Scanning Electron Microscope StudyofSaccharomyces cerevisiae Spheroplast Formation (original) (raw)
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Scanning electron microscopy of cells and protoplasts ofSaccharomyces rouxii
Current Microbiology, 1980
Cells of Saccharomyces rouxii from a normal broth culture were subjected to a high osmotic pressure (2 M KC1), fixed in 3% glutaraldehyde fortified with 2 M KC1, and then processed routinely for examination in a scanning electron microscope. Micrographs revealed birth and bud scars typical for the genus and an apparently undamaged surface topography. Protoplasts were prepared from the same material by digestion of cell walls with snail gut enzymes in the presence of 2 M KCI. Naked protoplasts were obtained and these exhibited surface invaginations. In addition, spheroidal protrusions were noted and these structures were equated with the periplasmic bodies previously described by transmission electron microscopy. The propensity for periplasmic body formation in Saccharomyces rouxii is contrasted with other Saccharomyces species and the circumstantial evidence that relates periplasmic bodies to cryptic fl-fructofuranosidase in S. rouxii is briefly discussed.
Journal of General Microbiology, 1976
A method is described for the preparation of spheroplasts in high yield from Schizosaccharomyces pombe, by treating cells grown in the presence of glucose and deoxyglucose with snail digestive enzymes. Gentle disruption of such spheroplasts yielded homogenates, from which marker enzymes for nuclei (NAD pyrophosphorylase) and mitochondria (cytochrome c oxidase activity and spectroscopically-detectable cytochromes a + a,) could be quantitatively sedimented by low-speed centrifugation. In contrast to previous findings with Saccharomyces carlsbergensis, cytochrome c oxidase and another mitochondrial enzyme, succinate dehydrogenase, were completely sedimentable by zonal centrifugation in sucrose gradients in the presence of either 2 mM-MgC1, or 0.4 mM-EDTA.
Journal of Bacteriology, 1979
Long, nonseptate filamentous cells consisting of 5 to 40 single-cell unit lengths were formed from Escherichia coli surface mutant ONT-3 by treatment with a sublethal concentration of sodium dodecyl sylfate. As distinct from several other elongated cells (e.g., thymine-starved filaments), it was found here that stable giant spheroplasts, 5 to 10 micrometers in diameter, were produced by the action of lysozyme in the presence of bovine serum albumin via the gradual fusion of distinct spheroplasting bulbs.
Archives of Biochemistry and Biophysics, 1979
Intact spheroplasts, vesicles obtained from French-press lysates (chromatophores), and spheroplast-derived vesicles were isolated from photosynthetically grown ceils of Rhodopseudomonas sphaeroides. Lysed spheroplasts showed specific activities of succinate, NADH, and L-lactate dehydrogenase which were eight-, six-, and seven-fold higher, respectively, than those of intact spheroplasts when ferricyanide was used as electron acceptor. Mg2+-ATPase activity of lysed spheroplasts, measured using an assay system coupled to the oxidation of NADH, was seven-fold higher than the activity of intact sheroplasts. Toluene-treated spheroplast-derived vesicles displayed higher succinate dehydrogenase (ferricyanide reduction) and Mg*+-ATPase activities than untreated vesicles whereas no differences were measured between untreated and toluene-treated chromatophores. However, NADH dehydrogenase (ferricyanide reduction) activities of both toluene-treated vesicles and chromatophores were higher than the activities of untreated vesicles and chromatophores. When chromatophores and spheroplast-derived vesicles were preincubated with trypsin, the L-lactate and succinate dehydrogenase activities of chromatophores were preferentially inactivated when phenazine methosulfate was used as electron acceptor. The data indicate that chromatophores are oriented in an opposite direction to the spheroplast-derived vesicles. At least 80% of the latter are oriented in a direction equivalent to the cytoplasmic membrane of intact cells and spheroplasts. Spheroplast-derived vesicles from cells grown with higher light intensities seem to be more uniformly oriented than those obtained from cells grown with lower light intensities.
Preparation of spheroplasts from the strict anaerobe Selenomonas ruminantium
Journal of Microbiological Methods, 1990
A method for the preparation of spheroplasts from Selenomonas ruminantium is described. The method is based on the lysozyme/EDTA method developed by Kaback and collaborators. The most important modification introduced consisted in the substitution of nitrilotriacetic acid for EDTA. The final yield of spheroplasts was ~ 80% of the original whole cell suspension. However, to achieve this yield, the inclusion of n-decanoic acid and higher yeast extract levels in the growth medium, as well as the substitution of mutanolysin for lysozyme and of nitrilotriacetic acid for EDTA during the incubation of cell suspensions at lower temperatures, were also all found necessary. Right-side-out vesicles were obtained from these spheroplasts according to the original method.
Enzymatic hydrolysis of the walls of yeasts cells and germinated fungal spores
Biochimica et Biophysica Acta (BBA) - General Subjects, 1977
Crude lytic enzymes (predominantly ~-(1-3)-glucanases from Oerskovia xanthineolytica, Basidiomycete QM 806 and Rhizopus arrhizus QM 1032 were tested against the walls of log-phase Saccharomyces cerevisiae and germinated spores of Aspergillus aculeatus, Aureobasidium pullulans, Myrothecium verrucaria, Rhizopus arrhizus and Trichoderma viride. Release of reducing sugars and formation of spheroplasts from viable cells were used as criteria to evaluate enzymatic activity. The enzyme preparation from Oerskovia was more active than the other two enzymes for formation of spheroplasts and release of reducing sugars from Au. pullulans, M. verrucaria, S. cerevisiae and T. viride. The basidiomycetous enzyme was also able to attack these viable cells and form spheroplasts, but the rate of release of reducing sugars and the extent of spheroplast formation was lower than for the Oerskovia enzyme. The enzyme from R. arrhizus did not attack viable cells extensively or release spheroplasts from the organisms tested. All three enzyme preparations released reducing sugars from Asp. aculeatus without the formation of spheroplasts. R. arrhizus was resistant to treatment by these enzymes. The crude Oerskovia enzyme exhibited an endo-splitting action pattern against laminarin and lichenin. The ratio of cell lytic activity to laminarinase activity was higher for the Oerskovia preparation than for the preparations from the other two organisms. The basidiomycete enzyme showed a predominantly exo-splitting action pattern against laminarin and did not attack lichenin to a great extent, but was capable of yeast wall lysis; The enzyme from R. arrhizus was predominantly endo-splitting against laminarin and lichenin, and thus similar to the Oerskovia enzyme. However, it 11 showed no action against viable cells. Chitinase assisted the Oershovia and basidiomycete enzyme preparations in the release of reducing sugars from Asp. aculeatus, M. verrucaria and T. viride. Chitosanase showed the greatest action towards R. arrhizus.