Water potential,glycerol synthesis,and water content of germinating Phycomyces spores

During early germination, the sporangiospores of Phycomyces blakesleeanus synthesized large amounts of glycerol. Glycerol started leaking out of the spores after some 20 min germination. Simultaneously the water content of the spores greatly increased. Water uptake was accompanied by disapperance of the phase contrast halo and an increase in spore cross-sectional area which all occurred during the same period between 10 and 30 min germination. When spores were incubated in 0.5 or 1 M sucrose, glycerol accumulated in the spores to much higher concentrations and the increase in cellular water content was greatly reduced and retarded. Glycerol synthesis and the concomitant lowering of spore osmotic potential was not the only mediator of spore swelling since equally important glycerol concentrations loaded into dormant spores did not cause spore water uptake or swelling. Also the swelling of the spores was less affected than water uptake by decreases in ambient water potential. Apparently also cell wall loosening was involved in the swelling phenomenon which might have important implications for cellular metabolism.     

Plasma membrane structures of Saccharomyces cerevisiae and Candida albicans revealed by freeze-fracturing before and after treatment with filipin

Abstract Plasma membrane structures of Saccharomyces cerevisiae and Candida albicans during growth were studied by means of freeze-fracturing before and after filipin treatment. Undifferentiated regions of the plasma membrane were severely deformed by filipin, indicating the existence of a high level of ergosterol. The plasma membrane of small buds was mildly deformed by filipin, which suggested the existence of a low level of ergosterol. The bottom part of invaginations and the plasma membrane of the neck between the mother cell and the bud usually lacked filipin-induced deformations. Constraints existed in these regions which might restrict the ability of filipin to deform the membrane.     

Effects of chlorhexidine diacetate on Candida albicans, C. glabrata and Saccharomyces cerevisiae

S.J. HIOM, J.R. FURR, A.D. RUSSELL AND J.R. DICKINSON, 1992. The effects of chlorhexidine diacetate (CHA) on Candida albicans, C. glabrata and wild-type and mannan, and permeability mutants of Saccharomyces cerevisiae have been studied. A CHA concentration of 10 μg/ml had little lethal activity against the Candida strains, but was more effective against S. cerevisiae. Concentrations of 100 and especially 1000 μg/ml brought about a much more rapid death of cells. 2-Mercaptoethanol enhanced the activity of CHA to some extent. Some of the mutant strains of S. cerevisiae were rather more sensitive than the wild-type strain. The age of cultures of C. albicans and C. glabrata influenced their response to CHA.     

Influence of salinity, nutrients and light on the germination and growth of Enteromorpha sp. spores

In many shallow coastal areas worldwide, several species of opportunistic macroalgae (mainly Chlorophyta) have an excessive growth, as a consequence of eutrophication processes. Therefore, bloom-forming macroalgae become the dominant primary producers within these coastal systems. However, frequently the ecology and the ecophysiology of adult macroalgae have been insufficient to explain their seasonal abundances. Thus, it is essential to understand the factors that regulate the germination and growth of spores of opportunistic green macroalgae. In the present work, we assessed the effects of nutrients (N and P), salinity and light on the germination and growth of Enteromorpha spores. Overall, the results highlight the fact that, such as for adult macroalgae, spore germination and growth are adversely affected by low salinities. Growth of the spores is significantly decreased at 5 psu, while salinities of 20 and, especially of 35 psu, clearly promote the spore growth. Additionally, Enteromorpha spores seem to be particularly sensitive to PO₄-P limitation and to NH₄-N toxicity, which suggests a higher sensitivity to the variation of external nutrient concentrations than adult macroalgae. The present results contribute to increase the understanding about the factors that control macroalgal growth at its early phases of development. In particular, the results suggest that the growth of spores from opportunistic green macroalgae is strongly salinity-dependent. Consequently, in highly hydrodynamic systems such as most shallow estuaries, salinity variations may play a determinant role in the yearly abundances of green macroalgae, since it controls macroalgal growth from the spores to the adults.     

Resistance of the neck plasma membrane between the mother and the bud of Saccharomyces cerevisiae and Candida albicans to amphotericin B-induced deformation

Abstract The polyene antibiotic, amphotericin B, at high concentrations (5–20 μg/ml) induced particle-free smooth areas in the plasma membranes of Saccharomyces cerevisiae and Candida albicans . These areas occured more or less over the entire plasma membrane of unbudded cells. In budded cells, however, the neck between the mother and bud did not undergo deformation. This suggests the strong interaction between the filamentous ring, which is firmly attached to the neck plasma membrane, and plasma membrane particles in the neck regions.     

Effects of chlorhexidine diacetate on Candida albicans, C. glabrata and Saccharomyces cerevisiae.

The effects of chlorhexidine diacetate (CHA) on Candida albicans, C. glabrata and wild-type and mannan, and permeability mutants of Saccharomyces cerevisiae have been studied. A CHA concentration of 10 micrograms/ml had little lethal activity against the Candida strains, but was more effective against S. cerevisiae. Concentrations of 100 and especially 1000 micrograms/ml brought about a much more rapid death of cells. 2-Mercaptoethanol enhanced the activity of CHA to some extent. Some of the mutant strains of S. cerevisiae were rather more sensitive than the wild-type strain. The age of cultures of C. albicans and C. glabrata influenced their response to CHA.     

The effect of cryptolepine on the morphology and survival of Escherichia coli, Candida albicans and Saccharomyces cerevisiae

The antimicrobial activity of the indoloquinoline alkaloid, cryptolepine, isolated from Cryptolepis sanguinolenta (Fam. Periplocaceae) was determined against selected micro-organisms. The minimum inhibitory concentration (MIC) ranges obtained, expressed as μg ml⁻¹, were: 5–10 for Saccharomyces cerevisiae NCPF 3139; 10–20 for S. cerevisiae NCPF 3178; 20–40 for Escherichia coli NCTC 10418; 40–80 for E. coli NCTC 11560, Candida albicans ATCC 10231 and C. tropicalis NCPF; and 80–160 for C. albicans NCPF 3242 and NCPF 3262.
Biocidal effects were noted at concentrations 2–4 times those of the MIC of the alkaloid following challenge with 10⁶ cfu ml⁻¹ of micro-organisms. Time-kill studies showed a reduction in viable count from 10⁶ to < 10 cfu ml⁻¹ in 4 h in C. albicans ATCC 10231 exposed to 320 μg ml⁻¹ of the agent; 3 log cycle reductions were recorded for the 6 h counts of E. coli NCTC 10418 and S. cerevisiae NCPF 3139 exposed to 40μg ml⁻¹ and 160 μg ml⁻¹ of the alkaloid respectively.
These results were consistent with findings using scanning electron microscopy. Exposure of cells to biocidal concentrations of cryptolepine produced filamentation prior to lysis in E. coli NCTC 10418 and extreme disturbance of surface structure, including partial and total collapse, followed by lysis in C. albicans ATCC 10231 and S. cerevisiae NCPF 3139.     

The Effect of Sodium Azide on the Thermotolerance of the Yeasts Saccharomyces cerevisiae and Candida albicans

The addition of sodium azide (a mitochondrial inhibitor) at a concentration of 0.15 mM to glucose-grown Saccharomyces cerevisiae or Candida albicans cells before exposing them to heat shock increased cell survival. At higher concentrations of azide, its protective effect on glucose-grown cells decreased. Furthermore, azide, even at low concentrations, diminished the thermotolerance of galactose-grown yeast cells. It is suggested that azide exerts a protective effect on the thermotolerance of yeast cells when their energy requirements are met by the fermentation of glucose. However, when cells obtain energy through respiratory metabolism, the azide inhibition of mitochondria enhances the damage inflicted on the cells by heat shock.     

The effects of sex and mutation rate on adaptation in test tubes and to mouse hosts by Saccharomyces cerevisiae

Some hypotheses for the evolution of sex focus on adaptation to changing or heterogeneous environments, but these hypotheses have rarely been tested. We tested for advantages of sex and of increased mutation rates in yeast strains in two contrasting environments: a standard and relatively homogeneous laboratory environment of minimal medium in test tubes, and the variable environment of a mouse brain experienced by pathogenic strains. Evolving populations were founded as equal mixtures of sexual and obligately asexual genotypes. In the sexuals, cycles of sporulation, meiosis, and mating were induced approximately every 50 mitotic generations, with the asexuals undergoing sporulation but not ploidy cycles or recombination. In both environments, replicate negative control populations established with the same pair of strains were propagated with neither mating nor meiosis. In test tubes with no sex induced, sexuals were fixed in all five replicates within 250 mitotic generations, whereas in mice with no sex induced, asexuals were fixed in all four replicates by 170 generations. Inducing sex altered these outcomes in opposite directions in test tubes and mice, decreasing the fixation frequencies of sexuals in test tubes but increasing them in mice. These contrasts with asexual controls suggest an advantage for sex in mice but not in test tubes, although there was no difference between test tubes and mice in the numbers of populations fixed-for sexuals. In analogous experiments testing for an advantage of increased mutation rates, wild-type genotypes became fixed at the expense of mutators in every replicate of both test tube and mouse populations, indicating a disadvantage for mutators in both environments. Increased rates of point mutation do not appear to accelerate adaptation.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

The antagonistic effect of Saccharomyces boulardii on Candida albicans filamentation, adhesion and biofilm formation

The dimorphic fungus Candida albicans is a member of the normal flora residing in the intestinal tract of humans. In spite of this, under certain conditions it can induce both superficial and serious systemic diseases, as well as be the cause of gastrointestinal infections. Saccharomyces boulardii is a yeast strain that has been shown to have applications in the prevention and treatment of intestinal infections caused by bacterial pathogens. The purpose of this study was to determine whether S. boulardii affects the virulence factors of C. albicans . We demonstrate the inhibitory effect of live S. boulardii cells on the filamentation (hyphae and pseudohyphae formation) of C. albicans SC5314 strain proportional to the amount of S. boulardii added. An extract from S. boulardii culture has a similar effect. Live S. boulardii and the extract from S. boulardii culture filtrate diminish C. albicans adhesion to and subsequent biofilm formation on polystyrene surfaces under both aerobic and microaerophilic conditions. This effect is very strong and requires lower doses of S. boulardii cells or concentrations of the extract than serum-induced filamentation tests. Saccharomyces boulardii has a strong negative effect on very important virulence factors of C. albicans , i.e. the ability to form filaments and to adhere and form biofilms on plastic surfaces.     

Effect of ozone on ATP,cytosolic enzymes and permeability of Saccharomyces cerevisiae

Treatment of a yeast suspension with ozone inactivates a number of cytosolic enzymes. Among 15 studied, the most drastic inactivation was found for glyceraldehyde-3-phosphate dehydrogenase and to lesser extents: NAD-glutamate dehydrogenase, pyruvate decarboxylase, phosphofructokinase-1 and NAD-alcohol dehydrogenase. Ozone treatment also effects the quantity of ATP and of other nucleoside triphosphates, reducing to about 50% of the initial value. The ATP missing in the cells appears in the medium. NAD and protein also accumulate in the medium suggesting that the yeast cells have been permeabilized. Permeabilization of the yeast cells by treatment with ozone preceeds the inactivation of glyceraldehyde-3-phosphate dehydrogenase and other cytosolic enzymes.Dedicated to Prof. Dr. B. Hess at the occasion of his 65th birthday     

A sensitive predictor for potential GPI lipid modification sites in fungal protein sequences and its application to genome-wide studies for Aspergillus nidulans, Candida albicans, Neurospora crassa, Saccharomyces cerevisiae and Schizosaccharomyces pombe

The fungal transamidase complex that executes glycosylphosphatidylinositol (GPI) lipid anchoring of precursor proteins has overlapping but distinct sequence specificity compared with the animal system. Therefore, a taxon-specific prediction tool for the recognition of the C-terminal signal in fungal sequences is necessary. We have collected a learning set of fungal precursor protein sequences from the literature and fungal proteomes. Although the general four segment scheme of the recognition signal is maintained also in fungal precursors, there are taxon specificities in details. A fungal big-Pi predictor has been developed for the assessment of query sequence concordance with fungi-specific recognition signal requirements. The sensitivity of this predictor is close to 90%. The rate of false positive prediction is in the range of 0.1%. The fungal big-Pi tool successfully predicts the Gas1 mutation series described by C. Nuoffer and co-workers, and recognizes that the human PLAP C terminus is not a target for the fungal transamidase complex. Lists of potentially GPI lipid anchored proteins for five fungal proteomes have been generated and the hits have been functionally classified. The fungal big-Pi prediction WWW server as well as precursor lists are available at     

The effects of temperature and pH on the ethanol tolerance of the wine yeasts, Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata

The influences of temperature and pH on the survival and growth of Saccharomyces cerevisiae, Candida stellata and Kloeckera apiculata were examined in the presence of ethanol concentrations between 2.5 and 15% v/v. At 15°C, the maximum concentrations of ethanol permitting the growth of S. cerevisiae, C. stellata and K. apiculata were 15%, 11% and 9%, respectively. These maximum concentrations were decreased at 10°C and 30°C. Cells of S. cerevisiae showed no loss in viability when incubated for 12 d at 10°C or 15°C in the presence of 15% ethanol but showed some loss at 30°C. Cells of C. stellata were tolerant of 12.5% ethanol at 10°C and 15°C but not at 30°C. Cells of K. apiculata were tolerant of 10–12.5% ethanol at 15°C but not at 10°C or 30°C. Sensitivity of the yeast cells to ethanol was marginally increased on decreasing the pH from 6-0 to 3–0.     

Uptake and cellular distribution of copper, cobalt and cadmium in strains of Saccharomyces cerevisiae cultured on elevated concentrations of these metals

Abstract Training by repeated culture on elevated concentrations of Co, Cu and Cd resulted in increased resistance to these metals in 3 strains of Saccharomyces cerevisiae . All the trained strains showed reduced uptake of these metals and altered intracellular distribution. Co-trained strains showed increased Co-accumulation in the vacuole, which was also the main compartment for Co in unstrained cells. Cd- and Cu-trained cells had increased amounts of these metals in the soluble fractions of the cell, but not specifically in the vacuole. These mechanisms appeared to be distinct and to vary in their stability following detraining on metal-free medium.     

Comparison of two cytochromes P-450 from Candida maltosa: primary structures, substrate specificities and effects of their expression in Saccharomyces cerevisiae on the proliferation of the endoplasmic reticulum

cDNAs were cloned, sequenced and expressed which encode two different cytochrome P-450 forms of the alkane-assimilating yeast Candida maltosa, designated as P-450Cm1 and P-450Cm2. The amino acid sequences deduced were about 55% identical. Expression in Saccharomyces cerevisiae resulted in the formation of intact microsomal P-450 systems catalyzing the hydroxylation of n-hexadecane and lauric acid with significantly different substrate preferences. A massive proliferation of the endoplasmic reticulum was observed in the S. cerevisiae cells which produced P-450. Depending on the P-450 form expressed, distinctly organized stacks of paired membranes appeared and occupied considerable areas of the cytoplasm. As shown by immunoelectron microscopy for P-450Cm1, the protein expressed was highly concentrated within these newly formed membrane structures.     

The effects of selenium,vitamin E and their combination on the composition of fatty acids and proteins in Saccharomyces cerevisiae

The aim of our studies was to test the effect and role of vitamin E and selenium supplements on yeast cell. In this study, the effects of selenium (Se), vitamin E (Vit. E), and their combination (Se plus Vit. E) on the composition of fatty acids and proteins were examined in Saccharomyces cerevisiae strains WET136 and 522. S. cerevisiae cells were grown up in YEPD medium supplemented with Se, Vit. E or their combination. It was found that the level of stearic acid was increased in all supplemented groups (p<0·05; p<0·001). The content of saturated and unsaturated fatty acids was decreased (p<0·05; p<0·01; p<0·001) in Vit. E and Vit. E plus Se supplemented S. cerevisiae. On the other hand, Se alone caused an increase (p<0·001) in the saturated fatty acids but a decrease (p<0·05; p<0·001) in the unsaturated fatty acids. Total proteins in S. cerevisiae were significantly increased (p<0·001) by Vit. E supplement. There was no significant change observed in S. cerevisiae supplemented with Se. These findings indicate that membrane composition of S. cerevisiae is affected by both Vit. E and Se supplements. © 1997 John Wiley & Sons, Ltd.     

Analyses of the effects of Rck2p mutants on Pbs2p<Superscript>DD</Superscript>-induced toxicity in<Emphasis Type="Italic"> Saccharomyces cervisiae</Emphasis> identify a MAP kinase docking motif,and unexpected functional inactivation due to acidic substitution of T379

Rck2p is a Ser/Thr kinase that binds to, and is activated by, Hog1p. Expression of the MAP kinase kinase Pbs2p^DD from a GAL1 -driven plasmid hyperactivates the HOG MAP kinase pathway, and leads to cessation of growth. This toxic effect is reduced by deletion of RCK2. We studied the structural and functional basis for the role of Rck2p in mediating the growth arrest phenotype associated with overexpression of Pbs2p^DD. Rck2p kinase activity is required for the effect, because Rck2p(487–610), as well as full-length Rck2p, is toxic with Pbs2p^DD, but kinase-defective versions of either protein with a K201R mutation are not. Thus, the C-terminal portion of Rck2p is not required provided the protein is activated by removal of the autoinhibitory domain. Relief of inhibition in Rck2p normally requires phosphorylation by Hog1p, and Rck2p contains a putative MAP kinase docking site (TILQR⁵⁸⁹R⁵⁹⁰KKVQ) in its C-terminal segment. The Rck2p double mutant R589A/R590A expressed from a centromeric plasmid did not detectably bind Hog1p-GFP and was functionally inactive in mediating the toxic effect of Pbs2p^DD, equivalent to an RCK2 deletion. However, overexpression of Rck2p R589A/R590A from a multicopy plasmid restored function. In contrast, RCK2-K201R acted as a multicopy suppressor of PBS2 ^DD, markedly reducing its toxicity. This suppressor activity required the K201R mutation, and the effect was largely lost when the docking site was mutated, suggesting suppression by inhibition of Hog1p functions. We also studied the effect of replacing the predicted T379 and established S520 phosphorylation sites in Rck2p by glutamic acid. Surprisingly, the T379E mutant markedly reduced Pbs2p^DD toxicity, and toxicity was only partially rescued by S520E. Rck2 T379E was sufficiently inactive in an rck2 strain to allow some cells to survive PBS2 ^DD toxicity even when overexpressed. The significance of these findings for our understanding of Rck2p function is discussed.Communicated by M. Collart