A simple method for bakers' yeast cell disruption using a three-phase fluidized bed equipped with an agitator

A three-phase fluidized bed equipped with a turbine agitator was utilized as a simple device for disrupting bakers' yeast cells (Saccharomyces cerevisiae). The degree of yeast cell disruption was evaluated based on the number of broken cells and its validity was confirmed by the total amount of crude soluble proteins released and by microscopic observation. It was found that the equipment could yield 90% of yeast cell disruption. With the presence of glass beads, the degree of cell disruption became higher as agitating speed is increased. The disruption enhancement would be attributed to the grinding effect resulting from the interaction between yeast cells and glass beads. One-thousand micrometers of glass beads yielded a higher degree of disruption than larger ones. An increase in liquid flow rate hindered the degree of disruption because of shorter contact time although the shear rates in the yeast suspension would become more rigorous.     

Rapid isolation of yeast genomic DNA: Bust n' Grab

Background

Mutagenesis of yeast artificial chromosomes (YACs) often requires analysis of large numbers of yeast clones to obtain correctly targeted mutants. Conventional ways to isolate yeast genomic DNA utilize either glass beads or enzymatic digestion to disrupt yeast cell wall. Using small glass beads is messy, whereas enzymatic digestion of the cells is expensive when many samples need to be analyzed. We sought to develop an easier and faster protocol than the existing methods for obtaining yeast genomic DNA from liquid cultures or colonies on plates.

Results

Repeated freeze-thawing of cells in a lysis buffer was used to disrupt the cells and release genomic DNA. Cell lysis was followed by extraction with chloroform and ethanol precipitation of DNA. Two hundred ng – 3 μg of genomic DNA could be isolated from a 1.5 ml overnight liquid culture or from a large colony. Samples were either resuspended directly in a restriction enzyme/RNase coctail mixture for Southern blot hybridization or used for several PCR reactions. We demonstrated the utility of this method by showing an analysis of yeast clones containing a mutagenized human β-globin locus YAC.

Conclusion

An efficient, inexpensive method for obtaining yeast genomic DNA from liquid cultures or directly from colonies was developed. This protocol circumvents the use of enzymes or glass beads, and therefore is cheaper and easier to perform when processing large numbers of samples.

     

一种简便的适用于酵母双杂交系统的酵母质粒提取方法

目的:建立一种适用于酵母双杂交系统的简便快捷的酵母质粒提取方法。方法:以酿酒酵母为供试材料,用玻璃珠振荡法破除酵母细胞壁,提取酵母总DNA,最后通过电转化大肠杆菌DH10B获得目的质粒。结果:粗提得到的质粒可直接转化DH10B,作为模板用于PCR分析及酵母双杂交后续的序列分析等,大大降低了工作量。结论:该方法简便快捷,经济实用,降低了成本,提高了效率,可以作为一种实验室酵母质粒提取方法。     

Comamonas testosteroni colony phenotype influences exopolysaccharide production and coaggregation with yeast cells.

A Comamonas testosteroni strain was isolated from activated sludge on the basis of its ability to coaggregate with yeast cells. On agar plates the following two types of colonies were formed: colonies with a mucoid appearance and colonies with a nonmucoid appearance. On plates this strain alternated between the two forms, making sectored colonies. In liquid medium with constant agitation no such change was observed. In the absence of agitation and in contact with a glass surface a culture with predominantly nonmucoid-colony-forming cells very rapidly shifted to a culture dominated by mucoid-colony-forming cells. In liquid medium the reverse was observed under stress conditions imposed by hydrogen peroxide, sodium dodecyl sulfate, or starvation. Nonmucoid cells formed very rapidly settling flocs with yeast cells, while coaggregation of mucoid cells with yeast cells did not occur. These findings may be relevant to the behavior of activated sludge microbial communities.     

Totally chlorine-free bleaching of flax pulp

Invertase and urease are enzyme entities highly associated with the cells of the astaxanthin-producer yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) during any stage of its cell growth cycle. In this study cellobiose was a more efficient carbon source than sucrose or its hexose counterparts for invertase expression. Extensive ultrasonication or abrasion with glass pearls were required in order to promote enzyme release. In contrast to the yeast whose growth declines above 27 degrees C, the released enzymes displayed a higher optimum temperature range when assayed in vitro. Isoforms from both enzymes could be resolved either by FPLC on DEAE-Sepharose or by an affinity approach on immobilized Concanavalin. The zymogram for invertase showed a pI somewhat less acidic than that of the similar enzyme from S. cerevisiae.     

Overexpression of multisubunit replication factors in yeast.

