Sensitive Yeast Cells Research Articles

Mapping the metal-catalytic site of a zinc-activated phytochelatin synthase

Phytochelatins are small enzyme-synthesized peptides that mediate tolerance to several heavy metals. To gain insight into the unusual metal catalytic site of the Euglena gracilis phytochelatin synthase (EgPCS), site-directed mutagenesis was performed on the C-terminal Cys/His (C/H) rich region 421-C-C-X-H-X-H-X-H-H-H-430. EgPCS mutants were analyzed at the cellular, kinetic and structural levels. Cd2+-resistance was conferred to Cd2+-sensitive yeast cells by EgPCS_wt (476aa) and truncated EgPCS_435 (435aa), but not by EgPCS_415 (415aa), suggesting that the resistance phenotype was linked to the enzyme C/H rich region. Moreover, full-length mutants (in which C and H residues in the C/H rich region were replaced by Ala) EgPCS_C2 (C421A/C422A) and EgPCS_C2H5 (C421A/C422A/H424A/H426A/H428A/H429A/H430A) did not provide Cd2+-resistance; in contrast, EgPCS_H2 (H424A/H426A), EgPCS_H3 (H428A/H429A/H430A), and EgPCS_H5 (H424A/H426A/H428A/H429A/H430A) mutants conferred similar Cd2+-tolerance like EgPCS_wt. Kinetic analysis showed that maximal rate (Vmax) for PC2 synthesis, affinity constants (Km Zn-GS2 or Km Cd-GS2) and catalytic efficiencies (Vmax/Km) were differentially impaired in the mutants, as compared to EgPCS_wt, with EgPCS_C2 being the most perturbed enzyme; however, the K0.5 values for GSH were not affected. All EgPCS mutants were predominantly monomeric. Far UV circular dichroism spectra and differential scanning calorimetry endotherms, indicated that alterations of the catalytic properties of EgPCS_C2 were not due to partial unfolding or destabilization of the native state of this mutant. The results indicated that C421/C422 and H424A/H426A/H428/H429/H430 are essential components of EgPCS for catalysis and activation by metal-substrate complexes.

Binding of the exocyst complex to the SNARE regulator Sec1

Vesicles in eukaryotic cells act as intracellular carriers to transport cargo between membrane‐bound organelles and the plasma membrane. Cellular trafficking is a highly regulated process that is critical for maintaining cellular growth, homeostasis, signaling and division. These cellular trafficking events ultimately culminate in membrane fusion, where secretory vesicles release their contents to the extracellular space. However, the molecular and structural underpinnings of trafficking and membrane fusion still remains misunderstood. The process of intracellular membrane fusion relies on several conserved protein families, including SNAREs, Sec1/Munc18 (SM) proteins, multi‐subunit tethering complexes (e.g. exocyst), and Rab GTPases. Each of these regulatory factors plays multiple roles in assisting vesicle docking and SNARE assembly, indicating a trafficking system with overlapping sets of reactions. Exocyst, a hetero‐octameric protein complex, is proposed to tether vesicles to the plasma membrane and promote specific SNARE‐mediated membrane fusion. Previous binding experiments using recombinant proteins revealed interactions between two exocytic SNARE proteins and Sec1 with a single subunit of exocyst, Sec6. Currently, Sec6 is proposed to bind both binary (Sso1:Sec9), and ternary (Sso1:Sec9:Snc2) SNARE complexes during SNARE complex assembly. The timing in which Sec1 binds to the ternary SNARE complex and exocyst to mediate membrane fusion is unknown. Temperature sensitive sec1 yeast cells are defective for cell growth, SNARE complex assembly, and secretion of protein and lipid cargo at the restrictive temperature. This evidence suggests that Sec1 may play both a general and a specific role during membrane trafficking. Recent structural evidence from a mutant “activated” yeast exocyst complex shows increased flexibility and dynamics of two subunits of exocyst, Exo70 and Sec6, which lead to increased binding to SNAREs compared to wild‐type exocyst. Our study aims to further investigate the mechanisms of SNARE assembly and membrane fusion, using biochemical and EM methods to characterize the interactions of yeast exocyst, Sec1 and SNARE proteins. Using purified forms of each of the proteins and complexes, interactions between Sec1, SNARE proteins, and exocyst (wild type and activated mutant) are being analyzed and quantified.

Analysis of Yeast Killer Toxin K1 Precursor Processing via Site-Directed Mutagenesis: Implications for Toxicity and Immunity.

K1 represents a heterodimeric A/B toxin secreted by virus-infected Saccharomyces cerevisiae strains. In a two-staged receptor-mediated process, the ionophoric activity of K1 leads to an uncontrolled influx of protons, culminating in the breakdown of the cellular transmembrane potential of sensitive cells. K1 killer yeast necessitate not only an immunity mechanism saving the toxin-producing cell from its own toxin but, additionally, a molecular system inactivating the toxic α subunit within the secretory pathway. In this study, different derivatives of the K1 precursor were constructed to analyze the biological function of particular structural components and their influence on toxin activity as well as the formation of protective immunity. Our data implicate an inactivation of the α subunit during toxin maturation and provide the basis for an updated model of K1 maturation within the host cell's secretory pathway.IMPORTANCE The killer phenotype in the baker's yeast Saccharomyces cerevisiae relies on two double-stranded RNA viruses that are persistently present in the cytoplasm. As they carry the same receptor populations as sensitive cells, killer yeast cells need-in contrast to various bacterial toxin producers-a specialized immunity mechanism. The ionophoric killer toxin K1 leads to the formation of cation-specific pores in the plasma membrane of sensitive yeast cells. Based on the data generated in this study, we were able to update the current model of toxin processing, validating the temporary inactivation of the toxic α subunit during maturation in the secretory pathway of the killer yeast.

Transcriptome Kinetics of Saccharomyces cerevisiae in Response to Viral Killer Toxin K1.

The K1 A/B toxin secreted by virus-infected Saccharomyces cerevisiae strains kills sensitive cells via disturbance of cytoplasmic membrane functions. Despite decades of research, the mechanisms underlying K1 toxicity and immunity have not been elucidated yet. In a novel approach, this study aimed to characterize transcriptome changes in K1-treated sensitive yeast cells in a time-dependent manner. Global transcriptional profiling revealed substantial cellular adaptations in target cells resulting in 1,189 differentially expressed genes in total. Killer toxin K1 induced oxidative, cell wall and hyperosmotic stress responses as well as rapid down-regulation of transcription and translation. Essential pathways regulating energy metabolism were also significantly affected by the toxin. Remarkably, a futile cycle of the osmolytes trehalose and glycogen was identified probably representing a critical feature of K1 intoxication. In silico analysis suggested several transcription factors involved in toxin-triggered signal transduction. The identified transcriptome changes provide valuable hints to illuminate the still unknown molecular events leading to K1 toxicity and immunity implicating an evolutionarily conserved response at least initially counteracting ionophoric toxin action.

Production of fluorescent and cytotoxic K28 killer toxin variants through high cell density fermentation of recombinant Pichia pastoris

BackgroundVirus infected killer strains of the baker’s yeast Saccharomyces cerevisiae secrete protein toxins such as K28, K1, K2 and Klus which are lethal to sensitive yeast strains of the same or related species. K28 is somewhat unique as it represents an α/β heterodimeric protein of the A/B toxin family which, after having bound to the surface of sensitive target cells, is taken up by receptor-mediated endocytosis and transported through the secretory pathway in a retrograde manner. While the current knowledge on yeast killer toxins is largely based on genetic screens for yeast mutants with altered toxin sensitivity, in vivo imaging of cell surface binding and intracellular toxin transport is still largely hampered by a lack of fluorescently labelled and biologically active killer toxin variants.ResultsIn this study, we succeeded for the first time in the heterologous K28 preprotoxin expression and production of fluorescent K28 variants in Pichia pastoris. Recombinant P. pastoris GS115 cells were shown to successfully process and secrete K28 variants fused to mCherry or mTFP by high cell density fermentation. The fluorescent K28 derivatives were obtained in high yield and possessed in vivo toxicity and specificity against sensitive yeast cells. In cell binding studies the resulting K28 variants caused strong fluorescence signals at the cell periphery due to toxin binding to primary K28 receptors within the yeast cell wall. Thereby, the β-subunit of K28 was confirmed to be the sole component required and sufficient for K28 cell wall binding.ConclusionSuccessful production of fluorescent killer toxin variants of S. cerevisiae by high cell density fermentation of recombinant, K28 expressing strains of P. pastoris now opens the possibility to study and monitor killer toxin cell surface binding, in particular in toxin resistant yeast mutants in which toxin resistance is caused by defects in toxin binding due to alterations in cell wall structure and composition. This novel approach might be easily transferable to other killer toxins from different yeast species and genera. Furthermore, the fluorescent toxin variants described here might likewise represent a powerful tool in future studies to visualize intracellular A/B toxin trafficking with the help of high resolution single molecule imaging techniques.

