Effect of 2-amino-2-thiazoline on the sulfhydryl group content of yeasts of various degrees of radiosensitivity

The influence of 2-amino-2-thiazoline on the level of total sulphydryl groups in the haploid and diploid yeast cells Saccharomyces cerevisiae and Pichia guilliermondii has been studied. The increase of radioresistance of the yeast cells has been shown to be connected with the growing content of the endogenous thiols.     

Transformation of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts by a plasmid carrying the ADE2 gene of Saccharomyces cerevisiae

Red adenine-dependent mutants of Hansenula polymorpha, Pichia guilliermondii, Williopsis saturnus yeasts have been transformed by the plasmid pYE (ADE2) 2 DNA containing ADE2 gene from Saccharomyces cerevisiae. The analysis of two P. guilliermondii Ade+-transformants has revealed the integration of pYE (ADE2)2 sequence into the recipient strain genome and partial restoration of the deficient function.     

The role of glutathione in natural and modified radioresistance of yeast cells

A study was made of the dependence of different natural and modified radioresistance upon glutathione content of yeast cells (Saccharomyces cerevisiae and Pichia guilliermondii). It was shown that glutathione was only involved in the formation of natural radioresistance in Saccharomyces cerevisiae cells. It was also shown that the increase in the radioresistance of yeast cells under the effect of 2-amino-2-thiazoline was accompanied by the increase in the level of total glutathione in them.     

Identification of the genes affecting the regulation of riboflavin synthesis in the flavinogenic yeast Pichia guilliermondii using insertion mutagenesis

Pichia guilliermondii is a representative of a group of so-called flavinogenic yeast species that overproduce riboflavin (vitamin B(2)) in response to iron limitation. Using insertion mutagenesis, we isolated P. guilliermondii mutants overproducing riboflavin. Analysis of nucleotide sequence of recombination sites revealed that insertion cassettes integrated into the genome disrupting P. guilliermondii genes similar to the VMA1 gene of Ashbya gossypii and Saccharomyces cerevisiae and FES1 and FRA1 genes of S. cerevisiae. The constructed P. guilliermondiiΔvma1-17 mutant possessed five- to sevenfold elevated riboflavin production and twofold decreased iron cell content as compared with the parental strain. Pichia guilliermondiiΔfra1-45 mutant accumulated 1.8-2.2-fold more iron in the cells and produced five- to sevenfold more riboflavin as compared with the parental strain. Both Δvma1-17 and Δfes1-77 knockout strains could not grow at 37 °C in contrast to the wild-type strain and the Δfra1-45 mutant. Increased riboflavin production by the wild-type strain was observed at 37 °C. Although the Δfes1-77 mutant did not overproduce riboflavin, it showed partial complementation when crossed with previously isolated P. guilliermondii riboflavin-overproducing mutant rib80-22. Complementation analysis revealed that Δvma1-17 and Δfra1-45 mutants are distinct from previously reported riboflavin-producing mutants hit1-1, rib80-22 and rib81-31 of this yeast.     

Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae.

Pichia anomala inhibits the growth of Penicillium roqueforti and Aspergillus candidus on agar. In this investigation, antagonistic activity on agar against 17 mold species was determined. The abilities of Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae to inhibit the growth of the mold Penicillium roqueforti in nonsterile high-moisture wheat were compared by adding 10(3) Penicillium roqueforti spores and different amounts of yeast cells per gram of wheat. Inoculated grain was packed in glass tubes, incubated at 25 degrees C with a restricted air supply, and the numbers of yeast and mold CFU were determined on selective media after 7 and 14 days. Pichia anomala reduced growth on agar plates for all of the mold species tested in a dose-dependent manner. Aspergillus fumigatus and Eurotium amstelodami were the most sensitive, while Penicillium italicum and Penicillium digitatum were the most resistant. Pichia anomala had the strongest antagonistic activity in wheat, with 10(5) and 10(6) CFU/g completely inhibiting the growth of Penicillium roqueforti. Inhibition was least pronounced at the optimum temperature (21 degrees C) and water activity (0.95) for the growth of Penicillium roqueforti. Pichia guilliermondii slightly reduced the growth of Penicillium roqueforti in wheat inoculated with 10(5) and 10(6) yeast CFU/g. S. cerevisiae inhibited mold growth only weakly at the highest inoculum level. Pichia anomala grew from 10(3) to 10(7) CFU/g of wheat in 1 week. To reach the same level, Pichia guilliermondii had to be inoculated at 10(4) CFU while S. cerevisiae required an inoculum of 10(5) CFU to reach 10(7) CFU/g of wheat.     

