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. 1989 Feb;171(2):1048–1054. doi: 10.1128/jb.171.2.1048-1054.1989

Mechanism for iron-regulated transcription of the Escherichia coli cir gene: metal-dependent binding of fur protein to the promoters.

D W Griggs 1, J Konisky 1
PMCID: PMC209700  PMID: 2644221

Abstract

The molecular basis for the greatly elevated expression of the cir gene (encoding the colicin I receptor) in cells unable to maintain a critical supply of intracellular iron was investigated by genetic and biochemical means. Deletion analysis of the cloned promoter region allowed delineation of sequences necessary for control of transcription initiating at the two promoters, P1 and P2. Gel retardation assays were used to demonstrate both binding of purified Fur (ferric uptake regulation) protein to the iron control region and lack of binding to DNA fragments which are not involved in cir regulation. An operator sequence spanning 43 to 47 base pairs and completely encompassing the two promoters was identified by DNase I protection experiments (footprinting), with binding occurring in a metal-dependent fashion. Thus, during iron-replete growth, Fur appears to act as a repressor of transcription by blocking formation of a DNA-RNA polymerase complex, analogous to the mechanism previously described for regulation of the aerobactin operon (V. de Lorenzo, S. Wee, M. Herrero, and J.B. Neilands, J. Bacteriol. 169:2624-2630, 1987). Characterized and putative Fur recognition sites from several genes were analyzed and classified by statistical methods.

