Genetically modified Escherichia coli for colorimetic detection of inorganic and organic Hg compounds

  • J. Klein
  • J. Altenbuchner
  • R. Mattes
Chapter
Part of the EXS book series (EXS, volume 80)

Summary

A sensitive colorimetric bacterial system was developed for the detection of Hg(II) and organomercury compounds. The bioactive species, a recombinant Escherichia coli, produces proportionally elevated levels of the enzyme β-galactosidase with increasing amounts of Hg. This is due to a reporter plasmid which carries a Hg(II)-inducible promoter (mer promoter) from the Hg resistance transposon Tn501 regulating the transcription of a promoterless lacZ gene. Additionally, a pMB1 origin of replication without the natural RNA polymerase start site is fused downstream of the mer promoter leading to a Hg(II)-inducible plasmid replication, which results in an improved signal-to-noise ratio. To enhance the sensitivity of this cellular biosensor, the transport proteins for Hg(II) uptake are constitutively produced by a helper plasmid. To enable the detection of organically bound Hg, the Streptomyces lividans organomercurial lyase, an enzyme which catalyses the cleavage of C-Hg-bonds of organomercurial compounds, is also provided by the helper pasmid. Hg(II) and phenylmercuric acetate (PMA) concentrations as low as 5x10-10 M (0.1 ppb) may be detected within a few minutes.

Keywords

Reporter Plasmid Helper Plasmid Transcriptional Fusion Miller Unit Organomercury Compound 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.
This is a preview of subscription content, log in to check access.

