Use of PMA1 as a Housekeeping Biomarker for Assessment of Toxicant-Induced Stress in Saccharomyces cerevisiae

The brewer's yeast Saccharomyces cerevisiae has emerged as a versatile and robust model system for laboratory use to study toxic effects of various substances. In this study, toxicant-induced stresses of pure compounds were investigated in Saccharomyces cerevisiae utilizing a destabilized version of the green fluorescent protein optimized for expression in yeast (yEGFP3) under control of the promoter of the housekeeping plasma membrane ATPase gene PMA1. The responses of the biomarker upon increasing test compound concentrations were monitored by determining the decrease in fluorescence. The reporter assay deployed a simple and robust protocol for the rapid detection of toxic effects within a 96-well microplate format. Fluorescence emissions were normalized to cell growth determined by absorption and were correlated to internal reference standards. The results were expressed as effective concentrations (EC₂₀). Dose-response experiments were conducted in which yeast cells were exposed in minimal medium and in the presence of 20% fetal calf serum to sublethal concentrations of an array of heavy metals, salt, and a number of stress-inducing compounds (Diclofenac, Lindane, methyl-N-nitro-N-nitrosoguanidine [MNNG], hydroxyurea, and caffeine). Long-term exposure (7 h) played a considerable role in the adaptive response to intoxication compared to early responses at 4 h exposure. The data obtained after 4 h of exposure and expressed as EC₂₀ were compared to 50% inhibitory concentration values derived from cell line and ecotoxicological tests. This study demonstrates the versatility of the novel biomarker to complement existing test batteries to assess contaminant exposure and effects.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

Phenotype character of the methylglyoxal resistance gene in Saccharomyces cerevisiae: expression in Escherichia coli and application to breeding wild-type yeast strains.

The gene responsible for the methylglyoxal resistance of Saccharomyces cerevisiae was cloned, and its phenotypic characteristics were investigated. S. cerevisiae cells with the gene could accumulate large amounts of glutathione in the medium and should remarkably high resistance to various toxic compounds such as methylglyoxal, tetramethylthiuram disulfide, iodoacetamide, and heavy-metal ions. The gene was also expressed in Escherichia coli cells, and the resistance of E. coli cells to toxic compounds also increased as observed for S. cerevisiae cells. The phenotypic characteristics of the gene were applicable to the selection of the transformants of wild-type yeast strains having no genetic markers.     

A prototype microfluidic chip using fluorescent yeast for detection of toxic compounds

A microfluidic chip has been developed to enable the screening of chemicals for environmental toxicity. The microfluidic approach offers several advantages over macro-scale systems for toxicity screening, including low cost and flexibility in design. This design flexibility means the chips can be produced with multiple channels or chambers which can be used to screen for different toxic compounds, or the same toxicant at different concentrations. Saccharomyces cerevisiae containing fluorescent markers are ideal candidates for the microfluidic screening system as fluorescence is emitted without the need of additional reagents. Microfluidic chips containing eight multi-parallel channels have been developed to retain yeast within the chip and allow exposure of them to toxic compounds. The recombinant yeast used was GreenScreentrade mark which expresses green fluorescent proteins when is exposed to genotoxins. After exposure of the yeast to target compounds, the fluorescence emission was detected using an inverted microscope. Qualitative and quantitative comparisons of the fluorescent emission were performed. Results indicated that fluorescent intensity per area significantly increases upon exposure to methyl-methanesulfonate, a well known genotoxic compound. The microfluidic approach reported here is an excellent tool for cell-based screening and detection of different toxicities. The device has the potential for use by industrial manufacturers to detect and reduce the production and discharge of toxic compounds, as well as to characterise already polluted environments.     

