Absence of cell wall chitin in Saccharomyces cerevisiae leads to resistance to Kluyveromyces lactis killer toxin

Kluyveromyces lactis killer toxin causes sensitive strains of a variety of yeasts to arrest at the G1 stage of the cell cycle, and to lose viability. We describe here the isolation and characterization of a class of recessive mutations in Saccharomyces cerevisiae that leads to toxin resistance and a temperature-sensitive phenotype. These mutant cells arrest growth at 37°C with a characteristic phenotype of elongated buds. Cloning of the gene complementing these defects revealed it to be CAL1, coding for chitin synthase 3 activity. Calcofluor staining of the mutant cells indicated that chitin is absent both at 23°C and 37°C. Given that the CAL1 activity is responsible for the synthesis of most of chitin in yeast cells, and that in its absence the cells are viable but resistant to the killer toxin, our results strongly suggest that chitin might represent the receptor for this killer toxin.     

A rapid method for localized mutagenesis of yeast genes.

We have developed a simple procedure for the localized mutagenesis of yeast genes. In this technique the region of interest is first amplified under mutagenic polymerase chain reaction (PCR) conditions. Cotransformation of the PCR product with a gapped plasmid containing homology to both ends of the PCR product allows in vivo recombination to repair the gap with the mutagenized DNA. This procedure is efficient, allows targeting of specific regions for mutagenesis, and requires no subcloning steps in Escherichia coli.     

Genome‐wide screen of Saccharomyces cerevisiae for killer toxin HM‐1 resistance

We screened a set of Saccharomyces cerevisiae deletion mutants for resistance to killer toxin HM‐1, which kills susceptible yeasts through inhibiting 1,3‐beta‐glucan synthase. By using HM‐1 plate assay, we found that eight gene‐deletion mutants had higher HM‐1‐resistance compared with the wild‐type. Among these eight genes, five—ALG3, CAX4, MNS1, OST6 and YBL083C—were associated with N‐glycan formation and maturation. The ALG3 gene has been shown before to be highly resistant to HM‐1. The YBL083C gene may be a dubious open reading frame that overlaps partially the ALG3 gene. The deletion mutant of the MNS1 gene that encodes 1,2‐alpha‐mannosidase showed with a 13‐fold higher HM‐1 resistance compared with the wild‐type. By HM‐1 binding assay, the yeast plasma membrane fraction of alg3 and mns1 cells had less binding ability compared with wild‐type cells. These results indicate that the presence of the terminal 1,3‐alpha‐linked mannose residue of the B‐chain of the N‐glycan structure is essential for interaction with HM‐1. A deletion mutant of aquaglyceroporin Fps1p also showed increased HM‐1 resistance. A deletion mutant of osmoregulatory mitogen‐activated protein kinase Hog1p was more sensitive to HM‐1, suggesting that high‐osmolarity glycerol pathways plays an important role in the compensatory response to HM‐1 action. Copyright © 2010 John Wiley & Sons, Ltd.     

Interaction of SMKT, a killer toxin produced by Pichia farinosa, with the yeast cell membranes.

SMKT (salt-mediated killer toxin), a killer toxin produced by the halotolerant yeast, Pichia farinosa, kills yeasts of several genera, including Saccharomyces cerevisiae. To elucidate the killing mechanism of SMKT, we examined the interaction of SMKT with membranes using liposomes. Leakage of calcein from calcein-entrapped liposomes was observed in the presence of SMKT. Destruction of liposomes was observed by dark-field microscopy. Comparison of intact S. cerevisiae cells with SMKT-treated cells by dark-field microscopy indicated that the spherical cell membrane is disrupted by SMKT. Using sodium carbonate extraction, we obtained direct evidence for the first time that SMKT is associated with the membrane of sensitive cells. Our results indicate that SMKT kills sensitive S. cerevisiae by interacting with the yeast cell membrane.     

Brownian motion of polyphosphate complexes in yeast vacuoles: characterization by fluorescence microscopy with image analysis

In the vacuoles of Saccharomyces cerevisiae yeast cells, vividly moving insoluble polyphosphate complexes (IPCs) <1 µm size, stainable by a fluorescent dye, 4′,6‐diamidino‐2‐phenylindole (DAPI), may appear under some growth conditions. The aim of this study was to quantitatively characterize the movement of the IPCs and to evaluate the viscosity in the vacuoles using the obtained data. Studies were conducted on S. cerevisiae cells stained by DAPI and fluorescein isothyocyanate‐labelled latex microspheres, using fluorescence microscopy combined with computer image analysis (ImageJ software, NIH, USA). IPC movement was photorecorded and shown to be Brownian motion. On latex microspheres, a methodology was developed for measuring a fluorescing particle's two‐dimensional (2D) displacements and its size. In four yeast cells, the 2D displacements and sizes of the IPCs were evaluated. Apparent viscosity values in the vacuoles of the cells, computed by the Einstein–Smoluchowski equation using the obtained data, were found to be 2.16 ± 0.60, 2.52 ± 0.63, 3.32 ± 0.9 and 11.3 ± 1.7 cP. The first three viscosity values correspond to 30–40% glycerol solutions. The viscosity value of 11.3 ± 1.7 cP was supposed to be an overestimation, caused by the peculiarities of the vacuole structure and/or volume in this particular cell. This conclusion was supported by the particular quality of the Brownian motion trajectories set in this cell as compared to the other three cells. Copyright © 2010 John Wiley & Sons, Ltd.     