Facile genetic and biochemical manipulation coupled with rapid cell growth and low cost of growth media has established the yeast Saccharomyces cerevisiae as a versatile workhorse. This article describes the use of yeast expression systems for the overproduction of complex multipolypeptide replication factors. The regulated overexpression of these factors in yeast provides for a readily accessible and inexpensive source of these factors in large quantities. The methodology is illustrated with the five-subunit replication factor C. Whole-cell extracts are prepared by blending yeast cells with glass beads or frozen yeast with dry ice. Procedures are described that maximize the yield of these factors while minimizing proteolytic degradation.     

Assessment of the intracellular pH of immobilized and continuously perfused yeast cells employing fluorescence ratio imaging analysis

The intracellular pH (pHin) of Saccharomyces cerevisiae was measured employing fluorescence ratio imaging microscopy (FRIM). The yeast cells were fluorescently labeled with the pH dependent probe 5(and-6)-carboxyfluorescein (cF) or 5(and-6)-carboxyfluorescein succinimidyl ester (cFSE), and subsequently attached to ferric nitrate pretreated glass slides. The labeled and adhered cells could still divide and were metabolically active. Measurement of the pHin was performed during continuous perfusion of the cells with buffer or medium. Cells labeled with cF are highly fluorescent and in non-energized cells the pHin could be easily measured. However, in energized yeast cells cF was accumulated in the vacuoles and/or exported to the extracellular environment, most likely by an energy-dependent transport system, thus limiting the time period over which the pHin can be effectively measured. Therefore, cFSE (which conjugates with aliphatic amines in the cytoplasm) was applied to prevent translocation of fluorescent probe to the vacuole and/or extracellular environment. The continuous perfusion in combination with the cFSE labeling of the immobilized cells was successfully applied to determine the effect of low and high pHin and addition of glucose on the pHin of individual yeast cells over a long time period.     

Mechanical disintegration of microorganisms in an industrial homogenizer

Disintegration of microorganisms in a continuously working industrial homogenizer has been studied. The homogenizer consists of rotating discs in a cylinder filled with glass beads. Different parameters for disintegration of baker's yeast were investigated. The disintegration process is a first-order reaction and it is influenced by the flow rate of the suspension and by the agitator speed. At a flow rate of 200 liters/hr about 85% of the yeast cells can be disrupted in a single pass through the disintegrator. This type of disintegrator can be used for disruption of cells in order to produce single-cell protein, active enzymes and other valuable cell components.     

Measurement of plasma membrane potentials of yeast cells with glass microelectrodes

Glass microelectrodes were used to measure the electrical potential difference (Δψ) across plasma membrane of the yeast Pichia humboldtii. The cells were captured in the neck of a glass microfunnel and impaled with a glass microelectrode. The measurements were reproducible and stable for several minutes. The highest Δψ values were obtained in cells metabolizing glucose at pH 6. Δψ in cells deenergized by uncouplers or in dead cells was reduced to about one third of the maximal value. This residual Δψ probably represented Donnan potential. Δψ also was reduced by increasing concentrations of K⁺ in the medium. Other monovalent cations were distinctly less effective: Li⁺ ⪡ Na⁺ < K⁺, and Ca²⁺ was without effect. These experiments prove the applicability of the electrophysiological technique on yeast cells and thus open the way for direct determination of the electrical component of the plasma membrane electrochemical proton gradient.     

Cellular immune response in Rhodnius prolixus: role of ecdysone in hemocyte phagocytosis

In this paper we investigate in vivo and in vitro effects of orally administered azadirachtin and ecdysone on the phagocytic responses of Rhodnius prolixus 5th-instar larval hemocytes to the yeast Saccharomyces cerevisiae. Groups of insects fed non-treated blood (control) and insects that received azadirachtin plus ecdysone in the blood meal were inoculated with yeast cells in the hemocele. The injected yeast cells disappeared rapidly from the hemolymph, being removed completely by 90min after inoculation. In the insects treated only with azadirachtin the clearance of free yeast circulating particles was significantly delayed compared to the two previously mentioned groups. It was demonstrated that the binding of yeast cells to hemocytes was reduced in the insects treated only with azadirachtin in comparison to both non-treated control and azadirachtin plus ecdysone-treated groups. Phagocytosis occurred when yeast cells were added to hemocyte monolayers prepared with hemolymph from blood fed insects, treated or not with azadirachtin plus ecdysone, so that yeast cells were rapidly bound to hemocytes and internalized in high numbers. By contrast, insects treated with azadirachtin exhibited a drastic reduction in the quantity of yeast cell-hemocyte binding and subsequent internalization. In all groups, the hemocytes attached to the glass slides were predominantly plasmatocytes. The magnitude and speed of the cellular response suggests that hemocyte phagocytosis is one of the main driving forces for the clearance of free circulating yeast cells from the hemolymph. We propose that ecdysone modulates phagocytosis in R. prolixus larvae, and that this effect is antagonized by azadirachtin.     

