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. 1994 Jan;60(1):357–362. doi: 10.1128/aem.60.1.357-362.1994

Pseudomonas putida Strains Which Constitutively Overexpress Mercury Resistance for Biodetoxification of Organomercurial Pollutants

Joanne M Horn 1,*, Maren Brunke 1, W-D Deckwer 1, Kenneth N Timmis 1
PMCID: PMC201314  PMID: 16349162

Abstract

Improved biocatalysts for mercury (Hg) remediation were generated by random mutagenesis of Pseudomonas putida with a minitransposon containing merTPAB, the structural genes specifying organomercury resistance. Subsequent selection for derivatives exhibiting elevated resistance levels to phenylmercury allowed the isolation of strains that constitutively express merTPAB at high levels, conferring the ability to cleave Hg from an organic moiety and reduce the freed Hg(II) to the less toxic elemental form, Hg0, at greater rates. Constitutive overexpression of merTPAB had no apparent effect on culture growth rates, even when Hg(II) was initially present at otherwise toxic concentrations. These properties were also combined with benzene and toluene catabolism, allowing detoxification of the metal component of phenyl mercuric acetate, as well as degradation of its aromatic moiety.

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Selected References

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  1. BAUCHOP T., ELSDEN S. R. The growth of micro-organisms in relation to their energy supply. J Gen Microbiol. 1960 Dec;23:457–469. doi: 10.1099/00221287-23-3-457. [DOI] [PubMed] [Google Scholar]
  2. Begley T. P., Walts A. E., Walsh C. T. Bacterial organomercurial lyase: overproduction, isolation, and characterization. Biochemistry. 1986 Nov 4;25(22):7186–7192. doi: 10.1021/bi00370a063. [DOI] [PubMed] [Google Scholar]
  3. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  4. Brown N. L., Ford S. J., Pridmore R. D., Fritzinger D. C. Nucleotide sequence of a gene from the Pseudomonas transposon Tn501 encoding mercuric reductase. Biochemistry. 1983 Aug 16;22(17):4089–4095. doi: 10.1021/bi00286a015. [DOI] [PubMed] [Google Scholar]
  5. Brown N. L., Misra T. K., Winnie J. N., Schmidt A., Seiff M., Silver S. The nucleotide sequence of the mercuric resistance operons of plasmid R100 and transposon Tn501: further evidence for mer genes which enhance the activity of the mercuric ion detoxification system. Mol Gen Genet. 1986 Jan;202(1):143–151. doi: 10.1007/BF00330531. [DOI] [PubMed] [Google Scholar]
  6. Brunke M., Deckwer W. D., Frischmuth A., Horn J. M., Lünsdorf H., Rhode M., Röhricht M., Timmis K. N., Weppen P. Microbial retention of mercury from waste streams in a laboratory column containing merA gene bacteria. FEMS Microbiol Rev. 1993 Jul;11(1-3):145–152. doi: 10.1111/j.1574-6976.1993.tb00278.x. [DOI] [PubMed] [Google Scholar]
  7. Figurski D. H., Helinski D. R. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci U S A. 1979 Apr;76(4):1648–1652. doi: 10.1073/pnas.76.4.1648. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Foster T. J., Ginnity F. Some mercurial resistance plasmids from different incompatibility groups specify merR regulatory functions that both repress and induce the mer operon of plasmid R100. J Bacteriol. 1985 May;162(2):773–776. doi: 10.1128/jb.162.2.773-776.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Fox B., Walsh C. T. Mercuric reductase. Purification and characterization of a transposon-encoded flavoprotein containing an oxidation-reduction-active disulfide. J Biol Chem. 1982 Mar 10;257(5):2498–2503. [PubMed] [Google Scholar]
  10. Franklin F. C., Bagdasarian M., Bagdasarian M. M., Timmis K. N. Molecular and functional analysis of the TOL plasmid pWWO from Pseudomonas putida and cloning of genes for the entire regulated aromatic ring meta cleavage pathway. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7458–7462. doi: 10.1073/pnas.78.12.7458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gibson D. T., Koch J. R., Kallio R. E. Oxidative degradation of aromatic hydrocarbons by microorganisms. I. Enzymatic formation of catechol from benzene. Biochemistry. 1968 Jul;7(7):2653–2662. doi: 10.1021/bi00847a031. [DOI] [PubMed] [Google Scholar]
  12. Griffin H. G., Foster T. J., Silver S., Misra T. K. Cloning and DNA sequence of the mercuric- and organomercurial-resistance determinants of plasmid pDU1358. Proc Natl Acad Sci U S A. 1987 May;84(10):3112–3116. doi: 10.1073/pnas.84.10.3112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kreuzer K., Pratt C., Torriani A. Genetic analysis of regulatory mutants of alkaline phosphatase of E. coli. Genetics. 1975 Nov;81(3):459–468. doi: 10.1093/genetics/81.3.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lund P. A., Brown N. L. Regulation of transcription in Escherichia coli from the mer and merR promoters in the transposon Tn501. J Mol Biol. 1989 Jan 20;205(2):343–353. doi: 10.1016/0022-2836(89)90345-8. [DOI] [PubMed] [Google Scholar]