酿酒酵母X330高浓度发酵时耐酒精性能的初步研究

在完全合成培养基条件下,就渗透压保护剂和营养物质对一株产高浓度酒精的酿酒酵母X330高浓度发酵时耐酒精性能的影响进行了初步研究。结果表明,与渗透压相比,营养缺乏对酿酒酵母高浓度发酵时酒精耐受性能可能起着更为关键和重要的作用。发酵培养基中各营养元素对耐酒精性能的影响不同,由高到低的顺序是酵母抽提物>蛋白胨>硫酸镁>维生素C=磷酸二氢钾>氯化钙=硫酸铵。渗透压保护剂(甘氨酸和脯氨酸)能有效提高菌体酒精耐受性能。当甘氨酸添加浓度为20mmol/L或脯氨酸添加浓度为10mmol/L时,发酵终点酒精浓度最高,菌体于30℃在18%(V/V)酒精冲击下的存活率最大,且均高于对照组(未添加甘氨酸且未添加脯氨酸)水平,但甘氨酸的促进作用强于脯氨酸。     

Saccharomyces cerevisiae cells harboring the gene encoding sarcotoxin IA secrete a peptide that is toxic to plant pathogenic bacteria.

Sarcotoxin IA is a cecropin-type antibacterial protein produced by the flesh fly, Sarcophaga peregrina. Similar to other bactericidal small proteins produced by insects, sarcotoxin IA is released into the hemolymph of larvae and nymphs upon mechanical injury or bacterial infection. The gene (sarco) that encodes this toxin was introduced into Saccharomyces cerevisiae yeast cells and was expressed under a constitutive yeast promoter. The transformed yeast cells were grown in a liquid medium, and a peptide with a similar molecular size to that of the mature sarcotoxin IA was detected in the medium by Western blot analysis. The secreted sarcotoxin-like peptide (SLP) had a potent cytotoxic effect against several bacteria, including plant pathogenic bacteria, similar to the toxic effects of the authentic sarcotoxin IA. Erwinia carotovora was more susceptible to the toxic medium than Pseudomonas solanacearum and Pseudomonas syringae pv. lachrymans. Thus, yeast may be used in the production of such proteins for employment against various bacterial pathogens.     

Toxicity and mutagenicity of selenium compounds in Saccharomyces cerevisiae

Selenium (Se) is an essential trace element for humans, animals and some bacteria which is important for many cellular processes. Se's bio-activity is mainly influenced by its chemical form and dose. The use of Se supplements in the human diet emphasizes the need to establish both the beneficial and detrimental doses of each Se compound. We have evaluated three different Se compounds, sodium selenite (SeL), selenomethionine (SeM) and Se-methylselenocysteine (SeMC), with respect to their potential DNA damaging effects. The budding yeast Saccharomyces cerevisiae was used as a model system to test the toxic and mutagenic effects as well as the DNA double-strand breakage potency of these Se compounds in both exponentially growing and stationary yeast cells. Only SeL manifested any significant toxic effects in the yeast which were more pronounced in the exponentially growing cells than in those cells in the stationary phase of growth. The toxic effects of SeL were however accompanied with the pro-mutagenic effects in the stationary cell phase of growth. The toxic and mutagenic effects of SeL are likely associated with the ability of this compound to generate DNA double-strand breaks (DSB). We also show that SeL significantly increased frame-shift mutations, especially 1-4 bp deletions, in the CAN1 mutational spectrum of the yeast genome when compared to untreated control. We propose that SeL is acting as an oxidizing agent in S. cerevisiae producing superoxide and oxidative damage to DNA accounting for the observed DSB and cell death.     

Adenosine accumulation in Saccharomyces cerevisiae cultured in medium containing low levels of adenine.

By monitoring the in vivo incorporation of low concentrations of radiolabeled adenine into acid-soluble compounds, we observed the unusual accumulation of two nucleosides in Saccharomyces cerevisiae that were previously considered products of nucleotide degradation. Under the culture conditions used in the present study, radiolabeled adenosine was the major acid-soluble intracellular derivative, and radiolabeled inosine was initially detected as the second most prevalent derivative in a mutant lacking adenine aminohydrolase. The use of yeast mutants defective in the conversion of adenine to hypoxanthine or to AMP renders very unlikely the possibility that the presence of adenosine and inosine is attributable to nucleotide degradation. These data can be explained by postulating the existence of two enzyme activities not previously reported in S. cerevisiae. The first of these activities transfers ribose to the purine ring and may be attributable to purine nucleoside phosphorylase (EC 2.4.2.1) or adenosine phosphorylase (EC 2.4.2.-). The second enzyme converts adenosine to inosine and in all likelihood is adenosine aminohydrolase (EC 3.5.4.4).     