Development of a transformation system for gene knock-out in the flavinogenic yeast Pichia guilliermondii

Pichia guilliermondii is a representative of a yeast species, all of which over-synthesize riboflavin in response to iron deprivation. Molecular genetic studies in this yeast species have been hampered by a lack of strain-specific tools for gene manipulation. Stable P. guilliermondii ura3 mutants were selected on the basis of 5'-fluoroorotic acid resistance. Plasmid carrying Saccharomyces cerevisiae URA3 gene transformed the mutant strains to prototrophy with a low efficiency. Substitution of a single leucine codon CUG by another leucine codon CUC in the URA3 gene increased the efficiency of transformation 100 fold. Deletion cassettes for the RIB1 and RIB7 genes, coding for GTP cyclohydrolase and riboflavin synthase, respectively, were constructed using the modified URA3 gene and subsequently introduced into a P. guilliermondii ura3 strain. Site-specific integrants were identified by selection for the Rib(-) Ura(+) phenotype and confirmed by PCR analysis. Transformation of the P. guilliermondii ura3 strain was performed using electroporation, spheroplasting or lithium acetate treatment. Only the lithium acetate transformation procedure provided selection of uracil prototrophic, riboflavin deficient recombinant strains. Depending on the type of cassette, efficiency of site-specific integration was 0.1% and 3-12% in the case of the RIB1 and RIB7 genes, respectively. We suggest that the presence of the ARS element adjacent to the 3' end of the RIB1 gene significantly reduced the frequency of homologous recombination. Efficient gene deletion in P. guilliermondii can be achieved using the modified URA3 gene of S. cerevisiae flanked by 0.8-0.9 kb sequences homologous to the target gene.     

Arsenite-induced lipid peroxidation in Saccharomyces cerevisiae

The ability of sodium arsenite at concentrations of 10(-2), 10(-4), and 10(-6) M to induce lipid peroxidation in Saccharomyces cerevisiae cells was studied. Arsenite at the concentrations 10(-2) and 10(-4) M enhanced lipid peroxidation and inhibited the growth of yeast cells. Enhanced lipid peroxidation likely induced oxidative damage to various cellular structures, which led to suppression of the metabolic activity of cells. Arsenite at the concentration 10(-6) M did not activate lipid peroxidation in cells. All of the tested arsenite concentrations inhibited the activity of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase in cells. The inference is made that the toxicity of arsenite may be related to its stimulating effect on intracellular lipid peroxidation.     

Mediated amperometry reveals two distinct modes of yeast responses to glucose

Mediated amperometry was exploited to monitor intracellular redox activity without cell disruption. Continuous measurements of menadione-mediated glucose currents at carbon paste electrodes with various immobilized intact wild type yeasts (Saccharomyces cerevisiae, Candida pulcherrima, Clavispora lusitaniae, Wickerhamomyces anomalus, Pichia guilliermondii, Kluyveromyces lactis var. lactis, Debaryomyces hansenii, Candida zeymolaydes and Candida tropicalis) revealed two distinct and previously unreported modes of development of the currents during the first 2 to 3 min. after subjection to glucose. A correlation among the values of the currents and the capacities of wild type yeasts to secrete various substances was observed.     

Comparative analysis of the structure of SUP2 genes in Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1 alpha-like protein factor, involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. A SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of temperature-sensitive sup2 mutation of S. cerevisiae. Nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82.4 kDa exceeding the SUP2 protein of S. cerevisiae for 6 kDa. The SUP2 gene product of P. pinus is similar to the Sup2 protein of S. cerevisiae by its structure and includes a highly conservative (76%) C-terminal region homologus to EF-1 alpha and a lowly conservative N-terminal region. The relation between the evolutionary conservativity of different regions of the Sup2 protein and their functional significance is discussed.     

Induction of lipid peroxidation during heavy metal stress in Saccharomyces cerevisiae and influence of plasma membrane fatty acid unsaturation.

The degree of plasma membrane fatty acid unsaturation and the copper sensitivity of Saccharomyces cerevisiae are closely correlated. Our objective was to determine whether these effects could be accounted for by differential metal induction of lipid peroxidation. S. cerevisiae S150-2B was enriched with the polyunsaturated fatty acids (PUFAs) linoleate (18:2) and linolenate (18:3) by growth in 18:2- or 18:3-supplemented medium. Potassium efflux and colony count data indicated that sensitivity to both copper (redox active) and cadmium (redox inactive) was increased in 18:2-supplemented cells and particularly in 18:3-supplemented cells. Copper- and cadmium-induced lipid peroxidation was rapid and associated with a decline in plasma membrane lipid order, detected by fluorescence depolarization measurements with the membrane probe trimethylammonium diphenylhexatriene. Levels of thiobarbituric acid-reactive substances (lipid peroxidation products) were up to twofold higher in 18:2-supplemented cells than in unsupplemented cells following metal addition, although this difference was reduced with prolonged incubation up to 3 h. Conjugated-diene levels in metal-exposed cells also increased with both the concentration of copper or cadmium and the degree of cellular fatty acid unsaturation; maximal levels were evident in 18:3-supplemented cells. The results demonstrate heavy metal-induced lipid peroxidation in a microorganism for the first time and indicate that the metal sensitivity of PUFA-enriched S. cerevisiae may be attributable to elevated levels of lipid peroxidation in these cells.     

