Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Research Paper
  • Published:

Tracking Recombinant Organisms in the Environment: β–Galactosidase as a Selectable Non–Antibiotic Marker for Fluorescent Pseudomonads

Bio/Technology volume 4, pages 439–444 (1986)Cite this article

Abstract

A sensitive, selectable marker system has been developed for fluorescent pseudomonads based on expression of the E. coli lac operon genes encoding β–galactosidase and lactose permease. Examination of over 500 fluorescent Pseudomonas isolates from soil samples revealed the constant absence of the o–nitrophenylgalactoside (ONPG) positive phenotype, and the inability to utilize lactose as a sole carbon source. Based on these observations broad host–range plasmids containing E. coli lacZ and lacY genes were constructed and stably introduced into native P. fluorescens strains to confer the ability to cleave the chromogenic substrate X–Gal and to grow on minimal lactose media. This marker system enabled the detection of Lac+ transformants at a sensitivity of <10 CFU/g soil.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Similar content being viewed by others

References

  1. Office of Technology Assessment. 1983. Commercial biotechnology: An international analysis. Washington, D.C.

  2. Gillett, J.W., Levin, S.A., Harwell, M.A., Andow, D.A., Alexander, M. and Stern, A.M. 1984. Potential impacts of environmental release of biotechnology products: Assessment, regulation, and research needs. Ecosystems Research Center, Cornell University, Ithaca, N.Y.

    Google Scholar 

  3. Brill, W.J. 1985. Safety concerns and genetic engineering in agriculture. Science 227:381–384.

    Article  PubMed  Google Scholar 

  4. Covello, V.T. and Fiksel, J.R. 1985. The suitability and applicability of risk assessment methods for environmental applications of biotechnology. National Science Foundation, Report No. NSF/PRA 8502286, Washington, D.C.

    Google Scholar 

  5. Schroth, M.N., Thomson, S.V., and Moller, W.J. 1979. Streptomycin resistance in Erwinia amylovora. Phytopathology 69:565–568.

    Article  CAS  Google Scholar 

  6. Burr, T.J., Schroth, M.N. and Suslow, T. 1978. Increased potato yield by treatment of seedpieces with specific strains of Pseudomonas fluorescens and P. putida. Phytopathology 68:1377–1383.

    Article  Google Scholar 

  7. Kloepper, J.W., Schroth, M.N., and Miller, T.D. 1980. Effects of rhizosphere colonization by plant growth-promoting rhizobacteria on potato plant development and yield. Phytopathology 70:1078–1082.

    Article  Google Scholar 

  8. Suslow, T.V. and Schroth, M.N. 1982. Rhizobacteria of sugar beets: Effects of seed application and root colonization on yield. Phytopathology 72:199–206.

    Article  Google Scholar 

  9. Hemming, B.C. and Drahos, D.J. 1984. β-Galactosidase, a selectable non-antibiotic marker for fluorescent pseudomonads. J. Cellular Biochemistry Supp. 8b:252.

    Google Scholar 

  10. Kalnins, A., Otto, K., Ruther, U., and Muller-Hill, B. 1983. Sequence of the lacZ gene of Escherichia coli. EMBO Journal 2:593–597.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Buchel, D.E., Gronenborn, B., and Muller-Hill, B. 1980. Sequence of the lactose permease gene. Nature 283:541–545.

    Article  CAS  PubMed  Google Scholar 

  12. Zabin, I. and Fowler, A.V. 1978. β-Galactosidase, the lactose permease protein, and thiogalactoside transacetylase, p. 89–121. In: The Operon. J. Miller and W. Resnikoff, (eds.) Cold Spring Harbor Laboratory, New York.

    Google Scholar 

  13. Teather, R.M., Bramhall, J., Riede, I., Wright, J.K., Furst, M., Aichele, G., Wilhelm, R., and Overath, P. 1980. Lactose carrier protein of Escherichia coli. Eur. J. Biochem 108:223–231.

    Article  CAS  PubMed  Google Scholar 

  14. Mieschendahl, M., Buchel, D., Bocklage, H., and Muller-Hill, B. 1981. Mutations in the lacY gene of Escherichia coli define functional organization of lactose permease. Proc. Natl. Acad. Sci. USA 78:7652–7656.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Weller, D.M. 1983. Colonization of wheat roots by a fluorescent pseudomonad suppressive to take-all. Phytopathology 73:1548–1553.