Expression of K1 Toxin Derivatives in Saccharomyces cerevisiae Mimics Treatment with Exogenous Toxin and Provides a Useful Tool for Elucidating K1 Mechanisms of Action and Immunity.

Killer toxin K1 is a heterodimeric protein toxin secreted by Saccharomyces cerevisiae strains infected with the M1 double-stranded RNA ‘killer’ virus. After binding to a primary receptor at the level of the cell wall, K1 interacts with its secondary plasma membrane receptor Kre1p, eventually leading to an ionophoric disruption of membrane function. Although it has been under investigation for decades, neither the particular mechanisms leading to toxicity nor those leading to immunity have been elucidated. In this study, we constructed derivatives of the K1α subunit and expressed them in sensitive yeast cells. We show that these derivatives are able to mimic the action of externally applied K1 toxin in terms of growth inhibition and pore formation within the membrane, leading to a suicidal phenotype that could be abolished by co-expression of the toxin precursor, confirming a mechanistic similarity of external and internal toxin action. The derivatives were successfully used to investigate a null mutant completely resistant to externally applied toxin. They provide a valuable tool for the identification of so far unknown gene products involved in K1 toxin action and/or immunity.

The Biology of Pichia membranifaciens Killer Toxins.

The killer phenomenon is defined as the ability of some yeast to secrete toxins that are lethal to other sensitive yeasts and filamentous fungi. Since the discovery of strains of Saccharomyces cerevisiae capable of secreting killer toxins, much information has been gained regarding killer toxins and this fact has substantially contributed knowledge on fundamental aspects of cell biology and yeast genetics. The killer phenomenon has been studied in Pichia membranifaciens for several years, during which two toxins have been described. PMKT and PMKT2 are proteins of low molecular mass that bind to primary receptors located in the cell wall structure of sensitive yeast cells, linear (1→6)-β-d-glucans and mannoproteins for PMKT and PMKT2, respectively. Cwp2p also acts as a secondary receptor for PMKT. Killing of sensitive cells by PMKT is characterized by ionic movements across plasma membrane and an acidification of the intracellular pH triggering an activation of the High Osmolarity Glycerol (HOG) pathway. On the contrary, our investigations showed a mechanism of killing in which cells are arrested at an early S-phase by high concentrations of PMKT2. However, we concluded that induced mortality at low PMKT2 doses and also PMKT is indeed of an apoptotic nature. Killer yeasts and their toxins have found potential applications in several fields: in food and beverage production, as biocontrol agents, in yeast bio-typing, and as novel antimycotic agents. Accordingly, several applications have been found for P. membranifaciens killer toxins, ranging from pre- and post-harvest biocontrol of plant pathogens to applications during wine fermentation and ageing (inhibition of Botrytis cinerea, Brettanomyces bruxellensis, etc.).

Antimicrobial properties and death-inducing mechanisms of saccharomycin, a biocide secreted by Saccharomyces cerevisiae.

We recently found that Saccharomyces cerevisiae (strain CCMI 885) secretes antimicrobial peptides (AMPs) derived from the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH) that are active against various wine-related yeast and bacteria. Here, we show that several other S. cerevisiae strains also secrete natural biocide fractions during alcoholic fermentation, although at different levels, which correlates with the antagonistic effect exerted against non-Saccharomyces yeasts. We, therefore, term this biocide saccharomycin. The native AMPs were purified by gel-filtration chromatography and its antimicrobial activity was compared to that exhibited by chemically synthesized analogues (AMP1 and AMP2/3). Results show that the antimicrobial activity of the native AMPs is significantly higher than that of the synthetic analogues (AMP1 and AMP2/3), but a conjugated action of the two synthetic peptides is observed. Moreover, while the natural AMPs are active at pH 3.5, the synthetic peptides are not, since they are anionic and cannot dissolve at this acidic pH. These findings suggest that the molecular structure of the native biocide probably involves the formation of aggregates of several peptides that render them soluble under acidic conditions. The death mechanisms induced by the AMPs were also evaluated by means of epifluorescence microscopy-based methods. Sensitive yeast cells treated with the synthetic AMPs show cell membrane disruption, apoptotic molecular markers, and internalization of the AMPs. In conclusion, our work shows that saccharomycin is a natural biocide secreted by S. cerevisiae whose activity depends on the conjugated action of GAPDH-derived peptides. This study also reveals that S. cerevisiae secretes GAPDH-derived peptides as a strategy to combat other microbial species during alcoholic fermentations.

