1887

Abstract

The ferric uptake regulator (Fur) of was expressed in in its native form and as a fusion to the maltose-binding protein (MBP). Fur from the MBP fusion bound to MBP after proteolytic cleavage, and the two could only be separated by partial unfolding. The refolded protein was in the same conformation as native protein (as judged by circular dichroism and fluorescence spectroscopies) and was fully active in DNA-binding assays. As-prepared native Fur contained small amounts of Zn that were easily removed by treatment with EDTA, and apo-protein could be reconstituted with approximately one Zn ion per monomer. Thus, the Fur can probably accommodate a single Zn ion bound to the metal-sensing site. The single cysteine residue of Fur aligns with a cysteine in other members of the Fur family that is essential for activity of the protein, and is believed to provide one of the ligands to a structural Zn ion. This cysteine residue was shown to be dispensable for the activity of Fur, which is consistent with the suggestion that the protein does not contain a structural Zn ion. Members of the Fur family contain a highly conserved His-His-Asp-His motif. Alanine substitutions of residues in this motif showed His-87 and His-89 of Fur to be essential for activity, whilst His-86 and Asp-88 are partially dispensable.

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2002-08-01
2024-12-09
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References

  1. Althaus E. W., Outten C. E., Olson K. E., Cao H., O’Halloran T. V. 1999; The ferric uptake regulation (Fur) repressor is a zinc metalloprotein. Biochemistry 38:6559–6569 [CrossRef]
    [Google Scholar]
  2. Andrews S. C., Harrison P. M., Guest J. R. 1989; Cloning, sequencing, and mapping of the bacterioferritin gene ( bfr ) of Escherichia coli K12. J Bacteriol 171:3940–3947
    [Google Scholar]
  3. Barton H. A., Johnson Z., Cox C. D., Vasil A. I., Vasil M. L. 1996; Ferric uptake regulator mutants of Pseudomonas aeruginosa with distinct alterations in the iron-dependent repression of exotoxin A and siderophores in aerobic and microaerobic environments. Mol Microbiol 21:1001–1017 [CrossRef]
    [Google Scholar]
  4. 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]
  5. Chai W., Stewart V. 1998; NasR, a novel RNA-binding protein, mediates nitrate-responsive transcription antitermination of the Klebsiella oxytoca M5al nasF operon leader in vitro . J Mol Biol 283:339–351 [CrossRef]
    [Google Scholar]
  6. Coy M., Neilands J. B. 1991; Structural dynamics and functional domains of the Fur protein. Biochemistry 30:8201–8210 [CrossRef]
    [Google Scholar]
  7. Coy M., Doyle C., Besser J., Neilands J. B. 1994; Site-directed mutagenesis of the ferric uptake regulation gene of Escherichia coli . Biometals 7:292–298
    [Google Scholar]
  8. Dietze E. C., Wang R. W., Lu A. Y. H., Atkins W. M. 1996; Ligand effects on the fluorescence properties of tyrosine-9 in alpha 1-1 glutathione S -transferase. Biochemistry 35:6745–6753 [CrossRef]
    [Google Scholar]
  9. 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]
  10. Garg R. P., Vargo C. J., Cui X., Kurtz D. M. Jr 1996; A [2Fe-2S] protein encoded by an open reading frame upstream of the Escherichia coli bacterioferritin gene. Biochemistry 35:6297–6301 [CrossRef]
    [Google Scholar]
  11. Gill S. C., von Hippel P. H. 1989; Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326 [CrossRef]
    [Google Scholar]
  12. 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]
  13. Groen B. H., Bloemendal M., Mulders J. W. M., Hadden J. M., Chapman D., Van Stokkum I. H. M., Van Grondelle R. 1996; Spectroscopic characterization of recombinant follicle stimulating hormone. Spectrochim Acta 52:1331–1346 [CrossRef]
    [Google Scholar]
  14. Hall H. K., Foster J. W. 1996; The role of Fur in the acid tolerance response of Salmonella typhimurium is physiologically and genetically separable from its role in iron acquisition. J Bacteriol 178:5683–5691
    [Google Scholar]
  15. Hantke K. 1987; Selection procedure for deregulated iron transport mutants ( fur) in Escherichia coli K12: fur not only affects iron metabolism. Mol Gen Genet 210:135–139 [CrossRef]
    [Google Scholar]
  16. Hassett D. J., Sokol P. A., Howell M. L., Ma J. F., Schweizer H. T., Ochsner U., Vasil M. L. 1996; Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J Bacteriol 178:3996–4003
    [Google Scholar]
  17. Hutchings M. I., Shearer N., Wastell S., van Spanning R. J. M., Spiro S. 2000; Heterologous NNR-mediated nitric oxide signaling in Escherichia coli . J Bacteriol 182:6434–6439 [CrossRef]
    [Google Scholar]
  18. 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]
  19. Ko Y. H., Thomas P. J., Delannoy M. R., Pedersen P. L. 1993; The cystic fibrosis transmembrane conductance regulator. Overexpression, purification, and characterization of wild type and delta F508 mutant forms of the first nucleotide binding fold in fusion with the maltose-binding protein. J Biol Chem 268:24330–24338
    [Google Scholar]
  20. Lakowicz J. R. 1983 Principles of Fluorescence Spectroscopy New York: Plenum Press;
    [Google Scholar]
  21. Lam M. S., Litwin C. M., Carroll P. A., Calderwood S. B. 1994; Vibrio cholerae fur mutations associated with loss of repressor activity: implications for structural-functional relationships of Fur. J Bacteriol 176:5108–5115
    [Google Scholar]
  22. Leoni L., Ciervo A., Orsi N., Visca P. 1996; Iron-regulated transcription of the pvdA gene in Pseudomonas aeruginosa : effect of Fur and PvdS on promoter activity. J Bacteriol 178:2299–2313
    [Google Scholar]
  23. Li J., Kustu S., Stewart V. 1994; In vitro interaction of nitrate-responsive regulatory protein NarL with DNA target sequences in the fdnG , narG , narK and frdA operon control regions of Escherichia coli K-12. J Mol Biol 241:150–165 [CrossRef]
    [Google Scholar]
  24. Litwin M., Calderwood S. B. 1993; Role of iron in regulation of virulence genes. Clin Microbiol Rev 6:137–149
    [Google Scholar]
  25. Massé E., Gottesman S. 2002; A small RNA regulates the expression of genes involved in iron metabolism in Escherichia coli . Proc Natl Acad Sci USA 99:4620–4625 [CrossRef]
    [Google Scholar]
  26. Michaud-Soret I., Adrait A., Jaquinod M., Forest E., Touati D., Latour J.-M. 1997; Electrospray ionization mass spectrometry analysis of the apo- and metal-substituted forms of the Fur protein. FEBS Lett 413:473–476 [CrossRef]
    [Google Scholar]
  27. Miller J. H. 1992 A Short Course in Bacterial Genetics Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  28. 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]
  29. Ochsner U. A., Johnson Z., Lamont I. L., Cunliffe H. E., Vasil M. L. 1996; Exotoxin A production in Pseudomonas aeruginosa requires the iron-regulated pvdS gene encoding an alternative sigma factor. Mol Microbiol 21:1019–1028 [CrossRef]
    [Google Scholar]
  30. Pace C. N., Scholtz J. M. 1997; Measuring the conformational stability of a protein. In Protein Structure: a Practical Approach pp 299–321 Edited by Creighton T. E. Oxford: IRL Press;
    [Google Scholar]
  31. Prince R. W., Cox C. D., Vasil M. L. 1993; Coordinate regulation of siderophore and exotoxin A production: molecular cloning and sequencing of the Pseudomonas aeruginosa fur gene. J Bacteriol 175:2589–2598
    [Google Scholar]
  32. Ratledge C., Dover L. G. 2000; Iron metabolism in pathogenic bacteria. Annu Rev Microbiol 54:881–941 [CrossRef]
    [Google Scholar]
  33. Santoro M. M., Bolen D. W. 1988; Unfolding free energy changes determined by the linear extrapolation method. 1. Unfolding of phenylmethanesulfonyl α-chymotrypsin using different denaturants. Biochemistry 27:8063–8068 [CrossRef]
    [Google Scholar]
  34. Soengas M. S., Mateo C. R., Salas M., Acuña A. U., Gutiérrez C. 1997; Structural features of ϕ29 single-stranded DNA-binding protein. I. Environment of tyrosines in terms of complex formation with DNA. J Biol Chem 272:295–302 [CrossRef]
    [Google Scholar]
  35. 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]
  36. Stojiljkovic I., Baumler A. J., Hantke K. 1994; Fur regulon in gram-negative bacteria. Identification and characterization of new iron-regulated Escherichia coli genes by a Fur titration assay. J Mol Biol 236:531–545 [CrossRef]
    [Google Scholar]
  37. Vasil M. L., Ochsner U. A. 1999; The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 34:399–413 [CrossRef]
    [Google Scholar]
  38. Wilderman P. J., Vasil A. I., Johnson Z., Wilson M. J., Cunliffe H. E., Lamont I. L., Vasil M. L. 2001; Characterization of an endoprotease (PrpL) encoded by a PvdS-regulated gene in Pseudomonas aeruginosa . Infect Immun 69:5385–5394 [CrossRef]
    [Google Scholar]
  39. Zheleznova E. L., Crosa J. H., Brennan R. G. 2000; Characterization of the DNA- and metal-binding properties of Vibrio anguillarum Fur reveals conservation of a structural Zn2+ ion. J Bacteriol 182:6264–6267 [CrossRef]
    [Google Scholar]
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