Comparison of glucose uptake kinetics in different yeasts.

The kinetics of glucose uptake were investigated in laboratory wild-type strains of Saccharomyces cerevisiae of differing genetic backgrounds, in other species of Saccharomyces, and in other yeasts, both fermentative and respiratory. All yeasts examined displayed more than one uptake system for glucose. Variations in apparent Km values, velocity of uptake, and effects of glucose concentration on carrier activity were observed. The three type strains for the species S. cerevisiae, Saccharomyces bayanus, and Saccharomyces carlsbergensis gave distinctive patterns, and each of the laboratory strains was similar to one or another of the type strains. Other fermentative yeasts (Pichia guillermondi and Pichia strasburgensis) regulated glucose uptake in a manner similar to that of Saccharomyces spp. Such was not true for the respiratory yeasts investigated, Pichia heedi and Yarrowia lipolytica, which did not demonstrate glucose repression of carrier activity; this finding suggests that this mechanism of control of transporter activity may be associated with fermentative ability.     

Glycerol production by yeasts under osmotic and sulfite stress.

The yeasts Saccharomyces cerevisiae, Candida boidinii, Pichia augusta, and Pichia anomala were tested for glycerol production both under osmotic stress and by addition of a sulfite-steering agent. The osmotic pressure was increased by employing glucose concentrations from 50 to 200 g/L and by supplementing with NaCl (40 g/L). Of all the yeasts, S. cerevisiae exhibited the highest level of osmotolerance. The increased osmotic pressure affected glycerol formation the most in C. boidinii. In both Pichia species, glycerol formation was not sufficiently induced when exposed to sugar and salt stress. The addition of 40 g/L Na2SO3 to the medium containing 100 g/L glucose shifted the metabolism of all yeasts towards glycerol formation. Saccharomyces cerevisiae achieved 68.6%, while C. boidinii reached 25.5% of the theoretical glycerol yield, respectively. The highest glycerol yield, 82.3% of the theoretical, was produced by S. cerevisiae under microaerophilic conditions.     

Effects of low electric treatment on yeast microflora

AIMS: To contribute to the understanding of phenomena related to different intensity electric current treatments on the growth and metabolism of selected micro-organisms using laboratory samples of pure and co-cultures (Saccharomyces cerevisiae strain 404 and Hanseniaspora guilliermondii strain 465). METHODS AND RESULTS: Low electric current (10, 30, 50 and 100 mA) was applied to prepared samples. Parameters, such as polarity, treatment duration (18-48 h) and type of inoculum yeast, were varied one at a time to highlight their cause-effect relationships. The effects on cell activity as well as microflora viability were assessed. Bioindicators capable of describing the phenomena caused by the electric current on the microflora were identified. CONCLUSIONS: Results demonstrated that a low voltage treatment using graphite electrodes had a greater effect on the viable S. cerevisiae strain 404 microflora. There was less bactericidal activity in the S. cerevisiae strain 404 than in the H. guilliermondii strain 465. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may be of significant importance in the development of new technological processes in the fields of agriculture and food, particularly new fermenting process controls.     

Selective sugar consumption by apiculate yeasts

Selective consumption of glucose and fructose among apiculate yeasts was evaluated. Results showed that Hanseniaspora guilliermondii and H. uvarum type strains were fructosophilic, unlike the other type strains. The difference in glucose and fructose use was confirmed in different media and throughout sugar consumption. Selective consumption of fructose is widely diffused among apiculate wine yeasts and could positively interfere with fermentation behaviour of Saccharomyces cerevisiae .     

Yeast associations with mosquitoes of the genus Aedes Mg. (Diptera, Culicidae) in the Tom-Ob river region

The inner microflora of blood-sucking mosquitoes genus Aedes inhabiting different biotops of aspen-birch woods of the Tomsc Priob region has been investigated. 120 strains of yeasts genera Pichia, Hansenula, Saccharomyces, Torulaspora, Metschnicowia, Rhodotorula, Cryptococcus, Candida and Aueobasidium have been isolated, the most number of species belong to the genus Pichia. Yeasts P. guilliermondii, A. pullulans and M. pulcherrima have been found during all periods of insects collection. The most number of yeasts species has been found in mosquitos of mass species--A. diantaeus, A. excrucians. A. punctor and A. communis. It is supposed that isolated yeasts are collected by mosquitoes off natural substrates in the course of their development.     