  16. Lund P. A., Brown N. L. Role of the merT and merP gene products of transposon Tn501 in the induction and expression of resistance to mercuric ions. Gene. 1987;52(2-3):207–214. doi: 10.1016/0378-1119(87)90047-3. [DOI] [PubMed] [Google Scholar]
  17. Miller V. L., Mekalanos J. J. A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol. 1988 Jun;170(6):2575–2583. doi: 10.1128/jb.170.6.2575-2583.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Misra T. K., Brown N. L., Fritzinger D. C., Pridmore R. D., Barnes W. M., Haberstroh L., Silver S. Mercuric ion-resistance operons of plasmid R100 and transposon Tn501: the beginning of the operon including the regulatory region and the first two structural genes. Proc Natl Acad Sci U S A. 1984 Oct;81(19):5975–5979. doi: 10.1073/pnas.81.19.5975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mukhopadhyay D., Yu H. R., Nucifora G., Misra T. K. Purification and functional characterization of MerD. A coregulator of the mercury resistance operon in gram-negative bacteria. J Biol Chem. 1991 Oct 5;266(28):18538–18542. [PubMed] [Google Scholar]
  20. Nakahara H., Kinscherf T. G., Silver S., Miki T., Easton A. M., Rownd R. H. Gene copy number effects in the mer operon of plasmid NR1. J Bacteriol. 1979 Apr;138(1):284–287. doi: 10.1128/jb.138.1.284-287.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nakahara H., Silver S., Miki T., Rownd R. H. Hypersensitivity to Hg2+ and hyperbinding activity associated with cloned fragments of the mercurial resistance operon of plasmid NR1. J Bacteriol. 1979 Oct;140(1):161–166. doi: 10.1128/jb.140.1.161-166.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ni'Bhriain N. N., Silver S., Foster T. J. Tn5 insertion mutations in the mercuric ion resistance genes derived from plasmid R100. J Bacteriol. 1983 Aug;155(2):690–703. doi: 10.1128/jb.155.2.690-703.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Nriagu J. O., Pacyna J. M. Quantitative assessment of worldwide contamination of air, water and soils by trace metals. Nature. 1988 May 12;333(6169):134–139. doi: 10.1038/333134a0. [DOI] [PubMed] [Google Scholar]
  24. Nucifora G., Chu L., Silver S., Misra T. K. Mercury operon regulation by the merR gene of the organomercurial resistance system of plasmid pDU1358. J Bacteriol. 1989 Aug;171(8):4241–4247. doi: 10.1128/jb.171.8.4241-4247.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Nucifora G., Silver S., Misra T. K. Down regulation of the mercury resistance operon by the most promoter-distal gene merD. Mol Gen Genet. 1989 Dec;220(1):69–72. doi: 10.1007/BF00260858. [DOI] [PubMed] [Google Scholar]
  26. O'Halloran T., Walsh C. Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization. Science. 1987 Jan 9;235(4785):211–214. doi: 10.1126/science.3798107. [DOI] [PubMed] [Google Scholar]
  27. Philippidis G. P., Malmberg L. H., Hu W. S., Schottel J. L. Effect of gene amplification on mercuric ion reduction activity of Escherichia coli. Appl Environ Microbiol. 1991 Dec;57(12):3558–3564. doi: 10.1128/aem.57.12.3558-3564.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Schottel J. L. The mercuric and organomercurial detoxifying enzymes from a plasmid-bearing strain of Escherichia coli. J Biol Chem. 1978 Jun 25;253(12):4341–4349. [PubMed] [Google Scholar]
  29. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  30. Stanisich V. A., Bennett P. M., Richmond M. H. Characterization of a translocation unit encoding resistance to mercuric ions that occurs on a nonconjugative plasmid in Pseudomonas aeruginosa. J Bacteriol. 1977 Mar;129(3):1227–1233. doi: 10.1128/jb.129.3.1227-1233.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Summers A. O. Organization, expression, and evolution of genes for mercury resistance. Annu Rev Microbiol. 1986;40:607–634. doi: 10.1146/annurev.mi.40.100186.003135. [DOI] [PubMed] [Google Scholar]
  32. Tezuka T., Tonomura K. Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62. J Bacteriol. 1978 Jul;135(1):138–143. doi: 10.1128/jb.135.1.138-143.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Young R. A., Davis R. W. Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1194–1198. doi: 10.1073/pnas.80.5.1194. [DOI] [PMC free article] [PubMed] [Google Scholar]

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