1887

Abstract

The iron-storage protein bacterioferritin (Bfr) from strain F62 was identified in cell-free extracts and subsequently purified by column chromatography. Gonococcal Bfr had an estimated molecular mass of 400 kDa by gel filtration; however, analysis by SDS-PAGE revealed that it was composed of 18 kDa (BfrA) and 22 kDa (BfrB) subunits. DNA encoding BfrB was amplified by PCR using degenerate primers derived from the N-terminal amino acid sequence of BfrB and from a C-terminal amino acid sequence of Bfr. The DNA sequence of was subsequently obtained by genome walking using single-specific-primer PCR. The two Bfr genes were located in tandem with an intervening gap of 27 bp. A potential Fur-binding sequence (12 of 19 bp identical to the consensus neisserial sequence) was located within the 5’ flanking region of in front of a putative −35 hexamer. The homology between the DNA sequences of and was 557%; the deduced amino acid sequences of BfrA (154 residues) and BfrB (157 residues) showed 397% identity, and showed 413% and 561% identity, respectively, to Bfr. Expression of recombinant BfrA and BfrB in strain DH5α was detected on Western blots probed with polyclonal anti- Bfr antiserum. Most Bfrs are homopolymers with identical subunits; however, the evidence presented here suggests that gonococcal Bfr was composed of two similar but not identical subunits, both of which appear to be required for the formation of a functional Bfr. A Bfr-deficient mutant was constructed by inserting the Ω fragment into the BfrB gene. The growth of the BfrB-deficient mutant in complex medium was reduced under iron-limited conditions. The BfrB-deficient mutant was also more sensitive to killing by HO and paraquat than the isogenic parent strain. These results demonstrate that gonococcal Bfr plays an important role in iron storage and protection from iron-mediated oxidative stress.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-145-10-2967
1999-10-01
2019-10-22
Loading full text...

Full text loading...

/deliver/fulltext/micro/145/10/1452967a.html?itemId=/content/journal/micro/10.1099/00221287-145-10-2967&mimeType=html&fmt=ahah

