1887

Abstract

Iron is essential for the survival of almost all organisms, although excess iron can result in the generation of free radicals which are toxic to cells. To avoid the toxic effects of free radicals, the concentration of intracellular iron is generally regulated by the ferric uptake regulator Fur in bacteria. The 150 aa ORF from was cloned into pRSETa, and the His-tagged fusion protein was purified by nickel affinity column chromatography. DNA binding activity of this protein was studied by an electrophoretic mobility shift assay using the end-labelled promoters P and P. The results showed a decrease in migration for both promoter DNAs in the presence of the Fur protein, and the change in migration was competitively inhibited with an excess of the same unlabelled promoters. No shift in migration was observed when a similar assay was performed using non-specific end-labelled DNA. The assay showed that binding of Fur to P or P was independent of iron or manganese ions, and was not inhibited in the presence of 2 mM EDTA. Inductively coupled plasma MS of the Fur protein showed no iron or manganese, but 0.48 mole zinc per mole protein was detected. A DNase I protection assay revealed that Fur specifically bound to and protected a 19 bp consensus Fur box sequence located in the promoters of and . There was no requirement for iron or manganese in this assay also. However, Northern blot analysis showed an increase in transcription under iron-restricted compared to high-level conditions. Thus, the study suggests that under conditions, the affinity of the Fur protein for the 19 bp Fur box sequence does not require iron, but iron availability regulates transcription . Thus, the regulation by Fur in this intracellular pathogen may be dependent on either the structure of the DNA binding domain or other intracellular factors yet to be identified.

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2007-04-01
2024-12-09
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References

  1. Althaus E. W., Outen C. E., Olson K. E., Cao H., O'Halloram T. V. 1999; The ferric uptake regulation (Fur) repressor is a zinc metalloprotein. Biochemistry 38:6559–6569 [CrossRef]
    [Google Scholar]
  2. Andre P., Oberle S., Specklin V., Lombard Y., Vidon D. J. 2003; Low-level iron-dependent mutants of Listeria monocytogenes and their virulence in macrophages. Can J Microbiol 49:78–84 [CrossRef]
    [Google Scholar]
  3. Andrews S. C., Robinson A. K., Rodríguez-Quiñones F. 2003; Bacterial iron homeostasis. FEMS Microbiol Rev 27:215–237 [CrossRef]
    [Google Scholar]
  4. Barchini E., Cowart R. E. 1996; Extracellular iron reductase activity produced by Listeria monocytogenes . Arch Microbiol 166:51–57 [CrossRef]
    [Google Scholar]
  5. Bishop D. K., Hinrichs D. J. 1987; Adoptive transfer of immunity to Listeria monocytogenes . The influence of in vitro stimulation on lymphocyte subset requirements. J Immunol 139:2005–2009
    [Google Scholar]
  6. Braun V. 1997; Avoidance of iron toxicity through regulation of bacterial iron transport. J Biol Chem 387:779–786
    [Google Scholar]
  7. Braun V. 2005; Bacterial iron transport related to virulence. Contrib Microbiol 12:210–233
    [Google Scholar]
  8. Brown J. S., Holden D. W. 2002; Iron acquisition by Gram-positive bacterial pathogens. Microbes Infect 4:1149–1156 [CrossRef]
    [Google Scholar]
