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J Bacteriol. Jul 1994; 176(13): 3944–3955.
PMCID: PMC205592

Promoter and operator determinants for fur-mediated iron regulation in the bidirectional fepA-fes control region of the Escherichia coli enterobactin gene system.

Abstract

The fepA-entD and fes-entF operons in the enterobactin synthesis and transport system are divergently transcribed from overlapping promoters, and both are inhibited by the Fur repressor protein under iron-replete conditions. A plasmid harboring divergent fepA'-phoA and fes-entF'-'lacZ fusions, both under the control of this bidirectional regulatory region, was constructed for the purpose of monitoring changes in expression of the two operons simultaneously. Deletion analysis, site-directed mutagenesis, and primer extension were employed to define both a single promoter governing the expression of fes-entF and two tandemly arranged promoters giving rise to the opposing fepA-entD transcript. A single Fur-binding site that coordinately regulates the expression of all transcripts emanating from this control region was identified by in vitro protection from DNase I nicking. The substitution of one base pair in the Fur recognition sequence relieved Fur repression but did not change the in vitro affinity of Fur for its binding site. Additional mutations in a limited region outside of the promoter determinants for either transcript inhibited expression of both fes and fepA. These observations suggest a mechanism of Fur-mediated regulation in this compact control region that may involve other regulatory components.