Experimente mit Hefe

Why bread dough rises, although yeast cannot metabolize starch: Experiments with yeast Yeast is ideal for simple experiments to visualize metabolic processes, such as those known from baking bread, for example. Thus the metabolism of different sugars to carbon dioxide can be qualitatively demonstrated by inflating balloons or quantitatively by gas production in a fermentation tube (glass airlock for fermentation). The experiments presented can be used to answer the question of why yeast is added to bread dough even though it is not able to convert the starch present in the flour.     

Calcium and Saccharomyces cerevisiae

The claim that Ca may be a dispensable element for yeast Saccharomyces cerevisiae has been reexamined. The cells of S. cerevisiae could grow in media which contained no added Ca and were deprived of contaminating Ca²⁺ by filtration through a Chelex 100 column. Also, the cells were able to grow in the presence of fairly high concentrations of EGTA. The apparent intracellular concentrations of Ca, assessed from the content of radioactive ⁴⁵Ca in cells preloaded with ⁴⁵CaCl₂, could vary within the range of approx. 2 nM to 2.8 mM, without adversively affecting growth or morphology of the cells. An extremely low affinity for Ca²⁺ of the system taking up Ca into the cells was corroborated. However, even the Chelex 100-treated media were found in contain 1–5 μM Ca when maintained in glass culture vessels. Also, the ability of the cells to take up Ca from a medium containing surplus of EGTA or EDTA was demonstrated. su14CEDTA, alone or in the presence of Ca, could also be transported into the cells. It has been inferred that Ca must be as essential for yeast as it is for other eucaryotic organisms. The omnipresence of contaminating Ca and peculiarities of the Ca transporting system, combined with an intricate intracellular compartmentation of Ca, would account for the impossibility to prove the importance of Ca for yeast by direct growth studies.     

Continuous High-resolution Microscopic Observation of Replicative Aging in Budding Yeast

We demonstrate the use of a simple microfluidic setup, in which single budding yeast cells can be tracked throughout their entire lifespan. The microfluidic chip exploits the size difference between mother and daughter cells using an array of micropads. Upon loading, cells are trapped underneath these micropads, because the distance between the micropad and cover glass is similar to the diameter of a yeast cell (3-4 μm). After the loading procedure, culture medium is continuously flushed through the chip, which not only creates a constant and defined environment throughout the entire experiment, but also flushes out the emerging daughter cells, which are not retained underneath the pads due to their smaller size. The setup retains mother cells so efficiently that in a single experiment up to 50 individual cells can be monitored in a fully automated manner for 5 days or, if necessary, longer. In addition, the excellent optical properties of the chip allow high-resolution imaging of cells during the entire aging process.     

RNA isolation from yeast using silica matrices.

RNA isolation from yeast is complicated by the need to initially break the cell wall. While this can be accomplished by glass bead disruption or enzyme treatment, these approaches result in DNA contamination and/or the need for incubation periods. We have developed a protocol for the isolation of RNA samples from yeast that minimizes degradation by RNases and incorporates two purification steps: acid phenol extraction and binding to a silica matrix. The procedure requires no precipitation steps, facilitating automation, and can be completed in less than 90 min. The RNA quality is ideal for microarray analysis.     

Yeast cell adhesion on oligopeptide modified surfaces

Self-assembling oligopeptides are novel materials with potential bioengineering applications; this paper explores the use of one of these oligopeptides, EAK 16 II, for modifying the surface properties of cell-supporting substrates. To characterize the surface properties, thermodynamic measurements of liquid contact angle and surface free energy were correlated to atomic force microscopy (AFM) observations. A critical concentration of 0.1 mg/ml was found necessary to completely modify the surface properties of the substrate with EAK 16 II. Adhesion of a yeast cell, Candida utilis, was modified by the coating of EAK 16 II on both hydrophobic (plastic) and hydrophilic (glass) surfaces: Cell coverage was slightly enhanced on the glass substrate, but decreased significantly on the plastic substrate. This indicates that the yeast cell adhesion was mainly determined via hydrophobic interactions between the substrate and the cell wall. However, on the EAK 16 II modified glass substrate, surface roughness might be a factor in causing a slightly larger cell adhesion than that on bare glass. The morphology of adhered cells was also obtained with AFM imaging, showing a depression at the center of the cell on all substrates. Small depressions on the oligopeptide-coated surfaces and plastic substrate may indicate good water-retaining ability by the cell. There was no apparent difference in cell adhesion and morphology among cells obtained from lag, exponential and stationary growth phases.     