Die Bildung von Killertoxin und die Beeinflussung der Gärung durch Apiculatus-Hefen

Formation of killer toxin by apiculate yeasts and interfernnce with fermentation Several strains of the frequently occurring apiculate yeast ( Hanseniaspora uvarum ) form a killer toxin (a protein) which inhibits a sensitive wine yea st ( Saccharomyces cereirisiae ). This toxin was partially concentrated by lyophilization and precipitation with e thanol. The toxin activity was determined with the agar diffusion method. The optimum of activity was observed at pH 4, but the killer toxin is also active at pH 3.5, the pH of grape must. The presence of apiculate killer yeasts s lowed the fermentation of a sensitive wine yeast ( S. cerevisiae 381). In mixed cultures of the killer strain H. uvarum 470 with S. cerevisiae the maximum inhibition of the latter was at 20 °C and at pH 3.5. Within 2 d about 95 % of the sensitive yeast cells were killed. Several days later the wine yeast resumed fermentation. The killer toxin delayed the normal fermentation by 10- 20 d. Depending on the ratio of cell numbers between the sensitive wine yeast and the apiculate killer yeast, the fermentation was suppressed for up to 15 d. When the cell number of wine yeasts was less than 0.01 % of the killer yeast cells ( H. uvarum 470) all sensitive cells were killed. Even at an inoculum of 106 cells/ml of S. cereirisiae a delay in the normal fermentation was observed when the sensitive yeast was added 2 d after the apiculate killer strain. By adding bentonite at a concentration of 0.01 % the interference of killer toxin with fermentation was prevented. The delay of normal fermentation which was caused by killer strains of H. uvarwn was not only observed in laboratory media but a lso in experiments with grape must.

TdKT, a new killer toxin produced by Torulaspora delbrueckii effective against wine spoilage yeasts

Microbiological spoilage is a major concern throughout the wine industry, and control tools are limited. This paper addresses the identification and partial characterization of a new killer toxin from Torulaspora delbrueckii with potential biocontrol activity of Brettanomyces bruxellensis, Pichia guilliermondii, Pichia manshurica and Pichia membranifaciens wine spoilage. A panel of 18 different wine strains of T. delbrueckii killer yeasts was analysed, and the strain T. delbrueckii NPCC 1033 (TdKT producer) showed a significant inhibitory effect on the growth of all different spoilage yeasts evaluated. The TdKT toxin was then subjected to a partial biochemical characterization. Its estimated molecular weight was >30kDa and it showed glucanase and chitinase enzymatic activities. The killer activity was stable between pH4.2 and 4.8 and inactivated at temperature above 40°C. Pustulan and chitin — but not other cell wall polysaccharides — prevented sensitive yeast cells from being killed by TdKT, suggesting that those may be the first toxin targets in the cell wall. TdKT provoked an increase in necrosis cell death after 3h treatment and apoptotic cell death after 24h showing time dependence in its mechanisms of action. Killer toxin extracts were active at oenological conditions, confirming their potential use as a biocontrol tool in winemaking.

Evidence for DNA Cleavage Caused Directly by a transfer RNA-Targeting Toxin

The killer yeast species Pichia acaciae produces a heteromeric killer protein, PaT, that causes DNA damage and arrests the cell cycle of sensitive Saccharomyces cerevisiae in the S phase. However, the mechanism by which DNA damage occurs remains elusive. A previous study has indicated that Orf2p, a subunit of PaT, specifically cleaves an anticodon loop of an S. cerevisiae transfer RNA (tRNAGln mcm5s2UUG). This finding raised a question about whether the DNA damage is a result of the tRNA cleavage or whether Orf2p directly associates with and cleaves the genomic DNA of sensitive yeast cells. We showed that Orf2p cleaves genomic DNA in addition to cleaving tRNA in vitro. This DNA cleavage requires the same Orf2p residue as that needed for tRNA cleavage, His299. The expression of Orf2p, in which His299 was substituted to alanine, abolished the cell cycle arrest of the host cell. Moreover, the translation impairment induced by tRNA cleavage enabled Orf2p to enter the nucleus, thereby inducing histone phosphorylation.