References

  1. Andrews, S. C., Harrison, P. M. & Guest, J. R. ( 1989; ). Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12. J Bacteriol 171, 3940-3947.
    [Google Scholar]
  2. Andrews, S. C., Smith, J. M. A., Yewdall, S. J., Guest, J. R. & Harrison, P. M. ( 1991; ). Bacterioferritins and ferritins are distantly related in evolution: conservation of ferroxidase-center residues. FEBS Lett 293, 164-168.[CrossRef]
    [Google Scholar]
  3. Andrews, S. C., Arosio, P., Bottke, W. & 7 other authors ( 1992; ). Structure, function, and evolution of ferritins. J Inorg Biochem 47, 161–174.[CrossRef]
    [Google Scholar]
  4. Andrews, S. C., Smith, J. M. A., Hawkins, C., Williams, J. M., Harrison, P. M. & Guest, J. R. ( 1993; ). Overproduction, purification and characterization of the bacterioferritin of Escherichia coli and a C-terminal extended variant. Eur J Biochem 213, 329-338.[CrossRef]
    [Google Scholar]
  5. Braun, V. ( 1997; ). Avoidance of iron toxicity through regulation of bacterial iron transport. Biol Chem 378, 779-786.
    [Google Scholar]
  6. Briat, J.-F. ( 1992; ). Iron assimilation and storage in prokaryotes. J Gen Microbiol 138, 2475-2483.[CrossRef]
    [Google Scholar]
  7. Brooks, B. W., Martin Young, N., Watson, D. C., Robertson, R. H., Sugden, E. A., Nielsen, K. H. & Becker, S. A. W. E. ( 1991; ). Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin. J Clin Microbiol 29, 1652-1658.
    [Google Scholar]
  8. Brun, N. E., Andrews, S. C., Guest, J. R., Harrison, P. M., Moore, G. R. & Thomson, A. J. ( 1995; ). Identification of the ferroxidase center of Escherichia coli bacterioferritin. Biochem J 312, 385-392.
    [Google Scholar]
  9. Cheesman, M. R., Brun, N. E., Kadir, F. H. & 7 other authors ( 1993; ). Haem and non-haem iron sites in Escherichia coli bacterioferritin: spectroscopic and model building studies. Biochem J 292, 47–56.
    [Google Scholar]
  10. Chung, M. C. M. ( 1985; ). A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin. Anal Biochem 148, 498-502.[CrossRef]
    [Google Scholar]
  11. Cristina, M., Pessolani, V., Smith, D. R., Rivoire, B., McCormick, J., Hefta, S. A., Cole, S. T. & Brennan, P. J. ( 1994; ). Purification, characterization, gene sequence, and significance of a bacterioferritin from Mycobacterium leprae. J Exp Med 180, 319-327.[CrossRef]
    [Google Scholar]
  12. Denoel, P. A., Zygmunt, M. S., Weynaants, V., Tibor, A., Lichtfouse, B., Briffeuil, P., Limet, J. N. & Letesson, J.-J. ( 1995; ). Cloning and sequencing of the bacterioferritin gene of Brucella melitensis 16M strain. FEBS Lett 361, 238-242.[CrossRef]
    [Google Scholar]
  13. Doig, P., Austin, J. W. & Trust, T. J. ( 1993; ). The Helicobacter pylori 19·6-kilodalton protein is an iron-containing protein resembling ferritin. J Bacteriol 175, 557-560.
    [Google Scholar]
  14. Dunford, H. B. ( 1987; ). Free radicals in iron-containing systems. Free Radic Biol Med 3, 405-421.[CrossRef]
    [Google Scholar]
  15. Evans, D. J.Jr, Evans, D. G., Lampert, H. C. & Nakano, H. ( 1995; ). Identification of four new prokaryotic bacterioferritins, from Helicobacter pylori, Anabaena variabilis, Bacillus subtilis and Treponema pallidum, by analysis of gene sequences. Gene 153, 123-127.[CrossRef]
    [Google Scholar]
  16. Flitter, R. W., Rowley, D. A. & Halliwell, B. ( 1983; ). Iron oxides in acid drainage environments and their association with bacteria. Chem Geol 74, 321-330.
    [Google Scholar]
  17. Ford, G. C., Harrison, P. M., Rice, D. W., Smith, J. M. A., Treffry, A., White, J. L. & Yariv, J. ( 1984; ). Ferritin: design and formation of an iron-storage molecule. Philos Trans R Soc Lond Ser B Biol Sci 304, 551-565.[CrossRef]
    [Google Scholar]
  18. Frolow, F., Kalb, A. J. & Yariv, J. ( 1994; ). Structure of a unique twofold symmetric heme-binding site. Struct Biol 1, 453-460.[CrossRef]
    [Google Scholar]
  19. Genco, C. A. & Desai, P. J. ( 1996; ). Iron acquisition in the pathogenic Neisseria. Trends Microbiol 4, 179-184.[CrossRef]
    [Google Scholar]
  20. Grossman, M. J., Hinton, S. M., Minak-Bernero, V., Slaughter, C. & Stiefel, E. I. ( 1992; ). Unification of the ferritin family of proteins. Proc Natl Acad Sci USA 89, 2419-2423.[CrossRef]
    [Google Scholar]
  21. Haber, F. & Weiss, J. ( 1934; ). The catalytic decomposition of hydrogen peroxide by iron salts. Proc R Soc Ser A 147, 332-333.[CrossRef]
    [Google Scholar]
  22. Halliwell, B. & Gutteridge, J. M. G. ( 1984; ). Oxygen toxicity, oxygen radicals, transition metals and disease. Biochem J 219, 1-12.
    [Google Scholar]
  23. Harker, A. R. & Wullstein, L. H. ( 1985; ). Evidence for two nonidentical subunits of bacterioferritin from Azotobacter vinelandii. J Bacteriol 162, 651-655.
    [Google Scholar]
  24. Harrison, P. M. & Arosio, P. ( 1996; ). The ferritins: molecular properties, iron storage function and cellular regulation. Biochim Biophys Acta 1275, 161-203.[CrossRef]
    [Google Scholar]