  9. Bsat N., Helmann J. D. 1999; Interaction of Bacillus subtilis Fur (ferric uptake repressor) with the dhb operator in vitro and in vivo . J Bacteriol 181:4299–4307
    [Google Scholar]
  10. Bsat N., Herbig A., Casillas-Martinez L., Setlow P., Helmann J. D. 1998; Bacillus subtilis contains multiple Fur homologs: identification of the iron uptake (Fur) and peroxide regulon (PerR) repressors. Mol Microbiol 29:189–198 [CrossRef]
    [Google Scholar]
  11. Chakraborty T. 1999; Molecular and cell biological aspects of infection by Listeria monocytogenes . Immunobiology 201:155–163 [CrossRef]
    [Google Scholar]
  12. Chatterjee S. S., Hossain H., Otten S., Kuenne C., Kuchimina K., Machata S., Domann E., Chakraborty T., Hain T. 2006; Intracellular gene expression profile of Listeria monocytogenes . Infect Immun 74:1323–1338 [CrossRef]
    [Google Scholar]
  13. Conte M. P., Longhi C., Polidoro M., Petrone G., Buonfiglio V., Di Santo S., Papi E., Seganti L., Viscaand P., Valenti P. 1996; Iron availability affects entry of Listeria monocytogenes into the enterocyte like cell Caco-2. Infect Immun 64:3925–3929
    [Google Scholar]
  14. Cossart P. 2002; Molecular and cellular basis of the infection by Listeria monocytogenes : an overview. Int J Med Microbiol 291:401–409
    [Google Scholar]
  15. Coulanges V., Andre P., Ziegler O., Buchheit L., Vidon D. J.-M. 1997; Utilization of iron-catecholamines complexes involving ferric reductase activity in Listeria monocytogenes . Infect Immun 65:2778–2785
    [Google Scholar]
  16. Coulanges V., Andre P., Vidon D. J.-M. 1998; Effect of siderophores, catecholamines, and catecholamine compounds on Listeria spp. Growth in iron-complexed medium. Biochem Biophys Res Commun 249:526–530 [CrossRef]
    [Google Scholar]
  17. Cowart R. E., Foster B. G. 1985; Differential effects of iron on the growth of Listeria monocytogenes : minimum requirements and mechanism of acquisition. J Infect Dis 151:721–730 [CrossRef]
    [Google Scholar]
  18. Coy M., Neilands J. B. 1991; Structural dynamics and functional domains of the Fur protein. Biochemistry 30:8201–8210 [CrossRef]
    [Google Scholar]
  19. Delany I., Pacheco A. B., Spohn G., Rappuoli R., Scarlato V. 2001; Iron-dependent transcription of the frpB gene of Helicobacter pylori is controlled by the Fur repressor protein. J Bacteriol 183:4932–4937 [CrossRef]
    [Google Scholar]
  20. Delany I., Spohn G., Rappuoli R., Scarlato V. 2003; An anti-repression Fur operator upstream of the promoter is required for iron-mediated transcriptional autoregulation in Helicobacter pylori . Mol Microbiol 50:1329–1338 [CrossRef]
    [Google Scholar]
  21. de Lorenzo V., Herrero M., Giovannini F., Neilands J. B. 1988; Fur (ferric uptake regulation) protein and CAP (catabolite-activator protein) modulate transcription of fur gene in Escherichia coli . Eur J Biochem 173:537–546 [CrossRef]
    [Google Scholar]
  22. Deneer H., Healey V., Boychuck I. 1995; Reduction of exogenous ferric iron by a surface-associated ferric reductase of Listeria spp. Microbiology 141:1985–1992 [CrossRef]
    [Google Scholar]
  23. Dorman C. J. 1994 Genetics of Bacterial Virulence Oxford: Blackwell Scientific Publications;
    [Google Scholar]
  24. Escolar L., Perez-Martin J., de Lorenzo V. 1999; Opening the iron-box: transcriptional metalloregulation by the Fur protein. J Bacteriol 181:6223–6229
    [Google Scholar]