A novel killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b

In our previous study, it was found that the killer toxin produced by the marine-derived yeast Wickerhamomyces anomalus YF07b has both killing activity and β-1,3-glucanase activity and the molecular mass of it is 47.0 kDa. In this study, the same yeast strain was found to produce another killer toxin which only had killing activity against some yeast strains, but had no β-1,3-glucanase activity and the molecular mass of the purified killer toxin was 67.0 kDa. The optimal pH, temperature and NaCl concentration for action of the purified killer toxin were 3.5, 16 °C and 4.0 % (w/v), respectively. The purified killer toxin could be bound by the whole sensitive yeast cells, but was not bound by manann, chitin and β-1,3-glucan. The purified killer toxin had killing activity against Yarrowia lipolytica, Saccharomyces cerevisiae, Metschnikowia bicuspidata WCY, Candida tropicalis, Candida albicans and Kluyveromyces aestuartii. Lethality of the sensitive cells treated by the newly purified killer toxin from W. anomalus YF07b involved disruption of cellular integrity by permeabilizing cytoplasmic membrane function.

Specific phase arrest of cell cycle restores cell viability against tRNA cleavage by killer toxin

Zymocin and PaT are killer toxins that induce cell cycle arrest of sensitive yeast cells in G1 and S phase, respectively. Recent studies have revealed that these two toxins cleave specific tRNAs, indicating that the cell growth impairment is due to the tRNA cleavage. Additionally, we have previously shown that the active domain of colicin D (D-CRD), which also cleaves specific Escherichia coli tRNAs, statically impairs growth when expressed in yeast cells. To verify that phase-specific cell cycle arrest is also induced by the expression of D-CRD, D-CRD and the subunits of zymocin and PaT that have tRNA cleaving activity were expressed in yeast cells and cell cycle status was analyzed. Our results indicate that phase-specific arrest does not commonly occur by tRNA cleavage, and it saves the cell viability. Furthermore, the extent of protein synthesis impairment may determine the phase specificity of cell cycle arrest.

Decondensation ofXenopussperm chromatin usingSaccharomyces cerevisiaewhole-cell extractsThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

Biochemical studies using highly condensed Xenopus sperm chromatin and protein extracts prepared from multiple systems have lead to the identification of conserved proteins involved in chromosome decondensation. However, mutations to these proteins are unavailable as the systems used are not amenable to genetic studies. We took a genetic approach to isolating chromosome decondensation mutants by incubating Xenopus sperm chromatin with whole-cell extracts prepared from the Hartwell library of random temperature sensitive (ts) yeast cells. We show that decondensation of Xenopus sperm chromatin using wild type yeast extracts was rapid, ATP- and extract-dependent, and resistant to heat, N-ethylmaleimide, protease K, RNase A, and micrococcal nuclease. From 100 mutant extracts screened, we obtained one strain, referred to as rmc4, that was chromosome decondensation defective. The mutant was slow growing and exhibited germination defects. Low concentrations of rmc4 extract would eventually decondense sperm heads, and fractionation of the mutant extract produced a decondensation competent fraction, suggesting the presence of an overactive inhibitor in rmc4 cells. We performed a multicopy suppressor screen that identified PDE2, a gene encoding a protein that inhibits protein kinase A (PKA) activity. As PKA was previously shown in human cells to maintain condensed chromatin, our results suggest that PKA activity is elevated in rmc4 cells, causing a decondensation defect. Thus, our experiments reveal that yeast encodes an evolutionarily conserved chromosome decondensation activity that can be genetically manipulated.