A rapid and selective assay for measuring cell surface hydrophobicity of brewer's yeast cells

A rapid and selective assay was developed to measure cell surface hydrophobicity of brewer's yeast cells. During this so-called magnobead assay, bottom-fermenting yeast cells adhere to paramagnetic, polystyrene-coated latex beads which can easily be removed from the cell suspension by using a (samarium-cobalt) magnet. At pH 4·5, electrostatic repulsion between yeast cells and latex beads was found to be minimal and yeast cell adhesion was predominantly based on hydrophobic interactions. The percentage of cells adhering to the beads could be calculated and provided a measure for cell surface hydrophobicity. Cell surface hydrophobicity measured by the magnobead assay was found to yield similar results, as did determination of contact angles of water droplets on a layer of yeast cells, a standard method for measuring surface hydrophobicity. However, the magnobead assay has the following advantages: (i) it is a quick and simple method, and, more significantly, (ii) hydrophobicity can be measured under physiological conditions. Use of the magnobead assay confirmed that a higher level of cell surface hydrophobicity is correlated with stronger flocculence of brewer's lager yeast cells.     

A spheroplast rate assay for determination of cell wall integrity in yeast

The rate of formation of spheroplasts of yeast can be used as an assay to study the structural integrity of cell walls. Lysis can be measured spectrophotometrically in hypotonic solution in the presence of Zymolyase, a mixture of cell wall-digesting enzymes. The optical density of the cell suspension decreases as the cells lyse. We optimized this assay with respect to enzyme concentration, temperature, pH, and growth conditions for several strains of Saccharomyces cerevisiae. The level of variability (standard deviation) was 1–5% between trials where the replications were performed on the same culture using enzyme prepared from the same lot, and 5–15% for different cultures of the same strain. This assay can quantitate differences in cell wall structure (1) between exponentially growing and stationary phase cells, (2) among different S. cerevisiae strains, (3) between S. cerevisiae and Candida albicans, (4) between parental and mutated lines, and (5) between drug- or chemically-treated cells and controls. © 1998 John Wiley & Sons, Ltd.     

A new method for quantitative determination of polysaccharides in the yeast cell wall. Application to the cell wall defective mutants of Saccharomyces cerevisiae

A reliable acid hydrolysis method for quantitative determination of the proportion of β-glucan, mannan and chitin in Saccharomyces cerevisiae cell wall is reported together with a simple extraction procedure to quantify within a standard error of less than 2% the proportion of the wall per gram of cell dry mass. This method is an optimized version of Saeman's procedure based on sulfuric acid hydrolysis of complex polysaccharides. It resulted in an almost complete release of glucose, mannose and glucosamine residues from cell wall polysaccharides. After complete removal of sulfate ions by precipitation with barium hydroxide, the liberated monosaccharides were separated and quantified by high performance anion-exchange chromatography with pulsed amperometric detection. The superiority of this method over the hydrolysis in either trifluoroacetic or hydrochloric acid resides in its higher efficiency regarding the release of glucose from β1,6-glucan and of glucosamine from chitin. The sulfuric acid method was successfully applied to determine the β-glucan, mannan and chitin contents in cell walls of genetically well-characterized yeast mutants defective in cell wall biosynthesis, and in Schizosaccharomyces pombe cell walls. The simplicity and reliability of this procedure make it the method of choice for the characterization of cell walls from S. cerevisiae mutants generated in the EUROFAN programme, as well as for other pharmacological and biotechnological applications. © 1998 John Wiley & Sons, Ltd.     

快速霉菌酵母测试片法与GB 4789.15—2016在3种类型食品中霉菌计数比较

目的比较快速霉菌酵母测试片(RYM)法和GB 4789.15—2016《食品安全国家标准食品微生物学检验霉菌和酵母计数》方法(以下简称国标法)测定3种类型食品样品中霉菌计数的一致性。方法选取90份糕点、面包、饼干样品,30份含乳饮料、植物蛋白饮料、固体饮料样品,30份保健食品样品并对3种类型食品样品进行人工污染得到的人工污染样品,利用RYM法和国标法进行霉菌计数的测定,检测结果通过配对资料t检验以及对数值差值绝对值(|dlog|)汇总分析进行统计。结果两种方法对3种类型食品样品以及人工污染样品检测结果在统计学意义上无显著性差异(P0.05);|dlog|≤0.50占比均为100.0%,表明两种方法的检测结果一致性较好。结论 RYM法和国标法对3种类型的食品样品及其人工污染样品中霉菌计数的检测结果一致性较好。     

A rapid method for the discrimination of genes encoding classical Shiga toxin (Stx) 1 and its variants, Stx1c and Stx1d, in Escherichia coli

Subtyping of Shiga toxin (Stx)-encoding genes by conventional polymerase chain reaction (PCR) is time-consuming. We developed a single step real-time fluorescence PCR with melting curve analysis to distinguish rapidly stx1 from its variants, stx1c and stx1d. Melting temperatures (Tm) of 206 Stx-producing Escherichia coli (STEC) identified to harbor stx1 or stx1c were analyzed using a specific hybridization probe over the variable region. 170 of 171 stx1-harboring STEC displayed Tm of 69 degrees C to 70 degrees C, whereas 34 of 35 strains containing stx1c had Tm of 65 degrees C-66 degrees C. This constant and reproducible difference of 4 degrees C demonstrated that melting curve analysis is a reliable technique to differentiate stx1 from stx1c. Two isolates displayed atypical Tm. Sequence analysis showed that one of them was 100% identical to stx1d within a 511 bp DNA stretch. Our data demonstrate that real-time PCR is a rapid and reliable tool to differentiate stx1 from stx1c and stx1d and to detect new stx1 variants. Because stx1-harboring STEC cause diarrhoea and hemolytic-uremic syndrome, whereas those containing stx1c are often shed asymptomatically, a rapid differentiation between stx1 and its variants using the procedure developed here has both clinical implications and a direct significance for the risk assessment analysis of STEC isolated from foods.