The effects of glucose and oxygen on the cytochromes and metabolic activity of yeast batch cultures. I. Saccharomyces spp.

Saccharomyces cerevisiae and Saccharomyces carlsbergensis were grown in batch culture with and without oxygen control. The concentrations of A-, B- and C-type cytochromes of both yeasts were dependent on the oxygen concentration during growth as well as on the initial glucose concentration of the growth medium. S. cerevisiae cytochromes were maximal after growth in low glucose and low oxygen; S. carlsbergensis cytochromes were maximal after growth in low glucose and high oxygen. Except when glucose was in very low concentration, its catabolism by S. carlsbergensis was directed predominantly towards ethanolic fermentation regardless of the oxygen concentration. Growth rate, total cell mass and yield were maximal, and anabolism was closely balanced with catabolism, when glucose and oxygen of S. carlsbergensis cultures were both high. Under these conditions neither catabolism, respiratory or ethanolic, nor glucose uptake were maximal.     

Three newly delimited species of Saccharomyces sensu stricto

Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species. S. cerevisiae and S. bayanus strains had only 22% of their genomes in common. S. pastorianus, with intermediate hybridization values between S. cerevisiae and S. bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species. S. pastorianus replaces S. carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904. These data do not agree with the results of traditional physiological tests.     

Characteristics of yeast causing clouding of dry white wines

Summary Fourteen cultures of yeast were isolated from dry white wines which contained a cloud and/or sediment. These organisms were identified asS. chevalieri Guilliermond (2 cultures),S. carlsbergensis var.monacensis (Hansen) Dekker (1),S. oviformis Osterwalder (6),S. cerevisiae (2),Pichia alcoholophila Klöcker (2), andCandida rugosa (Anderson) Diddens and Lodder (1).     

Restriction polymorphisms in the internal transcribed spacers and 5.8S rDNA ofSaccharomyces

Polymorphism in enzymatically amplified ribosomal DNA (rDNA) were examined in 18 strains ofSaccharomyces. Restriction patterns generated from the region spanning the internal transcribed spacers (ITS) and the 5.8S rDNA produced two clusters corresponding toS. bayanus andS. cerevisiae. The type culture ofS. carlsbergensis (ATCC 76529), which could not be separated from theS. cerevisiae group by small subunit (SSU) rDNA patterns, showed aScrfI profile that was distinct from all the other strains. The type culture ofs. intermedius var.turicensis (ATCC 76519) is assigned toS. bayanus on the basis of the combined results of SSU and ITS restriction analyses.S. kluyveri occurred at a separate branch of the distance tree and is unrelated to any of the strains. Results were in general agreement with reported DNA homologies and are discussed in relation to other molecular and genetic data.     

Influence of glucose concentration on the physiology and lipid composition of some yeasts

The fatty acid contents of the “Crabtree-positive” yeastsSaccharomyces cerevisiae, S. carlsbergensis andS. delbrueckii decreased with increasing concentrations of glucose in the medium. These lower values were due to a lower content of sterol esters and phosphatides inS. cerevisiae, and of sterol esters inS. carlsbergensis. In contrast the fatty acid contents of the Crabtree-negativeS. fragilis, Schwanniomyces occidentalis andCandida utilis increased with increasing concentrations of glucose and inCandida utilis this was due almost entirely to a higher content of triglycerides. This work was supported in part by grant B/SR/5780 from the Science Research Council. We are grateful to the Brewer's Society for a Research Scholarship to Mr. B. Johnson. We thank Mr. A. Bradley for competent technical assistance.     

Molecular Analysis of the α-Galactosidase MEL Genes in Yeast Saccharomyces sensu stricto

To infer the molecular evolution of yeast Saccharomyces sensu stricto from analysis of the -galactosidase MEL gene family, two new genes were cloned and sequenced from S. bayanus var. bayanus and S. pastorianus. Nucleotide sequence homology of the MEL genes of S. bayanus var. bayanus (MELb), S. pastorianus (MELpt), S. bayanus var. uvarum (MELu), and S. carlsbergensis (MELx) was rather high (94.1–99.3%), comparable with interspecific homology (94.8–100%) of S. cerevisiae MEL1-MEL11. Homology of the MEL genes of sibling species S. cerevisiae (MEL1), S. bayanus (MELb), S. paradoxus (MELp), and S. mikatae(MELj) was 76.2–81.7%, suggesting certain species specificity. On this evidence, the -galactosidase gene of hybrid yeast S. pastorianus (S. carlsbergensis) was assumed to originate from S. bayanus rather than from S. cerevisiae.     

