Yeast Growth Phase Research Articles

Canine serum immunoreactivity toM. pachydermatis in vitrois influenced by the phase of yeast growth

In western immunoblotting studies of canine sera using Malassezia pachydermatis extracts, the reported patterns of immunoreactivity vary between different laboratories. Because the duration of culture influences the antigenic composition of lipid-dependent Malassezia spp. when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity using canine sera. Extracts of M. pachydermatis CBS 1879 grown in Sabouraud's liquid medium at 37 degrees C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by sodium dodecyl sulphate polyacrylamide gel electrophoresis and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and 98 and 68 kDa bands were recognized by five sera. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera used recognized more bands in exponential phase (d2) extracts when compared with decline phase (d8-d10) extracts, and two of these sera showed most bands in stationary phase (d4-d6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. There is variation in antigenic expression in different growth phases of M. pachydermatis, which might explain discrepancies between previous laboratory studies of canine immunity to this yeast.

FC‐4 Canine serum immunoreactivity to Malassezia pachydermatis is influenced by the phase of growth in vitro

Western immunoblotting studies of canine sera using Malassezia pachydermatis extracts have shown that infected dogs commonly have antibodies that recognize multiple antigens. However, reported patterns of immunoreactivity vary between different laboratories. Since culture duration influences the antigenic composition of lipid‐dependent Malassezia when probed with human sera, we investigated whether the in vitro growth phase of M. pachydermatis influences immunoreactivity to canine sera. Extracts of M. pachydermatis CBS1879 grown in Sabouraud's liquid medium at 37°C for 2, 4, 6, 8 and 10 days were prepared by mechanical disruption, centrifugation, dialysis and lyophilization. Yeast growth phase was assessed by sequential colony counts and optical density measurements. Patterns of IgG immunoreactivity in high (n = 3) and low (n = 3) titre sera were compared using extracts prepared at each time point by SDS‐PAGE and western immunoblotting. Protein bands of 62 and 49 kDa were recognized by all sera, and five sera recognized 98 and 68 kDa bands. Proteins of 188, 66, 58, 57, 38, 28 and 17 kDa were only recognized by high titre sera. All high titre sera recognized more bands in exponential phase (day 2) extracts when compared with decline phase (days 8–10) extracts, and two of these sera showed most bands in stationary phase (days 4–6) extracts. Bands of 62 and 57 kDa were primarily detected in exponential and early stationary phase extracts. Antigenic variation in extracts of M. pachydermatis prepared during different growth phases might explain discrepancies between previous laboratory studies of immunity to this yeast. Funding: Government of Malaysia.

Influence of yeast quality on performance of gnotobiotically grown Artemia

Using axenically grown Artemia, a model system was developed to evaluate the effect of bacteria on the survival and development of this crustacean. Two strains of baker's yeast ( Saccharomyces cerevisiae) were used in all experiments as feed for Artemia: a wild-type strain and its mnn9 mutant, defective in the synthesis of mannoproteins in the outer cell wall. The genetic background, yeast growth phase and growth medium appeared to be important parameters determining the quality of yeast cells as feed for Artemia. A strong positive correlation between Artemia performance and the yeast cell wall chitin and glucan content was obtained, while the mannoprotein content was negatively correlated. Mnn9 yeast cells grown till exponential phase in minimal medium proved to be excellent feed for Artemia, yielding an average 95% survival and 4-mm growth after 6 days at 28 °C, which is comparable to the best results obtained with algal feed. The standard growth test yields highly reproducible results and can become an excellent tool to study the mode of action of bacteria. Furthermore, yeast cell viability and the method used to kill/sterilize the cells are important parameters influencing nauplii performance.

Polarized hyphal growth in Candida albicans requires the Wiskott-Aldrich Syndrome protein homolog Wal1p.

The yeast-to-hypha transition is a key feature in the cell biology of the dimorphic human fungal pathogen Candida albicans. Reorganization of the actin cytoskeleton is required for this dimorphic switch in Candida. We show that C. albicans WAL1 mutants with both copies of the Wiskott-Aldrich syndrome protein (WASP) homolog deleted do not form hyphae under all inducing conditions tested. Growth of the wild-type and wal1 mutant strains was monitored by in vivo time-lapse microscopy both during yeast-like growth and under hypha-inducing conditions. Isotropic bud growth produced round wal1 cells and unusual mother cell growth. Defects in the organization of the actin cytoskeleton resulted in the random localization of actin patches. Furthermore, wal1 cells exhibited defects in the endocytosis of the lipophilic dye FM4-64, contained increased numbers of vacuoles compared to the wild type, and showed defects in bud site selection. Under hypha-inducing conditions wal1 cells were able to initiate polarized morphogenesis, which, however, resulted in the formation of pseudohyphal cells. Green fluorescent protein (GFP)-tagged Wal1p showed patch-like localization in emerging daughter cells during the yeast growth phase and at the hyphal tips under hypha-inducing conditions. Wal1p-GFP localization largely overlapped with that of actin. Our results demonstrate that Wal1p is required for the organization of the actin cytoskeleton and hyphal morphogenesis in C. albicans as well as for endocytosis and vacuole morphology.

