Compositional, ultrastructural and nanotechnological characterization of the SMA strain of Saccharomyces pastorianus: Towards a more complete fermentation yeast cell analysis.

Nano scanning Auger microscopy (NanoSAM) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) have been used in materials science research for some time, but NanoSAM, in particular, has only recently been applied to biological specimens. Here, the first concurrent utilization of NanoSAM, TOF-SIMS and microscopic techniques for the examination of a standard beverage fermentation strain of Saccharomyces pastorianus uncovered the presence of intracellular networks of CO2 in fermenting cells. Respiring cells produced few bubbles and instead had large internal vacuolar structures. Transmission electron microscopy analysis also showed osmiophilic layers at the cell exterior of fermenting cells that became more prevalent with fermentation duration, while osmiophilic layers were largely absent in respiring cells. TOF-SIMS analysis showed a compositional difference at the exterior and interior of SMA cells and between fermenting and respiring cells. Fermenting cells also appeared to have different 3-OH oxylipin profiles compared to respiring cells based upon examination with immunofluorescence microscopy. The results of this work and further study using these materials science techniques will substantially enhance our understanding of the chemical, ultrastructural and metabolic changes that occur in fermentation yeasts.

Structure of the SLC4 transporter Bor1p in an inward-facing conformation.

Bor1p is a secondary transporter in yeast that is responsible for boron transport. Bor1p belongs to the SLC4 family which controls bicarbonate exchange and pH regulation in animals as well as borate uptake in plants. The SLC4 family is more distantly related to members of the Amino acid-Polyamine-organoCation (APC) superfamily, which includes well studied transporters such as LeuT, Mhp1, AdiC, vSGLT, UraA, SLC26Dg. Their mechanism generally involves relative movements of two domains: a core domain that binds substrate and a gate domain that in many cases mediates dimerization. To shed light on conformational changes governing transport by the SLC4 family, we grew helical membrane crystals of Bor1p from Saccharomyces mikatae and determined a structure at ∼6 Šresolution using cryo-electron microscopy. To evaluate the conformation of Bor1p in these crystals, a homology model was built based on the related anion exchanger from red blood cells (AE1). This homology model was fitted to the cryo-EM density map using the Molecular Dynamics (MD) Flexible Fitting method and then relaxed by all-atom MD simulation in explicit solvent and membrane. Mapping of water accessibility indicates that the resulting structure represents an inward-facing conformation. Comparisons of the resulting Bor1p model with the X-ray structure of AE1 in an outward-facing conformation, together with MD simulations of inward-facing and outward-facing Bor1p models, suggest rigid body movements of the core domain relative to the gate domain. These movements are consistent with the rocking-bundle transport mechanism described for other members of the APC superfamily.

FTIR spectroscopic discrimination of Saccharomyces cerevisiae and Saccharomyces bayanus strains.

In this study, we tested the potential of Fourier-transform infrared absorption spectroscopy to screen, on the one hand, Saccharomyces cerevisiae and non-S. cerevisiae strains and, on the other hand, to discriminate between S. cerevisiae and Saccharomyces bayanus strains. Principal components analysis (PCA), used to compare 20 S. cerevisiae and 21 non-Saccharomyces strains, showed only 2 misclassifications. The PCA model was then used to classify spectra from 14 Samos strains. All 14 Samos strains clustered together with the S. cerevisiae group. This result was confirmed by a routinely used electrophoretic pattern obtained by pulsed-field gel electrophoresis. The method was then tested to compare S. cerevisiae and S. bayanus strains. Our results indicate that identification at the strain level is possible. This first result shows that yeast classification and S. bayanus identification can be feasible in a single measurement.

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 different yeast strains and their culture conditions on the prevention of wine model solution browning by yeast lees.

The purpose of this work was to examine the possible involvement of yeast membrane components in the adsorption of browning compounds from oxidized white wine. For this purpose, different yeast strains and growth conditions (aerobiosis and anaerobiosis) were tested for their ability to prevent browning of two model solutions consisting of (+)-catechin/acetaldehyde and (+)-catechin/glyoxylic acid. The obtained results showed that the effects of yeast lees are different according to the type of the studied model solution and the growth conditions that affect both the quantity and the quality of membrane sterols of the yeasts. Moreover, in vitro experiments proved that yeast membrane sterols could be likely involved in the yeast's ability to adsorb polyphenolic compounds and mainly the colorless intermediate compounds of the browning reactions.

Interaction between Lactobacillus kefir and Saccharomyces lipolytica isolated from kefir grains: evidence for lectin-like activity of bacterial surface proteins.