    Article  Google Scholar 

  16. Weller, D.M. and Cook, R.J. 1982. Pseudomonads from take-all conducive and suppressive soils. Phytopathology 72:264.

  17. Weller, D.M. and Cook, R.J. 1983. Suppression of take-all of wheat by seed-treatment with fluorescent pseudomonads. Phytopathology 73:463–469.

    Article  Google Scholar 

  18. Ballard, R.W., Palleroni, N.J., Doudoroff, M., Stanier, R.Y., and Mandel, M. 1970. Taxonomy of the aerobic pseudomonads: Pseudomonas cepacia, P. marginala, P. alliicola and P. caryophylli. J. Gen. Microbiol. 60:199–214.

    Article  CAS  PubMed  Google Scholar 

  19. Palleroni, N.J. and Doudoroff, M. 1972. Some properties and taxonomic subdivisions of the genus Pseudomonas. Ann. Rev. Phytopathology 10:73–100.

    Article  Google Scholar 

  20. Stanier, R.Y., Palleroni, N.J., and Doudoroff, M. 1966. The aerobic pseudomonads: a taxonomic study. J. Gen. Microbiol. 43:159–271.

    Article  CAS  PubMed  Google Scholar 

  21. Okamura, Y., Shoda, M., and Shigezo, U. 1983. Activity and synthesis of β-galactosidase in various lactose utilizing bacteria. Agric. Biol. Chem. 47:1133–1134.

    CAS  Google Scholar 

  22. Analytab Products, Ayerst Laboratories, Inc. 1982. Analytical profile index, enterobacteriaceae and other gram negative bacteria. Plain-view, New York.

  23. Hayward, A.C. 1978. Occurrence of glycoside hydrolases in plant pathogenic and related bacteria. J. Appl. Bacteriol. 43:407–412.

    Article  Google Scholar 

  24. Bagdasarian, M., Lurz, R., Ruckert, 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 

  25. Casadaban, M.J., Chou, J., and Cohen, S.N. 1980. In vitro gene fusions that join an enzymatically active β-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J. Bacteriol. 143:971–980.

    CAS  PubMed  PubMed Central  Google Scholar 

  26. Maniatis, T., Fritsch, E., and Sambrook, J. 1982. Molecular Cloning, a Laboratory Manual. Cold Spring Harbor Laboratory, New York.

    Google Scholar 

  27. 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 

  28. Kawalek, M. and Schaad, N.W. 1985. β-galactosidase as a metabolic marker in Xanthomonas campestris pv. campestris. Phytopathology 75:1326.

    Google Scholar 

  29. Miller, J. 1972. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory, N.Y.

    Google Scholar 

  30. Newman, M.J., Foster, D.L., Wilson, T.H., Kaback, H.R. 1981. Purification and reconstitution of functional lactose carrier from E. coli. J. Biol. Chem. 256:11804–11808.

    CAS  PubMed  Google Scholar 

  31. Renart, J., Reiser, J., and Stark, G.R. 1979. Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: A method for studying antibody specificity and antigen structure. Proc. Natl. Acad. Sci. USA. 76:3116–3120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Gillaspie, A.G., Sasser, M., and Davis, M.J. 1984. Fatty acid profiles of bacteria causing ratoon stunting disease (RSD) of sugarcane and bermudagrass stunting disease (BSD). Phytopathology 74:880.

    Google Scholar 

  33. Drahos, D., Brackin, M.J., and Barry, G. 1985. Bacterial strain identification by comparative analysis of chromosomal DNA restriction patterns. Phytopathology 75:1381.

    Google Scholar 

  34. Krivi, G.G. and Rowold, E. 1984. Monoclonal antibodies to bovine somatotropin: Immunoadsorbent reagents for mammalian somatotropins. Hybridoma 3:151–162.

    Article  CAS  PubMed  Google Scholar 

  35. Cohen, G.N. and Monad, J. 1957. Bacterial permeases. Bacteriol. Rev. 21:169–194.

    CAS  PubMed  PubMed Central  Google Scholar 

  36. Marin, A. and Marshall, R.T. 1983. Characterization of glycosidases produced by Pseudomonas fluorescens 26. J. Food Protection 46:676–680.

