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.     

The action of visible radiation on the formation and properties of Saccharomyces ascospores

Summary Light in the blue and green bands of the spectrum inhibited ascospore formation in both Saccharomyces cerevisiae and S. carlsbergensis. Ascospores which were produced failed to stain with malachite green and mature spores formed in the dark lost their staining ability when exposed to light in these bands. It is thought that this is due to an alteration brought about in the molecular organization of the spore wall, so that the damaged walls become permeable to molecules of considerably greater size. Red light was without effect on S. cerevisiae, but stimulated sporulation in S. carlsbergensis. This response seemed to be strictly under the control of the yeast cell, and of a totally different nature from the injurious character of shorter wave lengths.     

Production of Nicotinamide Adenine Dinucleotide by Saccharomyces carlsbergensis

The accumulation of NAD was studied by culturing yeast in the presence of NAD precursors, Among the strains tested, Saccharomyces carlsbergensis showed the highest ability for the accumulation of NAD, Additions of pantothenate, inositol, zinc ion and fatty acids were effective for the accumulation of NAD. Under the optimal culture condition, NAD level in Saccharomyces carlsbergensis reached 42 mg per gram dry cells. Surfactants belonging to alkyl sulfate were effective on the leaking of the intracellular NAD, and about 75 mg of NAD per 100 ml was accumulated.     

Stoffwechselregulation in Durchfluß-systemen

Zusammenfassung Am Beispiel der unter kontinuierlicher Substratzufuhr erfolgenden Äthanol-Synthese in Hefe (Saccharomyces carlsbergensis) konnte gezeigt werden, daß unabhängig von der exogenen Substratmenge der Speicherstoffwechsel einer linearen Regelfunktion unterliegt. Das Verhältnis zwischen Äthanolbildung aus exogenem und endogenem Material ist derart ausbalanciert, daß die Gesamtbilanz erhalten bleibt, nach der 50–60% der zugeführten Glucosemenge im Äthanol wiederzufinden sind.

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Metabolic control in flow systemsII. Regulation of ethanol-synthesis and pool turnover in yeast under continuous substrate infusion

Summary The synthesis of ethanol under continuous infusion of d-glucose to anaerobic Saccharomyces carlsbergensis demonstrates very distinctly that turnover of the carbohydrate pool has the characteristics of a linear regulation, independent of the exogenous substrate amounts. The relation between glycolysis and pool turnover is balanced exctly so that 50–60% of glucose infused will be synthesized to ethanol.

     

Stoffwechsel-Regulation aerober Zellsuspensionen vonSaccharomyces carlsbergensis nach Glucosezugabe

Zusammenfassung Wird eine ausgehungerte aerobe Zellsuspension vonSaccharomyces carlsbergensis mit Glucose gefüttert, dann ist eine deutliche Stimulierung des O₂-Verbrauchs zu registrieren. Mit Hilfe verschiedener Meßmethoden wurde der Verlauf dieser nicht-linearen O₂-Abnahme analysiert und mit den gleichzeitig auftretenden Konzentrationsänderungen einiger glykolytischer Metabolite korreliert.Dabei zeigt sich, daß ca. 20–60 Sekunden nach Glucosezugabe die Rate des O₂-Verbrauchs abnimmt und gleichzeitig eine deutliche Äthanol-Synthese einsetzt, obgleich der O₂-Partialdruck zu diesem Zeitpunkt noch etwa halbmaximal ist.

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Metabolite-regulation of aerobic cell-suspension ofSaccharomyces carlsbergensis after feeding with glucose. I. Shift from respiration to aerobic fermentation

Summary After feeding with glucose, an aerobic starved cell-suspension ofSaccharomyces carlsbergensis shows an increasing of oxygen consumption. This stimulation is not linear during the transition from high to low oxygen-level.For this aerobic phase the regulated fluxes of some metabolites are analyzed. It could be shown that with highest oxygen consumption an ethanol synthesis is starting.