Several microbial interactions involving yeast and lactobacilli have been suggested in fermented products. Co-aggregation between Lactobacillus kefir and yeast Saccharomyces lipolytica isolated from kefir grains was studied by scanning electron microscopy and aggregation assays. Six out of twenty Lb. kefir strains were able to co-aggregate with Sacch. lipolytica CIDCA 812 and showed thermolabile non-covalently bound surface molecules involved in this interaction. Co-aggregation inhibition after Lb. kefir pre-treatment with 5 m-LiCl or 20 g SDS/l showed that bacterial S-layer proteins play an important role in this interaction. Presence of different sugar (mannose, sucrose and fructose) or yeast pre-treatment with sodium periodate inhibited co-aggregation between Lb. kefir and Sacch. lipolytica. Co-aggregating Lb. kefir strains were also able to agglutinate with human red blood cells and they lost this ability after treatment with 5 m-LiCl. These results and the capacity of purified S-layer proteins of Lb. kefir to haemagglutinate, strongly suggest that a lectin-like activity of bacterial surface proteins (S-layer) mediates the aggregation with yeast cells.

Formulations for protecting the probiotic Saccharomyces boulardii from degradation in acidic condition.

Saccharomyces boulardii is a nonpathogenic yeast with proven health benefits, some of them depending on its viability. However, the living yeast is sensitive to environmental conditions and its viability is less than 1% in the faeces after oral administration. Therefore, we assessed the survival conditions of S. boulardii in aqueous suspension and in its freeze-dried form and we formulated microspheres with the former and tablets with the latter in order to preserve the viability of the probiotic. While the viability of the yeast in aqueous suspension could be maintained for one year at -20 degrees C and +5 degrees C, increasing the temperature led to almost total mortality within 14 d at +40 degrees C and 4 d at +60 degrees C. The viability of the freeze-dried yeast was preserved for one year at +25 degrees C without moisture. With 75% relative humidity, the mortality was significant at 28 d at +25 degrees C and almost total within 1 d at +60 degrees C. In vitro, whereas less than 1% of non-encapsulated or non-tabletted S. boulardii survived after 120 min at pH 1.1, both formulations in microspheres and direct compression enabled to protect the yeast from degradation in HCl and to release it viable at pH 6.8. However, despite a similar release profile from both dosage forms, the compression led to a significant decrease in the viability of the freeze-dried yeast. In conclusion, although both formulations are efficient in protecting S. boulardii in acidic condition, microspheres provide a higher entrapment efficiency and a faster release of the viable probiotic in intestinal condition than matrix tablets.

Dependence of synergistic fungicidal activity of Cu2+ and allicin, an allyl sulfur compound from garlic, on selective accumulation of the ion in the plasma membrane fraction via allicin-mediated phospholipid peroxidation.

Allicin was effective in decreasing the lethal concentration of Cu (2+) against various fungal strains including a plant pathogen, Fusarium oxysporum, so that the minimum fungicidal concentration (MFC) of the ion for the fungus could be reduced to 2 % of that detected without allicin. In Saccharomyces cerevisiae, Cu (2+) was not apparently taken up by cells when added alone at a non-lethal concentration, whereas the ion was efficiently incorporated into cells in the presence of allicin, as in the case of cells treated with the ion at a lethal concentration. Although allicin likely increased cellular permeability to Cu (2+) due to its promotive effect on plasma membrane phospholipid peroxidation, these cell-surface events did not result in endogenous reactive oxygen species (ROS) production, a typical toxic effect of the ion. Cu (2+) was detected in the cytoplasmic fraction of cells that had been treated with the ion at a lethal concentration, whereas the ion was entrapped in the plasma membrane fraction upon their treatment with the ion at a low concentration in combination with allicin. Cu (2+) could be solubilized from the plasma membrane fraction by a procedure for the extraction of hydrophobic proteins rather than the extraction of phospholipids, suggesting its complexation with a plasma membrane protein as a result of allicin treatment. Such a subcellular localization of Cu (2+) resulted in the selective leakage of intracellular K (+), but not in the disruptive damage on the plasma membrane, and was considered to underlie the synergistic fungicidal activity of Cu (2+) and allicin.

Antimicrobial action of hydrolyzed chitosan against spoilage yeasts and lactic acid bacteria of fermented vegetables.

