Skip to main content
SpringerLink
Log in

Protoplast formation ofNeurospora crassa by an inducible enzyme system ofArthrobacter GJM-1

  • Published:
Current Microbiology Aims and scope Submit manuscript

Abstract

A novel method for preparation ofNeurospora crassa protoplasts has been developed based on treatment of hyphae with an inducible enzyme system ofArthrobacter GJM-1 that had been obtained by growing the bacterium onNeurospora cells as the sole carbon source. The lytic system was characterized for enzyme activities relevant to hydrolysis of major cell-wall components ofN. crassa, such as 1,3-β-d-glucanase, α-mannanase, chitinase, proteinase, α-amylase and β-d-glucosidase. The optimal conditions for protoplast formation from young hyphae ofN. crassa (10 mg dry weight) were 180 min treatment by 40 mg/ml of freeze-dried lytic system. Protoplasts which were released from both hyphal tips and sides were found not to contain cell-wall material, as judged by electron microscopy and Calcofluor binding. When freed of the lytic system, 50% of protoplasts regenerated a mycelium.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Literature Cited

  1. Biely P, Vrsanska M, Kratky Z (1980) Xylan-degrading enzymes of the yeastCryptococcus albidus: identification and cellular localization. Eur J Biochem 180:313–321

    Google Scholar 

  2. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein using the principle of protein-dye binding. Anal Biochem 72:248–254

    PubMed  Google Scholar 

  3. Brooks KM, Addison R, Scarborough G (1983) Isolation and characterization of plasma membranes from strains ofNeurospora crassa with wild-type morphology. J Biol Chem 258:13909–13919

    PubMed  Google Scholar 

  4. Brown JA, Ogawa K, Wood TM (1986) Studies on the preparation and regeneration of protoplasts from the cellulolytic fungusPenicillium pinophilum. Enzyme Microb Technol 9:527–532

    Google Scholar 

  5. Case ME, Schweizer M, Kushner SR, Gilles NH (1979) Efficient transformation ofNeurospora crassa utilizing hybrid plasmid DNA. Proc Natl Acad Sci USA 76:5259–5263

    PubMed  Google Scholar 

  6. Dubois MK, Gilles A, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugars and related substances. Anal Chem 28:350–356

    Google Scholar 

  7. Farkas V (1979) Biosynthesis of cell walls of fungi. Microbiol Rev 43:117–144

    PubMed  Google Scholar 

  8. Greco WR, Priore RL, Sharma M, Korytnyk W (1982) Ros-Fit: an enzyme kinetics nonlinear regression curve fitting package for a microcomputer. Comp Biomed Res 15:39–45

    Google Scholar 

  9. Jones GH, Ballou CE (1969) Studies on the structure of yeast mannan. 1. Purification and some properties of an α-mannosidase from anArthrobacter species. J Biol Chem 244:1043–1051

    PubMed  Google Scholar 

  10. Kevei F, Peberdy JF (1977) Interspecific hybridization betweenAspergillus nidulans andAspergillus rugulosus by fusion of somatic protoplasts. J Gen Microbiol 102:255–262

    Google Scholar 

  11. Maraz A, Ferenczy L (1980) Selective transfer of fungal cytoplasmic genetic elements by protoplast fusion. Curr Microbiol 4:343–345

    Google Scholar 

  12. Paleg LG (1959) Citric acid interference in the elimination of reducing sugars with alkaline copper reagents. Anal Chem 31:1902–1904

    Google Scholar 

  13. Pfenninger KH, Ellis L, Johnson MP, Freidman LB, Somlo S (1983) Nerve growth cones isolated from fetal rat brain. 1. Subcellular fractionation and characterization. Cell 35:573–584

    PubMed  Google Scholar 

  14. Quigley DR, Selitrennikoff CP (1984) β(1→3) Glucan synthase activity ofNeurospora crassa: kinetic analysis of negative effectors. Exp Mycol 8:320–333

    Google Scholar 

  15. Quigley DR, Jabri E, Selitrennikoff CP (1985) Protoplasts ofNeurospora crassa: α-amylase improves yield from wild-type hyphae. Exp Mycol 9:254–258

    Google Scholar 

  16. Quigley DR, Taft CS, Stark T, Selitrennikoff CP (1987) Optimal conditions for the release of protoplasts ofNeurospora using Novozym 234. Exp Mycol (in press)

  17. Rinderknecht H, Grokas MC, Silverman P, Haverback BJ (1968) A new ultrasensitive method for the determination of proteolytic activity. Clin Chim Acta 21:197–203

    PubMed  Google Scholar 

  18. Selitrennikoff CP, Bloomfield EC (1984) Formation and regeneration of protoplasts of wild-typeNeurospora crassa. Curr Microbiol 11:113–118

    Google Scholar 

  19. Shematek EM, Braatz JA, Cabib E (1980) Biosynthesis of the yeast cell wall. 1. Preparation and properties of β-(1→3) glucan synthase. J Biol Chem 255:888–894

    PubMed  Google Scholar 

  20. Vogel WJ (1956) A convenient growth medium forNeurospora. Microb Genet Bull 13:42–43

    Google Scholar 

  21. Vrsanska M, Kratky Z, Biely P (1977) Lysis of intact yeast cells and isolated cell walls by an inducible enzyme system ofArthrobacter GJM-1. J Allg Mikrobiol 17:391–402

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Cellular and Structural Biology, University of Colorado Health Sciences Center, 4200 East Ninth Avenue, Box B-111, 80262, Denver, Colorado, USA

    Maria Hrmova &  Claude P. Selitrennikoff

Authors
  1. Maria Hrmova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Claude P. Selitrennikoff
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hrmova, M., Selitrennikoff, C.P. Protoplast formation ofNeurospora crassa by an inducible enzyme system ofArthrobacter GJM-1. Current Microbiology 16, 33–38 (1987). https://doi.org/10.1007/BF01568166

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF01568166

Keywords

Search

Navigation