Gts1p Activates SNF1-dependent Derepression of HSP104 and TPS1 in the Stationary Phase of Yeast Growth

We previously reported that the GTS1 product, Gts1p, plays an important role in the regulation of heat tolerance of yeast under glucose-limited conditions in either batch or continuous culture. Here we show that heat tolerance was decreased in GTS1-deleted and increased in GTS1-overexpressing cells under glucose-derepressed conditions during the batch culture and that the disruption of SNF1, a transcriptional activator of glucose-repressible genes, diminished this effect of GTS1. Intracellular levels of Hsp104 and trehalose, which were reportedly required for the acquisition of heat tolerance in the stationary phase of cell growth, were affected in both GTS1 mutants roughly in proportion to the gene dosage of GTS1, whereas those of other Hsps were less affected. The mRNA levels of genes for Hsp104 and trehalose-6-phosphate synthase 1 changed as a function of GTS1 gene dosage. The Q-rich domain of Gts1p fused with the DNA-binding domain of LexA activated the transcription of the reporter gene LacZ, and Gts1p lacking the Q-rich domain lost the activation activity of HSP104 and TPS1. Furthermore, Gts1p bound to subunits of Snf1 kinase, whereas it did not bind to DNA. Therefore, we suggested that GTS1 increases heat tolerance by mainly activating Snf1 kinase-dependent derepression of HSP104 and TPS1 in the stationary phase of yeast growth.

Potential role for extracellular glutathione-dependent ferric reductase in utilization of environmental and host ferric compounds by Histoplasma capsulatum.

The mammalian host specifically limits iron during Histoplasma capsulatum infection, and fungal acquisition of iron is essential for productive infection. H. capsulatum expresses several iron acquisition mechanisms under iron-limited conditions in vitro. These components include hydroxamate siderophores, extracellular glutathione-dependent ferric reductase enzyme, extracellular nonproteinaceous ferric reductant(s), and cell surface ferric reducing agent(s). We examined the relationship between these mechanisms and a potential role for the extracellular ferric reductase in utilization of environmental and host ferric compounds through the production of free, soluble Fe(II). Siderophores and ferric reducing agents were coproduced under conditions of iron limitation. The H. capsulatum siderophore dimerum acid and the structurally similar basidiomycete siderophore rhodotorulic acid acted as substrates for the ferric reductase, and rhodotorulic acid removed Fe(III) bound by transferrin. The mammalian Fe(III)-binding compounds hemin and transferrin served both as substrates for the ferric reductase and as iron sources for yeast-phase growth at neutral pH. In the case of transferrin, there was a correlation between the level of iron saturation and efficacy for both of these functions. Our data are not consistent with an entirely pH-dependent mechanism of iron acquisition from transferrin, as has been suggested to occur in the macrophage phagolysosome. The foreign siderophore ferrioxamine B also acted as a substrate for the ferric reductase, while the foreign siderophore ferrichrome did not. Both ferrioxamine and ferrichrome served as iron sources for yeast- and mold-phase growth, the latter presumably by some other acquisition mechanism(s).

The estimation of the yeast growth phase by nonlinear dielectric properties of the measured electric current

Abstract We measured the activity of yeast in each growth phase by using nonlinear dielectric properties. In this paper, we used a 2-electrode system, rather than the previous 4-electrode system, to measure the current waves for the analysis of the phase angle of response waves and the facilitation of the measurements. As a result, in the exciting frequency and field, 14 Hz and 0.75 V cm or 1.25 V cm −1 , the 2nd harmonic of the response wave for cell suspensions showed a significant difference between the induction phase and the others. However, for the supernatant of suspensions, there was no difference in the power spectrum of the even-numbered harmonics. With the phase angle, the same tendency was observed in the power spectrum. When another combination of field and frequency (0.25 or 1.75 V cm −1 and 7, 28, 35 or 42 Hz) was applied, there were no significant differences in either the power spectrum or the phase angle. These results were different from those measured by a 4-electrode system, although the range of frequency and field are almost the same. However, we found that the 2nd harmonics in the induction phase were different from those in the other phases. We can say that this 2-electrode system is applicable for the estimation of the growth of yeast cells.

Acetaldehyde stimulation of the growth of Saccharomyces cerevisiae in the presence of inhibitors found inlignocellulose-to-ethanol fermentations

The addition of small quantities of acetaldehyde to fermentations containing inhibitory concentrations of furfural, acetate and other compounds typically present in lignocellulosic hydrolyzates significantly reduced the lag phase of yeast growth and stimulated ethanol production. Similar effects were observed when acetaldehyde (0.06 g l−1) was added to fermentations of a birch wood hydrolyzate produced by steam/acid pretreatment. Acetaldehyde addition appears to have potential as a low-cost alternative (or adjunct) to current procedures for medium detoxification in lignocellulose-to-ethanol fermentations, particularly those in which high inhibitor concentrations are generated through recycling of the culture broth. Journal of Industrial Microbiology & Biotechnology (2000) 25, 104–108.

Technological Approach to Improve Beer Flavor Stability: Adjustments of Wort Aeration in Modern Fermentation Systems Using the Electron Spin Resonance Method

From the point of view of improving beer flavor stability, wort aeration methods used in modern fermentation technology, such as high-gravity brewing and large cylindroconical tank systems, were investigated in detail using a novel electron spin resonance method, which could determine the endogenous antioxidant activity (EA) value. The results showed that the optimization of wort aeration methods in the multifilling fermentation systems (“Drauflassen”) was essential for controlling the EA value of the finished beer as well as fermentation performance. The key point in the optimization of wort aeration methods was to consider the yeast growth phase at the time of wort aeration and the quantitative ratio of additional aeration to the first aeration depending on the fermentation conditions. Based on the extensive series of studies on improving the oxidative flavor stability of beer, the relationships between the EA value and sulfite level in beer are considered. A strategy for improving oxidative flavor stability of beer is also proposed.

Formation of Hydrogen Sulfide and Glutathione During Fermentation of White Grape Musts

Volatile sulfur compounds (VSCs) and nonvolatile thiol-containing compounds (NVTCs), which are potential precursors of VSCs, were monitored by gas chromatography during fermentation of eight white grape musts: one Thompson Seedless, one Palomino, one Chenin blanc, two Sauvignon blancs, and three Chardonnays. The assimilable amino acid (EAA) concentration of the musts ranged from 480 mg/L to 2022 mg/L (mean = 1131 mg/L). Using a GC-flame photometric detector, three sulfur containing peaks were tentatively identified based on their retention times: hydrogen sulfide (H₂S), ethylmercaptan, dimethylsulfide, and one unknown peak (RT, 9.7 min) was detected. The predominant VSC, H₂S, was continuously produced throughout fermentation and was highest during rapid growth phase of yeast. All juices produced H₂S most rapidly during rapid yeast growth, but musts with low EAA generally produced higher levels of H₂S throughout the fermentation. Total H₂S, which ranged from 112 to 516 mg/L, was inversely correlated with concentration of assimilable amino acids (<i>p</i> &lt; 0.01) and with total nitrogen content (<i>p</i> &lt; 0.10). H₂S at the end of fermentation was negatively correlated with EAA (<i>p</i> &lt; 0.01) and positively with days to dryness (<i>p</i> &lt; 0.05). For all musts, concentration of NVTCs, primarily glutathione (GSH), steadily increased towards the end of fermentation. Final wine concentration of GSH (0.1 to 5.1 mg/L) was correlated with both total N (<i>p</i> &lt; 0.01) and EAA (<i>p</i> &lt; 0.05).

Differential inhibitory effects of protoberberines on sterol and chitin biosyntheses in Candida albicans.

The anti-Candida potentials of 12 Korean medicinal plants were explored: methanol extracts from Coptis rhizoma and Phellodendron amurense caused significant inhibition of growth of Candida albicans, Candida glabrata, Candida krusei and Candida parapsilosis. The predominant active components of the extracts were the protoberberines berberine and palmatine; the most potent inhibition of growth was exhibited by berberine on C. krusei (MIC <4 mg/L) and palmatine on C. parapsilosis (MIC 16 mg/L). Both berberine and palmatine inhibited the in-vivo rate of incorporation of L-[methyl-14C]methionine into C-24 of ergosterol in C. albicans (50% inhibition concentration (IC50 values), 25 microM and 300 microM, respectively); this result suggests that sterol 24-methyl transferase (24-SMT) is one of the cellular targets for the antifungal activity of the protoberberines. In-vitro 24-SMT activity in microsomes from the yeast growth form of C. albicans was inhibited by both berberine (inhibition constant (Ki) 232 microM) and palmatine (Ki 257 microM) in a non-competitive manner; inhibition of 24-SMT was more marked for the mycelial form than for the yeast growth form of this organism. Palmatine inhibited chitin synthase from both the yeast and mycelial growth phases of C. albicans in a non-competitive manner (Ki 780 microM). The effects of protoberberines, extracted from established medicinal plants, on both sterol and cell wall biosyntheses in pathogenic fungi indicate that the potential of these compounds, or their semi-synthetic derivatives, as a novel class of antifungal agents should be investigated more fully.

