THE BIOGENESIS OF MITOCHONDRIA : III. The Lipid Composition of Aerobically and Anaerobically Grown Saccharomyces cerevisiae as Related to the Membrane Systems of the Cells

The growth conditions known to influence the occurrence of mitochondrial profiles and other cell membrane systems in anaerobic cells of S. cerevisiae have been examined, and the effect of the several growth media on the lipid composition of the organism has been determined. The anaerobic cell type containing neither detectable mitochondrial profiles nor the large cell vacuole may be obtained by the culture of the organism on growth-limiting levels of the lipids, ergosterol, and unsaturated fatty acids. Under these conditions, the organism has a high content of short-chain saturated fatty acids (10:0, 12:0), phosphatidyl choline, and squalene, compared with aerobically grown cells, and it is especially low in phosphatidyl ethanolamine and the glycerol phosphatides (phosphatidyl glycerol + cardiolipin). The high levels of unsaturated fatty acids normally found in the phospholipids of the aerobic cells are largely replaced by the short-chain saturated acids, even though the phospholipid fraction contains virtually all of the small amounts of unsaturated fatty acid present in the anaerobic cells. Such anaerobic cells may contain as little as 0.12 mg of ergosterol per g dry weight of cells while the aerobic cells contain about 6 mg of ergosterol per g dry weight. Anaerobic cell types containing mitochondrial profiles can be obtained by the culture of the organism in the presence of excess quantities of ergosterol and unsaturated fatty acids. Such cells have increased levels of total phospholipid, ergosterol, and unsaturated fatty acids, although these compounds do not reach the levels found in aerobic cells. The level of ergosterol in anaerobic cells is markedly influenced by the nature of the carbohydrate in the medium; those cells grown on galactose media supplemented with ergosterol and unsaturated fatty acids have well defined mitochondrial profiles and an ergosterol content (2 mg per g dry weight of cells) three times that of equivalent glucose-grown cells which have poorly defined organelle profiles. Anaerobic cells which are low in ergosterol synthesize increased amounts of squalene.     

The influence of conditions of growth on the endogenous metabolism of Saccharomyces cerevisiae: effect on protein,carbohydrate, sterol and fatty acid content and on viability

The nature of the endogenous reserves of Saccharomyces cerevisiae was examined with respect to conditions of growth, specifically extremes of oxygen tension and carbon source. Cells were grown in batch culture at 30 C under aerobic conditions on a galactose or glucose carbon source and under anaerobic conditions on glucose. The greatest effect of growth conditions on the chemical composition of the cells was on their fatty acid and sterol content.Cells grown under both aerobic and anaerobic conditions mobilised concurrently protein, glycogen, trehalose and fatty acids during a period of 72 hours' starvation under aerobic conditions. The viability of both types of the aerobically grown cells declined to 75% during this period and was not influenced by the initial fatty acid and sterol content of the cells. Cells grown anaerobically showed a more rapid decline in viability which was only 17% after 72 hours' starvation. This loss of viability was not due to a lack of available endogenous reserves but was probably due to an impaired membrane function caused by a deficiency of sterols and unsaturated fatty acids.     

Construction of squalene-accumulatingSaccharomyces cerevisiae mutants by gene disruption through homologous recombination

Saccharomyces cerevisiae synthesizes ergosterol via squalene, but squalene is hardly detected in aerobically grown cells. To obtain a stable squalene-accumulating yeast strain, we attempted to disrupt a gene required in the conversion of squalene to ergosterol, by homologous recombination with a short piece of the gene fragment conjugated with an integration plasmid vector carrying theLEU2 gene. Two mutants that required ergosterol at least for fast growth were isolated. In an aerobic cultivation and with ergosterol supplementation, the two mutants accumulated squalene up to 5 mg/g dry cells. Southern hybridization analysis indicated that both mutants had acquired the vector DNA integrated in the same gene, or nearby genes, on chromosome 12.     

Regulation of sterol biosynthesis inSaccharomyces cerevisiae

Sterol synthesis inSaccharomyces cerevisiae was primarily controlled by the growth rate. At low specific growth rates the intermediates of ergosterol biosynthesis prevailed in cells. At the same time, the total sterol content reached about 6% of dry matter whereas the content of ergosterol was only 2–2.5%, which seems to be the maximum value forS. cerevisiae. After esterification with fatty acids these sterol intermediates are stored in lipid globules together with reserve triacylglycerols. The sporulatingS. cerevisiae cells contained 3.5% sterols and 1.5% ergosterol of dry matter.     

Effects of Inositol Deficiency on Respiratory and Fermentative Activities of Inositol-exacting Yeast,Schizosaccharomyces pombe

Inositol deficiency of Schizosaccharomyces pombe did not induce significant change of contents of various cellular components except for phospholipids and inositol. The most remarkable decrease in inositol content by the deficiency occurred in the mitochondrial fraction. Electronmicroscopic observation of the inositol-deficient cells of Sch. pombe showed no remarkable thickening of cell wall as occurred in the inositol-exacting mutant of Saccharomyces cerevisiae Strain A–21–20.

Marked loss of fermentative activity under the aerobic condition was caused from inositol deficiency without significant change of activities of respiration and anaerobic fermentation. This seemed to indicate elevated regulatory control of the fermentative activity by oxygen in the inositol-deficient Sch. pombe.

Phosphorylative activities of intact cells and the isolated mitochondria coupled with oxidation was also remarkably suppressed by inositol deficiency.     

Occurrence of cytochromes in R and S yeast mutants of the genusSaccharomyces

Visible region of an absorption spectrum was followed in cells of original strains and of rough mutants ofSaccharomyces cerevisiae andS. cerevisiae var.ellipsoideus. It was found that there are no substantial differences in relative content of cytochromesb andc in aerobically grown rough and smooth yeast forms, in spite of the fact that both forms differ substantially in the metabolic oxygen quotient. If the cytochromes present were not reduced in washed cells by dithionite or by substrate addition, the rough forms exhibited a lower cytochrome b:c ratio than the smooth forms. Under anaerobic conditions of cultivation, the rough forms retained a typical aerobic spectrum, lacking, however, the cytochromea and a₃ band; the ratio of cytochromesb andc was changed in favour of cytochromeb (from the original 1.7: 1 up to 3.4: 1). The inability of the rough mutants to produce anaerobic cytochrome spectrum represented by cytochrome b₁ was connected with their inability to reproduce under anaerobic conditions.     

Steroid interference with antifungal activity of polyene antibiotics

Wide differences exist among the polyene antibiotics, nystatin, rimocidin, filipin, pimaricin, and amphotericin B, with reference to steroid interference with their antifungal activities against Candida albicans. Of the numerous steroids tested, ergosterol was the only one which effectively antagonized the antifungal activity of all five polyene antibiotics. The antifungal activities of nystatin and amphotericin B were the least subject to vitiation by the addition of steroids other than ergosterol, and those of filipin, rimocidin, and pimaricin were the most sensitive to interference. Attempts to delineate the structural requirements of steroids possessing polyene-neutralizing activity in growing cultures of C. albicans are discussed. The ultraviolet absorbance of certain antibiotic steroid combinations was also studied.     

Respiration and NADH-Oxidation of the Roots of Flood-Tolerant and Flood-Intolerant Senecio Species as Affected by Anaerobiosis

Respiration was measured under anaerobiosis in the roots of two Senecio species: S. aquaticus Hill, which is flood-tolerant, and S. jacobaea L., which is flood-intolerant. NADH-oxidation under anaerobiosis was measured in roots of S. aquaticus, S. jacobaea and S. vulgaris L., which is also flood-intolerant. Protein content of S. aquaticus was about 15% higher under anaerobiosis. At 20°C respiration of the roots of S. aquaticus was 50% inhibited under anaerobiosis, while an almost complete inhibition occurred in the roots of S. jacobaea. The activities of nitrate reductase, glutamate dehydrogenase and lactate dehydrogenase were considerably higher in the roots of S. aquaticus grown under anaerobic conditions than in roots grown under aerobic conditions. In S. jacobaea glutamate dehydrogenase activity was lower and in S. vulgaris nitrate reductase was lower and glutamate dehydrogenase activity was higher in roots grown under anaerobic conditions. The possible role of these enzymes for metabolism under anaerobic conditions by oxidizing a surplus of NADH is discussed. Since oxidative phosphorylation is 50% inhibited under anaerobiosis, ATP has to be generated in a different way. It is argued that maintenance of the ATP-level may be compensated by way of the enzymes mentioned above, in combination with a modified glucose utilization.     

Oxygen Requirements of the Food Spoilage Yeast Zygosaccharomyces bailii in Synthetic and Complex Media

Most yeast species can ferment sugars to ethanol, but only a few can grow in the complete absence of oxygen. Oxygen availability might, therefore, be a key parameter in spoilage of food caused by fermentative yeasts. In this study, the oxygen requirement and regulation of alcoholic fermentation were studied in batch cultures of the spoilage yeast Zygosaccharomyces bailii at a constant pH, pH 3.0. In aerobic, glucose-grown cultures, Z. bailii exhibited aerobic alcoholic fermentation similar to that of Saccharomyces cerevisiae and other Crabtree-positive yeasts. In anaerobic fermentor cultures grown on a synthetic medium supplemented with glucose, Tween 80, and ergosterol, S. cerevisiae exhibited rapid exponential growth. Growth of Z. bailii under these conditions was extremely slow and linear. These linear growth kinetics indicate that cell proliferation of Z. bailii in the anaerobic fermentors was limited by a constant, low rate of oxygen leakage into the system. Similar results were obtained with the facultatively fermentative yeast Candida utilis. When the same experimental setup was used for anaerobic cultivation, in complex YPD medium, Z. bailii exhibited exponential growth and vigorous fermentation, indicating that a nutritional requirement for anaerobic growth was met by complex-medium components. Our results demonstrate that restriction of oxygen entry into foods and beverages, which are rich in nutrients, is not a promising strategy for preventing growth and gas formation by Z. bailii. In contrast to the growth of Z. bailii, anaerobic growth of S. cerevisiae on complex YPD medium was much slower than growth in synthetic medium, which probably reflected the superior tolerance of the former yeast to organic acids at low pH.     

Comparative responses of the yeast mutant strain GL7 to lanosterol,cycloartenol, and cyclolaudenol

Under anaerobic growth conditions the isomeric 4,4′,14-trimethylcholestane derivatives lanosterol and, more efficiently, cycloartenol satisfy the sterol requirement of the yeast sterol auxotroph $\text{Saccharomyces cerevisiae}$ strain GL7. Aerobic mutant growth is supported only by cycloartenol and not by lanosterol, suggesting different structural requirements for aerobic and anaerobic cells. It is proposed that the non-planar conformation imposed by the 9,19-cyclopropane ring of cycloartenol moderates the adverse membrane effects of the nuclear methyl groups at C-4 and C-14. Under both aerobic and anaerobic conditions cyclolaudenol, a C-24-methyl derivative of cycloartenol, is a significantly more effective sterol source for strain GL7 than cycloartenol. This result is in keeping with the predominance of C-24-methyl sterols (ergosterol) in wild-type yeast.     

Metabolic flux analysis of the sterol pathway in the yeast Saccharomyces cerevisiae

The yeast Saccharomyces cerevisiae is a useful model system for examining the biosynthesis of sterols in eukaryotic cells. To investigate underlying regulation mechanisms, a flux analysis of the ergosterol pathway was performed. A stoichiometric model was derived based on well known biochemistry of the pathway. The model was integrated in the Software COMPFlux which uses a global optimization algorithm for the estimation of intracellular fluxes. Sterol concentration patterns were determined by gas chromatography in aerobic and anaerobic batch cultivations, when the sterol metabolism was suppressed due to the absence of oxygen. In addition, the sterol concentrations were observed in a cultivation which was shifted from anaerobic to aerobic growth conditions causing the sterol pools in the cell to be filled. From time-dependent flux patterns, possible limitations in the pathway could be localized and the esterification of sterols was identified as an integral part of regulation in ergosterol biosynthesis.     

Environmentally-induced changes in the neutral lipids and intracellular vesicles of Saccharomyces cerevisiae and Kluyveromyces fragilis

Saccharomyces cerevisiae, grown aerobically or anaerobically under conditions which induce a requirement for a sterol and an unsaturated fatty acid, synthesized approximately the same amounts of neutral lipid and intracellular low-density vesicles, although the neutral lipids in aerobically-grown cells contained more esterified sterol and less triacylglycerol than those in anaerobically-grown cells. Kluyveromyces fragilis synthesized much less neutral lipid and a smaller quantity of low-density vesicles than S. cerevisiae whether grown at 30°C (generation time 1.1 h) or 20°C (generation time 2.1 h). Both yeasts synthesized highly saturated triacylglycerols, relatively unsaturated phospholipids, and esterified sterols with an intermediate degree of unsaturation irrespective of the conditions under which they were grown. Free sterols in the yeasts were rich in ergosterol and 22(24)-dehydroergosterol, while the esterified sterol fractions were richer in zymosterol.     

