1887

Abstract

Evidence of a direct association between ferri-exochelin, the major extracellular siderophore of , and a 29 kDa protein has been obtained by three separate methods. (1) Direct binding of Fe(III)-exochelin by the 29 kDa protein in an envelope preparation from iron-deficient cells was demonstrated by the extraction of a complex with the non-denaturing detergent CHAPS, and subsequent CHAPS-PAGE and autoradiography. (2) Affinity chromatography on a chemically synthesized ferri-exochelin-Sepharose 4B matrix led to the retention of the 29 kDa protein and a 25 kDa protein. The smaller protein was partially eluted with 1mM ferri-exochelin although it did not form a stable complex with ferri-exochelin. The 29 kDa protein could not be eluted from the affinity matrix with 1mM ferri-exochelin either alone or with 1 M NaCl. Only 2% (w/v) SDS could do this, but resulted in protein denaturation. (3) Incubation of Fe-exochelin with CHAPS-solubilized envelope proteins in free solution followed by ion-exchange chromatography resolved three radioactive peaks; subsequent analysis by SDS-PAGE showed that the peak with the highest Fe-binding activity per unit protein contained both the 29 and 25 kDa proteins. A direct association was demonstrated between the 29 kDa protein and Fe-exochelin by gel filtration. The evidence suggests that the 29 kDa iron-regulated envelope protein of is a ferri-exochelin-binding protein and that the 25 kDa protein, which corresponds in size to a previously reported iron-regulated envelope protein in this bacterium, may have a role in the formation or maintenance of this complex. Proteins extracted from the cell envelope of iron-deficient with CHAPS were dialysed to remove the detergent, incorporated into liposome suspensions and then incubated with Fe(III)-exochelin. This increased the retention of Fe by 133-fold compared to proteins not placed in liposomes. Retention of Fe was dependent upon the protein loading of the liposomes. Gel filtration confirmed that the iron was retained by these vesicles and even after dialysis the majority of Fe was still retained by the vesicles. Re-solubilization of the labelled proteo-liposomes in various detergents gave limited recovery of a ferri-exochelin-protein complex. Attempts to resolve this complex by Triton X-100 PAGE led to separation of the two entities. The complex was stable, however, in a CHAPS-PAGE system.

Loading

Article metrics loading...

/content/journal/micro/10.1099/13500872-142-6-1521
1996-06-01
2021-10-17
Loading full text...

Full text loading...

/deliver/fulltext/micro/142/6/mic-142-6-1521.html?itemId=/content/journal/micro/10.1099/13500872-142-6-1521&mimeType=html&fmt=ahah

