Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1987 Sep;53(9):2013–2020. doi: 10.1128/aem.53.9.2013-2020.1987

Development of a defined medium and two-step culturing method for improved exotoxin A yields from Pseudomonas aeruginosa.

I I Blumentals 1, R M Kelly 1, M Gorziglia 1, J B Kaufman 1, J Shiloach 1
PMCID: PMC204050  PMID: 2445288

Abstract

A two-step method is described for the production of exotoxin A by Pseudomonas aeruginosa in which a defined growth medium is modified for the toxin production phase. As a result, specific exotoxin A yields comparable to those obtained with complex media were achieved. In the development of this two-step process, several divalent metallic cations (Ca2+, Cu2+, and Mn2+), in addition to iron, were found to inhibit the yield of exotoxin A while Ca2+ and glycerol were found to increase yields. Northern blot analysis of total RNA isolates suggests that these effects on exotoxin A yields are based on events at the transcription level.

Full text

PDF

Images in this article

Selected References

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

  1. Bjorn M. J., Iglewski B. H., Ives S. K., Sadoff J. C., Vasil M. L. Effect of iron on yields of exotoxin A in cultures of Pseudomonas aeruginosa PA-103. Infect Immun. 1978 Mar;19(3):785–791. doi: 10.1128/iai.19.3.785-791.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. CLARKE G. D. The effect of cobaltous ions on the formation of toxin and coproporphyrin by a strain of Corynebacterium diphtheriae. J Gen Microbiol. 1958 Jun;18(3):708–719. doi: 10.1099/00221287-18-3-708. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. DeBell R. M. Production of exotoxin A by Pseudomonas aeruginosa in a chemically defined medium. Infect Immun. 1979 Apr;24(1):132–138. doi: 10.1128/iai.24.1.132-138.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Gray G. L., Vasil M. L. Isolation and genetic characterization of toxin-deficient mutants of Pseudomonas aeruginosa PAO. J Bacteriol. 1981 Aug;147(2):275–281. doi: 10.1128/jb.147.2.275-281.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. 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]
  9. Lehrach H., Diamond D., Wozney J. M., Boedtker H. RNA molecular weight determinations by gel electrophoresis under denaturing conditions, a critical reexamination. Biochemistry. 1977 Oct 18;16(21):4743–4751. doi: 10.1021/bi00640a033. [DOI] [PubMed] [Google Scholar]
  10. Liu P. V. Exotoxins of Pseudomonas aeruginosa. I. Factors that influence the production of exotoxin A. J Infect Dis. 1973 Oct;128(4):506–513. doi: 10.1093/infdis/128.4.506. [DOI] [PubMed] [Google Scholar]
  11. Mills J. T., Dodel A. W., Kass E. H. Regulation of staphylococcal toxic shock syndrome toxin-1 and total exoprotein production by magnesium ion. Infect Immun. 1986 Sep;53(3):663–670. doi: 10.1128/iai.53.3.663-670.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Murata R., Soda S., Yamamoto A., Ito A. Further investigations on the influence of inorganic cations on growth and toxin production by Clostridium perfringens PB6K. Jpn J Med Sci Biol. 1968 Feb;21(1):55–70. doi: 10.7883/yoken1952.21.55. [DOI] [PubMed] [Google Scholar]
  13. Paddon C. J., Hartley R. W. Cloning, sequencing and transcription of an inactivated copy of Bacillus amyloliquefaciens extracellular ribonuclease (barnase). Gene. 1985;40(2-3):231–239. doi: 10.1016/0378-1119(85)90045-9. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Pollack M. The role of exotoxin A in pseudomonas disease and immunity. Rev Infect Dis. 1983 Nov-Dec;5 (Suppl 5):S979–S984. doi: 10.1093/clinids/5.supplement_5.s979. [DOI] [PubMed] [Google Scholar]
  16. Tiwari N. P., Campbell J. J. Enzymatic control of the metabolic activity of Pseudomonas aeruginosa grown in glucose or succinate media. Biochim Biophys Acta. 1969 Dec 30;192(3):395–401. doi: 10.1016/0304-4165(69)90388-2. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  19. VAN HEYNINGEN W. E. The neurotoxin of Shigella shigae. 5. The effect of iron and related metals on the growth, respiration and toxin production of Shigella shigae and related organisms. Br J Exp Pathol. 1955 Aug;36(4):373–380. [PMC free article] [PubMed] [Google Scholar]
  20. Vasil M. L., Chamberlain C., Grant C. C. Molecular studies of Pseudomonas exotoxin A gene. Infect Immun. 1986 May;52(2):538–548. doi: 10.1128/iai.52.2.538-548.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES