Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1990 Feb 11;18(3):619–624. doi: 10.1093/nar/18.3.619

Identification of the metalloregulatory element of the plasmid-encoded arsenical resistance operon.

M J San Francisco 1, C L Hope 1, J B Owolabi 1, L S Tisa 1, B P Rosen 1
PMCID: PMC333470  PMID: 2408017

Abstract

The regulatory region of the plasmid-encoded arsenical resistance (ars) operon was cloned as a 727-bp EcoRI-HindIII fragment. When cloned into a promoter probe vector this fragment conferred arsenite inducible tetracycline resistance in Escherichia coli, indicating that the fragment carried a regulatory gene, the arsR gene. A single region corresponding to -35 and -10 promoter recognition sites was identified. The transcriptional start site of the mRNA was determined by primer extension. The sequence has an open reading frame for a potential 13,179 Da polypeptide, termed the ArsR protein. The fragment was cloned into a temperature regulated expression vector. A protein with an apparent molecular mass of about 12 kDa was induced by either temperature or arsenite. This protein was purified and used to produce antibodies specific for the ArsR protein.

Full text

PDF
624

Images in this article

622Image
on p.622
623Image
on p.623
623Image
on p.623
623Image
on p.623

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  2. Chen C. M., Misra T. K., Silver S., Rosen B. P. Nucleotide sequence of the structural genes for an anion pump. The plasmid-encoded arsenical resistance operon. J Biol Chem. 1986 Nov 15;261(32):15030–15038. [PubMed] [Google Scholar]
  3. Chen C. M., Mobley H. L., Rosen B. P. Separate resistances to arsenate and arsenite (antimonate) encoded by the arsenical resistance operon of R factor R773. J Bacteriol. 1985 Feb;161(2):758–763. doi: 10.1128/jb.161.2.758-763.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Gold L., Pribnow D., Schneider T., Shinedling S., Singer B. S., Stormo G. Translational initiation in prokaryotes. Annu Rev Microbiol. 1981;35:365–403. doi: 10.1146/annurev.mi.35.100181.002053. [DOI] [PubMed] [Google Scholar]
  5. Goodwin T. W., Morton R. A. The spectrophotometric determination of tyrosine and tryptophan in proteins. Biochem J. 1946;40(5-6):628–632. doi: 10.1042/bj0400628. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  7. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  8. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  9. Mobley H. L., Chen C. M., Silver S., Rosen B. P. Cloning and expression of R-factor mediated arsenate resistance in Escherichia coli. Mol Gen Genet. 1983;191(3):421–426. doi: 10.1007/BF00425757. [DOI] [PubMed] [Google Scholar]
  10. Mobley H. L., Rosen B. P. Energetics of plasmid-mediated arsenate resistance in Escherichia coli. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6119–6122. doi: 10.1073/pnas.79.20.6119. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Remaut E., Tsao H., Fiers W. Improved plasmid vectors with a thermoinducible expression and temperature-regulated runaway replication. Gene. 1983 Apr;22(1):103–113. doi: 10.1016/0378-1119(83)90069-0. [DOI] [PubMed] [Google Scholar]
  13. Rosen B. P., Borbolla M. G. A plasmid-encoded arsenite pump produces arsenite resistance in Escherichia coli. Biochem Biophys Res Commun. 1984 Nov 14;124(3):760–765. doi: 10.1016/0006-291x(84)91023-4. [DOI] [PubMed] [Google Scholar]
  14. Rosen B. P., Weigel U., Karkaria C., Gangola P. Molecular characterization of an anion pump. The arsA gene product is an arsenite(antimonate)-stimulated ATPase. J Biol Chem. 1988 Mar 5;263(7):3067–3070. [PubMed] [Google Scholar]
  15. Rosenberg M., Court D. Regulatory sequences involved in the promotion and termination of RNA transcription. Annu Rev Genet. 1979;13:319–353. doi: 10.1146/annurev.ge.13.120179.001535. [DOI] [PubMed] [Google Scholar]
  16. Russel M., Model P. Replacement of the fip gene of Escherichia coli by an inactive gene cloned on a plasmid. J Bacteriol. 1984 Sep;159(3):1034–1039. doi: 10.1128/jb.159.3.1034-1039.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. San Francisco M. J., Tisa L. S., Rosen B. P. Identification of the membrane component of the anion pump encoded by the arsenical resistance operon of R-factor R773. Mol Microbiol. 1989 Jan;3(1):15–21. doi: 10.1111/j.1365-2958.1989.tb00098.x. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Silver S., Budd K., Leahy K. M., Shaw W. V., Hammond D., Novick R. P., Willsky G. R., Malamy M. H., Rosenberg H. Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus. J Bacteriol. 1981 Jun;146(3):983–996. doi: 10.1128/jb.146.3.983-996.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Silver S., Keach D. Energy-dependent arsenate efflux: the mechanism of plasmid-mediated resistance. Proc Natl Acad Sci U S A. 1982 Oct;79(20):6114–6118. doi: 10.1073/pnas.79.20.6114. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Summers W. C. A simple method for extraction of RNA from E. coli utilizing diethyl pyrocarbonate. Anal Biochem. 1970 Feb;33(2):459–463. doi: 10.1016/0003-2697(70)90316-7. [DOI] [PubMed] [Google Scholar]
  22. Tabor S., Richardson C. C. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1074–1078. doi: 10.1073/pnas.82.4.1074. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wray W., Boulikas T., Wray V. P., Hancock R. Silver staining of proteins in polyacrylamide gels. Anal Biochem. 1981 Nov 15;118(1):197–203. doi: 10.1016/0003-2697(81)90179-2. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES