Potassium requirements ofSaccharomyces cerevisiae

The growth rate ofSaccharomyces cerevisiae was dependent on K⁺ content in culture medium in a certain range of K⁺ concentrations. Above the upper limit of the range, growth did not respond to K⁺ increase, and below the lower limit, yeast died. Rb⁺ and Na⁺ enhanced growth in the range of K⁺ dependence and decreased the K⁺ concentration below which cells died. Both Rb⁺ and Na⁺ became toxic above a certain Rb⁺/K⁺ and Na⁺/K⁺ cellular ratio.     

The chitin-glucan complex ofSaccharomyces cerevisiae

Differentiation of the cell wall ofSaccharomyces cerevisiae at the site of the future bud was followed. A lentil-like structure originates on the inner side of the cell wall during the first phase. At the same time, an electron-dense layer occurs at the boundary between the inner layer of the cell wall and the lentil-like structure. During the second phase granular material is accumulated at the lower side of the lentil-like structure. During the third phase the lentil-like structure is split apart due to proliferation of the granular material resulting in formation of the base of the encircling region. The marked electron-dense layer observed from the first phase is attached to the surface of the encircling region during differentiation of the latter. During the budding proper the outer layers of the cell wall protrude and the end of the encircling region, together with the adjacent electron-dense layer, acquire their definitive appearance of rings, observed as marked electron-transparent and electron-dense tears on ultrathin sections.     

The chitin-glucan complex ofSaccharomyces cerevisiae

Fractions of the cell wall ofSaccharomyces cerevisiae where a α-chtin-glucan composition was established, were examined under the electron microscope as well as with phase fluorescence. The results indicate that the α-chitin-glucan complex shows a fibrillar arrangement and is localized in the socalled encircling region of the bud scar which has a tear-like appearance and is electron-transparent in ultrathin sections. There are indications of the presence of chitin even in the primary septum.     

The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii

Summary The digestion ofSaccharomyces cerevisiae byAcanthamoeba castellanii, at different times after feeding, has been examined by cytochemical techniques at electron microscope level and by measurement of yeast viability. The measurement of viability, combined with cytochemistry is presented as a novel method of examining the progress of digestion. Particular attention has been given to the temporal development of digestion.Vacuoles, probably primary lysosomes, have been identified containing acid phosphatase activity within minutes of feeding and these accumulate around and fuse with phagocytic vacuoles. Acid phosphatase levels in the digestive vacuoles appeared highest at 20 to 40 minutes. Yeast digestion was observed and yeast viability began to decline at this time. Mixing of autophagic and heterophagic material was also observed. At least half of the yeast population was still viable after 90 minutes.Our method (p-nitrophenyl phosphate) of enzyme localization has demonstrated plasma membrane associated acid phosphatase activity.     

Freeze-drying characteristics ofSaccharomyces cerevisiae andSaccharomyces carlsbergensis

The dispersal of yeast clumps to the unicellular state by certain sugars, does not increase the percentage survival after freeze-drying. Neither are those changes in cell-wall composition which occur upon ageing of the cell, and which are detectable by means of snail-gut enzymes, related to this cellular property. However, pre-treatment, with -mercaptoethanol, of a strain ofSaccharomyces carisbergensis increased the survival rate. This may be due to the reduction of certain sites in the cell wall. The oxygen consumption of yeast cultures before and after freeze-drying, agree with the hypothesis that low viabilities can arise from localized cellular damage which prevents cell reproduction by budding.     

Limited proteolysis ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Incubation ofSaccharomyces cerevisiae phosphoenolpyruvate carboxykinase with trypsin under native conditions cases a time-dependent loss of activity and the production of protein fragments. Cleavage sites determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis and sequence analyses identified protease-sensitive peptide bonds between amino acid residues at positions 9–10 and 76–77. Additional fragmentation sites were also detected in a region approximately 70–80 amino acids before the carboxyl end of the protein. These results suggest that the enzyme is formed by a central compact domain comprising more than two thirds of the whole protein structure. From proteolysis experiments carried out in the presence of substrates, it could be inferred that CO₂ binding specifically protects position 76–77 from trypsin action. Intrinsic fluorescence measurements demonstrated that CO₂ binding induces a protein conformational change, and a dissociation constant for the enzyme CO₂ complex of 8.2±0.6 mM was determined     

Temperature-sensitive flocculation during growth ofSaccharomyces cerevisiae

Summary Cell wall surface proteins were extracted from a temperature-sensitive flocculent strain ofSaccharomyces cerevisiae. Electrophoretic analysis identified two protein bands (28 and 43 kDa) present when grown at 21°C. These proteins were initially absent when the strain was grown at 37°C, but intensified after 6 days of growth concomitant with the onset of flocculation.     

Computer control for continuous cultivation ofSaccharomyces cerevisiae

Summary An on-line respiratory quotient control system has been developed for the continuous cultivation of baker's yeast. This system is based on moving identification of the microbial dynamics. The optimal dilution rate that was selected as the control variable was determined by minimizing a performance index. Without resorting to complicated microbial analysis, a simple and practical moving model is obtained by continually updating the input and output data. The experimental results indicate the satisfactory controllability of the present system and the possible extention of the proposed method to other bioprocesses.     

Regulation of citrate synthase activity ofSaccharomyces cerevisiae

Citrate synthase activity ofSaccharomyces cerevisiae was determined by a radioactive assay procedure and the reaction product,¹⁴C-citric acid, was identified by chromatographic techniques. ATP, d-ATP, GTP and NADPH were most inhibitory to the citrate synthasein vitro. The activity was inhibited to a lesser extent by ADP, UTP, and NADP whereas, AMP and CTP were much less inhibitory. NADH, like NAD, glutamic acid, glutamine, arginine, ornithine, proline, aspartic acid and α-ketoglutarate exhibited no inhibition. These results have been discussed in the light of the role of citrate synthase for the energy metabolism and glutamic acid biosynthesis.     

Morphological behaviour ofSaccharomyces cerevisiae during continuous fermentation

Summary Saccharomyces cerevisiae were observed to undergo drastic morphological changes when grown in continuous culture. Peak elongations and minimum cell volumes were found at intermediate dilution rates when it was believed the insitu glucose concentration was at its lowest. The shapes and sizes were reproducible and have been quantified at two different glucose feed concentrations.     

Electron microscopy of germinating ascospores ofSaccharomyces cerevisiae

The wall of mature ascospores ofSaccharomyces cerevisiae showed in sections under the electron microscope a dark outer layer and a lighter inner layer. The latter was composed of a greyish inner part and a light outer part. During germination, the spore grew out at one side and the dark outer layer was broken. Of the light inner layer, the inner greyish part became the wall of the vegetative cell, but the extented part of the cell had a new wall.     

Expression and purification of ubiquitin-specific protease (UBP1) ofSaccharomyces cerevisiae in recombinantEscherichia coli

Truncated form of UBP1, an ubiquitin-specific protease ofSaccharomyces cerevisiae, was overexpressed inEscherichia coli. The hexahistidine residue (His₆) was fused to the N-terminus of truncated UBP1 and the corresponding recombinant protein was purified with high yield by immobilized metal affinity chromatography. The truncated form of UBP1 protein was functional to cleave ubiquitinated human growth hormone as substrate. Effects of pH and temperature were investigated in order to optimize deubiquitinating reactions for the truncated UBP1. Optimum temperature and pH for the cleavage reaction were 40°C and pH 8.0, respectively.     

Transfer of isolated nuclei into protoplasts ofSaccharomyces cerevisiae

Cell nuclei were prepared from protoplasts of an adenine-requiring strain ofSaccharomyces cerevisiae, then purified in a discontinuous sucrose gradient, and applied to protoplasts of a recipient strain auxotrophic for uracil, leucine, and histidine. The transfer of the isolated nuclei into protoplasts was induced with polyethylene glycol. The main products of nuclear transfer in young complemented colonies were heterokaryons giving rise to parental type spontaneuos segregants on nutritionally complete medium. After several passages in minimal medium, however, the prototrophic colonies consisted exclusively of stable heterozygous diploid cells.     

Flocculation of industrial and laboratory strains ofSaccharomyces cerevisiae

Summary A comparative study has been made of different laboratory and industrial wild-type strains ofSaccharomyces cerevisiae in relation to their flocculation behavior. All strains were inhibited by mannose and only one by maltose. In regard to the stability of these characters in the presence of proteases and high salt concentrations, a relevant degree of variation was found among the strains. This was to such an extent that it did not allow their inclusion in the Flol or NewFlo phenotypes. Genetic characterization of one wild-type strain revealed that the flocculation-governing gene was allelic toFLO1 found in genetic strains.This paper is dedicated to Professor Herman Jan Phaff in honor of his 50 years of active research which still continues.     

Ploidy reduction usingp-Fluorophenylalanine of fusion products ofSaccharomyces cerevisiae

Fusion of yeast protoplasts was induced in the presence of polyethylene glycol and Ca++ ions. Two auxotrophic complementingSaccharomyces cerevisiae mutant strains were used in fusion experiments. One diploid and several polyploid fusion products were selected by complementation in minimal medium. The assessment of ploidy has been based on the DNA content of the parental cells and fusion products, assayed with the diphenylamine method. Treating the fusion product cells withp-Fluorophenylalanine (p-FPA), parentalhis andleu markers could not be recovered. Instead, a strong reduction of polyploid fusion product cell DNA content was evident. The analysis of meiotic products after hybridizing one fusion product with a prototrophicSaccharomyces cerevisiae standard strain led to the recovery of thehis parental marker. Preliminary evidence thatp-Fluorophenylalanine could be used as a diploidizing agent towards polyploid strains ofSaccharomyces cerevisiae is reported.     

Enzyme repression in the arginine pathway ofSaccharomyces cerevisiae

Enzyme repression in the arginine pathway ofSaccharomyces cerevisiae was demonstrated by comparison of specific enzyme activities in yeast grown with and without arginine in various mineral salts media. Of the enzymes tested only ornithine transcarbamoylase was found to be repressed by exogenous arginine. Acetylornithine-glutamate transacetylase and argininosuccinate lyase were not affected. No relationship between specific enzyme activities and intracellular arginine concentration was observed.During the adaptation of yeast grown in a medium supplemented with amino acids to a mineral salts medium, the enzymes ornithine transcarbamoylase and argininosuccinate lyase were not derepressed beyond their specific activities normally present in yeast grown in mineral salts media. Neither were the arginine-degrading enzymes arginase and ornithine transaminase broken down during this adaptation.Thanks are due to Professor E. G. Mulder and to Professor H. Veldkamp for stimulatory discussions; to the Heineken's Brouwerij, Rotterdam, and to the Landbouwhogeschoolfonds for research grants.     

Repetitive sequences in nuclear deoxyribonucleic acid ofSaccharomyces cerevisiae

Nuclear DNA isolated from aSaccharomyces cerevisiae ρ mutant was studied for the presence of repetitive sequences. A main-band DNA preparation free of rRNA genes and 2-μm plasmid DNA was prepared by density gradient centrifugation in Cs₂SO₄−Ag⁺. A fast renaturing fraction was obtained from this mainband DNA by 3 cycles of reassociation at a low C₀t value (0.2). This fraction reassociated 10 times faster than the bulk of the main-band DNA. Its sequences comprised about 3% of the genome and showed a considerable heterogeneity in respect to repetitiveness. The relationship of this fraction to the repetitive transposable elements recently found in yeast cells is discussed.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

Effects ofBacillus intermedius ribonuclease on the properties ofSaccharomyces cerevisiae

Effects ofBacillus intermedius ribonuclease on the physiological, biochemical, and consumer properties of baker’s yeastSaccharomyces cerevisiae were studied. This enzyme improved the yeast raising strength and increased the cell tolerance to various adverse factors. The antistress effect of RNase correlated ith an earlier start of the stationary growth phase and increased trehalose pool.     

Characterisation ofSaccharomyces cerevisiae genes encoding ribosomal protein YL6

We have characterised aSaccharomyces cerevisiae cDNA (cDNA13), originally isolated on the basis of the short half-life of the corresponding mRNA. We show here that its sequence is closely related to that of the genes encoding ribosomal proteins K37, KD4 and K5 ofSchizosaccharomyces pombe. ‘mRNA13’ also behaves like other mRNAs encoding ribosomal proteins, in that its abundance increases sharply when glucose is added to cells grown on ethanol (nutrient-up shift), and declines when cells are subjected to a mild heat-shock. Unspliced mRNA13 accumulates when cells bearing a temperature-sensitive splicing mutation are grown at the restrictive temperature. The gene(s) corresponding to cDNA13, like other ribosomal protein genes ofS. cerevisiae, thus contain an intron. Southern blot analysis indicates the presence of two separate loci related to cDNA13 in theS. cerevisiae genome. From the sequence of one of these, a complete polypeptide sequence was deduced. The first 40 amino acids are identical to those of YL6, aS. cerevisiae ribosomal protein characterised only by N-terminal protein sequence analysis. There is clear evidence within the genomic sequence for the predicted intron, and for elements similar to those that regulate expression of otherS. cerevisiae ribosomal protein genes.