The antimicrobial properties of various chitosan-lactate polymers (ranging from 0.5 to 1.2 MDa in molecular weight) against two yeasts isolated from fermented vegetables and against three lactic acid bacteria from a mixed starter for sauerkraut on methylene blue agar (MBA) and in vegetable juice medium (VJM) were investigated. Chitosan-lactate reduced the growth of all microorganisms in solid (MBA) as well as in liquid (VJM) medium. In MBA, a concentration of 5 g/liter was needed to inhibit the growth of Saccharomyces bayanus, while 1 g/liter was sufficient to inhibit the growth of Saccharomyces unisporus. Lactic acid bacteria were also inhibited in this range of concentrations. The low-molecular-weight chitosan-lactate DP3 (0.5 kDa) was most efficient in solid medium (MBA), and inhibitory activities decreased with increasing hydrolysate lengths. In liquid medium (VJM), 0.5 g of chitosan-lactate per liter reduced the growth rates for both yeasts, but 10 g/liter was insufficient to prevent yeast growth. Intermediate-molecular-weight chitosan-lactate (5 kDa) was more efficient than chitosan of low molecular weight. Native chitosan (1.2 MDa) showed no inhibition in either medium. Microscopic examination of S. unisporus Y-42 after treatment with chitosan-lactate DP25 showed agglutination of a refractive substance on the entire cell wall, suggesting an interaction between chitosan and the cell wall. When chitosanase was added to the culture media containing chitosan-lactate, refractive substances could not be observed.

A comparison of the yeast and rabbit 80 S ribosome reveals the topology of the nascent chain exit tunnel, inter-subunit bridges and mammalian rRNA expansion segments.

Protein synthesis in eukaryotes is mediated by both cytoplasmic and membrane-bound ribosomes. During the co-translational translocation of secretory and membrane proteins, eukaryotic ribosomes dock with the protein conducting channel of the endoplasmic reticulum. An understanding of these processes will require the detailed structure of a eukaryotic ribosome. To this end, we have compared the three-dimensional structures of yeast and rabbit ribosomes at 24 A resolution. In general, we find that the active sites for protein synthesis and translocation have been highly conserved. It is interesting that a channel was visualized in the neck of the small subunit whose entrance is formed by a deep groove. By analogy with the prokaryotic small subunit, this channel may provide a conserved portal through which mRNA is threaded into the decoding center. In addition, both the small and large subunits are built around a dense tubular network. Our analysis further suggests that the nascent chain exit tunnel and the docking surface for the endoplasmic reticulum channel are formed by this network. We surmise that many of these features correspond to rRNA, based on biochemical and structural data. Ribosomal function is critically dependent on the specific association of small and large subunits. Our analysis of eukaryotic ribosomes reveals four conserved inter-subunit bridges with a geometry similar to that found in prokaryotes. In particular, a double-bridge connects the small subunit platform with the interface canyon on the large subunit. Moreover, a novel bridge is formed between the platform and the base of the L1 domain. Finally, size differences between mammalian and yeast large subunit rRNAs have been correlated with five expansion segments that form two large spines and three extended fingers. Overall, we find that expansion segments within the large subunit rRNA have been incorporated at positions distinct from the active sites for protein synthesis and translocation.

Mitochondria--tool for taxonomic identification of yeasts from Saccharomyces sensu stricto complex.

Mitochondrial genomes of Saccharomyces and close relatives previously used for transplacement of mitochondria to S. cerevisiae were examined. The origins of replication in mitochondrial DNA, the presence of nuclear and mitochondrial polymorphic loci and the ability to produce mitochondrial respiration-deficient mutants were used to reclassify some collection yeasts and to assign others into four separate subgroups. The first included isolates identical to Saccharomyces cerevisiae (S. italicus, S. oviformis, S. chevalieri and S. capensis) which possess 5 or more replication origins. The second group consists of S paradoxus (var douglasii) mitochondrial genome with the equal number of ori sequences but incompatible mitochondria. The third group represents Saccharomyces sensu stricto petite-positive species (S. carlsbergensis, S. heterogenicus, S. uvarum, S. willianus) with 1-2 origins of replication significantly different from S. cerevisiae. In addition, the locus between tRNA(fMet) and tRNA(Pro) is about one-half of the 1400 bp members of S. cerevisiae complex. The last group includes isolates that do not belong to Saccharomyces sensu stricto group as they are petite-negative and devoid of any S. cerevisiae-like replication origins.

Three-dimensional architecture of the isolated yeast nuclear pore complex: functional and evolutionary implications.

We have calculated a three-dimensional map of the yeast nuclear pore complex (yNPC) from frozen-hydrated specimens, thereby providing a direct comparison with the vertebrate NPC. Overall, the smaller yNPC is comprised of an octagonal inner spoke ring that is anchored within the nuclear envelope by a novel membrane-interacting ring. In addition, a cylindrical transporter is located centrally within the spokes and exhibits a variable radial expansion in projection that may reflect gating. The inner spoke ring, a transmembrane spoke domain, and the transporter are conserved between yeast and vertebrates; hence, they are required to form a functional NPC. However, significant alterations in NPC architecture have arisen during evolution that may be correlated with differences in nuclear transport regulation or mitotic behavior.

Uptake of yeast (Saccharomyces boulardii) in normal and rotavirus treated intestine.