The Transcriptional Response of Saccharomyces cerevisiae to Pichia membranifaciens Killer Toxin

The transcriptional response of Saccharomyces cerevisiae to Pichia membranifaciens killer toxin (PMKT) was investigated. We explored the global gene expression responses of the yeast S. cerevisiae to PMKT using DNA microarrays, real time quantitative PCR, and Northern blot. We identified 146 genes whose expression was significantly altered in response to PMKT in a non-random functional distribution. The majority of induced genes, most of them related to the high osmolarity glycerol (HOG) pathway, were core environmental stress response genes, showing that the coordinated transcriptional response to PMKT is related to changes in ionic homeostasis. Hog1p was observed to be phosphorylated in response to PMKT implicating the HOG signaling pathway. Individually deleted mutants of both up- (99) and down-regulated genes (47) were studied for altered sensitivity; it was observed that the deletion of up-regulated genes generated hypersensitivity (82%) to PMKT. Deletion of down-regulated genes generated wild-type (36%), resistant (47%), and hypersensitive (17%) phenotypes. This is the first study that shows the existence of a transcriptional response to the poisoning effects of a killer toxin.

The Kluyveromyces lactis γ-toxin targets tRNA anticodons

Kluyveromyces lactis killer strains secrete a heterotrimeric toxin (zymocin), which causes an irreversible growth arrest of sensitive yeast cells. Despite many efforts, the target(s) of the cytotoxic gamma-subunit of zymocin has remained elusive. Here we show that three tRNA species tRNA(Glu)(mcm(5)s(2)UUC), tRNA(Lys)(mcm(5)s(2)UUU), and tRNA(Gln)(mcm(5)s(2)UUG) are the targets of gamma-toxin. The toxin inhibits growth by cleaving these tRNAs at the 3' side of the modified wobble nucleoside 5-methoxycarbonylmethyl-2-thiouridine (mcm(5)s(2)U). Transfer RNA lacking a part of or the entire mcm(5) group is inefficiently cleaved by gamma-toxin, explaining the gamma-toxin resistance of the modification-deficient trm9, elp1-elp6, and kti11-kti13 mutants. The K. lactis gamma-toxin is the first eukaryotic toxin shown to target tRNA.

Genetic, Biochemical, and Transcriptional Responses of Saccharomyces cerevisiae to the Novel Immunomodulator FTY720 Largely Mimic Those of the Natural Sphingolipid Phytosphingosine

Sphingolipids are signaling molecules that influence diverse cellular functions from control of the cell cycle to degradation of plasma membrane proteins. The synthetic sphingolipid-like compound FTY720 is an immunomodulating agent in clinical trials for transplant graft maintenance. In this report, we compare the effects of the natural yeast sphingolipid phytosphingosine with FTY720 in Saccharomyces cerevisiae. We show that the multicopy suppressor genes that induce growth resistance to FTY720 also confer resistance to growth-inhibitory concentrations of phytosphingosine. In addition, mutants for ubiquitination pathway proteins are shown to be resistant to the growth-inhibiting effect of both FTY720 and phytosphingosine. We observe fewer similarities between sphingosine and FTY720 than between FTY720 and phytosphingosine as revealed by genetic studies. Yeast cells lacking the specific sphingosine kinase LCB4 are sensitive to phytosphingosine and FTY720 but resistant to sphingosine, suggesting that FTY720 and phytosphingosine have a more related mechanism of action. Gene expression profile comparisons of sensitive and resistant yeast cells exposed to FTY720 and phytosphingosine highlight a number of similarities. In response to treatment with these compounds, approximately 77% of the genes that are regulated >2-fold by FTY720 also respond to phytosphingosine in the same direction in the parent strain. In addition, a close inspection of TAT1 and TAT2 transporters following exposure to phytosphingosine indicates that TAT1 protein is degraded in a similar fashion upon treatment with FTY720 and phytosphingosine. There were differences, however, with respect to the TAT2 protein level and the expression profiles of a subset of genes. The genetic, transcriptional, and biochemical data together indicate that FTY720 and phytosphingosine influence similar pathways in yeast cells. These findings offer further insights into the physiological pathways influenced by these compounds in all eukaryotic cells and help us to understand the therapeutic consequences of FTY720 in humans.

New Type of Ethanol Microbial Biosensor Based on a Highly Sensitive Amperometric Oxygen Electrode and Yeast Cells

A microbial biosensor for ethanol determination was prepared by immobilization of yeast cells of Saccharomyces ellipsoideus on the surface of an oxygen electrode with electrolyte in nonaqueous medium. Analytical determination is based on the respiratory activity of the microorganism in presence of the analyte. Response time of approximately 5 min for steady-state method and 2 min for kinetic method was registered. A lower detection limit of 6 μM was achieved comparing with previously reported biosensor. The major interfering compound in ethanol assay was glucose. Use of a second PTFE membrane to cover the biocatalytical yeast layer allowed developing a highly selective biosensor for ethanol.