    Article  CAS  Google Scholar 

  37. Gorbach, S.L. 1971. Intestinal microflora. Gastroenterology 60:1110–1131.

    CAS  PubMed  Google Scholar 

  38. Stotzky, G. and Babich, H. 1984. Fate of genetically-engineered microbes in natural environments. Recombinant DNA Technical Bulletin 7:163–188.

    CAS  PubMed  Google Scholar 

  39. Cole, M.A. and Elkan, G.H. 1979. Multiple antibiotic resistance in Rhizobium japonicum. Appl. Environ. Microbiol. 37:867–870.

    CAS  PubMed  PubMed Central  Google Scholar 

  40. Kelch, W.J. and Lee, J.S. 1978. Antibiotic resistance patterns of gram-negative bacteria isolated from environmental sources. Appl. Environ. Microbiol. 36:450–456.

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Kloepper, J.W., Leong, J., Teintze, M., and Schroth, M.N. 1980. Pseudomonas siderophores: A mechanism explaining disease-suppressive soils. Current Microbiology 4:317–320.

    Article  CAS  Google Scholar 

  42. Godwin, D. and Slater, J.H. 1979. The influence of the growth environment on the stability of a drug resistance plasmid in Escherichia coli K-12. J. Gen. Microbiol. 111:201–210.

    Article  CAS  PubMed  Google Scholar 

  43. Kloepper, J.W. and Scroth, M.N. 1981. Relationship of in vitro antibiosis of plant growth-promoting rhizobacteria to plant growth and the displacement of root microflora. Phytopathology 71:1020–1024.

    Article  Google Scholar 

  44. Cook, R.J. and Baker, K.F. 1983. The nature and practice of biological control of plant pathogens. The American Phytopathological Society, St. Paul, Minnesota.

  45. Chopra, I., Shales, S.W., Ward, M.W., and Wallace, L.J. 1981. Reduced expression of Tn10-mediated tetracycline resistance in Escherichia coli containing more than one copy of the transposon. J. Gen. Microbiol. 126:45–54.

    CAS  PubMed  Google Scholar 

  46. Levy, S.B. 1984. Resistance to the tetracyclines, p. 191–240. In: Antimicrobial Drug Resistance. L. E. Bryan (ed.) Academic Press, Inc. Orlando, Florida.

    Google Scholar 

  47. Kolata, G. 1985. How safe are engineered organisms? Science 229:34–35.

    Article  CAS  PubMed  Google Scholar 

  48. Barry, G.F. 1986. The permanent insertion of foreign genes into the chromosome of soil bacteria. Bio/Technology (May).

  49. Matteucci, M.D. and Caruthers, M.H. 1981. Synthesis of deoxyolionucleotides on a polymer support. J. Am. Chem. Soc. 103:3185–3191.

    Article  CAS  Google Scholar 

  50. Guerry, P., Embden, J.V., and Falkow, S. 1974. Molecular nature of two nonconjugative plasmids carrying drug resistance genes. J. Bacteriol. 117:619–630.

    CAS  PubMed  PubMed Central  Google Scholar 

  51. Messing, F., Crea, R., and Seeburg, P.H. 1981. A system for shotgun DNA sequencing. Nucleic Acids Res. 9:309–318.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. King, E.O., Ward, M.K., and Raney, D.E. 1954. Two simple media for the demonstration of pycocyanin and fluorescein. J. Lab. Clin. Med. 44:301–307.

    CAS  PubMed  Google Scholar 

  53. Casadaban, M. and Cohen, S. 1980. Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J. Mol. Biol. 138:179–207.

    Article  CAS  PubMed  Google Scholar 

  54. Poindron, P., Bourguignat, A., Scheid, F., and Lombard, Y. 1977. The orthonitrophenyl-β-D-galactoside hydrolase from Pseudomonas aeruginosa (0:11 serotypes) is a superficial enzyme. Can. J. Microbiol. 23:798–810.

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biological Sciences, Monsanto Co., 700 Chesterfield Village, St. Louis, MO 63198

    David J. Drahos, Bruce C. Hemming & Sylvia McPherson

Authors
  1. David J. Drahos
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bruce C. Hemming
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sylvia McPherson
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Drahos, D., Hemming, B. & McPherson, S. Tracking Recombinant Organisms in the Environment: β–Galactosidase as a Selectable Non–Antibiotic Marker for Fluorescent Pseudomonads. Nat Biotechnol 4, 439–444 (1986). https://doi.org/10.1038/nbt0586-439

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nbt0586-439

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing