1887

Abstract

Iron is an essential nutrient for the survival and pathogenesis of bacteria, but relatively little is known regarding its transport and regulation in staphylococci. Based on the known sequences of ferric-uptake regulatory () genes from several Gram-positive and Gram-negative bacteria, a fragment containing the homologue was cloned from a genomic library of RN450. Nucleotide sequence analysis of this fragment revealed the presence of a 447 bp ORF that encodes a putative 149 aa polypeptide with an apparent molecular mass of 17 kDa. A putative ferrichrome-uptake () operon, containing the conserved Fur-binding sequences (Fur box) in the promoter region, was also cloned from the same library. To characterize the impact of Fur on the operon, was cloned, overexpressed as a His-tagged protein and purified by Ni-affinity column chromatography. The recombinant protein was digested with enterokinase to remove the His tag. Electrophoretic mobility-shift assays indicated that Fur binds to the promoter region of the operon in the presence of divalent cations. Fur also interacted with the promoter region of the recently reported operon that has been proposed to constitute a siderophore-transport system in . The DNase I-protection assay revealed that Fur specifically binds to the Fur box located in the promoter region of the operon. The primer-extension reaction indicated that the transcription-start site of the operon was located inside the Fur box. partially complemented a mutation in . The data suggest that Fur regulates iron-transport processes in .

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-3-659
2000-03-01
2019-10-21
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/3/1460659a.html?itemId=/content/journal/micro/10.1099/00221287-146-3-659&mimeType=html&fmt=ahah

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. Augustin, J., Rosenstein, R., Wieland, B., Schneider, U., Schnell, N., Engelke, G., Entian, K. D. & Götz, F. ( 1992; ). Genetic analysis of epidermin biosynthetic genes and epidermin-negative mutants of Staphylococcus epidermidis. Eur J Biochem 204, 1149-1154.[CrossRef]
    [Google Scholar]
  3. Bagg, A. & Neilands, J. B. ( 1987; ). 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 26, 5471-5477.[CrossRef]
    [Google Scholar]
  4. Braun, V. ( 1997; ). Avoidance of iron toxicity through regulation of bacterial iron transport. Biol Chem 378, 779-786.
    [Google Scholar]
  5. 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]
  6. Bsat, N., Herbig, A., Casillas-Martinez, L., Setlow, P. & Helmann, J. D. ( 1998; ). Bacillus subtilis contains multiple Fur homologues: identification of the iron uptake (Fur) and peroxidase regulon (PerR) repressors. Mol Microbiol 29, 189-198.[CrossRef]
    [Google Scholar]
  7. Chen, L., James, L. P. & Helmann, J. D. ( 1993; ). Metalloregulation in Bacillus subtilis: isolation and characterization of two genes differentially repressed by metal ions. J Bacteriol 175, 5428-5437.
    [Google Scholar]
  8. Cockayne, A., Hill, P. J., Powell, N. B., Bishop, K., Sims, C. & Williams, P. ( 1998; ). Molecular cloning of a 32 kilodalton lipoprotein component of a novel iron-regulated Staphylococcus epidermidis ABC transporter. Infect Immun 66, 3767-3774.
    [Google Scholar]
  9. Cook, W. J., Kar, S. R., Taylor, K. B. & Hall, L. M. ( 1998; ). Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins. J Mol Biol 275, 337-346.[CrossRef]
    [Google Scholar]
  10. Courcol, R. J., Trivier, D., Bissinger, M. C., Martin, G. R. & Brown, M. R. ( 1997; ). Siderophore production by Staphylococcus aureus and identification of iron-regulation proteins. Infect Immun 65, 1944-1948.
    [Google Scholar]
  11. Crosa, J. H. ( 1997; ). Signal transduction and transcriptional and post-transcriptional control of iron-regulated genes in bacteria. Microbiol Mol Biol Rev 61, 319-336.
    [Google Scholar]
  12. Dorman, C. J. ( 1994; ). Virulence gene regulatory networks. In Genetics of Bacterial Virulence, pp. 204-259. Edited by C. J. Dorman. London: Blackwell Scientific Publications.
  13. Endo, G. & Silver, S. ( 1995; ). CadC, the transcriptional regulatory protein of the cadmium-resistance system of Staphylococcus aureus plasmid pI258. J Bacteriol 177, 4437-4441.
    [Google Scholar]
  14. Escolar, L., Pérez-Martı́n, L. & de Lorenzo, V. ( 1998; ). Binding of the Fur (ferric-uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence. J Mol Biol 283, 537-547.[CrossRef]
    [Google Scholar]
  15. Foster, J. W. ( 1991; ). Salmonella acid shock proteins are required for the adaptive acid tolerance response. J Bacteriol 173, 6896-6902.
    [Google Scholar]
  16. Gaballa, A. & Helmann, J. D. ( 1998; ). Identification of a zinc-specific metalloregulatory protein, Zur, controlling zinc transport operons in Bacillus subtilis. J Bacteriol 180, 5815-5821.
    [Google Scholar]
  17. Guerinot, M. L. ( 1994; ). Microbial iron transport. Annu Rev Microbiol 48, 743-772.[CrossRef]
    [Google Scholar]
  18. Haag, H., Fiedler, H. P., Meiwes, J., Drechsel, H., Jung, G. & Zahner, H. ( 1994; ). Isolation and biological characterization of staphyloferrin B, a compound with siderophore activity from staphylococci. FEMS Microbiol Lett 115, 125-130.[CrossRef]
    [Google Scholar]
  19. 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]
  20. Halliwell, B. & Gutteridge, J. M. ( 1984; ). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219, 1-14.
    [Google Scholar]
  21. Heidrich, C., Hantke, K., Bierbaum, G. & Sahl, H. G. ( 1996; ). Identification and analysis of a gene encoding a Fur-like protein of Staphylococcus epidermidis. FEMS Microbiol Lett 140, 253-259.[CrossRef]
    [Google Scholar]
  22. Heinrich, J. H., Gatlin, L. E., Kunsch, C., Choi, G. H. & Hanson, M. S. ( 1999; ). Identification and characterization of SirA, an iron-regulated protein from Staphylococcus aureus. J Bacteriol 181, 1436-1443.
    [Google Scholar]
  23. Hill, P. J., Cockayne, A., Landers, P., Morrissey, J. A., Sims, C. M. & Williams, P. ( 1998; ). SirR, a novel iron-dependent repressor in Staphylococcus epidermidis. Infect Immun 66, 4123-4129.
    [Google Scholar]
  24. 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 the structural-functional relationships of Fur. J Bacteriol 176, 5108-5115.
    [Google Scholar]
  25. Leblanc, B. & Moss, T. ( 1994; ). DNase I footprinting. In DNA–Protein Interactions: Principles and Protocols, pp. 1-10. Edited by G. G. Kneale. Totowa, NJ: Humana Press.
  26. Lindsay, J. A. & Riley, T. V. ( 1994; ). Staphylococcal iron requirements, siderophore production, and iron-regulated protein expression. Infect Immun 62, 2309-2314.
    [Google Scholar]
  27. Litwin, C. M. & Calderwood, S. B. ( 1993a; ). Role of iron in the regulation of virulence genes. Clin Microbiol Rev 6, 137-149.
    [Google Scholar]
  28. Litwin, C. M. & Calderwood, S. B. ( 1993b; ). Cloning and genetic analysis of the Vibrio vulnificus fur gene and construction of a fur mutant by in vivo marker exchange. J Bacteriol 175, 706-715.
    [Google Scholar]
  29. de Lorenzo, V., Giovannini, F., Herrero, M. & Neilands, J. B. ( 1988; ). Metal ion regulation of gene expression: Fur repressor–operator interaction at the promoter region of the aerobactin system of pColV-K30. J Mol Biol 203, 875-884.[CrossRef]
    [Google Scholar]
  30. Mademidis, A. & Koster, W. ( 1998; ). Transport activity of FhuA, FhuC, FhuD and FhuB derivatives in a system free of polar effects, and stoichiometry of components involved in ferrichrome uptake. Mol Gen Genet 258, 156-165.[CrossRef]
    [Google Scholar]
  31. Meiwes, J., Fiedler, H. P., Haag, H., Zahner, H., Konetschny-Rapp, S. & Jung, G. ( 1990; ). Isolation and characterization of staphyloferrin A, a compound with siderophore activity from Staphylococcus hyicus DSM 20459. FEMS Microbiol Lett 55, 201-205.
    [Google Scholar]
  32. Modun, B., Evans, R. W., Joannou, C. L. & Williams, P. ( 1998; ). Receptor-mediated recognition and uptake of iron from human transferrin by Staphylococcus aureus and Staphylococcus epidermidis. Infect Immun 66, 3591-3596.