Organization and evolution of the mitochondrial genome of yeast

Summary The mitochondrial genome of yeast (S. cerevisiae orS. carlsbergensis) appears to be formed by 60–70 genetic units, each one of which is formed by (1) a GC-rich sequence, possibly having a regulatory role; (2) a gene, and (3) an AT-rich spacer, which probably is not transcribed. Recombination in this genome appears to underlie a number of important phenomena. The organization of the mitochondrial genome of yeast and these recombinational events are discussed in relationship with the organization and evolution of the nuclear genome of eukaryotes.     

Induction of cytoplasmic respiratory deficient mutants in yeast by the folic acid analogue, methotrexate

Summary The folic acid analogue, methotrexate, was found to be an effective inducer of the cytoplasmic petite mutation in different strains of S. carlsbergensis and S. cerevisiae. The induction varies considerably from strain to strain and also with growth conditions under which the experiments are carried out. Determination of mitochondrial protein synthesis in the presence of methotrexate shows a considerable decrease in incorporation of radioactive leucine before any significant induction of petites has taken place.These results and a comparison with results from other laboratories obtained with different drugs provide the basis for a proposed mechanism of petite induction by methotrexate.     

Effects of Fusariotoxin T-2 on Saccharomyces cerevisiae and Saccharomyces carlsbergensis

A Fusarium metabolite, T-2 toxin, inhibits the growth of Saccharomyces carlsbergensis and Saccharomyces cerevisiae. The growth inhibitory concentrations of T-2 toxin were 40 and 100 μg/ml, respectively, for exponentially growing cultures of the two yeasts. S. carlsbergensis was more sensitive to the toxin and exhibited a biphasic dose-response curve. Addition of the toxin at 10 μg/ml of S. carlsbergensis culture resulted in a retardation of growth as measured turbidimetrically, after only 30 to 40 min. This action was reversible upon washing the cells free of the toxin. The sensitivity of the yeasts to the toxin was dependent upon the types and concentrations of carbohydrates used in the growth media. The sensitivity of the cells to the toxin decreased in glucose-repressed cultures. These results suggest that T-2 toxin interferes with mitochondrial functions of these yeasts.     

Visualization of chromosome-like structures in protoplasts of the yeast

Structures resembling chromosomes have been observed in protoplasts of Saccharomyces carlsbergensis during mitotic and meiotic division. The stage of visualization is prolonged in meiosis, and bivalents can be counted. The haploid chromosome number of S. carlsbergensis is between 22–26.     

Structure and organization of rhodophyte and chromophyte plastid genomes: implications for the ancestry of plastids

Summary Recombinational repair is the means by which DNA double-strand breaks (DSBs) are repaired in yeast. DNA divergence between chromosomes was shown previously to inhibit repair in diploid G1 cells, resulting in chromosome loss at low nonlethal doses of ionizing radiation. Furthermore, 15–20% divergence prevents meiotic recombination between individual pairs of Saccharomyces cerevisiae and S. carlsbergensis chromosomes in an otherwise S. cerevisiae background. Based on analysis of the efficiency of DSB-induced chromosome loss and direct genetic detection of intragenic recombination, we conclude that limited DSB recombinational repair can occur between homoeologous chromosomes. There is no difference in loss between a repair-proficient Pms⁺ strain and a mismatch repair mutant, pms1. Since DSB recombinational repair is tolerant of diverged DNAs, this type of repair could lead to novel genes and altered chromosomes. The sensitivity to DSB-induced loss of 11 individual yeast artificial chromosomes (YACs) containing mouse or human (chromosome 21 or HeLa) DNA was determined. Recombinational repair between a pair of homologous HeLa YACs appears as efficient as that between homologous yeast chromosomes in that there is no loss at low radiation doses. Single YACs exhibited considerable variation in response, although the response for individual YACs was highly reproducible. Based on the results with the yeast homoeologous chromosomes, we propose that the potential exists for intra- YAC recombinational repair between diverged repeat DNA and that the extent of repair is dependent upon the amount of repeat DNA and the degree of divergence. The sensitivity of YACs containing mammalian DNA to ionizing radiation-induced loss may thus be an indicator of the extent of repeat DNA.     

Cell growth restoration and high level protein expression by the promoter of hexose transporter, HXT7, from Saccharomyces cerevisiae

The promoters of high-affinity hexose transporter, HXT6 and HXT7, are sufficient for complementary expression of invertase to restore the growth of Saccharomyces cerevisiae in raffinose medium. The HXT7 promoter produced higher invertase activity at 139- and 30-fold of the basal activity in strains GN 3C.2 and W303-1, respectively. In addition, the HXT7 promoter expressed 3- to 9-fold more of enhanced green fluorescent protein than that of the constitutive ADH1 in three different S. cerevisiae strains, even during short-term incubation in glucose medium. Therefore, HXT7 promoter could be used for heterologous protein expression in S. cerevisiae.     

Food borne bacterial models for detection of benzo[a]pyrene‐DNA adducts formation using RAPD‐PCR

Random amplified polymorphic DNA (RAPD) PCR is a feasible method to evaluate genotoxin‐induced DNA damage and mutations. In this study, Lactobacillus plantarum ATCC 14917T, Enterococcus faecium DSMZ 20477T, Escherichia coli PQ37 and Saccharomyces cerevisiae S441 were screened for DNA genetic alterations by DNA fingerprinting using M13 and LA1 primers after treatment with three compounds forming covalent adducts with DNA [benzo[a]pyrenediol epoxide (BPDE), methyl methanesulfonate and 1,2,3,4‐diepoxybutane (DEB)]. M13 RAPD fingerprinting revealed that the total number of bands decreased in all treated DNA compared to control samples and generally the lost bands were characterized by high molecular weight. Some extra bands were detected for L. plantarum and E. faecium, while in E. coli and S. cerevisiae DNAs BPDE and DEB treatments did not result in new extra bands. Besides qualitatively analysis, cluster analysis based on Unweighted Pair‐Group Method with Average algorithm was performed to compare DNA fingerprints before and after treatments. This analysis confirmed the absence of significant differences between negative controls and treated DNA in S. cerevisiae and E. coli however the disappearance of some bands can be detected. The data indicate that this approach can be used for DNA damage detection and mutations induced by genotoxic compounds and highlighted the possible use of L. plantarum and E. faecium M13 based fingerprinting as reference for hazard identification in risk assessment.     

Der osmotische Wert als begrenzender Faktor für das Wachstum und die Gärung von Hefen

Zusammenfassung Von 10 osmotoleranten und 2 nichtosmotoleranten Hefen wurden die maximal verträglichen osmotischen Werte für Gärung und Wachstum in Lösungen von Fructose, Galactose, Maltose, Xylose, Lutrol, NaCl, KCl, Ammoniumchlorid, Ammonsulfat und Lithiumchlorid kryoskopisch und durch isotherme Destillation bestimmt. Sowohl osmotolerante als auch nichtosmotolerante Hefen zeigten im Wachstum auf Nähragar mit 2% bzw. 50% Glucose keinen Unterschied. Die osmotischen Grenzwerte osmotoleranter Hefen für die Gärung lagen in Fructoselösungen zwischen 238 at (Saccharomyces rosei) und 620 at (S. mellis und S. heterogenicus). Die entsprechenden Werte nichtosmotoleranter Hefen waren 160 at bei S. cerevisiae und 124 at bei S. carlsbergensis. Die Grenzwerte für das Wachstum waren meist niedriger als für die Gärung. Alle untersuchten Hefen tolerierten die höchsten osmotischen Werte in Fructoselösungen. In Neutralsalzlösungen stieg die Toleranz der Hefen entsprechend der Hofmeisterschen Reihe an. Die geringe Verträglichkeit konzentrierter Natriumchloridlösungen konnte durch steigende Mengen Fructose erhöht werden. Der gleiche Effekt wurde mit den von den untersuchten Hefen nicht vergärbaren Zuckern Galactose und Lactose erreicht. Die unterschiedliche Toleranz einer Hefe gegenüber verschiedenen Substanzen wird diskutiert und als Ursache eine verschieden starke Permeation und unterschiedliche Wirkung der gelösten Teilchen auf das Zellplasma angenommen.

---

The osmotic pressure as limiting factor for growth and fermentation of yeasts

Summary The limit values of osmotic pressure were investigated which ten osmotolerant and two nonosmotolerant yeasts supported in solutions of sugars (fructose, maltose, xylose) and salts (lutrol, sodium chloride, potassium chloride, ammonium chloride, ammonium sulphate and lithium chloride) during fermentation and growth by cryoscopy and isothermal destillation. Osmotolerant and nonosmotolerant yeasts did not show any different reaction when growing on agar media containing 2% resp. 50% glucose. The limit values for fermentation of osmotolerant yeasts in fructose solutions were found to be from 238 atm (Saccharomyces rosei) up to 620 atm (S. mellis and S. heterogenicus). The corresponding values of nonosmotolerant yeasts were 160 atm with S. cerevisiae and 124 atm with S. carlsbergensis. The limit values for growth were mostly lower than for fermentation. The highest osmotic values tolerated were found in solutions of fructose. The salt tolerance increased in accordance with the rows of Hofmeister. The low compatibility of concentrated solutions of sodium chloride could be raised by small amounts of fructose. The same effect was caused by lactose and galactose, two sugar types which the yeasts under study do not ferment. Yeast's different tolerance of concentrated solutions of various substances is discussed. The cause is supposed to be a various permeation and a different effect of the dissolved particles on the cytoplasm.

