Advertisement

Archives of Microbiology

, Volume 152, Issue 1, pp 25–32 | Cite as

Characterization of a proteinaceous extracellular coat synthesized by the “slime” variant of Neurospora crassa

  • J. P. Martinez
  • M. L. Gil
  • M. Casanova
  • M. Rico
  • R. Sentandreu
  • J. Ruiz-Herrera
Original Papers

Abstract

Cells of the “slime” strain of Neurospora crassa synthesize a coherent extracellular material which remains attached to the cell surface, but is released into the liquid medium by shaking. The material was purified and studied by different criteria. By electron microscopy it appears as long wavy sheets which strongly bind concanavalin A, but not wheat germ agglutinin, and maintain their integrity in the absence of structural polysaccharides. Analysis of the purified material revealed that it was free of contaminating membranes; it contained more than 70% protein, 1% neutral sugars (glucose, mannose, fucose and galactose), less than 2% lipids and ca. 4% not-characterized hexosaminelike compounds. Its polypeptide pattern as determined by PAGE was complex. The significance of this material is discussed.

Key words

Neurospora crassa “Slime” variant Cell coat Exocellular proteins Glycoproteins 

Abbreviations used

Au-WGA

colloidal gold-wheat germ agglutinin

Endo H

endo-β-N-acetylglucosaminidase H

Fe-Con A

ferritin labcled concanavalin A

FITC-Con A

fluorescent concanavalin A

PEM

proteinaceous extracellular material

PMSF

phenylmethyl sulfphonyl fluoride

BSDA

bovine serum albumin

CTAB

cetyltrimethyl-ammonium bromide

DOC-Na

sodium deoxychlate

DTT

dl-dithiothreitol

EDTA

ethylene diamine tetracetic acid

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Bartnicki-Garcia S, Bracker CE, Lippman E, Ruiz-Herrera J (1984) Chitosomes from the wall-less slime mutant of Neurospora crassa. Arch Microbiol 139:105–112Google Scholar
  2. Bigger CH, White MR, Braymer HD (1972) Ultrastructure and invertase secretion of the slime mutant of Neurospora crassa. J Gen Microbiol 71:159–166Google Scholar
  3. Bitter T, Muir HM (1962) A modified uronic acid carbazol reaction. Anal Biochem 4:330–334Google Scholar
  4. Bowman EJ (1983) Comparison of the vacuolar membrane ATPase of Neurospora crassa with the mitochondrial and plasma membrane ATPases. J Biol Chem 258:15238–15244Google Scholar
  5. Burnett JH (1979) Aspects of the structure and growth of hyphal walls. In: Burnett JH, Trinci APJ (eds) Fungal walls and hyphal growth. Cambridge University Press, Cambridge London New York Melbourne, pp 1–25Google Scholar
  6. Burnette WN (1981) “Western blotting”: electrophoretic transfer of proteins from sodium dodecyl sulfate-polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem 112:195–203Google Scholar
  7. Casanova M, Martinez JP, Gil ML, Sentandreu R, Ruiz-Herrera J (1987), Different molecular forms of invertase in the slime variant of Neurospora crassa: comparison with the wild-type strain. J Gen Microbiol 133:2447–2456Google Scholar
  8. Casanova M, Martinez ML, Gil ML, Sentandreu R, Ruiz-Herrera J (1989) Self-assembly properties of the proteinaceous coat secreted by the “slime” variant of Neurospora crassa. Arch Microbiol 152:33–38Google Scholar
  9. Dimler RJ, Schaeffer WC, Wise CS, Rist CE (1952) Quantitative paper chromatography of d-glucose and its oligosaccharides. Anal Chem 24:1411–1414Google Scholar
  10. Dow JM, Carreon RR, Villa VD (1981) Role of membranes of mycelial Mucor rouxii in synthesis and secretion of cell wall matrix polymers. J Bacteriol 145:272–279Google Scholar
  11. Emerson S (1963) Slime, a plasmodioid variant of Neurospora crassa. Genetica 34:162–182Google Scholar
  12. Gascon S, Lampen JO (1968) Purification of the internal invertase of yeast. J Biol Chem 243:1567–1572Google Scholar
