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Absence of persistence and transfer of genetic material by recombinantEscherichia coli in conventional, antibiotic-treated mice

  • Environmental Fate Issues: In Vivo Studies
  • Published:
Journal of Industrial Microbiology

Summary

Strain BST-1 is a derivative ofEscherichia coli K-12 that carries a plasmid designated pURA-4 and is the expression system used by The Upjohn Company in the production of recombinant bovine somatotropin (rbSt). This plasmid also encodes an ampicillin resistance gene. The plasmidless carrier strain, BST-1C, contains a gene for tetracycline resistance which is provided by the chromosomal insertion of the transposon Tn10. Therefore, BST-1 is resistant to ampicillin and tetracycline, while BST-1C is resistant only to tetracycline. The Food and Drug Administration requested that we conduct an environmental assessment study to monitor the ‘persistence of the recombinant live K-12E. coli organism compared to the hostE. coli organism’. In addition, we were requested to monitor ‘the potential transfer of genetic material from (our) recombinant organism to the indigenous microflora’ of the mouse gastrointestinal (GI) tract. The differences in persistence were determined by monitoring shedding of BST-1 and BST-1C in the feces of conventionally reared, outbred mice inoculated with either of the two strains. Even with antibiotic selective pressure applied (tetracycline in the water), BST-1 did not persist as well as the non-plasmid carrying parental stain, BST-1C. In the gene transfer experiments, transfer of pURA-4 was monitored by the appearance of the ampicillin resistance marker and/or by hybridization assays for the rbSt gene in indigenous, mouse-colonizingE. coli strains which had been made streptomycin resistant. At the limit of detection, no transfer of pURA-4 was detected either in vitro or in vivo. These data support an interpretation that BST-1 does not present an environmental hazard as measured by colonization/persistence in the gut of conventionally reared mammals.

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References

  1. -. 1984. Official Methods of Analysis. 14th edn. pp. 813–818, AOAC, Arlington.

  2. Anderson, E.S. 1975. Viability of, and transfer of a plasmid fromE. coli K-12 in the human intestine. Nature (Lond.) 255: 502–504.

    Google Scholar 

  3. Barry, B., C.L. Squires and C. Squires. 1979. Control features within rplJL-rpoBC transcription unit ofEscherichia coli. Proc. Natl. Acad. Sci. USA 76: 4922–4926.

    PubMed  Google Scholar 

  4. Bennett, P.M. 1991. Transposable elements and transposition in bacteria. In: Modern Microbial Genetics (U.N. Streips and R.E. Yasbin, eds.), pp. 323–364, Wiley-Liss, Inc., New York.

    Google Scholar 

  5. Bogosian, G. and J.F. Kane. 1991. Fate of recombinantEscherichia coli K-12 strains in the environment. Adv. Appl. Microbiol. 36: 87–131.

    PubMed  Google Scholar 

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

    PubMed  Google Scholar 

  7. Boyd, A.C., J.A.K. Archer and D.J. Sherratt. 1989. Characterization of the ColE1 mobilization region and its protein products. Mol. Gen. Genet. 217: 488–498.

    PubMed  Google Scholar 

  8. Clark, A.J. and G.J. Warren. 1979. Conjugal transmission of plasmids. Annu. Rev. Genet. 13: 99–125.

    PubMed  Google Scholar 

  9. Cohen, P.S., R.W. Pilsucki, M.L. Myhal, C.A. Rosen, D.C. Laux and V.J. Cabelli. 1979. Colonization potentials of male and femaleE. coli K12 strains,E. coli B and human fecalE. coli strains in the mouse GI tract. Recomb. DNA Tech. Bull. 2: 106–113.

    Google Scholar 

  10. Corpet, D.E., S. Lumeau and F. Corpet. 1989. Minimum antibiotic levels for selecting a resistance plasmid in a gnotobiotic animal model. Antimicrob. Agents Chemother. 33: 535–540.

    PubMed  Google Scholar 

  11. Coughter, J.P. and G.J. Stewart. 1989. Genetic exchange in the environment. Antonie van Leeuwenhoek J. Microbiol. 55: 15–22.

    PubMed  Google Scholar 

  12. Curtiss, R., III. 1981. Gene transfer. In: Manual of Methods for General Bacteriology (P. Gerhardt, R.G.E. Murray, R.N. Costilow, E.W. Nester, W.A. Wood, N.R. Krieg, and G.B. Phillips, eds.), pp. 243–265, American Society for Microbiology, Washington, DC.

