1887

Abstract

needs to synthesize haem in order to replicate intracellularly and to produce virulence in mice. Thus, to gain insight into the pathogenesis of the bacterium, regulatory proteins of the haem biosynthetic pathway were sought. An iron response regulator (Irr) from , which is a close relative of , was previously described as being involved in the coordination of haem biosynthesis and iron availability. The genome was searched for an orthologue gene, and the gene was cloned, sequenced and disrupted. A null mutant was constructed that accumulated protoporphyrin IX under conditions of iron deprivation. This phenotype was overcome by a complementing plasmid carrying the wild-type . Purified recombinant Irr behaved as a stable dimer and bound haem. Interestingly, , Irr was only detected in cells obtained from iron-limited cultures and the protein downregulated its own transcription. Through fusion, it was demonstrated that iron did not regulate transcription. The data reported show that Irr is a homodimeric protein that is accumulated in iron-limited cells, controls its own transcription and downregulates the biosynthesis of haem precursors.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.28213-0
2005-10-01
2019-11-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/151/10/3427.html?itemId=/content/journal/micro/10.1099/mic.0.28213-0&mimeType=html&fmt=ahah

References

  1. Almiron, M., Martinez, M., Sanjuan, N. & Ugalde, R. A. ( 2001; ). Ferrochelatase is present in Brucella abortus and is critical for its intracellular survival and virulence. Infect Immun 69, 6225–6230.[CrossRef]
    [Google Scholar]
  2. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. ( 1990; ). Basic local alignment search tool. J Mol Biol 215, 403–410.[CrossRef]
    [Google Scholar]
  3. Becker, A., Schmidt, M., Jager, W. & Puhler, A. ( 1995; ). New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. Gene 162, 37–39.[CrossRef]
    [Google Scholar]
  4. Bellaire, B. H., Elzer, P. H., Hagius, S., Walker, J., Baldwin, C. L. & Roop, R. M., 2nd ( 2003; ). Genetic organization and iron-responsive regulation of the Brucella abortus 2,3-dihydroxybenzoic acid biosynthesis operon, a cluster of genes required for wild-type virulence in pregnant cattle. Infect Immun 71, 1794–1803.[CrossRef]
    [Google Scholar]
  5. Cox, R. & Charles, H. P. ( 1973; ). Porphyrin-accumulating mutants of Escherichia coli. J Bacteriol 113, 122–132.
    [Google Scholar]
  6. 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]
  7. Frustaci, J. M. & O'Brian, M. R. ( 1992; ). Characterization of a Bradyrhizobium japonicum ferrochelatase mutant and isolation of the hemH gene. J Bacteriol 174, 4223–4229.
    [Google Scholar]
  8. Guo, B., Phillips, J. D., Yu, Y. & Leibold, E. A. ( 1995; ). Iron regulates the intracellular degradation of iron-regulatory protein 2 by the proteasome. J Biol Chem 270, 21645–21651.[CrossRef]
    [Google Scholar]
  9. Hamza, I., Chauhan, S., Hassett, R. & O'Brian, M. R. ( 1998; ). The bacterial Irr protein is required for coordination of heme biosynthesis with iron availability. J Biol Chem 273, 21669–21674.[CrossRef]
    [Google Scholar]
  10. 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]
  11. Kovach, M. E., Elzer, P. H., Hill, D. S., Robertson, G. T., Farris, M. A., Roop, R. M., 2nd & Peterson, K. M. ( 1995; ). Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166, 175–176.[CrossRef]
    [Google Scholar]
  12. Lestrate, P., Delrue, R. M., Danese, I. & 7 other authors ( 2000; ). Identification and characterization of in vivo attenuated mutants of Brucella melitensis. Mol Microbiol 38, 543–551.[CrossRef]