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Herrn Prof. Dr.A. Betz danke ich an dieser Stelle für viele wertvolle Hinweise zur Interpretation der hier beschriebenen Ergebnisse. Die gewissenhafte technische Assistenz erfolgte durch FrauR. Hinrichs. Die Durchführung dieser Arbeit wurde durch Sachbeihilfen der Stiftung Volkswagenwerk und der Deutschen Forschungsgemeinschaft ermöglicht.     

The genusSaccharomyces (Meyen) Reess

Twenty-nine different strains of several fermentation type II species of the genusSaccharomyces, whose main common characteristic was complete fermentation of raffinose, were studied. These can be regarded as the most highly developed species of the genusSaccharomyces. Their common and differential morphological, physiological, biochemical and serological characteristics were studied. In addition to the usual criteria, factors which played a role in the history and formation of these species were taken into account. Large species were compared with typical strains chosen on the basis of statistical typing, e.g.Saccharomyces carlsbergensis Hansen, and small species were compared with type strains, e.g.Saccharomyces logos van Laer et Den. andSaccharomyces uvarum Beijer, as nomenelatural types. It was found that the whole group could be divided into two categories, one grouped roundSaccharomyces carlsbergensis Hansen and the other roundSaccharomyces uvarum Beijer., which overlapped at the place occupied bySaccharomyces logos van Laer et Den. Another group of intermediate strains isolated from grapes came betweenSaccharomyces logos andSaccharomyces uvarum. In future, closer attention will have to be paid to strains which do not give a serological reaction with serum against the typical speciesSaccharomyces carlsbergensis, i. e. to the speciesSaccharomyces monacensis Hansen andSaccharomyces mandshuricus Saito.     

Yeasts of the Miami,Florida, Area

The examination of a total of 301 yeast cultures collected from 22 water samples in the Miami River has demonstrated the presence of 18 different yeasts, including 6 sporogenous and 12 asporogenous species.The more frequent species were Sacch. carlsbergensis (100%), Cand. tropicalis (31%), Pichia fermentans (22%) Torulopsis glabrata (18%) and Cryptococcus albidus (18%).Comparison of Miami River studies with earlier collections made in Key Biscayne soil as well as in Biscayne Bay reveals the occurrence of common species in all three habitats.     

Sterol-binding polysaccharides of Rhizopus arrhizus, Penicillium roquefortii and Saccharomyces carlsbergensis

Polysaccharides that bind with sterols and render them water-soluble were isolated from two mycelial fungi, Rhizopus arrhizus and Penicillium roquefortii and a yeast Saccharomyces carlsbergensis. The polysaccharides from R. arrhizus and S. carlsbergensis were accompanied by small quantities of phosphorus, protein and lipid, none of which significantly influenced the binding of sterol to polysaccharide. The chemical composition and sterol-binding properties of the polysaccharides from the filamentous species were almost identical, but differed significantly from those of the yeast polysaccharide. The principal sterol-binding polysaccharide of S. carlsbergensis was identified as a mannan and that of the filamentous fungi as a glucan(s). The binding capacity of the purified yeast polysaccharide was almost two-fold greater than that of R. arrhizus and P. roquefortii.     

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.     

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).     

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.     

Dual control of invertase Biosynthesis in chemostat culture

In a previous study on the chemostat culture of Saccharomyces carlsbergensis, maximum invertase specific activity was observed at an intermediate dilution rate. A possible regulation mechanism, assuming there are simultaneous effects of induction and repression on two sites of the operator loci for invertase formation, is proposed which might account for the observed curve of the dilution rate effect.     

Dark-recovery of Gamma- and UV-irradiated Saccharomyces carlsbergensis at 4° C

Experiments were carried out to find the effect of dark-holding in distilled water at 4° C on recovery of Gamma- and UV-irradiated cells of a haploid strain ofSaccharomyces carlsbergensis. It was found that there was an appreciable increase in survival of the irradiated cells following 4 to 24 hours' holding while no increase in the number of control cells was observed following similar treatment. It is suggested that some common type of damage induced by both Gamma- and UV-radiations inS. carlsbergensis may be repaired efficiently under metabolic conditions associated with growth at 4° C in distilled water.     