    Google Scholar 

  13. Feinberg, A.P. and B. Vogelstein. 1983. A technique for radiolabeling DNA restriction fragments to high specific activity. Anal. Biochem. 132: 6–13.

    PubMed  Google Scholar 

  14. Finegold, S.M., H.R. Attebery and V.L. Sutter. 1974. Effect of diet on human fecal flora: comparison of Japanese and American Diets. Am. J. Clin. Nutr. 27: 1456–1469.

    PubMed  Google Scholar 

  15. Finnegan, J. and D. Sherratt. 1982. Plasmid ColE1 conjugal mobility: The nature ofbom, a region required incis for transfer. Mol. Gen. Genet. 185: 344–351.

    PubMed  Google Scholar 

  16. Freter, R., R.R. Freter and H. Brickner. 1983. Experimental and mathematical models ofEscherichia coli plasmid transfer in vitro and in vivo. Infect. Immun. 39: 60–84.

    PubMed  Google Scholar 

  17. George, S.E., M.J. Kohan, B.B. Walsh and L.D. Claxton. 1989. Acute colonization study of polychlorinated biphenyldegrading pseudomonades in the mouse intestinal tract: comparisons of single and multiple exposures. Environ. Toxicol. Chem. 1: 123–131.

    Google Scholar 

  18. Gibbons, R.J., S.S. Socransky, S. deAranjo and J. Van Houte. 1964. Studies of predominant cultivated microbiota of dental plaques. Arch. Oral Biol. 9: 365–370.

    Google Scholar 

  19. Guyer, M.S. 1978. The γδ sequence of F is an insertion sequece. J. Mol. Biol. 126: 347–365.

    PubMed  Google Scholar 

  20. Lee, A. 1985. Neglected niches; the microbial ecology of the gastrointestinal tract. Adv. Microb. Ecol. 8: 115–162.

    Google Scholar 

  21. Levy, S.B. 1984. Survival of plasmids inEscherichia coli. In: Genetic Manipulation: Impact on Man and Society (W. Arber, K. Illmensee, W.U. Peacock and P. Starlinger, eds.), pp. 19–28, ICSU Press, Paris.

    Google Scholar 

  22. Levy, S.B. and B. Marshall. 1979. Survival ofE. coli hostvector systems in the human intestinal tract. Recomb. DNA Tech. Bull. 2: 77–80.

    Google Scholar 

  23. Levy, S.B. and B. Marshall. 1981. Risk assessment studies ofE. coli host-vector systems. Recomb. DNA Tech. Bull. 4: 91–98.

    PubMed  Google Scholar 

  24. Levy, S.B., B. Marshall, D. Rowse-Eagle, and A. Onderdonk. 1980. Survival ofEscherichia coli host-vector systems in the mammalian intestine. Science 209: 391–394.

    PubMed  Google Scholar 

  25. Maniatis, T., E.F., Fritsch and J. Sambrook. 1982. Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Press, Cold Spring Harbor, NY.

    Google Scholar 

  26. Margolin, P. 1987. Generalized transduction. In:Escherichia coli andSalmonella typhimurium: Cellular and Molecular Biology, Vol. 2 (F.C. Neidhardt, J.L. Ingraham, K.B. Low, B. Magasanik, M. Schaechter, H.E. Umbarger, eds.), pp. 1154–1168, American Society for Microbiology, Washington, DC.

    Google Scholar 

  27. Marshall, B., S. Schluederberg, C. Tachibana and S.B. Levy. 1981. Survival and transfer in the human gut of poorly mobilizable (pBR322) and of transferable plasmids from the same carrierE. coli. Gene 14: 145–154.

    PubMed  Google Scholar 

  28. Miller, J.H. 1972. Experiments in Molecular Genetics. Cold Springer Harbor Press, Cold Springer Harbor, NY.

    Google Scholar 

  29. Orbach, M.J. and E.N. Jackson. 1982. Transfer of chimeric plasmids amongSalmonella typhimurium strains by P22 transduction. J. Bacteriol. 149: 985–994.

    PubMed  Google Scholar 

  30. Paul, J.H., M.E. Frischer and J.M. Thurmond. 1991. Gene transfer in marine water column and sediment microcosms by natural plasmid transformation. Appl. Environ. Microbiol. 57: 1509–1515.

    Google Scholar 

  31. Ryder, T.B., D.B. Davison, J.I. Rosen, E. Ohtsubo and H. Ohtsubo. 1982. Analysis of plasmid genome evolution based on nucleotide-sequence comparison of two related plasmids ofEscherichia coli. Gene 17: 299–310.