    [Google Scholar]
  13. Lopez-Goñi, I., Moriyón, I. & Neilands, J. B. ( 1992; ). Identification of 2,3-dihydroxybenzoic acid as a Brucella abortus siderophore. Infect Immun 60, 4496–4503.
    [Google Scholar]
  14. Miller, J. H. ( 1992; ). A Short Course in Bacterial Genetics. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  15. O'Brian, M. R. & Thony-Meyer, L. ( 2002; ). Biochemistry, regulation and genomics of haem biosynthesis in prokaryotes. Adv Microb Physiol 46, 257–318.
    [Google Scholar]
  16. Ogawa, K., Sun, J., Taketani, S. & 8 other authors ( 2001; ). Heme mediates derepression of Maf recognition element through direct binding to transcription repressor Bach1. EMBO J 20, 2835–2843.[CrossRef]
    [Google Scholar]
  17. Oka, A., Sugisaki, H. & Takanami, M. ( 1981; ). Nucleotide sequence of the kanamycin resistance transposon Tn903. J Mol Biol 147, 217–226.[CrossRef]
    [Google Scholar]
  18. Paulsen, I. T., Seshadri, R., Nelson, K. E. & 28 other authors ( 2002; ). The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc Natl Acad Sci U S A 99, 13148–13153.[CrossRef]
    [Google Scholar]
  19. Pfeifer, K., Kim, K. S., Kogan, S. & Guarente, L. ( 1989; ). Functional dissection and sequence of yeast HAP1 activator. Cell 56, 291–301.[CrossRef]
    [Google Scholar]
  20. Pohl, E., Haller, J. C., Mijovilovich, A., Meyer-Klaucke, 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, 903–915.[CrossRef]
    [Google Scholar]
  21. Qi, Z. & O'Brian, M. R. ( 2002; ). Interaction between the bacterial iron response regulator and ferrochelatase mediates genetic control of heme biosynthesis. Mol Cell 9, 155–162.[CrossRef]
    [Google Scholar]
  22. Qi, Z., Hamza, I. & O'Brian, M. R. ( 1999; ). Heme is an effector molecule for iron-dependent degradation of the bacterial iron response regulator (Irr) protein. Proc Natl Acad Sci U S A 96, 13056–13061.[CrossRef]
    [Google Scholar]
  23. Sambrook, J., Fritsch, E. F. & Maniatis, T. ( 1989; ). Molecular Cloning: a Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  24. Smith, L. D. & Ficht, T. A. ( 1990; ). Pathogenesis of Brucella. Crit Rev Microbiol 17, 209–230.[CrossRef]
    [Google Scholar]
  25. 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]
  26. Ugalde, R. A. ( 1999; ). Intracellular lifestyle of Brucella spp. Common genes with other animal pathogens, plant pathogens, and endosymbionts. Microbes Infect 1, 1211–1219.[CrossRef]
    [Google Scholar]
  27. Wexler, M., Todd, J. D., Kolade, O., Bellini, D., Hemmings, A. M., Sawers, G. & Johnston, A. W. ( 2003; ). Fur is not the global regulator of iron uptake genes in Rhizobium leguminosarum. Microbiology 149, 1357–1365.[CrossRef]
    [Google Scholar]
  28. Woodcock, D. M., Crowther, P. J., Doherty, J., Jefferson, S., DeCruz, E., Noyer-Weidner, M., Smith, S. S., Michael, M. Z. & Graham, M. W. ( 1989; ). Quantitative evaluation of Escherichia coli host strains for tolerance to cytosine methylation in plasmid and phage recombinants. Nucleic Acids Res 17, 3469–3478.[CrossRef]
    [Google Scholar]
  29. Yang, J., Ishimori, K. & O'Brian, M. R. ( 2005; ). Two heme binding sites are involved in the regulated degradation of the bacterial iron response regulator (Irr) protein. J Biol Chem 280, 7671–7676.[CrossRef]
    [Google Scholar]
  30. Zhang, L. & Guarente, L. ( 1995; ). Heme binds to a short sequence that serves a regulatory function in diverse proteins. EMBO J 14, 313–320.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.28213-0
Loading
/content/journal/micro/10.1099/mic.0.28213-0
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