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. 1996 Oct 1;24(19):3714–3721. doi: 10.1093/nar/24.19.3714

Zn2+-sensing by the cyanobacterial metallothionein repressor SmtB: different motifs mediate metal-induced protein-DNA dissociation.

J S Turner 1, P D Glands 1, A C Samson 1, N J Robinson 1
PMCID: PMC146171  PMID: 8871549

Abstract

SmtB is a member of a family of repressors which dissociate from DNA in the presence of metals; Zn2+ being the most potent inducer of metallothionein gene (smtA) transcription in vivo. In Synechococcus PCC 7942 cells devoid of chromosomal smtB, four plasmid-encoded mutants of SmtB (C61S, T11S/C14S, C121S and H105R/H106R) repressed lacZ expression driven by the smtA operator-promoter. Gel retardation assays with extracts from the complemented cells detected multiple SmtB-dependent complexes similar to those obtained with extracts from wild-type cells or with recombinant-SmtB. Elevated [Zn2+] alleviated repression in vivo by all of the mutants except H105R/H106R. These His residues (one or both) are therefore essential for Zn2+-sensing while, contrary to expectations, Cys residues are not. Hence different motifs facilitate metal-induced DNA-dissociation by SmtB and ArsR (the related oxyanion-sensing repressor), presumably generating variety in the spectra of metals sensed. Nucleotides and amino acids involved in DNA-SmtB interaction have been further defined/inferred and we also confirm that additional unknown factors form specific associations with the smt operator-promoter in elevated [Zn2+].