     

Inhibition of Protein Translocation in Permeabilized Cells of Schizosaccharomyces pombe by Puromycin

To investigate protein translocation in eukaryotes, we reconstituted a protein translocation system using the permeabilized spheroplasts (P-cells) of the fission yeast Schizosaccharomyces pombe. The precursor of a sex pheromone of Saccharomyces cerevisiae, prepro-α-factor, was translocated across the endoplasmic reticulum (ER) of S. pombe posttranslationally, and glycosylated to the same extent as in the ER of S. cerevisiae. This suggested that the size of N-linked core-oligosaccharide in the ER of S. pombe is similar to that in S. cerevisiae. This translocation into the ER of S. pombe was inhibited by puromycin, but the translocation in the P-cells of S. cerevisiae was not inhibited. This difference in sensitivity to puromycin was due to the membrane but not the cytosolic fraction. Our results suggested that the translocation machinery of S. pombe was sensitive to puromycin and different from that of S. cerevisiae.     

Physiological and genetic stability of hybrids of industrial wine yeasts Saccharomyces sensu stricto complex

Aims: The aim of this study was to examine the physiological and genetic stability of hybrids of industrial wine yeasts Saccharomyces sensu stricto complex subjected to acidic stress during fermentation. Methods and Results: Laboratory‐constructed yeast hybrids, one intraspecific Saccharomyces cerevisiae × S. cerevisiae and three interspecific S. cerevisiae ×Saccharomyces bayanus, were subcultured in aerobic or anaerobic conditions in media with or without l ‐malic acid. Changes in the biochemical profiles, karyotypes and mitochondrial DNA profiles of the segregates were assessed after 50–190 generations. All yeast segregates showed a tendency to increase the range of the tested compounds utilized as sole carbon sources. Interspecific hybrids were alloaneuploid and their genomes tended to undergo extensive rearrangement especially during fermentation. The karyotypes of segregates lost up to four and appearance up to five bands were recorded. The changes in their mtDNA patterns were even broader reaching 12 missing and six additional bands. These hybrids acquired the ability to sporulate and significantly changed their biochemical profiles. The alloaneuploid intraspecific S. cerevisiae hybrid was characterized by high genetic stability despite the phenotypic changes. l ‐malic acid was not found to affect the extent of genomic changes of the hybrids, which suggests that their demalication ability is combined with resistance to acidic stress. Conclusions: The results reveal the plasticity and extent of changes of chromosomal and mitochondrial DNA of interspecific hybrids of industrial wine yeast especially under anaerobiosis. They imply that karyotyping and restriction analysis of mitochondrial DNA make it possible to quickly assess the genetic stability of genetically modified industrial wine yeasts but may not be applied as the only method for their identification and discrimination. Significance and Impact of the Study: Laboratory‐constructed interspecific hybrids of industrial strains may provide a model for studying the adaptive evolution of wine yeasts under fermentative stress.     

Production of phenylacetylcarbinol by various yeast species

Measurements of tolerance to the addition of benzaldehyde were carried out with six yeast species:Hansenula anomala, Brettanomyces vini Peynaud et Domercq, strain X,Saccharomyces carlsbergensis, Saccharomyces cerevisiae R XII,Saccharomyces ellipsoideus andTorula utilis. The techniques used were: comparison of evolution of carbon dioxide with and without benzaldehyde and production of phenylacetylcarbinol after a single addition of benzaldehyde (0.2%) and after four additions (total of 0.8%). The highest decarboxylase activity in the presence of benzaldehyde was found withHansenula anomala, Saccharomyces carlsbergensis andSaccharomyces cerevisiae. InHansenula anomala, benzaldehyde caused a 16% inhibition of fermentation, in the other cultures inhibition lay between 35.5 and 63.2%. After a single addition of benzaldehyde the greatest amount of phenylacetylcarbinol was formed byHansenula anomala, Saccharomyces carlsbergensis andSaccharomyces cerevisiae. The yield of phenylacetylcarbinol in these cases was between 46.0% and 51.5 weight% (calculated on added benzaldehyde). During fementation with a higher benzaldehyde concentration,Saccharomyces carlsbergensis utilized 70% aldehyde for the formation of phenylacetylcarbinol while the rest was mostly reduced to benzylalcohol. Phenylacetylcarbinol was estimated after extraction of the fermentation medium with ether polarographically and polarimetrically, benzaldehyde was estimated polarographically directly in the medium.     

Resistance to heavy metals by some Nigerian yeast strains

The heavy metal resistance of yeasts isolated from sugary substrates such as orange, palm wine and pineapple and identified asSaccharomyces carlsbergensis andS. cerevisiae was studied. The yeast isolates were tested against different concentrations of cadmium, copper, manganese, silver and zinc salts ranging from 1 to 20 mmol/L. Local yeasts showed resistance to 3–15 mmol/L cadmium, 18–20 copper, 16–20 manganese, 1–9 silver and 16–19 for zinc. The significance of the results is discussed in relation to the effects of heavy metals on growth of microorganisms and selection of yeasts for the brewing industry in Nigeria.