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Enzymatic Hydrolysis of Organophosphates: Cloning and Expression of a Parathion Hydrolase Gene from Pseudomonas diminuta

Bio/Technology volume 3, pages 567–571 (1985)Cite this article

Abstract

Pseudomonas diminuta strain MG hydrolyzes parathion to diethylthiophosphoric acid and p-nitrophenol. The esterase responsible for this reaction is encoded by a gene located on a plasmid termed pCMS1. The gene was cloned into plasmid pBR322 and the broad host range cloning vector pKT230. Enzyme activity was detected in Escherichia coli strains that contained the recombinant plasmids. A 1.5 kilobase BamHI fragment with single restriction sites for SalI, PstI and XhoI was shown to direct the synthesis of the enzyme. The 1.5 kilobase BamHI fragment was inserted into the high expression vector pUC7, and the resulting recombinant, pCMS40, was used to construct pKT230 derivatives containing the parathion hydrolase gene. Subsequent transfer of the recombinant plasmids into a cured derivative of P. diminuta MG and a p-nitrophenol-utilizing strain of Pseudomonas resulted in the isolation of transconjugants that exhibited parathion hydrolase activity. The highest enzyme activity was observed with P. diminuta MG.

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References

  1. Munnecke, D.M. 1981. The use of microbial enzymes for pesticide detoxification, p. 251–270. In: Microbial degradation of xenobiotics and recalcitrant compounds. Leisinger, T., Cook, A.M., Hütter, R., and Nüesch, J. (eds.), Academic Press Inc., London.

    Google Scholar 

  2. Dagley, S. 1984. Introduction, p. 1–11. In: Microbial degradation of organic compounds. Gibson, D.T. (ed.), Marcel Dekker Inc., New York.

    Google Scholar 

  3. Munnecke, D.M. and Hsieh, D.P.H. 1976. Pathways of microbial metabolism of parathion. Appl. Environ. Microbiol. 31: 63–69.

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Serdar, C.M., Gibson, D.T., Munnecke, D.M. and Lancaster, J.H. 1982. Plasmid involvement in parathion hydrolysis by Pseudomonas diminuta. Appl. Environ. Microbiol. 44: 246–249.

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Munnecke, D.M. 1976. Enzymatic hydrolysis of organophosphate insecticides, a possible pesticide disposal method. Appl. Environ. Microbiol. 32: 7–13.

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Munnecke, D.M. Enzymatic detoxification of waste organophosphate pesticides. J. Agric. Food Chem. 28: 105–111.

    Article  CAS  Google Scholar 

  7. Simpson, J.R. and Evans, W.C. 1953. The metabolism of nitrophenols by certain bacteria. Biochem. J. 55: xxiv.

    CAS  PubMed  Google Scholar 

  8. Spain, J.C., Wyss, O. and Gibson, D.T. 1979. Enzymatic oxidation of p-nitrophenol. Biochem. Biophys. Res. Commun. 88: 634–641.

    Article  CAS  PubMed  Google Scholar 

  9. Cook, A.M., Daughton, C.G. and Alexander, M. 1978. Phosphorus-containing pesticide breakdown products: Quantitative utilization as phosphorus sources by bacteria. Appl. Environ. Microbiol. 36: 668–672.

    CAS  PubMed  PubMed Central  Google Scholar 

  10. Haas, D. 1983. Genetic aspects of biodegradation by pseudomonads. Experientia 39: 1199–1213.

    Article  CAS  PubMed  Google Scholar 

  11. Boyer, H.W. and Roulland-Dussoix, D. 1969. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J. Mol. Biol. 41: 459–472.

    Article  CAS  PubMed  Google Scholar 

  12. Hohn, B. 1979. In vitro packaging of λ and cosmid DNA. Meth. Enzymol. 68: 299–309.

    Article  CAS  Google Scholar 

  13. Amann, E., Brosius, J. and Ptashne, M. 1983. Vectors bearing a hybrid trp-lac promoter useful for regulated expression of cloned Genes in Escherichia coli. Gene 25: 167–178.

    Article  CAS  PubMed  Google Scholar 

  14. Bolivar, F., Rodriguez, R.L., Greene, P.J., Betlach, M.C., Heyneker, H.L., Boyer, H.W., Crosa, J.H. and Falkow, S. 1977. Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2: 95–113.