References

  1. Cavinato A.G., Macleod R.M., Ahmed M.S. A nondenaturing gel electrophoresis system for the purification of membrane proteins. Prep Biocbem 1988; 18:205–215
    [Google Scholar]
  2. Cherayil B.J., Young R.A. A 28-kDa protein from Mycobacterium leprae is a target of the human antibody response in lepromatous leprosy. J Immunol 1988; 141:4370–4375
    [Google Scholar]
  3. Dale J.W., Patki A. Mycobacterial gene expression and regulation. In Molecular Biology of the Mycobacteria 1990 Edited by McFadden J. London: Academic Press; pp 173–198
    [Google Scholar]
  4. Dover L.G. Studies in the iron uptake mechanism of Mycobacterium smegmatis: identification of the iron uptake system 1995 PhD thesis, University of Hull;
    [Google Scholar]
  5. Fiss E.H., Yu S., Jacobs W.R. Identification of genes involved in the sequestration of iron in mycobacteria: the ferric exochelin biosynthetic and uptake pathways. Mol Microbiol 1994; 14:557–569
    [Google Scholar]
  6. Guerinot M.L. Microbial iron transport. Annu Rev Microbiol 1994; 48:743–772
    [Google Scholar]
  7. Hall R.M., Sritharan M., Messenger A.J.M., Ratledge C. Iron transport in Mycobacterium smegmatis: occurrence of iron-regulated envelope proteins as potential receptors for iron uptake. J Gen Microbiol 1987; 133:2107–2114
    [Google Scholar]
  8. Heinrichs D.E., Young L., Poole K. Pyochelin-mediated iron transport in Pseudomonas aeruginosa: involvement of a high-molecular-mass outer membrane protein. Infect Immun 1991; 59:3680–3684
    [Google Scholar]
  9. Henderson D.P., Payne S.M. Vibrio cholerae iron transport systems: roles of heme and siderophore iron transport in virulence identification of a gene associated with multiple iron transport systems. Infect Immun 1994; 62:5120–5125
    [Google Scholar]
  10. Lane S.J., Marshall P.S., Upton R.J., Ratledge C., Ewing M. Novel extracellular mycobactins, the carboxymycobactins from Mycobacterium avium. Tetrahedron Eett 1995; 36:4129–4132
    [Google Scholar]
  11. Messenger A.J.M., Ratledge C. Iron transport in Mycobacterium smegmatis: uptake of iron from ferric citrate. I Bacteriol 1982; 149:131–135
    [Google Scholar]
  12. Meyer J.M., Hohnadel D., Kahn A., Cornelis P. Pyoverdine-facilitated iron uptake in Pseudomonas aeruginosa: immunological characterization of the ferripyoverdine receptor. Mol Microbiol 1990; 4:1401–1405
    [Google Scholar]
  13. Neilands J.B., Kanopka K., Schwyn B., Coy M., Francis R.T., Paw B.H., Bagg A. Comparative biochemistry of microbial iron assimilation. In Iron Transport in Microbes 1987 Edited by Winkelmann G., Van Der Helm D., Neilands J.B. Weinheim: VCH Publishers; Plants and Animals, pp 3–33
    [Google Scholar]
  14. Ratledge C., Hall M.J. Influence of metal ions on the formation of mycobactin and salicylic acid in Mycobacterium smegmatis grown in static culture. J Bacteriol 1971; 108:314–319
    [Google Scholar]
  15. Ratledge C., Patel P.V., Mundy J. Iron transport in Mycobacterium smegmatis: the location of mycobactin by electron microscopy. J Gen Microbiol 1982; 128:1559–1565
    [Google Scholar]
  16. Roberts M., Wooldridge K.G., Gavine H., Kuswandi S.I., Williams P.H. Inhibition of biological activities of the aerobactin receptor protein in rough strains of Escherichia coli by polyclonal antiserum raised against native protein. J Gen Microbiol 1989; 135:2387–2398
    [Google Scholar]
  17. Schneider R., Hantke K. Iron-hydroxamate uptake systems in bacillus subtilis: identification of a lipoprotein as a part of a binding protein-dependent transport system. Mol Microbiol 1993; 8:111–121
    [Google Scholar]
  18. Sharman G.J., Williams D.H., Ewing D.F., Ratledge C. Isolation, purification and structure of exochelin MS, the extracellular siderophore from Mycobacterium smegmatis. Biochem J 1995; 305:187–196
    [Google Scholar]
  19. Sritharan M., Ratledge C. Iron regulated envelope proteins of mycobacteria grown in vitro and their occurrence in Mycobacterium avium and Mycobacterium leprae grown in vivo. BioMetals 1990; 2:203–208
    [Google Scholar]
  20. Stephenson M.C., Ratledge C. Iron transport in Mycobacterium smegmatis: uptake of iron from ferriexochelin-exochelin. J Gen Microbiol 1979; 110:193–202
    [Google Scholar]
  21. Stephenson M.C., Ratledge C. Specificity of exochelins for iron transport in three species of mycobacteria. J Gen Microbiol 1980; 116:521–523
    [Google Scholar]
  22. Wheeler P.R., Ratledge C. Metabolism of Mycobacterium tuberculosis. In Tuberculosis: Pathogenesis Protection and Control 1994 Edited by Bloom B.R. Washington, DC: American Society for Microbiology; pp 353–385
    [Google Scholar]
  23. Wooldridge K.G., Morrissey J.A., Williams P.H. Transport of ferric-aerobactin into the periplasm and cytoplasm of Escherichia coli K12: role of envelope-associated proteins and effect of endogenous siderophores. J Gen Microbiol 1992; 138:597–603
    [Google Scholar]
  24. Zhou X.H., Van Der Helm D. A novel purification of ferric citrate receptor (Fee A) from Escherichia coli UT 5600 and further characterization of its binding activity. BioMetals 1993; 6:37–44
    [Google Scholar]
  25. Zhou X.H., Van Der Helm D., Adjimani J. Purification of outer membrane iron transport receptors from Escherichia coli by fast protein liquid chromatography: Fep A and Fee A. BioMetals 1993; 6:25–35
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/13500872-142-6-1521
Loading
/content/journal/micro/10.1099/13500872-142-6-1521
Loading

Data & Media loading...

Most cited this month Most Cited RSS feed

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