A binary screening assay for pro-oestrogens in food: metabolic activation using hepatic microsomes and detection with oestrogen sensitive recombinant yeast cells

An assay, employing microsomes prepared from rat liver and a recombinant cell bioassay (RCBA) expressing the human oestrogen receptor (α) linked to a reporter gene, was evaluated for the detection of pro-oestrogens in food using methoxychlor and mestranol as model compounds. Bio-activation of the hop phytoestrogen isoxanthohumol to the potent oestrogen 8-prenylnaringenin was also investigated. The oestrogenic potency values for reference standards determined with the RCBA (17 β-oestradiol = 100%) were: methoxychlor 0.0025%, mestranol 1.3%, isoxanthohumol 0.001%, and for their potential respective metabolites were: bishydroxymethoxychlor 0.015%, 17 α-ethynyl oestradiol 69% and 8-prenylnaringenin 0.4%. Incubation of methoxychlor and mestranol (10 μ M) with microsomes prepared from the liver of rats treated with Aroclor 1254 significantly increased (p < 0.001) their oestrogenic potency from 0.0021 and 2.4% to 0.015 and 8.3%, respectively. In contrast, the potency of the hop phytoestrogen isoxanthohumol was unchanged. Metabolites were identified by UV-HPLC-MS/MS as monohydroxy methoxychlor and HPTE from methoxychlor, and the major metabolite of mestranol was 17 α-ethynyl oestradiol. There was no evidence for the metabolism of isoxanthohumol. Mestranol was also activated by microsomes induced with saline (control), β -napthoflavone, 3-methylcholantherene, isoniazid or pregnenolone-16α -carbonitrile, but not phenobarbitone. These studies demonstrate the principle for use of a binary assay system for the detection of pro-oestrogens and indicate the potential value for risk assessment of endocrine disrupting chemicals.

Involvement of β-Glucans in the Wide-Spectrum Antimicrobial Activity of Williopsis saturnus var. mrakii MUCL 41968 Killer Toxin

Williopsis saturnus var. mrakii MUCL 41968 secretes a 85-kDa glycoprotein killer toxin (WmKT) that displays a cytocidal activity against a wide range of microorganisms, making WmKT a promising candidate for the development of new antimicrobial molecules. Although the killing mechanism of WmKT is still unknown, the toxin was recently proposed to bind to the surface of sensitive microorganisms through the recognition of beta-glucans. Indeed, Saccharomyces cerevisiae strains sensitive to the toxin become resistant when mutated in their beta-glucan synthesis pathway. To investigate the interaction of WmKT with beta-glucans, we examined in agar diffusion assays the WmKT activity in the presence of enzymes displaying beta-glucanase activity. The toxin activity was also investigated using spheroplasts derived from sensitive yeast cells. The hydrolytic activity of WmKT was studied using specific glucosidase inhibitors as well as various sugar molecules covalently linked to p-nitrophenyl as potential substrates. Finally, the ultrastructural modifications induced by WmKT activity on sensitive yeasts were assessed by scanning electron microscopy. The data reported here support the hypothesis that WmKT binds to sensitive cells using surface-exposed beta-glucans. Indeed beta-glucanase exerts an antagonistic effect on WmKT activity and spheroplasts derived from WmKT-sensitive yeast cells are shown to be resistant to WmKT, suggesting that cell wall beta-glucans are required for WmKT lethal effect. Because WmKT exhibits amino acid sequence similarities with proteins suspected to be glucanase, we also investigated the effect of castanospermine, a potent glucosidase inhibitor, on WmKT activity. Castanospermine completely abolished WmKT killer activity as well as its hydrolytic enzymatic activity against p-nitrophenyl beta-D-glucopyranoside. The scanning electron microscopy analysis of sensitive yeast cells treated with the toxin reveals that WmKT causes cell wall modifications similar to those observed with zymolyase. The results reported in this study show that WmKT activity requires an interaction between the mycocin and the cell wall beta-glucans. Moreover, they indicate that WmKT acts on sensitive yeast cells through a hydrolytic activity directed against cell wall beta-glucans that disrupts the yeast cell wall integrity leading to death.