    PubMed  Google Scholar 

  32. Sambrook, J., E.F. Fritsch and T. Maniatis. 1989. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

    Google Scholar 

  33. Schmidt, C. and H. Schmieger. 1984. Selective transduction of recombinant plasmids with clonedpac sites bySalmonella phage P22. Mol. Gen. Genet. 196: 123–128.

    PubMed  Google Scholar 

  34. Shoemaker, N.B., C. Getty, J.F. Gardner and A.A. Salyers. 1986. Tn4351 transposes inBacteroides spp. and mediates the integration of plasmid R751 into theBacteroides chromosome. J. Bacteriol. 165: 929–936.

    PubMed  Google Scholar 

  35. Smith, C.A. and C.M. Thomas. 1989. Relationships and evolution of IncP plasmids. In: Promiscuous Plasmids of Gram-Negative Bacteria (Thomas, C.M., ed.), pp. 57–77, Academic Press, New York.

    Google Scholar 

  36. Smith, H.W. 1975. Survival of orally administeredE. coli K-12 in the alimentary tract of man. Nature (Lond.) 255: 500–502.

    Google Scholar 

  37. Smith, H.W. 1978. Is it safe to useEscherichia coli K12 in recombinant DNA experiments? J. Infect. Dis. 137: 655–660.

    PubMed  Google Scholar 

  38. Squires, C., A. Krauner, G. Barry, W.F. Shen and C.L. Squires. 1981. Nucleotide sequence at the end of the gene for the RNA polymerase β′ subunit (rpoC). Nucleic Acid. Res. 9: 6827–6840.

    PubMed  Google Scholar 

  39. Stewart, G.J. and C.A. Carlson. 1986. The biology of natural transformation. Annu. Rev. Microbiol. 40: 211–235.

    PubMed  Google Scholar 

  40. Stewart, G.J. and C.D. Sinigalliano. 1990. Detection of horizontal gene transfer by natural transformation in native and introduced species of bacteria in marine and synthetic sediments. Appl. Environ. Microbiol. 56: 1818–1824.

    Google Scholar 

  41. Stotzky, G. and H. Babich. 1984. Fate of geneticallyengineered microbes in natural environments. Recomb. DNA Tech. Bull. 7: 163–188.

    PubMed  Google Scholar 

  42. Takahashi, H. and H. Saito. 1982. High-frequency transduction of pBR322 by cytosine-substituted T4 bacteriophage: evidence for encapsulation and transfer of head-to-tail plasmid concatemers. Plasmid 8: 29–35.

    PubMed  Google Scholar 

  43. Tomich, C-S.C., E.R. Olson, P.S. Kaytes, S.K. Rockenbach and N.T. Hatzenbulher. 1989. Effects of nucleotide sequences directly downstream from the AUG on the expression of bovine somatotropin inE. coli. Nucleic Acids Res. 17: 3179–3197.

    PubMed  Google Scholar 

  44. Uhlin, B.E.R., S. Molin, P. Gustafsson and K. Nordstrom. 1979. Plasmids with temperature dependent copy number for amplification of cloned genes and their products. Gene 6: 91–106.

    PubMed  Google Scholar 

  45. Uhlin, B.E. and K. Nordstrom. 1977. R plasmid gene dosage effects inEscherichia coli K-12: copy mutants of the R plasmid R1drd-19. Plasmid 1: 1–17.

    PubMed  Google Scholar 

  46. Uhlin, B.E. and K. Nordstrom. 1978. A runaway-replication mutant of plasmid R1drd-19: Temperature-dependent loss of copy number control. Molec. Gen. Genet. 165: 167–179.

    PubMed  Google Scholar 

  47. Willetts, N. 1988. Conjugation. Methods Microbiol. 21: 49–77.

    Google Scholar 

  48. Williams, P.H. 1977. Plasmid transfer in the human alimentary tract. FEMS Microbiol. Lett. 2: 91–95.

    Google Scholar 

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Authors and Affiliations

  1. The Upjohn Company, 7923-190-083, Kalamazoo, MI, USA

    Robert J. Yancey Jr., Susan F. Kotarski, Kerry K. Thurn, Robert A. Lepley & John E. Mott

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  1. Robert J. Yancey Jr.
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Yancey, R.J., Kotarski, S.F., Thurn, K.K. et al. Absence of persistence and transfer of genetic material by recombinantEscherichia coli in conventional, antibiotic-treated mice. Journal of Industrial Microbiology 11, 259–271 (1993). https://doi.org/10.1007/BF01569599

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