  25. Fisher C. W., Martin S. E. 1999; Effects of iron and selenium on the production of catalase, superoxide dismutase, and listeriolysin O in Listeria monocytogenes . J Food Prot 62:1206–1209
    [Google Scholar]
  26. Fuangthong M., Herbig A. F., Bsat N., Helmann J. D. 2002; Regulation of the Bacillus subtilis fur and perR genes by PerR: not all members of the PerR regulon are peroxide inducible. J Bacteriol 184:3276–3286 [CrossRef]
    [Google Scholar]
  27. Geoffroy C., Gaillard J. L., Alouf J. E., Berche P. 1987; Purification, characterization, and toxicity of the sulfhydryl-activated hemolysin listeriolysin O from Listeria monocytogenes . Infect Immun 55:1641–1646
    [Google Scholar]
  28. Glaser P., Frangeul L., Buchrieser C., Rusniok C., Amend A., Baquero F., Berche P., Bloecker H., Brandt P. & other authors 2001; Comparative genomics of Listeria species. Science 294:849–852
    [Google Scholar]
  29. Gonzalez de Peredo A., Saint-Pierre C., Adrait A., Jacquamet L., Latour J. M., Michaud-Soret I., Forest E. 1999; Identification of the two zinc-bound cysteines in the ferric uptake regulation protein from Escherichia coli : chemical modification and mass spectrometry analysis. Biochemistry 38:8582–8589 [CrossRef]
    [Google Scholar]
  30. Gonzalez de Peredo A., Saint-Pierre C., Latour J. M., Michaud-Soret I., Forest E. 2001; Conformational changes of the ferric uptake regulation protein upon metal activation and DNA binding; first evidence of structural homologies with the diphtheria toxin repressor. J Mol Biol 310:83–91 [CrossRef]
    [Google Scholar]
  31. Gray M. J., Freitag N. E., Boor K. J. 2006; How the bacterial pathogen Listeria monocytogenes mediates the switch from environmental Dr. Jekyll to pathogenic Mr. Hyde. Infect Immun 74:2505–2512 [CrossRef]
    [Google Scholar]
  32. Griffiths E. 1999; Iron in biological systems. In Iron and Infection , 2nd edn. pp 1–26 Edited by Bullen J. J. Griffiths E. New York: Wiley;
    [Google Scholar]
  33. Gutteridge J. M. C., Rowley D. A., Halliwell B. 1982; Superoxide dependent formation of hydroxyl radical and lipid peroxidation in the presence of iron salt: detection of catalytic iron and antioxidant activity in extracellular fluids. Biochem J 206:605–609
    [Google Scholar]
  34. Hamza I., Hassett R., O'Brian M. R. 1999; Identification of a functional fur gene in Bradyrhizobium japonicum . J Bacteriol 181:5843–5846
    [Google Scholar]
  35. Hamza I., Qi Z., King N. D., O'Brian M. R. 2000; Fur-independent regulation of iron metabolism by irr in Bradyrhizobium japonicum . Microbiology 146:669–676
    [Google Scholar]
  36. Hartford T., O'Brien S., Andrew P. W., Jones D. J., Roberts I. S. 1993; Utilization of transferrin-bound iron by Listeria monocytogenes . FEMS Microbiol Lett 108:311–318 [CrossRef]
    [Google Scholar]
  37. Hernandez J. A., Bes M. T., Fillat M. F., Neira J. L., Peleato M. L. 2002; Biochemical analysis of the recombinant Fur (ferric uptake regulator) protein from Anabaena PCC 7119: factors affecting its oligomerization state. Biochem J 366:315–322
    [Google Scholar]
  38. Imlay J. A., Chin S. M., Linn S. 1988; Toxic DNA damage by hydrogen peroxide through the Fenton reaction in vivo and in vitro . Science 240:640–642 [CrossRef]
    [Google Scholar]
  39. Jacquamet L., Aberdam D., Adrait A., Hazemann J. L., Latour J. M., Michaud-Soret I. 1998; X-ray absorption spectroscopy of a new zinc site in the Fur protein from Escherichia coli . Biochemistry 37:2564–2571 [CrossRef]