Water-soluble immunomodulator produced by Trichosporon pullulans yeast

An extracellular glycoprotein exhibiting immune activity was produced by Trichosporon pullulans yeast grown in a defined ethanol-containing medium. The glycoprotein had a molecular mass of 100–150 kDa. The carbohydrate portion of the glycoprotein consisted of mannose as a predominant component, glucose, and a small amount of arabinose. This glycoprotein was named MAG. Immunological studies showed that MAG exhibited a high immunomodulating activity, revealed distinct adjuvant properties, had a wide therapeutic index, and increased non-specific resistance of mammals to infection. In batch culture, the most active MAG production (36·4 mgl 1) coincided with the phase of active yeast growth. In a chemostat under nitrogen limitation, the rate of the MAG synthesis increased with increasing a specific growth rate.

Production of Erythritol by Newly Isolated Osmophilic Trichosporon sp.

One strain of osmophilic Trichosporon sp. which produces highest amounts of erythritol, was isolated from honeycomb. This strain produced highest amounts of erythritol with total glucose utilization in fermentation medium at 35°C and a temperature of 30°C results in an insufficient glucose utilization and lower production of erythritol, but higher growth of cell mass as compared with fermentation temperature of 35°C. An efficient fermentation medium for production of erythritol by the strain was mixture of 30% glucose and 4% corn steep liquor, and using this culture medium, the strain produced 141 g/l of erythritol at 35°C after 3 days of fermentation in 5001-fermentor. It was also found that yield of microbial cells (Yx/S) to yield of erythritol (Ye/S) was 1:1 during phase of yeast growth and the proportion was changes to 1:10 during stationary phase.

Rapid fermentation of beer using an immobilized yeast multistage bioreactor system

A multistage bioreactor system for rapid beer fermentation was developed. The main fermentation process, which conventionally requires 7 d, could be shortened to 2 d by this system. The concentration of esters and higher alcohols are major factors in brewery fermentation, their production being closely related to the yeast growth phase. Yeast metabolism was successfully subdivided into a growth and a restricted phase through a combination of a continuous stirredtank reactor (CSTR) and an immobilized yeast packed-bed reactor (PBR). Production of higher alcohols was high in the CSTR because of its association with the level of biosynthetic activityde novo. A small amount was also produced in the PBR, however, possibly a result of an overflow in carbohydrate metabolism. Ester formation mainly occurred in the PBR, a linear increase in the level of ester being observed with flow through the PBR. The reactor system control strategy was to maximize the level of both higher alcohol and ester formation. The CSTR/PBR control range, based on extract consumption, was varied between 1:1 and 1:2. A ratio of 1:1 tended to create a flat beer, whereas a ratio 1:2 gave a beer of richer quality. Amino acid uptake by the yeast directly contributed to a reduction in the wort pH, whereas no relation could be observed between the level of organic acid production and pH.

Morphogenesis and adhesion of Aureobasidium pullulans

Two strains of the dimorphic fungus Aureobasidium pullulans were grown in liquid and on solid media varying in carbon and nitrogen content, and on leaf surfaces. Hyphae were observed in all systems but comprised a very low proportion (often below quantitative detection) of the total biomass. In liquid media, hyphae were found sparsely and only in the wash-zone on walls of the culture flasks. Yeast phase growth (blastospores) occurred in pH-buffered media that were nutrient balanced, or continuously carbon-limited (fed-batch culture), or carbon-exhausted (batch culture). Blastospores exposed to conditions with limited nitrogen but sufficient organic carbon, or to acidified media, converted to swollen cells and chlamydospores. The latter morphotypes accumulated carbon internally as lipid granules, and then externally as capsular and soluble extracellular polysaccharide. They were cohesive and also adhered more strongly to cellulose membranes overlying agar media or to leaves than did blastospores. Pullulanase treatment diminished the capsules, cohesion, and adhesion. Addition of soluble extracellular polysaccharide to blastospores enhanced their adhesion to leaves. We conclude that extracellular polysaccharide can play a role in adhesion of A. pullulans.Key words: phylloplane, leaf surface, epiphytic growth, extracellular polysaccharide, cell attachment.