The dynamic behaviour of yeast cells immobilised in porous glass studied by membrane mass spectrometry

Membrane mass spectrometry (MMS) with reduced sample withdrawal has been used to investigate the metabolic activity of yeast cells immobilised in porous glass. An adapted MS membrane inlet reactor with a polyethylene terephthalate barrier membrane has been constructed for this purpose. In a first experiment, the mass transport of O₂ in a porous glass disc under well-defined experimental conditions has been studied by determining the apparent effective diffusion coefficient. The behaviour of immobilised Saccharomyces cerevisiae has been monitored by the MMS measurement of O₂ and CO₂ after applying a step in glucose concentration. Free-cell kinetic parameters were used in a dynamic reaction-diffusion model to simulate the O₂ consumption curve. The theoretical and experimental curve showed comparable behaviour, which means that the immobilisation of yeast cells in porous glass has no substantial effect on its growth kinetics.     

Isolation of Polyribosomes from Yeast During Sporulation and Vegetative Growth

Exponentially growing and sporulating cells of Saccharomyces cerevisiae have been subjected to a variety of conditions which mechanically disrupt the cell in an effort to establish conditions which permit the recovery of intact polyribosomes. Grinding cells for 10 s with glass beads in a Bronwill cell homogenizer was sufficiently gentle to yield a polyribosome content in exponentially growing cells which was similar to values obtained from yeast spheroplasts. Polyribosome patterns in sporulating yeast were similar to those from exponentially growing cells. This technique is fast, reproducible over a wide range of cell concentrations, and eliminates the need to make spheroplasts to recover intact polyribosomes.     

Motile properties of the kinesin-related Cin8p spindle motor extracted from Saccharomyces cerevisiae cells

We have developed microtubule binding and motility assays for Cin8p, a kinesin-related mitotic spindle motor protein from Saccharomyces cerevisiae. The methods examine Cin8p rapidly purified from crude yeast cell extracts. We created a recombinant form of CIN8 that fused the biotin carrying polypeptide from yeast pyruvate carboxylase to the carboxyl terminus of Cin8p. This form was biotinated in yeast cells and provided Cin8p activity in vivo. Avidin-coated glass surfaces were used to specifically bind biotinated Cin8p from crude extracts. Microtubules bound to the Cin8p-coated surfaces and moved at 3.4 +/- 0.5 micrometer/min in the presence of ATP. Force production by Cin8p was directed toward the plus ends of microtubules. A mutation affecting the microtubule-binding site within the motor domain (cin8-F467A) decreased Cin8p's ability to bind microtubules to the glass surface by >10-fold, but reduced gliding velocity by only 35%. The cin8-3 mutant form, affecting the alpha2 helix of the motor domain, caused a moderate defect in microtubule binding, but motility was severely affected. cin8-F467A cells, but not cin8-3 cells, were greatly impaired in bipolar spindle forming ability. We conclude that microtubule binding by Cin8p is more important than motility for proper spindle formation.     

Receptor elements for biosensors in two ways of methylotrophic yeast immobilization

Receptor elements for biosensors based on Hansenula polymorpha NCYC 495 ln yeast cells for ethanol assay were developed using two ways of cell immobilization, i.e., physical adsorption on a glass fiber membrane and covalent binding on a modified nitrocellulose membrane. The linear diapason of ethanol assays for a biosensor based on yeast cells adsorbed on glass fiber was 0.05–1.18; for a biosensor based on yeasts immobilized on a nitrocellulose membrane, 0.2–1.53 mM. Receptor elements based on sorbed cells possessed 2.5 times higher long-term stability. The time response was 1.5 times less for cells immobilized using DEAE-dextran and benzoquinone. The results of ethyl alcohol assays using biosensors based on cells immobilized via adsorption and covalent binding, as well as using the standard areometric method, had high correlation coefficients (0.998 and 0.997, respectively, for the two ways of immobilization). The results indicate the possibility to consider the described models of receptor elements for biosensors as prototypes for experimental samples for practical use.     

A glass bead treatment facilitating the fixation and infiltration of yeast and other refractory cells for electron microscopy

Summary Physical removal of the cell wall of yeast and other fungal cells, by rapid mixing of the cells with glass beads after preliminary fixation in glutaraldehyde or formalin, removes the cell wall barrier to fixation and/or infiltration of the cells for electron microscopy. The technique has been used on a variety of fungal cells which have been difficult to fix for electron microscopy, and appears to have wide applicability.