    [Google Scholar]
  40. Jin B., Newton S. M., Shao Y., Jiang X., Charbit A., Klebba P. E. 2006; Iron acquisition systems for ferric hydroxamates, haemin and haemoglobin in Listeria monocytogenes . Mol Microbiol 59:1185–1198 [CrossRef]
    [Google Scholar]
  41. Kathariou S. 2002; Listeria monocytogenes virulence and pathogenicity, a food safety perspective. J Food Prot 65:1811–1829
    [Google Scholar]
  42. Kyte J., Doolittle R. F. 1982; A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132 [CrossRef]
    [Google Scholar]
  43. Leblanc B., Moss T. 1994; DNase I footprinting. In DNA–Protein Interactions: Principles and Protocols. Methods in Molecular Biology pp 1–10 Edited by Kneale G. G. New Jersey: Humana Press;
    [Google Scholar]
  44. Lewin A. C., Doughty P. A., Flegg L., Moore G., Spiro S. 2002; The ferric uptake regulator of Pseudomonas aeruginosa has no essential cysteine residues and does not contain a structural zinc ion. Microbiology 148:2449–2456
    [Google Scholar]
  45. Litwin C. M., Calderwood S. B. 1993; Role of iron in the regulation of virulence genes. Clin Microbiol Rev 6:137–149
    [Google Scholar]
  46. Litwin C. M., Boyko S. A., Calderwood S. B. 1992; Cloning, sequencing, and transcriptional regulation of the Vibrio cholerae fur gene. J Bacteriol 174:1897–1903
    [Google Scholar]
  47. McHugh J. P., Rodriguez-Quinones F., Abdul-Tehrani H., Svistunenko D. A., Poole R. K., Cooper C. E., Andrews S. C. 2003; Global iron-dependent gene regulation in Escherichia coli . A new mechanism for iron homeostasis. J Biol Chem 278:29478–29486 [CrossRef]
    [Google Scholar]
  48. Mikael L. G., Pawelek P., Labrie J., Sirois M., Coulton J. W., Jacques M. 2002; Molecular cloning and characterization of the ferric hydroxamate uptake ( fhu ) operon in Actinobacillus pleuropneumoniae . Microbiology 148:2869–2882
    [Google Scholar]
  49. Miller R. A., Britigan B. E. 1997; Role of oxidants in microbial pathophysiology. Clin Microbiol Rev 34:2593–2594
    [Google Scholar]
  50. Newton S. M., Klebba P. E., Raynaud C., Shao Y., Jiang X., Dubail I., Archer C., Frehel C., Charbit A. 2005; The svpA–srtB locus of Listeria monocytogenes : Fur-mediated iron regulation and effect on virulence. Mol Microbiol 55:927–940
    [Google Scholar]
  51. Nienaber A., Hennecke H., Fischer H.-M. 2001; Discovery of a haem uptake system in the soil bacterium Bradyrhizobium japonicum . Mol Microbiol 41:787–800
    [Google Scholar]
  52. Ochsner U. A., Vasil A. I., Vasil M. L. 1995; Role of the ferric uptake regulator of Pseudomonas aeruginosa in the regulation of siderophores and exotoxin A expression: purification and activity on iron-regulated promoters. J Bacteriol 177:7194–7201
    [Google Scholar]
  53. Park S. Y., Kelminson K. L., Lee A. K., Zhang P., Warner R. E., Rehkopf D. H., Calderwood S. B., Koehler J. E. 2001; Identification, characterization, and functional analysis of a gene encoding the ferric uptake regulation protein in Bartonella species. J Bacteriol 183:5751–5755 [CrossRef]
    [Google Scholar]
  54. Pohl E., Haller J., Mijovilovich A., Meyer-Klauke W., Garman E., Vasil M. L. 2003; Architecture of a protein central to iron homeostasis: crystal structure and spectroscopic analysis of the ferric uptake regulator. Mol Microbiol 47:903–905 [CrossRef]
    [Google Scholar]