BACKGROUND: There has recently been a growing interest in the use of the non-pathogenic yeast Saccharomyces boulardii, in the treatment of gastrointestinal disorders, including diarrhoea. The full effects of administration of the yeast are not fully understood. AIMS: To investigate the morphological effects of inoculated S boulardii on mouse intestinal villi, both in control animals and those treated with rotavirus. METHODS: Seven day old BALB/c seronegative mice were intubated with either rotavirus (30 microliters orally) or S boulardii (1.5 g/kg) or both rotavirus and S boulardii administered together. Control animals were given saline only. Animals were killed by decapitation 48 hours post-treatment. The middle region of the small intestine was studied using light microscopy and transmission and scanning electron microscopy, including backscattered electron imaging. RESULTS: Animals treated with rotavirus with or without S boulardii developed severe diarrhoea and showed morphological villous changes such as stromal separation and increased epithelial vacuolation. Specimens treated with S boulardii contained yeast particles within the mucosal tissues. CONCLUSION: The administration of S boulardii did not influence the changes produced by rotavirus, but yeast particles appeared to be taken up by the villous mucosa, with the predominant route apparently being uptake between adjacent epithelial cells.

Ethanol production by a mixed culture of flocculent strains of Zymomonas mobilis and Saccharomyces sp.

Pure and mixed cultures of Zymomonas mobilis and Saccharomyces sp. were tested for the production of ethanol using sucrose as the carbon source. Both strains, isolated from spontaneously fermenting sugar-cane juice, are flocculent and alcohol-tolerant. The best results were obtained using a mixed culture, with a yield of 0.5 g ethanol/g sugar consumed and a volumetric productivity of 1.5 g ethanol l-1 h-1. No levan was produced even if a sucrose-based medium was used.

Synonomy of the yeast genera Saccharomyces Meyen ex Hansen and Pachytichospora van der Walt.

The type strains and other strains of the phenotypically similar taxa Saccharomyces castelli Capriotti, Saccharomyces dairensis Naganishi, and Pachytichospora transvaalensis van der Walt were studied by comparing the ascospore morphologies of these organisms by examining ultrathin sections by transmission electron microscopy. The results of this study and another investigation of DNA base sequence homology demonstrated that the monotypic genus Pachytichospora van der Walt is invalid. We propose that the species Saccharomyces transvaalensis van der Walt should be reinstated.

Purification and some properties of membrane-bound and soluble pyrophosphatases of yeast vacuoles.

The membrane-bound and soluble pyrophosphatase (PPase) activities of Saccharomyces carlsbergensis vacuoles are determined by the functioning of special enzymes and are not due to non-specific PPi hydrolysis by other vacuolar phosphohydrolases. The molecular mass of the membrane-bound PPase is apparently 120,000 and its molecule consists of three subunits with Mr = 41,000. Soluble PPase has a molecular mass of about 82,000 and includes three subunits with Mr = 28,000. Both enzymes are glycoproteins. The vacuolar membrane-bound PPase is a proton pump.

Debaryomyces udenii, sp. nov. (Saccharomycetaceae), a new species from soil.

A new, soil-associated species of the genus Debaryomyces, D. udenii, is described. The species is characterized by pusticulate rather than verrucate ascospores, and slowly lytic asci.

Fibrous component of yeast mitochondria.

An intramitochondrial fibrous component (IMF) was consistently detected by electron microscopy in all eight strains of yeast examined, either respiratory competent or deficient, and including two species, Saccharomyces cerevisiae and S. uvarum. IMF was always found assembled into a layer 20 nm in thickness. In respiratory-deficient mutants, multiple IMF layers roll up concentrically to give rise to cylindrical inclusion bodies which attain several micrometers in length. In normal respiring yeasts, IMF occurs in close association with cristae to elaborate composite structures in which multiple parallel IMF layers are sandwiched between a pair of cristae. Evidence is presented to demonstrate the extramitochondrial origin of IMF.

Interaction with mitochondrial membranes of a synthetic peptide with a sequence common to extra peptides of mitochondrial precursor proteins.

A hepta-peptide, Arg-Leu-Leu-Pro-Ser-Leu-Gly, which has a sequence involved in the extra peptides of mitochondrial proteins, was synthesized chemically. The peptide was found to bind specifically to mitochondria, but not to microsomes. The binding was blocked by pretreatment of mitochondria with trypsin but was not affected by the presence of apocytochrome c. The synthetic peptide inhibited the binding to mitochondria of the precursor protein of ATPase inhibitor, which was synthesized in vitro, but did not inhibit that of the precursor of the 9 K stabilizing factor, which has an entirely different extra-peptide sequence. The peptide also did not inhibit the binding of apocytochrome c. These results suggest the existence of a common protein receptor on mitochondrial membranes that facilitates entrance of a group of mitochondrial precursor proteins, including pre-ATPase inhibitor.