    [Google Scholar]
  33. Neilands, J. B. ( 1995; ). Siderophores: structure and function of microbial iron transport compound. J Biol Chem 270, 26723-26726.[CrossRef]
    [Google Scholar]
  34. Novick, R. P. ( 1990; ). The staphylococcus as a genetic system. In Molecular Biology of the Staphylococci, pp. 1-40. Edited by R. P. Novick. New York: VCH.
  35. Pohl, E., Holmes, R. K. & Hol, W. G. J. ( 1999; ). Crystal structure of the iron-dependent regulator (IdeR) from Mycobacterium tuberculosis shows both metal-binding sites fully occupied. J Mol Biol 285, 1145-1156.[CrossRef]
    [Google Scholar]
  36. 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]
  37. Ramadurai, L., Lockwood, K. J., Nadakavukaren, M. J. & Jayaswal, R. K. ( 1999; ). Characterization of a chromosomally encoded glycylglycine endopeptidase of Staphylococcus aureus. Microbiology 145, 801-808.[CrossRef]
    [Google Scholar]
  38. Rose, T. M., Schultz, E. R., Henikoff, J. G., Pietrokovski, S., MacCallum, C. M. & Henikoff, S. ( 1998; ). Consensus-degenerate hybrid oligonucleotide primers for amplification of distantly related sequences. Nucleic Acids Res 26, 1628-1635.[CrossRef]
    [Google Scholar]
  39. Rowland, B. M., Grossman, T. H., Osburne, M. S. & Taber, H. W. ( 1996; ). Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes. J Bacteriol 178, 119-123.
    [Google Scholar]
  40. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  41. Schäffer, S., Hantke, K. & Braun, V. ( 1985; ). Nucleotide sequence of the iron regulatory gene fur. Mol Gen Genet 200, 110-113.[CrossRef]
    [Google Scholar]
  42. Schmitt, M. P., Predich, M., Doukhan, L., Smith, I. & Holmes, R. K. ( 1995; ). Characterization of an iron-dependent regulatory protein (IdeR) of Mycobacterium tuberculosis as a functional homolog of the diphtheria toxin repressor (DtxR) from Corynebacterium diphtheriae. Infect Immun 63, 4284-4289.
    [Google Scholar]
  43. Singh, V. K., Xiong, A., Usgaard, T. R., Chakrabarti, S., Deora, R., Misra, T. K. & Jayaswal, R. K. ( 1999; ). ZntR is an autoregulatory protein and negatively regulates the chromosomal zinc resistance operon znt of Staphylococcus aureus. Mol Microbiol 33, 200-207.[CrossRef]
    [Google Scholar]
  44. Stojiljkovic, I., Bäumler, A. J. & Hantke, K. ( 1994; ). Fur regulon in Gram-negative bacteria: identification and characterization of new iron-regulated Escherichia coli genes by the Fur titration assay. J Mol Biol 236, 531-545.[CrossRef]
    [Google Scholar]
  45. Tao, X., Schiering, N., Zeng, H. Y., Ringe, D. & Murphy, J. R. ( 1994; ). Iron, DtxR, and the regulation of diphtheria toxin expression. Mol Microbiol 14, 191-197.[CrossRef]
    [Google Scholar]
  46. Tardat, B. & Touati, D. ( 1993; ). Iron and oxygen regulation of Escherichia coli MnSOD expression: competition between the global regulators Fur and ArcA for binding to DNA. Mol Microbiol 9, 53-63.[CrossRef]
    [Google Scholar]
  47. Thompson, J. D., Higgins, D. G. & Gibson, T. J. ( 1994; ). clustal w: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22, 4673-4680.[CrossRef]
    [Google Scholar]
  48. Trivier, D. & Courcol, R. J. ( 1996; ). Iron depletion and virulence in Staphylococcus aureus. FEMS Microbiol Lett 141, 117-127.[CrossRef]
    [Google Scholar]
  49. Xiong, A. & Jayaswal, R. K. ( 1998; ). Molecular characterization of a chromosomal determinant conferring resistance to zinc and cobalt ions in Staphylococcus aureus. J Bacteriol 180, 4024-4029.
    [Google Scholar]
  50. 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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-3-659
Loading
/content/journal/micro/10.1099/00221287-146-3-659
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error