芝麻香型白酒大曲中酵母菌的分离与鉴定

为了开发利用白酒大曲中的酵母菌资源,采用稀释涂布平板法,从芝麻香型白酒大曲中分离得到15株酵母菌株。利用26S rRNA基因序列分析技术对其进行分类鉴定。结果表明,其中6株酵母菌为酿酒酵母（Saccharomyces cerevisiae）,6株为库德里阿兹威氏毕赤酵母（Pichia kudriavzevii）,3株为热带假丝酵母（Candida tropicalis）;并对代表菌株Y1、Y2及Y4进行了形态观察;最后通过随机扩增多态性DNA标记（random amplified polymorphic DNA, RAPD）对分离得到的酵母菌进行分型,表明6株酿酒酵母分属于5个株型,6株库德里阿兹威氏毕赤酵母分属于5个株型,3株热带假丝酵母分属于3个株型。     

Occurrence and diversity of yeasts involved in fermentation of West African cocoa beans

Samples of cocoa beans were taken on two separate occasions during heap and tray fermentations in Ghana, West Africa. In total 496 yeast isolates were identified by conventional microbiological analyses and by amplification of their ITS1-5.8S rDNA-ITS2 regions. For important species the identifications were confirmed by sequencing of the D1/D2 domain of the 5' end of the large subunit (26S) rDNA. Assimilations of organic acids and other carbon compounds were conducted. For dominant yeasts intraspecies variations were examined by determination of chromosome length polymorphism (CLP) using pulsed-field gel electrophoresis. For the heap fermentations maximum yeast cell counts of 9.1 x 10(7) were reached, whereas maximum yeast counts of 6.0 x 10(6) were reached for the tray fermentations. Candida krusei was found to be the dominant species during heap fermentation, followed by P. membranifaciens, P. kluyveri, Hanseniaspora guilliermondii and Trichosporon asahii, whereas Saccharomyces cerevisiae and P. membranifaciens were found to be the dominant species during tray fermentation followed by low numbers of C. krusei, P. kluyveri, H. guilliermondii and some yeast species of minor importance. For isolates within all dominant species CLP was evident, indicating that several different strains are involved in the fermentations. Isolates of C. krusei, P. membranifaciens, H. guilliermondii, T. asahii and Rhodotorula glutinis could be found on the surface of the cocoa pods and in some cases on the production equipment, whereas the origin of e.g. S. cerevisiae was not indicated by the results obtained. In conclusion, the results obtained show that fermentation of cocoa beans is a very inhomogeneous process with great variations in both yeast counts and species composition. The variations seem to depend especially on the processing procedure, but also the season and the post-harvest storage are likely to influence the yeast counts and the species composition.     

The identification and characterization of osmotolerant yeast isolates from chemical wastewater evaporation ponds

Ramat Hovav is a major chemical industrial park manufacturing pharmaceuticals, pesticides, and various aliphatic and aromatic halogens. All wastewater streams are collected in large evaporation ponds. Salinity in the evaporation ponds fluctuates between 3% (w/v) and saturation and pH values range between 2.0 and 10.0. We looked for microorganisms surviving in these extreme environmental conditions and found that 2 yeast strains dominate this biotope. 18S rDNA sequence analysis identified the isolates as Pichia guilliermondii and Rhodotorula mucilaginosa. Both isolates grew in NaCl concentrations ranging up to 3.5 M and 2.5 M, respectively, and at a pH range of 2-10. There was a distinct difference between the Rhodotorula and Pichia strains and S. cerevisiae RS16 that served as a control strain with respect to accumulation of osmoregulators and internal ion concentrations when exposed to osmotic stress. The Pichia and Rhodotorula strains maintained high glycerol concentration also in media low in NaCl. Utilization of various carbon sources was examined. Using a tetrazolium-based assay we show that the Rhodotorula and Pichia strains are capable of utilizing a wide range of different carbon sources including anthracene, phenanthrene, and other cyclic aromatic hydrocarbons.     

Cloning and characterization of the Pichia pastoris MET2 gene as a selectable marker

We describe the isolation and characterization of a new biosynthetic gene, MET2, from the methylotrophic yeast Pichia pastoris. The predicted product of PpMET2 is significantly similar to its Saccharomyces cerevisiae counterpart, ScMET2, which encodes homoserine-O-transacetylase. The ScMET2 was able to complement the P. pastoris met2 strain; however, the converse was not true. Expression vectors based on PpMET2 for the intracellular and secreted production of foreign proteins and corresponding auxotrophic strains were constructed and tested for use in heterologous expression. The expression vectors and corresponding strains provide greater flexibility when using P. pastoris for recombinant protein expression.