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Altenbuchner, J., Choi, C.L., Glinstedt, J., Schmitt, R. and Richmond, M.H. (1981) The transposon Tn501 (Hg) andTn7727 (Tc) are related. Genet. Res. Camb. 37: 285-289.Google Scholar
  2. Aschner, M. and Aschner, J.L. (1990) Mercury neurotoxicity: mechanisms of blood-brain barrier transport. Neurosci. Biobehav. Rev. 14: 169-174.Google Scholar
  3. Bloom, N. and Fitzgerald, W.F. (1988) Determination of volatile merury species at the pico- gramm level by low-temperature gas chromatography with cold-vapour atomic fluorescence detection. Anal. Chim. Acta 208:151 -161.Google Scholar
  4. 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.Google Scholar
  5. 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.Google Scholar
  6. Chang, A.C.Y, and Cohen, S.N. (1978) Construction and characterization of amplifiable multi-copy DNA cloning vehicles derived from the pl5A cryptic miniplasmid. J. Bacteriol. 134: 1141 - 1156.PubMedGoogle Scholar
  7. Chung, C.T., Niemela, S.L. and Miller, R.H. (1989) One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc. Natl. Acad. Sei. USA 86: 2172-2175Google Scholar
  8. Condee, C.W. and Summers, A.O. (1992) A mer-lux transcriptional fusion for real-time exami-nation of in vitro gene expression kinetics and promoter response to altered superhelicity. J. Bacteriol. 174: 8094 - 8101.PubMedGoogle Scholar
  9. De Flora, S., Benicelli, C. and Bagnasco, M. (1994) Genotoxicity of mercury compounds. A review. Mut. Res. 317: 57-79.Google Scholar
  10. De la Cruz, F. and Glinstedt, J. (1982) Genetic and molecular characterization of Tn27, a multiple resistance transposon from R100.1. J. Bacteriol. 151: 222 - 228.PubMedGoogle Scholar
  11. Klein, J. (1992) Quecksilbernachweis über Reporterenzyme- Biosonde und Enzymsensor. Ph.D. thesis, University of Stuttgart, Germany.Google Scholar
  12. Klein, J., Altenbuchner, J. and Mattes, R. (1989) Mercury detection with transcriptional fusions 150in Tn507. 13th Workshop on Procaryotic genetics, Disentis, CH (13-16. September, 1989 ).Google Scholar
  13. Klein, J., Altenbuchner, J. and Mattes, R. (1991) A new method to detect mercury using bacteria as biosensor. In: M. Reuss, H. Chmiel, E.-D. Gilles and H.-J. Knackmuss (eds): Biochemical Engineering - Stuttgart. Gustav Fischer, Stuttgart, New York, pp 323 - 326.Google Scholar
  14. Lund, P.A. and Brown, N.L. (1987) Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene 52: 207 - 214.PubMedCrossRefGoogle Scholar
  15. Lund, P. A. and Brown, N.L. (1989) Regulation of transcription in Escherichia coli from the mer and merR promoters in the transposon Tn501. J. Mol. Biol. 205: 343-353.Google Scholar
  16. Marsh, J.L., Erfle, M. and Wykes, E.J. (1984) The pIC plasmid and phage vectors with versatile cloning sites for recombinant selection by insertional inactivation. Gene 32: 481 - 485.PubMedCrossRefGoogle Scholar
  17. McKenney, K., Shimatake, H., Court, D., Schmeissner, U., Brady, C. and Rosenberg, M. (1981) A system to study promoter and terminator signals recognized by Escherichia coli RNA polymerase In: J.G. Chirikjan andT.S. Papas (eds): Gene amplification and analysis, Vol II: Analysis of nucleic acids by enzymatic methods. Elsevier-North Holland Press, Amsterdam, pp 383 - 415.Google Scholar
  18. Messer, W. and Vielmetter, W. (1965) High resolution colony staining for the detection of bacterial growth requirement mutants using naphthol a20-dye techniques. Biochem. Biophys. Res. Commun. 21: 182-186.Google Scholar
  19. Miller, J.H. (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.Google Scholar
  20. Nakahara, H., Silver, S., Miki, T. and Rownd, R.H. (1979) Hypersensitivity to Hg(II) and hyper- binding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1. J. Bacteriol. 140: 161 - 166.PubMedGoogle Scholar
  21. O’Halloran, T.V, Frantz, B., Shin, M.K., Ralston, D.M. and Wright, J.G. (1989) The MerR heavy metal receptor mediates positive interaction in a topologically novel transcription complex. Cell 56: 110 - 129.Google Scholar
  22. Omang, S.H. (1971) Determination of mercury in natural waters and effluents by flameless atomic absorption spectrophotometry. Anal. Chim. Acta 53: 415-420.Google Scholar
  23. Panayotatos, N. (1984) DNA replication regulated by the priming promoter. Nucl. Acids Res. 6: 2641-2648.Google Scholar
  24. Park, S.-J., Wireman, J. and Summers, A.O. (1992) Genetic analysis of the Tn21 mer operator- promoter. J. Bacteriol. 174: 2160 - 2171.PubMedGoogle Scholar
  25. Ralston, D.M. and O’Halloran, T.V (1990) Ultrasensitivity and heavy metal selectivity of the allosterically modulated MerR transcription complex. Proc. Natl. Acad. Sci. USA 87: 3846-3850.Google Scholar
  26. Robinson, J.B. andTuovinen, O.H. (1984) Mechanisms of microbial resistance and detoxifica¬tion of mercury and organomercury compounds: Physiological, biochemical and genetic analyses. Microbiol. Rev. 48: 95-124.Google Scholar
  27. Ross, W., Park, S.-J. and Summers, A.O. (1989) Genetic analysis of transcriptional activation and repression in the Tn21 mer operon. J. Bacteriol. 171: 4009 - 4018.PubMedGoogle Scholar
  28. Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular cloning: a laboratory manual. 2nd edn. Cold Spring Harbor Laboratory Press, New York.Google Scholar
  29. Sedlmeier, R. and Altenbuchner, J. (1992) Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans. Mol. Gen. Genet. 236: 76-85.Google Scholar
  30. Selifonova, O., Burlage, R. and Barkay, T. (1993) Bioluminescent sensors for detection of bio- available Hg(II) in the environment. Appl. Environ. Microbiol. 59: 3083-3090.Google Scholar
  31. Silver, A. and Walderhaug, M. (1992) Gene regulation of plasmid- and chromosome-determined inorganic ion transport in bacteria. Microbiol. Rev. 56: 195-228.Google Scholar
  32. Silver, S., Misra, T.K. and Laddaga, R.A. (1989) DNA sequence analysis of bacterial toxic heavy metal resistances. Biol. Trace Elem. Res. 21: 145-163.Google Scholar
  33. Summers, A.O. (1986) Organization, expression and evolution of genes for mercury resistance. Annu. Rev. Microbiol. 40: 607-634.Google Scholar
  34. Tescione, L. and Belfort, G. (1993) Construction and evaluation of a metal ion biosensor. Bio- technol. Bioeng. 42: 945-952.Google Scholar
  35. Ubben, D. and Schmitt, R. (1987) A transposable promoter and transposable promoter probes derived fromTn 1721. Gene 53: 127 - 134.PubMedCrossRefGoogle Scholar
  36. Vieira, J. and Messing, J. (1982) The pUC plasmids and M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19: 259 - 268.PubMedCrossRefGoogle Scholar
  37. Wylie, D.E., Carlson, L.D., Carlson, R., Wagner, F.W. and Schuster, S.M. (1991) Detection of mercuric ions in water by ELISA with a mercury-specific antibody. Anal. Biochem. 194: 381-387.Google Scholar
  38. Yanisch-Perron, C., Vieira, J. and Messing, J. (1985) Improved Ml3 phage-cloning vectors and host strains: nucleotide sequences of the M13mpl8 and pUC vectors. Gene 33: 103 - 119.PubMedCrossRefGoogle Scholar

Copyright information

© Birkhäuser Verlag 1997

Authors and Affiliations

  • J. Klein
    • 1
  • J. Altenbuchner
    • 1
  • R. Mattes
    • 1
  1. 1.Institute of Industrial GeneticsUniversity of StuttgartStuttgartGermany

Personalised recommendations