  13. Geoghegan W, Ackerman GA (1977) Adsorption of horseradish peroxidase, ovomucoid and antiimmunoglobulin to colloidal gold for the indirect detection of concanavalin A, wheat germ agglutinin and goat antihuman immunoglobulin G on cell surfaces at the electron microscopic level: a new method, theory and application. J Histochem Cytochem 25:1187–1200Google Scholar
  14. Hawkes R (1982) Identification of concanavalin A-binding proteins after sodium dodecyl sulphate-gel electrophoresis and protein blotting. Anal Biochem 123:143–146Google Scholar
  15. Laemmli VK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  16. Leal-Morales CA, Ruiz-Herrera J (1985) Alterations in the biosynthesis of chitin and glucan in the slime mutant of Neurospora crassa. Exp Mycol 9:28–38Google Scholar
  17. Livingston LR (1969) Locus-specific changes in cell wall composition characteristic of osmotic mutants of Neurospora crassa. J Bacteriol 99:85–90Google Scholar
  18. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  19. Makarow M (1985) Endocytosis in Saccharomyces cerevisiae: internalization of enveloped viruses into sphaeroplasts. EMBO j 4:1855–1860Google Scholar
  20. Metzenberg RL (1963) The localization of β-fructofuranosidase in Neurospora. Biochim Biophys Acta 77:455–465Google Scholar
  21. Millette CF, Scott BK (1984) Identification of spermatogenic cell plasma membrane glycoproteins by two dimensional electrophoresis and lectin blotting. J Cell Sci 65:233–248Google Scholar
  22. Opheim DJ (1978) Alpha-d-mannosidase of Saccharomyces cerevisiae, characterization and modulation of activity. Biochim Biophys Acta 524:121–130Google Scholar
  23. Rico H, Miragall F, Sentandreu R (1985) Abnormal formation of Candida albicans walls produced by calcofluor white: an ultrastructural and stereologic study. Exp Mycol 9:241–253Google Scholar
  24. rondle CJM, Morgan WTJ (1955) The determination of glucosamine and galactosamine. Biochem J 61:586Google Scholar
  25. Ruiz-Herrera J, Martinez JP, Casanova M, Gil ML, Sentandreu R (1987) Separation of chitosomes and secretory vesicles from the “slime” variant of Neurospora crassa. Arch Microbiol 149:156–162Google Scholar
  26. Ruperez P, Gomez-Miranda B, Leal JA (1983) Extracellular β-malonoglucan from Penicillium erythromellis. Trans Br Mycol Soc 80:313–318Google Scholar
  27. Ruperez P, Moya A, Leal JA (1986) Cell wall polysaccharides from Talaromyces species. Arch Microbiol 146:250–255Google Scholar
  28. Scarborough GA (1975) Isolation and characterization of Neurospora crassa plasma membranes. J Biol Chem 250:1106–1111Google Scholar
  29. Scarborough GA (1978) The Neurospora plasma membrane: a new experimental system for investigating eukaryote surface, membrane structure and function. Meth Cell Biol 20:117–133Google Scholar
  30. Selitrennikoff CP (1979) Chitin, synthetase activity from the slime variant of Neurospora crassa. Biochim Biophys Acta 571:224–232Google Scholar
  31. Spurr AR (1969) A low-viscosity epoxy resin embedding medium for electron microscopy. J Ultrastruct Res 26:31–43Google Scholar
  32. Trevelyan WE, Procter DP, Harrison JS (1950) Detection of sugars on paper chromatograms. Nature 166:444–445Google Scholar
  33. Trevithick JR, Galsworthy PR (1977) Morphology of slime variants of Neurospora crassa growing on a glass surface in liquid medium. Arch Microbiol 115:109–118Google Scholar
  34. Van Winkle WB (1969) Preliminary observations of the ultrastructure of the slime mutant. Neurospora Newslett 14:5Google Scholar
  35. Vogel HJ (1956) A convenient growth medium for Neurospora (medium N). Microbiol Gen Bull 13:42–43Google Scholar
  36. Woodward VW, Woodward CK (1968) The care and feeding of slime. Neurospora Newslett 13:18Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • J. P. Martinez
    • 1
  • M. L. Gil
    • 1
  • M. Casanova
    • 1
  • M. Rico
    • 1
  • R. Sentandreu
    • 1
  • J. Ruiz-Herrera
    • 1
  1. 1.Department de Microbiologia, Facultat de FarmaciaUniversitat de ValenciaValenciaSpain

Personalised recommendations