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Selected References

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

  1. Bagg A., Neilands J. B. Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli. Biochemistry. 1987 Aug 25;26(17):5471–5477. doi: 10.1021/bi00391a039. [DOI] [PubMed] [Google Scholar]
  2. Berg O. G., von Hippel P. H. Selection of DNA binding sites by regulatory proteins. II. The binding specificity of cyclic AMP receptor protein to recognition sites. J Mol Biol. 1988 Apr 20;200(4):709–723. doi: 10.1016/0022-2836(88)90482-2. [DOI] [PubMed] [Google Scholar]
  3. Berg O. G., von Hippel P. H. Selection of DNA binding sites by regulatory proteins. Trends Biochem Sci. 1988 Jun;13(6):207–211. doi: 10.1016/0968-0004(88)90085-0. [DOI] [PubMed] [Google Scholar]
  4. Bindereif A., Neilands J. B. Promoter mapping and transcriptional regulation of the iron assimilation system of plasmid ColV-K30 in Escherichia coli K-12. J Bacteriol. 1985 Jun;162(3):1039–1046. doi: 10.1128/jb.162.3.1039-1046.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Buck M., Ames B. N. A modified nucleotide in tRNA as a possible regulator of aerobiosis: synthesis of cis-2-methyl-thioribosylzeatin in the tRNA of Salmonella. Cell. 1984 Feb;36(2):523–531. doi: 10.1016/0092-8674(84)90245-9. [DOI] [PubMed] [Google Scholar]
  6. Buck M., Griffiths E. Iron mediated methylthiolation of tRNA as a regulator of operon expression in Escherichia coli. Nucleic Acids Res. 1982 Apr 24;10(8):2609–2624. doi: 10.1093/nar/10.8.2609. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Calderwood S. B., Mekalanos J. J. Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid. J Bacteriol. 1988 Feb;170(2):1015–1017. doi: 10.1128/jb.170.2.1015-1017.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Calderwood S. B., Mekalanos J. J. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J Bacteriol. 1987 Oct;169(10):4759–4764. doi: 10.1128/jb.169.10.4759-4764.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Coulton J. W., Mason P., Cameron D. R., Carmel G., Jean R., Rode H. N. Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12. J Bacteriol. 1986 Jan;165(1):181–192. doi: 10.1128/jb.165.1.181-192.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Crosa J. H. The relationship of plasmid-mediated iron transport and bacterial virulence. Annu Rev Microbiol. 1984;38:69–89. doi: 10.1146/annurev.mi.38.100184.000441. [DOI] [PubMed] [Google Scholar]
  11. Garner M. M., Revzin A. A gel electrophoresis method for quantifying the binding of proteins to specific DNA regions: application to components of the Escherichia coli lactose operon regulatory system. Nucleic Acids Res. 1981 Jul 10;9(13):3047–3060. doi: 10.1093/nar/9.13.3047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Griggs D. W., Tharp B. B., Konisky J. Cloning and promoter identification of the iron-regulated cir gene of Escherichia coli. J Bacteriol. 1987 Dec;169(12):5343–5352. doi: 10.1128/jb.169.12.5343-5352.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hantke K. Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet. 1981;182(2):288–292. doi: 10.1007/BF00269672. [DOI] [PubMed] [Google Scholar]
  14. Klebba P. E., McIntosh M. A., Neilands J. B. Kinetics of biosynthesis of iron-regulated membrane proteins in Escherichia coli. J Bacteriol. 1982 Mar;149(3):880–888. doi: 10.1128/jb.149.3.880-888.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lundrigan M. D., Kadner R. J. Nucleotide sequence of the gene for the ferrienterochelin receptor FepA in Escherichia coli. Homology among outer membrane receptors that interact with TonB. J Biol Chem. 1986 Aug 15;261(23):10797–10801. [PubMed] [Google Scholar]
  16. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  17. McDougall S., Neilands J. B. Plasmid- and chromosome-coded aerobactin synthesis in enteric bacteria: insertion sequences flank operon in plasmid-mediated systems. J Bacteriol. 1984 Jul;159(1):300–305. doi: 10.1128/jb.159.1.300-305.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McLennan B. D., Buck M., Humphreys J., Griffiths E. Iron-related modification of bacterial transfer RNA. Nucleic Acids Res. 1981 Jun 11;9(11):2629–2640. doi: 10.1093/nar/9.11.2629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Neilands J. B. Microbial envelope proteins related to iron. Annu Rev Microbiol. 1982;36:285–309. doi: 10.1146/annurev.mi.36.100182.001441. [DOI] [PubMed] [Google Scholar]
  20. Pabo C. O., Sauer R. T. Protein-DNA recognition. Annu Rev Biochem. 1984;53:293–321. doi: 10.1146/annurev.bi.53.070184.001453. [DOI] [PubMed] [Google Scholar]
  21. Pressler U., Staudenmaier H., Zimmermann L., Braun V. Genetics of the iron dicitrate transport system of Escherichia coli. J Bacteriol. 1988 Jun;170(6):2716–2724. doi: 10.1128/jb.170.6.2716-2724.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Reznikoff W. S., Siegele D. A., Cowing D. W., Gross C. A. The regulation of transcription initiation in bacteria. Annu Rev Genet. 1985;19:355–387. doi: 10.1146/annurev.ge.19.120185.002035. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sauer M., Hantke K., Braun V. Ferric-coprogen receptor FhuE of Escherichia coli: processing and sequence common to all TonB-dependent outer membrane receptor proteins. J Bacteriol. 1987 May;169(5):2044–2049. doi: 10.1128/jb.169.5.2044-2049.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Schneider T. D., Stormo G. D., Gold L., Ehrenfeucht A. Information content of binding sites on nucleotide sequences. J Mol Biol. 1986 Apr 5;188(3):415–431. doi: 10.1016/0022-2836(86)90165-8. [DOI] [PubMed] [Google Scholar]
  26. Schäffer S., Hantke K., Braun V. Nucleotide sequence of the iron regulatory gene fur. Mol Gen Genet. 1985;200(1):110–113. doi: 10.1007/BF00383321. [DOI] [PubMed] [Google Scholar]
  27. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  28. Worsham P. L., Konisky J. Effect of growth temperature on the acquisition of iron by Salmonella typhimurium and Escherichia coli. J Bacteriol. 1984 Apr;158(1):163–168. doi: 10.1128/jb.158.1.163-168.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Yanofsky C. Attenuation in the control of expression of bacterial operons. Nature. 1981 Feb 26;289(5800):751–758. doi: 10.1038/289751a0. [DOI] [PubMed] [Google Scholar]
  30. de Lorenzo V., Bindereif A., Paw B. H., Neilands J. B. Aerobactin biosynthesis and transport genes of plasmid ColV-K30 in Escherichia coli K-12. J Bacteriol. 1986 Feb;165(2):570–578. doi: 10.1128/jb.165.2.570-578.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. de Lorenzo V., Wee S., Herrero M., Neilands J. B. Operator sequences of the aerobactin operon of plasmid ColV-K30 binding the ferric uptake regulation (fur) repressor. J Bacteriol. 1987 Jun;169(6):2624–2630. doi: 10.1128/jb.169.6.2624-2630.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]

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