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  • Armstrong SK, Pettis GS, Forrester LJ, McIntosh MA. The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product. Mol Microbiol. 1989 Jun;3(6):757–766. [PubMed]
  • Bachmann BJ. Linkage map of Escherichia coli K-12, edition 8. Microbiol Rev. 1990 Jun;54(2):130–197. [PMC free article] [PubMed]
  • Bagg A, Neilands JB. Mapping of a mutation affecting regulation of iron uptake systems in Escherichia coli K-12. J Bacteriol. 1985 Jan;161(1):450–453. [PMC free article] [PubMed]
  • Bagg A, Neilands JB. Molecular mechanism of regulation of siderophore-mediated iron assimilation. Microbiol Rev. 1987 Dec;51(4):509–518. [PMC free article] [PubMed]
  • Beck CF, Warren RA. Divergent promoters, a common form of gene organization. Microbiol Rev. 1988 Sep;52(3):318–326. [PMC free article] [PubMed]
  • Brickman E, Beckwith J. Analysis of the regulation of Escherichia coli alkaline phosphatase synthesis using deletions and phi80 transducing phages. J Mol Biol. 1975 Aug 5;96(2):307–316. [PubMed]
  • Brickman TJ, Ozenberger BA, McIntosh MA. Regulation of divergent transcription from the iron-responsive fepB-entC promoter-operator regions in Escherichia coli. J Mol Biol. 1990 Apr 20;212(4):669–682. [PubMed]
  • Calderwood SB, Mekalanos JJ. Iron regulation of Shiga-like toxin expression in Escherichia coli is mediated by the fur locus. J Bacteriol. 1987 Oct;169(10):4759–4764. [PMC free article] [PubMed]
  • Calderwood SB, Mekalanos JJ. 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. [PMC free article] [PubMed]
  • Casadaban MJ, Chou J, Cohen SN. In vitro gene fusions that join an enzymatically active beta-galactosidase segment to amino-terminal fragments of exogenous proteins: Escherichia coli plasmid vectors for the detection and cloning of translational initiation signals. J Bacteriol. 1980 Aug;143(2):971–980. [PMC free article] [PubMed]
  • Clemens KE, Pintel DJ. The two transcription units of the autonomous parvovirus minute virus of mice are transcribed in a temporal order. J Virol. 1988 Apr;62(4):1448–1451. [PMC free article] [PubMed]
  • Cohen SN, Chang AC, Hsu L. Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc Natl Acad Sci U S A. 1972 Aug;69(8):2110–2114. [PMC free article] [PubMed]
  • Collado-Vides J, Magasanik B, Gralla JD. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev. 1991 Sep;55(3):371–394. [PMC free article] [PubMed]
  • Compan I, Touati D. Interaction of six global transcription regulators in expression of manganese superoxide dismutase in Escherichia coli K-12. J Bacteriol. 1993 Mar;175(6):1687–1696. [PMC free article] [PubMed]
  • de Lorenzo V, Wee S, Herrero M, Neilands JB. 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. [PMC free article] [PubMed]
  • de Lorenzo V, Giovannini F, Herrero M, Neilands JB. Metal ion regulation of gene expression. Fur repressor-operator interaction at the promoter region of the aerobactin system of pColV-K30. J Mol Biol. 1988 Oct 20;203(4):875–884. [PubMed]
  • De Lorenzo V, Herrero M, Giovannini F, Neilands JB. Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli. Eur J Biochem. 1988 May 2;173(3):537–546. [PubMed]
  • Fleming TP, Nahlik MS, McIntosh MA. Regulation of enterobactin iron transport in Escherichia coli: characterization of ent::Mu d(Apr lac) operon fusions. J Bacteriol. 1983 Dec;156(3):1171–1177. [PMC free article] [PubMed]
  • Fleming TP, Nahlik MS, Neilands JB, McIntosh MA. Physical and genetic characterization of cloned enterobactin genomic sequences from Escherichia coli K-12. Gene. 1985;34(1):47–54. [PubMed]
  • Fouser LA, Friesen JD. Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing. Cell. 1986 Apr 11;45(1):81–93. [PubMed]
  • Fried M, Crothers DM. Equilibria and kinetics of lac repressor-operator interactions by polyacrylamide gel electrophoresis. Nucleic Acids Res. 1981 Dec 11;9(23):6505–6525. [PMC free article] [PubMed]
  • Garner MM, 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. [PMC free article] [PubMed]
  • Goldberg MB, Boyko SA, Calderwood SB. Transcriptional regulation by iron of a Vibrio cholerae virulence gene and homology of the gene to the Escherichia coli fur system. J Bacteriol. 1990 Dec;172(12):6863–6870. [PMC free article] [PubMed]
  • Griggs DW, Konisky J. Mechanism for iron-regulated transcription of the Escherichia coli cir gene: metal-dependent binding of fur protein to the promoters. J Bacteriol. 1989 Feb;171(2):1048–1054. [PMC free article] [PubMed]
  • Hantke K. Regulation of ferric iron transport in Escherichia coli K12: isolation of a constitutive mutant. Mol Gen Genet. 1981;182(2):288–292. [PubMed]
  • Hantke K. Cloning of the repressor protein gene of iron-regulated systems in Escherichia coli K12. Mol Gen Genet. 1984;197(2):337–341. [PubMed]
  • Hassan HM, Sun HC. Regulatory roles of Fnr, Fur, and Arc in expression of manganese-containing superoxide dismutase in Escherichia coli. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3217–3221. [PMC free article] [PubMed]
  • Hawley DK, McClure WR. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. [PMC free article] [PubMed]
  • Kunkel TA. Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc Natl Acad Sci U S A. 1985 Jan;82(2):488–492. [PMC free article] [PubMed]
  • Lundrigan MD, Kadner RJ. 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]
  • Manoil C, Beckwith J. TnphoA: a transposon probe for protein export signals. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8129–8133. [PMC free article] [PubMed]
  • Nahlik MS, Brickman TJ, Ozenberger BA, McIntosh MA. Nucleotide sequence and transcriptional organization of the Escherichia coli enterobactin biosynthesis cistrons entB and entA. J Bacteriol. 1989 Feb;171(2):784–790. [PMC free article] [PubMed]
  • Neilands JB. Iron absorption and transport in microorganisms. Annu Rev Nutr. 1981;1:27–46. [PubMed]
  • Niederhoffer EC, Naranjo CM, Bradley KL, Fee JA. Control of Escherichia coli superoxide dismutase (sodA and sodB) genes by the ferric uptake regulation (fur) locus. J Bacteriol. 1990 Apr;172(4):1930–1938. [PMC free article] [PubMed]
  • Norrander J, Kempe T, Messing J. Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 1983 Dec;26(1):101–106. [PubMed]
  • Ozenberger BA, Brickman TJ, McIntosh MA. Nucleotide sequence of Escherichia coli isochorismate synthetase gene entC and evolutionary relationship of isochorismate synthetase and other chorismate-utilizing enzymes. J Bacteriol. 1989 Feb;171(2):775–783. [PMC free article] [PubMed]
  • Pettis GS, Brickman TJ, McIntosh MA. Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region. Identification of a unique iron-regulated bidirectional promoter. J Biol Chem. 1988 Dec 15;263(35):18857–18863. [PubMed]
  • Pettis GS, McIntosh MA. Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene. J Bacteriol. 1987 Sep;169(9):4154–4162. [PMC free article] [PubMed]
  • Prince RW, Storey DG, Vasil AI, Vasil ML. Regulation of toxA and regA by the Escherichia coli fur gene and identification of a Fur homologue in Pseudomonas aeruginosa PA103 and PA01. Mol Microbiol. 1991 Nov;5(11):2823–2831. [PubMed]
  • Ross W, Gosink KK, Salomon J, Igarashi K, Zou C, Ishihama A, Severinov K, Gourse RL. A third recognition element in bacterial promoters: DNA binding by the alpha subunit of RNA polymerase. Science. 1993 Nov 26;262(5138):1407–1413. [PubMed]
  • Shea CM, McIntosh MA. Nucleotide sequence and genetic organization of the ferric enterobactin transport system: homology to other periplasmic binding protein-dependent systems in Escherichia coli. Mol Microbiol. 1991 Jun;5(6):1415–1428. [PubMed]
  • Tardat B, Touati D. Two global regulators repress the anaerobic expression of MnSOD in Escherichia coli::Fur (ferric uptake regulation) and Arc (aerobic respiration control). Mol Microbiol. 1991 Feb;5(2):455–465. [PubMed]
  • Tardat B, Touati D. Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA. Mol Microbiol. 1993 Jul;9(1):53–63. [PubMed]
  • Wee S, Neilands JB, Bittner ML, Hemming BC, Haymore BL, Seetharam R. Expression, isolation and properties of Fur (ferric uptake regulation) protein of Escherichia coli K 12. Biol Met. 1988;1(1):62–68. [PubMed]

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