A positive effect of Propionibacterium freudenreichii on the growth of Saccharomyces cerevisiae during their cocultivation

The growth pattern of Saccharomyces cerevisiae and Propionibacterium freudenreichii ssp. shermanii (P. shermanii; propionic acid bacteria, PABs) during cocultivation in liquid media depended on the ratio of the cells in the inoculum. An increase in the growth rate of S. cerevisiae was observed at a PAB to yeast ratio of approximately 3 : 1; higher ratios exerted adverse effects on yeast growth. The culture liquid of 18- to 24-h (young) cultures of PABs stimulated yeast growth. Although yeast growth-stimulating exometabolites of PABs were not high-molecular-weight compounds, they were thermolabile. When present in the medium at concentrations of up to 1.5%, the antimicrobial agent sodium propionate did not interfere with S. cerevisiae growth; however, it completely inhibited the growth of B. subtilis at a concentration of 0.2%.     

Antimicrobial activity of aroma compounds against Saccharomyces cerevisiae and improvement of microbiological stability of soft drinks as assessed by logistic regression

The combined effects of a mild heat treatment (55 degrees C) and the presence of three aroma compounds [citron essential oil, citral, and (E)-2-hexenal] on the spoilage of noncarbonated beverages inoculated with different amounts of a Saccharomyces cerevisiae strain were evaluated. The results, expressed as growth/no growth, were elaborated using a logistic regression in order to assess the probability of beverage spoilage as a function of thermal treatment length, concentration of flavoring agents, and yeast inoculum. The logit models obtained for the three substances were extremely precise. The thermal treatment alone, even if prolonged for 20 min, was not able to prevent yeast growth. However, the presence of increasing concentrations of aroma compounds improved the stability of the products. The inhibiting effect of the compounds was enhanced by a prolonged thermal treatment. In fact, it influenced the vapor pressure of the molecules, which can easily interact within microbial membranes when they are in gaseous form. (E)-2-Hexenal showed a threshold level, related to initial inoculum and thermal treatment length, over which yeast growth was rapidly inhibited. Concentrations over 100 ppm of citral and thermal treatment longer than 16 min allowed a 90% probability of stability for bottles inoculated with 10(5) CFU/bottle. Citron gave the most interesting responses: beverages with 500 ppm of essential oil needed only 3 min of treatment to prevent yeast growth. In this framework, the logistic regression proved to be an important tool to study alternative hurdle strategies for the stabilization of noncarbonated beverages.     

Design and application of a biosensor for monitoring toxicity of compounds to eukaryotes

Here we describe an alternative approach to currently used cytotoxicity analyses through applying eukaryotic microbial biosensors. The yeast Saccharomyces cerevisiae was genetically modified to express firefly luciferase, generating a bioluminescent yeast strain. The presence of any toxic chemical that interfered with the cells' metabolism resulted in a quantitative decrease in bioluminescence. In this study, it was demonstrated that the luminescent yeast strain senses chemicals known to be toxic to eukaryotes in samples assessed as nontoxic by prokaryotic biosensors. As the cell wall and adaptive mechanisms of S. cerevisiae cells enhance stability and protect from extremes of pH, solvent exposure, and osmotic shock, these inherent properties were exploited to generate a biosensor that should detect a wide range of both organic and inorganic toxins under extreme conditions.     

