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. 1988 Nov;170(11):5385–5388. doi: 10.1128/jb.170.11.5385-5388.1988

Determination of the amino acid change responsible for the nontoxic, cross-reactive exotoxin A protein (CRM 66) of Pseudomonas aeruginosa PAO-PR1.

M J Wick 1, B H Iglewski 1
PMCID: PMC211620  PMID: 3141388

Abstract

Analysis of purified exotoxin A from parental Pseudomonas aeruginosa PAO1 and mutant strain PAO-PR1, which produces enzymatically inactive exotoxin A (CRM 66), revealed that CRM 66 lost 90% of parental enzymatic activity. Nucleotide sequence analysis of cloned exotoxin A genes showed a single amino acid substitution in CRM 66. Position 426 in the mature protein of parental (PAO1) exotoxin A is histidine, whereas in CRM 66, it is tyrosine.

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Selected References

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  1. Allured V. S., Collier R. J., Carroll S. F., McKay D. B. Structure of exotoxin A of Pseudomonas aeruginosa at 3.0-Angstrom resolution. Proc Natl Acad Sci U S A. 1986 Mar;83(5):1320–1324. doi: 10.1073/pnas.83.5.1320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brandhuber B. J., Allured V. S., Falbel T. G., McKay D. B. Mapping the enzymatic active site of Pseudomonas aeruginosa exotoxin A. Proteins. 1988;3(3):146–154. doi: 10.1002/prot.340030303. [DOI] [PubMed] [Google Scholar]
  3. Carroll S. F., Collier R. J. Active site of Pseudomonas aeruginosa exotoxin A. Glutamic acid 553 is photolabeled by NAD and shows functional homology with glutamic acid 148 of diphtheria toxin. J Biol Chem. 1987 Jun 25;262(18):8707–8711. [PubMed] [Google Scholar]
  4. Chou P. Y., Fasman G. D. Conformational parameters for amino acids in helical, beta-sheet, and random coil regions calculated from proteins. Biochemistry. 1974 Jan 15;13(2):211–222. doi: 10.1021/bi00699a001. [DOI] [PubMed] [Google Scholar]
  5. Cryz S. J., Jr, Friedman R. L., Iglewski B. H. Isolation and characterization of a Pseudomonas aeruginosa mutant producing a nontoxic, immunologically crossreactive toxin A protein. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7199–7203. doi: 10.1073/pnas.77.12.7199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cryz S. J., Jr, Iglewski B. H. Toxoids of Pseudomonas aeruginosa toxin A: production by chemical and genetic means. Rev Infect Dis. 1983 Nov-Dec;5 (Suppl 5):S992–S997. doi: 10.1093/clinids/5.supplement_5.s992. [DOI] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  9. Frank D. W., Iglewski B. H. Kinetics of toxA and regA mRNA accumulation in Pseudomonas aeruginosa. J Bacteriol. 1988 Oct;170(10):4477–4483. doi: 10.1128/jb.170.10.4477-4483.1988. [DOI] [PMC free article] [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. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Guidi-Rontani C., Collier R. J. Exotoxin A of Pseudomonas aeruginosa: evidence that domain I functions in receptor binding. Mol Microbiol. 1987 Jul;1(1):67–72. doi: 10.1111/j.1365-2958.1987.tb00528.x. [DOI] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. 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]
  16. Hwang J., Fitzgerald D. J., Adhya S., Pastan I. Functional domains of Pseudomonas exotoxin identified by deletion analysis of the gene expressed in E. coli. Cell. 1987 Jan 16;48(1):129–136. doi: 10.1016/0092-8674(87)90363-1. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. 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]
  19. 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]
  20. 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]
  21. Leppla S. H., Martin O. C., Muehl L. A. The exotoxin P. aeruginosa: a proenzyme having an unusual mode of activation. Biochem Biophys Res Commun. 1978 Mar 30;81(2):532–538. doi: 10.1016/0006-291x(78)91567-x. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. 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]
  24. 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]
  25. Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Ohman D. E., Burns R. P., Iglewski B. H. Corneal infections in mice with toxin A and elastase mutants of Pseudomonas aeruginosa. J Infect Dis. 1980 Oct;142(4):547–555. doi: 10.1093/infdis/142.4.547. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Pustell J., Kafatos F. C. A convenient and adaptable package of computer programs for DNA and protein sequence management, analysis and homology determination. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):643–655. doi: 10.1093/nar/12.1part2.643. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. 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]
  32. 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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