin production by clinical isolates of pseudomonas aeruginosa. Infect Immun. 1977 Mar;15(3):776–780. doi: 10.1128/iai.15.3.776-780.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Royle P. L., Matsumoto H., Holloway B. W. Genetic circularity of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol. 1981 Jan;145(1):145–155. doi: 10.1128/jb.145.1.145-155.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sanai Y., Morihara K., Tsuzuki H., Homma J. Y., Kato I. Proteolytic cleavage of exotoxin A from Pseudomonas aeruginosa: formation of an ADP-ribosyltransferase active fragment by the action of Pseudomonas elastase. FEBS Lett. 1980 Oct 20;120(1):131–134. doi: 10.1016/0014-5793(80)81063-5. [DOI] [PubMed] [Google Scholar]
  30. Soldati L., Crockett R., Carrigan J. M., Leisinger T., Holloway B. W., Haas D. Revised locations of the hisI and pru (proline utilization) genes on the Pseudomonas aeruginosa chromosome map. Mol Gen Genet. 1984;193(3):431–436. doi: 10.1007/BF00382080. [DOI] [PubMed] [Google Scholar]
  31. Stanisich V., Holloway B. W. Conjugation in Pseudomonas aeruginosa. Genetics. 1969 Feb;61(2):327–339. doi: 10.1093/genetics/61.2.327. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Sutcliffe J. G. Complete nucleotide sequence of the Escherichia coli plasmid pBR322. Cold Spring Harb Symp Quant Biol. 1979;43(Pt 1):77–90. doi: 10.1101/sqb.1979.043.01.013. [DOI] [PubMed] [Google Scholar]
  33. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. 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]
  35. Young L. S. The role of exotoxins in the pathogenesis of Pseudomonas aeruginosa infections. J Infect Dis. 1980 Oct;142(4):626–630. doi: 10.1093/infdis/142.4.626. [DOI] [PubMed] [Google Scholar]
  36. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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