Antimicrobial and Biological Effects of Bomphos and Phomphos on Bacterial and Yeast Cells

In this study, the antimicrobial effects of monophosphazenes such as SM, BOMPHOS, and PHOMPHOS were examined on bacterial and yeast strains. In addition, the biological effects of these compounds were tested on the Saccharomyces cerevisiae and Candida albicans cells. The SM has an antimicrobial effect on the bacterial and yeast strains within the range of 100 and 1500 μg. When the concentration was increased, the inhibition zone expanded on the growth media (P < 0.01; P < 0.001). Like SM, BOMPHOS molecule has antimicrobial activity on the bacterial and yeast cells. The most effective concentrations of BOMPHOS on the microorganisms were observed by 1500 μg (P < 0.001). The PHOMPHOS did not effect on the bacterial and yeast cells between 100 and 1000 range, but it has an antimicrobial effect in 1500 μg. In vitro media, the biological effects of these molecules were compared with vitamin E, melatonin, and fish oil on the yeast cells. In S. cerevisiae growth media, the cell densities were increased SM, BOMPHOS, and PHOMPHOS after 20, 30, and 45 h. The highest increase in the cell density were observed in media of BOMPHOS. In C. albicans growth media, the cell density was increased by melatonin after 20, 30, and 45 h, but were decreased by other supplemental groups. Lipid level of S. cerevisiae was reduced by administered 300 and 1000 μg vitamin E and fish oil (P < 0.01). In addition, the lipid level of the same yeast cell were diminished by the 1000 μg melatonin and 300 μg PHOMPHOS (P < 0.05, P < 0.01). The lipid level of C. albicans were increased by vitamin E and BOMPHOS and fish oil, but was decreased with PHOMPHOS (P < 0.01). In conclusion, while high concentration of PHOMPHOS has antimicrobial effects on the bacterial and yeast cells, the SM and BOMPHOS have antimicrobial effects in all the concentrations. PHOMPHOS decreased the lipid level of C. albicans, but BOMPHOS increased in the the same yeast cell. In addition, the antioxidants such as vitamin E, melatonin, and fish oils have affected on the lipid synthesis of yeast cells. Copyright 2000 Academic Press.     

Highly conserved toxicity of Saccharomyces cerevisiae Rap1p

Budding yeast ( Saccharomyces cerevisiae ) Rap1p has been expressed in fission yeast ( Schizosaccharo-myces pombe ) under the control of the regulatable fructose bisphosphatase ( fbp ) promoter. When the fbp promoter was derepressed, cells containing the complete RAP1 gene failed to show any significant growth, suggesting that Rap1p is toxic. A derivative of Rap1p that has a temperature-sensitive mutation in the DNA-binding domain was not toxic in cells grown at 37°C, a temperature at which DNA binding by rap1p ^ts is severely inhibited. Removal of a short region downstream of the DNA-binding domain, including a region previously shown to be essential for Rap1p toxicity in budding yeast, also abolished the toxic effect. The toxic effect of Rap1p has therefore been conserved between two distantly related yeasts. In budding yeast, overexpression of Rap1p also caused changes to the lengths of the telomeric repeats. No effects on telomeres were detected in fission yeast.     

Toxic effects caused by heavy metals in the yeast Saccharomyces cerevisiae: a comparative study

The decreasing order of toxicity of select heavy metals on the yeast Saccharomyces cerevisiae, in 10 mM MES (2-(N-morpholino)ethanesulfonic acid) pH buffer at pH 6.0, was found to be copper, lead, and nickel. Heavy metal (200 microM) induced a decrease in the number of viable cells by about 50% in the first 5 min for copper and in 4 h for lead, while nickel was not toxic up to a 200 microM concentration over a period of 48 h. Glucose (25 mM) strongly enhanced the toxic effect of 50 microM copper but had little or no effect on the toxicity of 200 microM lead or nickel. Copper, lead, and nickel induced the leakage of UV260-absorbing compounds from cells with different kinetics. The addition of 0.5 mM calcium, before addition of 200 microM copper, showed a protective action against cell death and decreased the release of UV-absorbing compounds, while no effect was observed against lead or nickel toxic effects. Copper complexation capacities of the filtrates of cells exposed for 2 h in 200 microM copper and 24 h in 200 microM lead were 51 and 14 microM, respectively. The implication of the complexation shown by these soluble compounds in the bioavailability of heavy metals is discussed.     