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Selected References

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  1. Bairoch A. A possible mechanism for metal-ion induced DNA-protein dissociation in a family of prokaryotic transcriptional regulators. Nucleic Acids Res. 1993 May 25;21(10):2515–2515. doi: 10.1093/nar/21.10.2515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Barbosa M. S., Lowy D. R., Schiller J. T. Papillomavirus polypeptides E6 and E7 are zinc-binding proteins. J Virol. 1989 Mar;63(3):1404–1407. doi: 10.1128/jvi.63.3.1404-1407.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chung C. T., Niemela S. L., Miller R. H. One-step preparation of competent Escherichia coli: transformation and storage of bacterial cells in the same solution. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2172–2175. doi: 10.1073/pnas.86.7.2172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Endo G., Silver S. CadC, the transcriptional regulatory protein of the cadmium resistance system of Staphylococcus aureus plasmid pI258. J Bacteriol. 1995 Aug;177(15):4437–4441. doi: 10.1128/jb.177.15.4437-4441.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Erbe J. L., Taylor K. B., Hall L. M. Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals. Nucleic Acids Res. 1995 Jul 11;23(13):2472–2478. doi: 10.1093/nar/23.13.2472. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Higuchi R., Krummel B., Saiki R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res. 1988 Aug 11;16(15):7351–7367. doi: 10.1093/nar/16.15.7351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Huckle J. W., Morby A. P., Turner J. S., Robinson N. J. Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Mol Microbiol. 1993 Jan;7(2):177–187. doi: 10.1111/j.1365-2958.1993.tb01109.x. [DOI] [PubMed] [Google Scholar]
  8. Ivey D. M., Guffanti A. A., Shen Z., Kudyan N., Krulwich T. A. The cadC gene product of alkaliphilic Bacillus firmus OF4 partially restores Na+ resistance to an Escherichia coli strain lacking an Na+/H+ antiporter (NhaA). J Bacteriol. 1992 Aug;174(15):4878–4884. doi: 10.1128/jb.174.15.4878-4884.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ji G., Silver S. Regulation and expression of the arsenic resistance operon from Staphylococcus aureus plasmid pI258. J Bacteriol. 1992 Jun;174(11):3684–3694. doi: 10.1128/jb.174.11.3684-3694.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kaneko T., Tanaka A., Sato S., Kotani H., Sazuka T., Miyajima N., Sugiura M., Tabata S. Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. I. Sequence features in the 1 Mb region from map positions 64% to 92% of the genome. DNA Res. 1995 Aug 31;2(4):153-66, 191-8. doi: 10.1093/dnares/2.4.153. [DOI] [PubMed] [Google Scholar]
  11. Kondorosi E., Pierre M., Cren M., Haumann U., Buiré M., Hoffmann B., Schell J., Kondorosi A. Identification of NolR, a negative transacting factor controlling the nod regulon in Rhizobium meliloti. J Mol Biol. 1991 Dec 20;222(4):885–896. doi: 10.1016/0022-2836(91)90583-r. [DOI] [PubMed] [Google Scholar]
  12. Lebrun M., Audurier A., Cossart P. Plasmid-borne cadmium resistance genes in Listeria monocytogenes are similar to cadA and cadC of Staphylococcus aureus and are induced by cadmium. J Bacteriol. 1994 May;176(10):3040–3048. doi: 10.1128/jb.176.10.3040-3048.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Morby A. P., Turner J. S., Huckle J. W., Robinson N. J. SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. Nucleic Acids Res. 1993 Feb 25;21(4):921–925. doi: 10.1093/nar/21.4.921. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Robinson N. J., Gupta A., Fordham-Skelton A. P., Croy R. R., Whitton B. A., Huckle J. W. Prokaryotic metallothionein gene characterication and expression: chromosome crawling by ligation-mediated PCR. Proc Biol Sci. 1990 Dec 22;242(1305):241–247. doi: 10.1098/rspb.1990.0130. [DOI] [PubMed] [Google Scholar]
  15. Rosenstein R., Nikoleit K., Götz F. Binding of ArsR, the repressor of the Staphylococcus xylosus (pSX267) arsenic resistance operon to a sequence with dyad symmetry within the ars promoter. Mol Gen Genet. 1994 Mar;242(5):566–572. doi: 10.1007/BF00285280. [DOI] [PubMed] [Google Scholar]
  16. Rosenstein R., Peschel A., Wieland B., Götz F. Expression and regulation of the antimonite, arsenite, and arsenate resistance operon of Staphylococcus xylosus plasmid pSX267. J Bacteriol. 1992 Jun;174(11):3676–3683. doi: 10.1128/jb.174.11.3676-3683.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. San Francisco M. J., Hope C. L., Owolabi J. B., Tisa L. S., Rosen B. P. Identification of the metalloregulatory element of the plasmid-encoded arsenical resistance operon. Nucleic Acids Res. 1990 Feb 11;18(3):619–624. doi: 10.1093/nar/18.3.619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Scanlan D. J., Bloye S. A., Mann N. H., Hodgson D. A., Carr N. G. Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters. Gene. 1990 May 31;90(1):43–49. doi: 10.1016/0378-1119(90)90437-v. [DOI] [PubMed] [Google Scholar]
  19. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  20. Sedlmeier R., Altenbuchner J. Cloning and DNA sequence analysis of the mercury resistance genes of Streptomyces lividans. Mol Gen Genet. 1992 Dec;236(1):76–85. doi: 10.1007/BF00279645. [DOI] [PubMed] [Google Scholar]
  21. Shi W., Dong J., Scott R. A., Ksenzenko M. Y., Rosen B. P. The role of arsenic-thiol interactions in metalloregulation of the ars operon. J Biol Chem. 1996 Apr 19;271(16):9291–9297. doi: 10.1074/jbc.271.16.9291. [DOI] [PubMed] [Google Scholar]
  22. Shi W., Wu J., Rosen B. P. Identification of a putative metal binding site in a new family of metalloregulatory proteins. J Biol Chem. 1994 Aug 5;269(31):19826–19829. [PubMed] [Google Scholar]
  23. Shimizu T., Hiyama T., Ikeuchi M., Inoue Y. Nucleotide sequence of a metallothionein gene of the thermophilic cyanobacterium Synechococcus vulcanus. Plant Mol Biol. 1992 Nov;20(3):565–567. doi: 10.1007/BF00040616. [DOI] [PubMed] [Google Scholar]
  24. Turner J. S., Morby A. P., Whitton B. A., Gupta A., Robinson N. J. Construction of Zn2+/Cd2+ hypersensitive cyanobacterial mutants lacking a functional metallothionein locus. J Biol Chem. 1993 Feb 25;268(6):4494–4498. [PubMed] [Google Scholar]
  25. Turner J. S., Robinson N. J., Gupta A. Construction of Zn2+/Cd(2+)-tolerant cyanobacteria with a modified metallothionein divergon: further analysis of the function and regulation of smt. J Ind Microbiol. 1995 Mar-Apr;14(3-4):259–264. doi: 10.1007/BF01569937. [DOI] [PubMed] [Google Scholar]
  26. Williams S. G., Attridge S. R., Manning P. A. The transcriptional activator HlyU of Vibrio cholerae: nucleotide sequence and role in virulence gene expression. Mol Microbiol. 1993 Aug;9(4):751–760. doi: 10.1111/j.1365-2958.1993.tb01735.x. [DOI] [PubMed] [Google Scholar]
  27. Wu J., Rosen B. P. Metalloregulated expression of the ars operon. J Biol Chem. 1993 Jan 5;268(1):52–58. [PubMed] [Google Scholar]
  28. Xu C., Shi W., Rosen B. P. The chromosomal arsR gene of Escherichia coli encodes a trans-acting metalloregulatory protein. J Biol Chem. 1996 Feb 2;271(5):2427–2432. doi: 10.1074/jbc.271.5.2427. [DOI] [PubMed] [Google Scholar]
  29. van der Plas J., Hegeman H., de Vrieze G., Tuyl M., Borrias M., Weisbeek P. Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin. Gene. 1990 Oct 30;95(1):39–48. doi: 10.1016/0378-1119(90)90411-j. [DOI] [PubMed] [Google Scholar]

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