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.     

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.     

Participation of vacuoles in regulation of levels of K+, Mg2+ and orthophosphate ions in cytoplasm of the yeast Saccharomyces carlsbergensis

Intracellular distributions of K⁺, Mg²⁺ and orthophosphate under various conditions of cultivation or incubation of the yeast Saccharomyces carlsbergensis were studied by differential extraction of ion pools. The decisive role of vacuolar compartmentation of ions in regulation of K⁺, Mg²⁺ and orthophosphate levels in the yeast cytoplasm was shown. The content of intracellular K⁺ and Mg²⁺ in yeast increased or decreased primarily depending on the increase or decrease in the vacuolar ion pool. The levels of K⁺ and Mg²⁺ in the cytoplasm were practically unchanged. Vacuoles were involved in regulation of Mn²⁺ concentration in the cytoplasm of the yeast S. carlsbergensis accumulating this ion in the presence of glucose. Alongside the vacuolar compartmentation, the chemical compartmentation, i. e. formation of bound Mg²⁺, Mn²⁺ and K⁺ was, evidently, also involved in the control of ion levels in the cytoplasm. The orthophosphate level in the yeast cytoplasm was regulated by its accumulation in vacuoles and biosynthesis of inorganic polyphosphates in these organelles. The biosynthesis of low-molecular weight polyphosphates occurred parallel to the accumulation of Mg²⁺ or Mn²⁺ in vacuoles, thus confirming the availability of the other mechanism for the transport of these ions through the tonoplast differing from the transport mechanism through the plasmalemma.     

Invertase biosynthesis by Saccharomyces carlsbergensis in batch and continuous cultures

The biosynthesis of invertase by Saccharomyces carlsbergensis LAM 1068 was studied in relation to its glucose effect at both unsteady and steady states of growth. Experimental correlations between the dilution rate and invertase specific activity (E/X) in chemostat, cultures led to an optimum for the enzyme synthesis at a particular intermediate growth rate. The value of E/X increased from 1.1 (U/mg biomass) in batch cultures to 13 (U/mg biomass) in chemostat cultures. A mutant strain A3 showed the highest value for E/X = 25 (U/mg biomass) at high dilution rates where glucose repression was observed with the wild strain.     

Über den Einfluß von 3,4-Benzpyren auf den Sprossungsrhythmus von Hefezellen

Zusammenfassung Fine einstündige Inkubation von Saccharomyces carlsbergensis-Zellen in einer 0,0125% igen 3,4-Benzpyren-Glycerin-Serumlösung führt zu einer deutlichen Verzögerung des Sprossungsrhythmus, besonders desÜbergangs von der 1. in die 2. Generation. Entsprechende Pyren-, 20-Methylcholanthren-und 9,10-Dimethyl-1,2-benzanthracen-Lösungen zeigen dagegen keinen derartigen Effekt.     

Characterization of a regulatory mutant of fructose 1,6-bisphosphatase in Saccharomyces carlsbergensis.

Summary The fdp mutation has been localized on the genome of Saccharomyces carlsbergensis, on chromosome II, between lys2 and tyr1, at a map distance of 31 centimorgan from lys2.Since the fdp mutant does not grow on glucose, fructose, mannose and sucrose, hexose transport and a number of enzymes of carbon metabolism were tested, but no significant differences could be found between the wild type and the mutant. Only the regulatory properties of glycogen synthetase are changed in the mutant, but it is doubtfull whether this can explain its phenotype.The disorganization of carbon metabolism of the mutant upon addition of glucose to the medium was analyzed in more detail. The most prominent feature observed until now is the accumulation of free glucose and hexose phosphates in the cell. This result indicates that somehow the feedback control between hexose transport and metabolism is impaired. Hexose phosphates are known to be toxic to many cells, including yeast. Therefore, accumulation of hexose phosphates in the presence of glucose in the medium, can explain the absence of growth on this carbon source.