Bacterial toxin-antitoxin gene system as containment control in yeast cells

The potential of a bacterial toxin-antitoxin gene system for use in containment control in eukaryotes was explored. The Escherichia coli relE and relB genes were expressed in the yeast Saccharomyces cerevisiae. Expression of the relE gene was highly toxic to yeast cells. However, expression of the relB gene counteracted the effect of relE to some extent, suggesting that toxin-antitoxin interaction also occurs in S. cerevisiae. Thus, bacterial toxin-antitoxin gene systems also have potential applications in the control of cell proliferation in eukaryotic cells, especially in those industrial fermentation processes in which the escape of genetically modified cells would be considered highly risky.     

Functional co-expression of xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 and UDP-glucuronosyltransferase 1A6, in yeast microsomes

Xenobiotic Phase I and Phase II reactions in hepatocytes occur sequentially and cooperatively during the metabolism of various chemical compounds including drugs. In order to investigate the sequential metabolism of 7-ethoxycoumarin (7EC) as model substrate in vitro, xenobiotic metabolizing enzymes, rat cytochrome P450 1A1 (P450 1A1) and UDP-glucuronosyltransferase 1A6 (UGT1A6) were co-expressed in Saccharomyces cerevisiae AH22. Rat P450 1A1 and yeast NADPH-P450 reductase were expressed on a multicopy plasmid (pGYR1) in the yeast. Rat UGT1A6 cDNA with a yeast alcohol dehydrogenase I promoter and terminator was integrated into yeast chromosomal DNA to achieve the stable expression. Co-expression of P450 1A1 and UGT1A6 in yeast microsomes was confirmed by immunoblot analysis. Protease treatment of the microsomes showed the correct topological orientation of UGT to the membranes. The metabolism of 7EC to 7-hydroxycoumarin (7HC) and its glucuronide in yeast microsomes was analyzed by reverse phase HPLC. In a co-expression system containing 7EC, NADPH and UDP-glucuronic acid, glucuronide formation was detected after a lag phase, following the accumulation of 7HC. In the case of P450 1A1 and UGT1A6, efficient coupling of hydroxylation and glucuronidation in 7EC metabolism was not observed in the co-expression system. This P450 and UGT co-expression system in yeast allows the sequential biotransformation of xenobiotics to be simulated in vitro.     

Cell density-dependent linoleic acid toxicity to Saccharomyces cerevisiae

Since the discovery of the apoptotic pathway in Saccharomyces cerevisiae, several compounds have been shown to cause apoptosis in this organism. While the toxicity of polyunsaturated fatty acids (PUFA) peroxides towards S. cerevisiae has been known for a long time, studies on the effect of nonoxidized PUFA are scarce. The present study deals specifically with linoleic acid (LA) in its nonoxidized form and investigates its toxicity to yeast. Saccharomyces cerevisiae is unable to synthesize PUFA, but can take up and incorporate them into its membranes. Reports from the literature indicate that LA is not toxic to yeast cells. However, we demonstrated that yeast cell growth decreased in cultures treated with 0.1 mM LA for 4 h, and 3-(4,5 dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide reduction (a measure of respiratory activity) decreased by 47%. This toxicity was dependent on the number of cells used in the experiment. We show apoptosis induction by LA concomitant with increases in malondialdehyde, glutathione content, activities of catalase and cytochrome c peroxidase, and decreases in two metabolic enzyme activities. While the main purpose of this study was to show that LA causes cell death in yeast, our results indicate some of the molecular mechanisms of the cell toxicity of PUFA.     

Expression of functional soluble forms of human beta-1, 4-galactosyltransferase I, alpha-2,6-sialyltransferase, and alpha-1, 3-fucosyltransferase VI in the methylotrophic yeast Pichia pastoris

The cDNAs encoding soluble forms of human beta-1, 4-galactosyltransferase I (EC 2.4.1.22), alpha-2,6-sialyltransferase (EC 2.4.99.1), and alpha-1,3-fucosyltransferase VI (EC 2.4.1.65), respectively, have been expressed in the methylotrophic yeast Pichia pastoris. The vector pPIC9 was used, which contains the N-terminal signal sequence of Saccharomyces cerevisiae alpha-factor to allow entry into the secretory pathway. The recombinant enzymes had similar kinetic properties as their native counterparts. Their identity was confirmed by Western blotting. Recombinant enzymes may be used for in vitro synthesis of oligosaccharides.