  55. Polidoro M., De Biase D., Montagnini B., Guarrera L., Cavallo S., Valenti P., Stefanini S., Chiancone E. 2002; The expression of the dodecameric ferritin in Listeria spp. is induced by iron limitation and stationary growth phase. Gene 296:121–128 [CrossRef]
    [Google Scholar]
  56. Portnoy D. A., Auerbuch V., Glomski I. J. 2002; The cell biology of Listeria monocytogenes infection: the intersection of bacterial pathogenesis and cell-mediated immunity. J Cell Biol 158:409–414 [CrossRef]
    [Google Scholar]
  57. Rea R. B., Gahan R. C., Hill C. 2004; Disruption of putative regulatory loci in Listeria monocytogenes demonstrates a significant role for Fur and PerR in virulence. Infect Immun 72:717–727 [CrossRef]
    [Google Scholar]
  58. Rodriguez G. M., Voskuil M. I., Gold B., Schoolnik G. K., Smith I. 2002; ideR , an essential gene in Mycobacterium tuberculosis : role of IdeR in iron-dependent gene expression, iron metabolism, and oxidative stress response. Infect Immun 70:3371–3381 [CrossRef]
    [Google Scholar]
  59. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  60. Schiering N., Tao X., Zeng H., Murphy J. R., Petsko G. A., Ringe D. 1995; Structures of the Apo- and the metal ion-activated forms of the diphtheria tox repressor from Corynebacterium diphtheriae . Proc Natl Acad Sci U S A 92:9843–9850 [CrossRef]
    [Google Scholar]
  61. Simon N., Coulanges V., Andre P., Vidon D. J.-M. 1995; Utilization of exogenous siderophores and natural catechols by Listeria monocytogenes . Appl Environ Microbiol 61:1643–1645
    [Google Scholar]
  62. Smith A., Hooper N. I., Shipulina N., Morgan W. T. 1996; Heme binding by a bacterial repressor protein, the gene product of the ferric uptake regulation ( fur ) gene of Escherichia coli . J Protein Chem 15:575–583 [CrossRef]
    [Google Scholar]
  63. Stojiljkovic I., Hantke K. 1995; Functional domains of the Escherichia coli ferric uptake regulator protein (Fur. Mol Gen Genet 247:199–205 [CrossRef]
    [Google Scholar]
  64. Storz G., Imlay J. 1999; Oxidative stress. Curr Opin Microbiol 2:188–194 [CrossRef]
    [Google Scholar]
  65. Sword C. P. 1966; Mechanisms of pathogenesis in Listeria monocytogenes infection I. Influence of iron. J Bacteriol 92:536–542
    [Google Scholar]
  66. Tai S. S., Lee C.-J., Winter R. E. 1993; Hemin utilization is related to virulence of Streptococcus pneumoniae . Infect Immun 61:5401–5405
    [Google Scholar]
  67. Vazquez-Boland J. A., Kuhn M., Berche P., Chakraborty T., Dominguez-Bernal G., Goebel W., Gonzalez-Zorn B., Wehland J., Kreft J. 2001; Listeria pathogenesis and molecular virulence determinants. Clin Microbiol Rev 14:584–640 [CrossRef]
    [Google Scholar]
  68. Weinberg E. D. 1978; Iron and infection. Microbiol Rev 42:45–66
    [Google Scholar]
  69. Xiong A., Singh V. K., Cabrera G., Jayaswal R. K. 2000; Molecular characterization of a ferric uptake regulator, Fur, from Staphylococcus aureus . Microbiology 145:801–808
    [Google Scholar]
  70. Yanisch-Perron C., Vieira J., Messing J. 1985; Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119 [CrossRef]
    [Google Scholar]
  71. Zhu K., Bayles D. O., Xiong A., Jayaswal R. K., Wilkinson B. J. 2005; Precursor and temperature modulation of fatty acid composition and growth of Listeria monocytogenes cold-sensitive mutants with transposon-interrupted branched-chain alpha-keto acid dehydrogenase. Microbiology 151:615–623 [CrossRef]
    [Google Scholar]
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