    Article  CAS  PubMed  Google Scholar 

  15. Bagdasarian, M., Lurz, R., Rückert, B., Franklin, F.C.H., Bagdasarian, M.M., Frey, J. and Timmis, K.N. 1981. Specific-purpose plasmid cloning vectors II. Broad host range, high copy number, RSF1010-derived vectors, and a host-vector system for Gene cloning in Pseudomonas. Gene 16: 237–247.

    Article  CAS  PubMed  Google Scholar 

  16. Vieira, J. and Messing, J. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259–268.

    Article  CAS  PubMed  Google Scholar 

  17. Figurski, D.H. and Helinski, D.R. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA. 76: 1648–1652.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bachmann, B.J. 1983. Linkage map of Escherichia coli K-12, edition 7. Microbiol. Rev. 47: 180–230.

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Stüber, D. and Bujard, H. 1981. Organization of transcriptional signals in plasmids pBR322 and pACYC184. Proc. Natl. Acad. Sci. USA. 78: 167–171.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Inouye, S., Ebina, Y., Nakazawa, A. and Nakazawa, T. 1984. Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida. Proc. Natl. Acad. Sci. USA. 18: 1688–1691.

    Article  Google Scholar 

  21. Inouye, S., Nakazawa, A. and Nakazawa, T. 1984. Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida. Gene 29: 323–330.

    Article  CAS  PubMed  Google Scholar 

  22. West, R.W. and Rodriguez, R.L. 1982. Construction and characterization of E. coli promoter-probe plasmid vectors III. pBR322 derivatives with deletions in the tetracycline resistance promoter region. Gene 20: 291–304.

    Article  CAS  PubMed  Google Scholar 

  23. Zech, R. and Wigand, K.D. 1975. Organophosphate-detoxicating enzymes in E. coli. Gelfiltration and isoelectric focusing of DFPase, paraoxonase and unspecific phosphohydrolases. Experientia 31: 157–158.

    Article  CAS  PubMed  Google Scholar 

  24. Stanier, R.Y., Palleroni, N.J. and Doudoroff, M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43: 149–271.

    Article  Google Scholar 

  25. Kado, C.I. and Liu, S.-T. 1981. Rapid procedure for detection and isolation of large and small plasmids. J. Bacteriol. 145: 1365–1373.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Hansen, J.B. and Olsen, R.H. 1978. Isolation of large bacterial plasmids and characterization of the P2 incompatibility group plasmids pMG1 and pMG5. J. Bacteriol. 135: 227–238.

    CAS  PubMed  PubMed Central  Google Scholar 

  27. Holmes, D.S. and Quigley, M. 1981. A rapid boiling method for the preparation of bacterial plasmids. Anal. Biochem. 114: 193–197.

    Article  CAS  PubMed  Google Scholar 

  28. Humpreys, G.O., Willshaw, G.A. and Anderson, E.S. 1975. A simple method for the preparation of large quantities of pure plasmid DNA. Biochim. Biophys. Acta. 383: 457–463.

    Article  Google Scholar 

  29. Meyers, J.A., Sanchez, D., Elwell, L.P. and Falkow, S. 1976. Simple agarose gel electrophoretic method for the identification and characterization of plasmid deoxyribonucleic acid. J. Bacteriol. 127: 1529–1537.

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Cohen, S.N., Chang, A.C.Y. and Hsu, L. 1972. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA. 69: 2110–2114.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ditta, G., Stanfield, S., Corbin, D. and Helinski, D.R. 1980. Broad host range DNA cloning system for gram-negative bacteria: Construction of a Gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: 7347–7351.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Bradford, M.M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72: 248–254.

    Article  CAS  PubMed  Google Scholar 

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  1. Center for Applied Microbiology and Department of Microbiology, The University of Texas at Austin, Austin, Texas, 78712

    Cüneyt M. Serdar & David T. Gibson

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  2. David T. Gibson
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Serdar, C., Gibson, D. Enzymatic Hydrolysis of Organophosphates: Cloning and Expression of a Parathion Hydrolase Gene from Pseudomonas diminuta. Nat Biotechnol 3, 567–571 (1985). https://doi.org/10.1038/nbt0685-567

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