Cell wall deficiency in "slime" strains of Neurospora crassa: osmotic inhibition of cell wall synthesis and beta-D-glucan synthase activity.
1. The RCP-3 S/H mutant of Neurospora crassa was obtained by vegetative selection in medium of high osmolarity of a mycelial form of an fz, sg, os-1 ("slime"-like) segregant. The mutant exhibits spheroplast-hyphal dimorphism conditioned by the osmolarity of the culture medium (Pietro et al. (1990). Journal of General Microbiology, 136: 121-129). The carbohydrate composition of the cell wall of the mutant was different from that of the wild type in the absence of an alkali-soluble galactosaminoglycan polymer. Furthermore the mutant cell wall had a somewhat lower content of beta-glucan relative to that of chitin. 2. Increasing concentrations of sorbitol in the culture medium of the mutant inhibited by 10-fold the formation of cell wall relative to total biomass. The cell wall of the mutant cultured in the presence of sorbitol lacked mannose- and galactose-containing polymers, and also showed progressively lower amounts of beta-glucan relative to chitin. 3. The activity of membrane-bound (1-3)-beta-D-glucan synthase from the mutant grown in the absence of sorbitol shared several properties with the wild type enzyme (i.e., Km app., Vmax, stability at 30 degrees C, activation by GTP gamma S, and dissociability by treatment with NaCl and Tergitol NP-40 into a membrane-bound catalytic center and a GTP-binding activating protein). On the other hand, the enzyme from the mutant but not that from the wild type was inactivated by about 15% by treatment with NaCl and detergent. 4. At high concentrations of sorbitol (1.0 M) the RCP-3 S/H mutant exclusively produced spheroplasts devoid of (1-3)-beta-D-glucan synthase activity. The defect was at the level of the membrane-bound catalytic center. The activity of the GTP-binding activating factor was apparently normal in these cells. 5. These results suggest that the definitive loss of cell wall in the N. crassa "slime" RCP-3 S/H mutant was due to a defect in (1-3)-beta-D-glucan synthase activity which was exaggerated in the presence of high osmolyte concentrations.
da-Silva
M M
MM
Departamento de Biologia, Faculdade de Filosofia, Universidade de São Paulo, Brasil.
Polizeli
M L
ML
Jorge
J A
JA
Terenzi
H F
HF
D002473:Cell Wall / Q000187:drug effects / Q000648:ultrastructure; D003470:Culture Media; D004234:Diuretics, Osmotic / Q000494:pharmacology; D005947:Glucose / Q000378:metabolism*; D005964:Glucosyltransferases / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D009154:Mutation / Q000187:drug effects; D009492:Neurospora crassa / Q000378:metabolism* / Q000648:ultrastructure; D013012:Sorbitol / Q000494:pharmacology
D003470:Culture Media; D004234:Diuretics, Osmotic; D013012:Sorbitol; D006160:Guanosine Triphosphate; D005964:Glucosyltransferases; C040232:glucan synthase; D005947:Glucose