  25. Hudson, A. J., Andrews, S. C., Hawkins, C., Williams, J. M., Izuhara, M., Meldrum, F. C., Mann, S., Harrison, P. M. & Guest, J. R. ( 1993; ). Overproduction, purification and characterization of the Escherichia coli ferritin. Eur J Biochem 218, 985-995.[CrossRef]
    [Google Scholar]
  26. Inglis, N. F., Stevenson, K., Hosie, A. H. F. & Sharp, J. M. ( 1994; ). Complete sequence of the gene encoding the bacterioferritin subunit of Mycobacterium avium subspecies silvaticum. Gene 150, 205-206.[CrossRef]
    [Google Scholar]
  27. Izuhara, M., Takamune, K. & Takata, M. ( 1991; ). Cloning and sequencing of an Escherichia coli K12 gene which encodes a polypeptide having similarity to the human ferritin H subunit. Mol Gen Genet 225, 510-513.
    [Google Scholar]
  28. Kadir, F. H. A. & Moore, G. R. ( 1990; ). Bacterial ferritin contains 24 haem groups. FEBS Lett 271, 141-143.[CrossRef]
    [Google Scholar]
  29. Klausner, R. D., Rouault, T. A. & Harford, J. B. ( 1993; ). Regulating the fate of mRNA: the control of cellular iron metabolism. Cell 72, 19-28.[CrossRef]
    [Google Scholar]
  30. Kurokwa, T., Fukumori, Y. & Yamanaka, T. ( 1989; ). Nitrobacter winogradskyi cytochrome b-559: a nonhaem iron-containing cytochrome related to bacterioferritin. Biochim Biophys Acta 976, 135-139.[CrossRef]
    [Google Scholar]
  31. Laulhère, J.-P., Labourè, A.-M., Van Wuytswinkel, O., Gagnon, J. & Briat, J. -F. ( 1993; ). Purification, characterization and function of bacterioferritin from the cyanobacterium Synechocystis P. C. C. 6803. Biochem J 281, 785-793.
    [Google Scholar]
  32. Mietzner, T. G., Luginbuhl, G. H., Sandstrom, E. C. & Morse, S. A. ( 1984; ). Identification of an iron-regulated 37,000-dalton protein in the cell envelope of Neisseria gonorrhoeae. Infect Immun 45, 410-416.
    [Google Scholar]
  33. Moore, G. R., Mann, S. & Bannister, J. V. ( 1986; ). Isolation and properties of the complex nonheme-iron-containing cytochrome b 557 (bacterioferritin) from Pseudomonas aeruginosa. J Inorg Biochem 28, 329-336.[CrossRef]
    [Google Scholar]
  34. Moore, G. R., Kadir, F. H. A., Al-Massad, F. K., Brun, N. E., Thomson, A. J., Greenwood, C., Keen, J. F. & Findlay, J. B. C. ( 1994; ). Structural heterogeneity of Pseudomonas aeruginosa bacterioferritin. Biochem J 304, 493-497.
    [Google Scholar]
  35. Penfold, C. N., Ringeling, P. L., Davy, S. L., Moore, G. R., McEwan, A. G. & Spiro, S. ( 1996; ). Isolation, characterization and expression of the bacterioferritin gene of Rhodobacter capsulatus. FEMS Microbiol Lett 139, 143-148.[CrossRef]
    [Google Scholar]
  36. Rocha, E. R., Andrews, S. C., Keen, J. N. & Brock, L. H. ( 1992; ). Isolation of a ferritin from Bacteroides fragilis. FEMS Microbiol Lett 95, 207-212.[CrossRef]
    [Google Scholar]
  37. Sanger, F., Nicklen, S. & Coulson, A. R. ( 1977; ). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74, 5463-5467.[CrossRef]
    [Google Scholar]
  38. Shyamala, V. & Ames, G. ( 1989; ). Genome walking by single-specific-primer polymerase chain reaction: SSP-PCR. Gene 84, 1-8.[CrossRef]
    [Google Scholar]
  39. Smith, J. M. A., Quirk, A. V., Plank, R. W. H., Diffin, F. M., Ford, G. C. & Harrison, P. M. ( 1988; ). The identity of Escherichia coli bacterioferritin and cytochrome b 1. Biochem J 255, 737-740.
    [Google Scholar]
  40. Stiefel, E. I. & Watt, G. D. ( 1979; ). Azotobacter cytochrome b 557·5 is a bacterioferritin. Nature 279, 81-83.[CrossRef]
    [Google Scholar]
  41. Theil, E. C. ( 1987; ). Ferritin: structure, gene regulation, and cellular function in animals, plants, and microorganisms. Annu Rev Biochem 56, 289-315.[CrossRef]
    [Google Scholar]
  42. Touati, D., Jacques, M., Tardat, B., Buchard, L. & Despied, S. ( 1995; ). Lethal oxidative damage and mutagenesis are generated by iron in fur mutants of Escherichia coli: protective role of superoxide dismutase. J Bacteriol 177, 2305-2314.
    [Google Scholar]
  43. Wai, S. N., Takata, T., Takada, A., Hamasaki, N. & Amako, K. ( 1995; ). Purification and characterization of ferritin from Campylobacter jejuni. Arch Microbiol 164, 1-6.[CrossRef]
    [Google Scholar]
  44. Wai, S. N., Nakayama, K., Umene, K., Moriya, T. & Amako, K. ( 1996; ). Construction of a ferritin-deficient mutant of Campylobacter jejuni: contribution of ferritin to iron storage and protection against oxidative stress. Mol Microbiol 20, 1127-1134.[CrossRef]
    [Google Scholar]
  45. Wooldridge, K. G. & Williams, P. H. ( 1993; ). Iron uptake mechanisms of pathogenic bacteria. FEMS Microbiol Rev 12, 325-348.[CrossRef]
    [Google Scholar]
  46. Yariv, J., Kalb, A. J., Serling, R., Bauminger, E. R., Cohen, S. G. & Ofer, S. ( 1981; ). The composition and the structure of bacterioferritin of Escherichia coli. Biochem J 197, 171-175.
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-145-10-2967
Loading
/content/journal/micro/10.1099/00221287-145-10-2967
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