Beta-Galactosidase of Kluyveromyces lactis (Lac4p) as reporter of gene expression in Candida albicans and C. tropicalis.

Vectors containing fusions of the Candida albicans ACT promoter to heterologous genes were constructed and transformed into a C. albicans host strain. beta-Galactosidase (Lac4p) activity was detected in transformants carrying an ACT fusion to the Kluyveromyces lactis LAC4 gene, while fusions to the Escherichia coli lacZ gene and to other heterologous genes were not expressed. Lac4p was also produced by C. tropicalis transformants carrying the ACT/LAC4 fusion. Plasmids in transformed C. albicans strains were present either as free multimers in high copy number or, more frequently, integrated into the genome in low copy number yielding high and low LAC4 mRNA and Lac4p expression levels, respectively. Lac4p-expressing transformants of C. tropicalis, but not of C. albicans, were able to utilize lactose as sole carbon source. An ACT/LAC4 fusion was not differentially expressed during the yeast and hyphal growth phases of C. albicans, indicating that the ACT promoter is not regulated during morphogenesis. These results define the first reporter gene system for convenient monitoring of gene expression in Candida species.

Expression of chitin synthase genes during yeast and hyphal growth phases of Candida albicans.

Chitin, the beta 1,4-linked polymer of N-acetylglucosamine, is a fibrous polysaccharide that in many yeasts helps to maintain the structure of the mother-bud junction and in filamentous fungi is often the major supporting component of the cell wall. We have previously described a Candida albicans chitin synthase, CHS1. The DNA and derived protein sequences of a second gene, CHS2, are presented and compared with previously published gene sequences. Northern blot analysis shows that strikingly different levels of synthase 1 and 2 expression occur during yeast and hyphal phases of Candida growth.

Urea Accumulation in Fermenting Grape Juice

Previously identified factors of yeast strain, arginine and high amino acid concentration that enhanced the accumulation of urea in fermenting grape juice were verified. It was determined that aeration of the ferment was required, and its timing important, for a significant accumulation of urea in wine. Continuous aeration caused a rapid initial accumulation of urea in the fermenting juice during the yeast growth phase, followed by a rapid and complete utilization of the urea, resulting in a minimal concentration in the final wine. Limited aerobic conditions (initial-aerobic followed by anaerobic fermentation) caused a similar rapid initial accumulation of urea, but the urea was then only partially utilized during the latter stage of fermentation. This resulted in a high concentration of urea in the final wine: the concentration being higher in a wine made from a nitrogen-rich Chardonnay juice than in that from a lower-nitrogen Rhine Riesling juice. Only very low concentrations of urea accumulated in the wine when anaerobic ferments were aerated during the latter stage of fermentation. Supplementing the ferment with excess ammonia (1 g/L) prevented accumulation of a high concentration of urea even in the presence of excess arginine and limited aeration.

Involvement of calcium, calmodulin and protein phosphorylation in morphogenesis of Candida albicans

N-Acetyl-D-glucosamine-induced germ tube formation in Candida albicans at 37 degrees C was accompanied by an increase in the rate of protein phosphorylation. The calmodulin antagonist trifluoperazine and the Ca2+ ionophore A23187, which inhibited germ tube formation, also reduced the rate of phosphorylation. The rate of phosphorylation was also reduced when cells were incubated at 25 degrees C, which favoured yeast-phase growth. Two-dimensional SDS-PAGE analysis of phosphoproteins from germ-tube-forming and yeast cells revealed two germ-tube-specific and three yeast-specific phosphoproteins. Germ tubes and hyphae had more calmodulin activity than yeast cells, irrespective of the germ-tube-inducing condition used. As a first step towards understanding the inhibitory effect of trifluoperazine on germ tube formation, calmodulin from C. albicans was purified to homogeneity. It was heat stable, and displayed a pronounced Ca2(+)-induced shift in electrophoretic mobility.

Effect of Sugar Transport Inactivation in Saccharomyces cerevisiae on Sluggish and Stuck Enological Fermentations

Sluggish and stuck (i.e., very delayed or incomplete) fermentations have been often observed in wine making. Some of them appeared to be associated with insufficient levels of yeast nutrients such as assimilable nitrogen. In these conditions, sugar transport catabolite inactivation, which is triggered by the protein synthesis arrest, may account in part for the inhibition of fermentation. Moreover, this mechanism of inhibition may explain the failure of added ammoniacal nitrogen to nitrogen-limited musts to restore or elevate rate of fermentation after the early yeast growth phase.