Regulation of the synthesis of mitochondrial enzymes and cytochromes

Summary The possibility that decreased mitochondrial function in anaerobic cultures of Saccharomyces cerevisiae is due to catabolite repression rather than anaerobiosis has been examined using a glucose-limited chemostat. Respiration, cytochromes, ubiquinone and a number of soluble and bound mitochondrial enzymes were measured in cells and cell-free homogenates. Derepression by growth in the chemostat under anaerobic conditions resulted in only small increases in the activity of bound enzymes, and in the amount of ubiquinone and respiration, compared with cells grown batch-wise (repressed). The extent of these increases was much smaller than that seen when cells were grown under aerobic conditions whether repressed or derepressed.     

Pyruvate decarboxylases from the petite-negative yeast<Emphasis Type="Italic"> Saccharomyces kluyveri</Emphasis>

Saccharomyces kluyveri is a petite-negative yeast, which is less prone to form ethanol under aerobic conditions than is S. cerevisiae. The first reaction on the route from pyruvate to ethanol is catalysed by pyruvate decarboxylase, and the differences observed between S. kluyveri and S. cerevisiae with respect to ethanol formation under aerobic conditions could be caused by differences in the regulation of this enzyme activity. We have identified and cloned three genes encoding functional pyruvate decarboxylase enzymes ( PDC genes) from the type strain of S. kluyveri (Sk-PDC11, Sk-PDC12 and Sk-PDC13). The regulation of pyruvate decarboxylase in S. kluyveri was studied by measuring the total level of Sk-PDC mRNA and the overall enzyme activity under various growth conditions. It was found that the level of Sk-PDC mRNA was enhanced by glucose and oxygen limitation, and that the level of enzyme activity was controlled by variations in the amount of mRNA. The mRNA level and the pyruvate decarboxylase activity responded to anaerobiosis and growth on different carbon sources in essentially the same fashion as in S. cerevisiae. This indicates that the difference in ethanol formation between these two yeasts is not due to differences in the regulation of pyruvate decarboxylase(s), but rather to differences in the regulation of the TCA cycle and the respiratory machinery. However, the PDC genes of Saccharomyces/Kluyveromyces yeasts differ in their genetic organization and phylogenetic origin. While S. cerevisiae and S. kluyveri each have three PDC genes, these have apparently arisen by independent duplications and specializations in each of the two yeast lineages.Communicated by C. P. Hollenberg     

Role of anaerobiosis in virulence of Salmonella typhimuirium

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen. In the present study, a virulent strain of S. typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S. typhimurium. Anaerobically grown S. typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically. Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro. Anaerobic S. typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria. LD₅₀ of anaerobic S. typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria. Cell surface hydrophobicity of anaerobic S. typhimurium was also found to be significantly higher than aerobic bacteria. Thus, it appears that exposure of S. typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.     

Influence of growth rate on the accumulation of ergosterol in yeast-cells in a phosphate limited continuous culture

Summary The influence of the growth rate on the accumulation of ergosterol inSaccharomyces cerevisiae was studied with glucose and ethanol as substrates under P-limitation in chemostat experiments. In cultures with glucose as carbon source a decrease in ergosterol content with dilution rates up to 0.08 h^–1 was observed, whereas above this dilution rate an increase in ergosterol content occurred. Similar but less marked effects were attained with ethanol as carbon source. A maximum specific rate of ergosterol synthesis of about 2.4 mg per h and g dry cell mass was calculated for phosphorus limited cultures.     

Effects of growth conditions on mitochondrial morphology inSaccharomyces cerevisiae

Effects of growth conditions on mitochondrial morphology were studied in livingSaccharomyces cerevisiae cells by vital staining with the fluorescent dye dimethyl-aminostyryl-methylpyridinium iodine (DASPMI), fluorescence microscopy, and confocal-scanning laser microscopy. Cells from respiratory, ethanol-grown batch cultures contained a large number of small mitochondria. Conversely, cells from glucose-grown batch cultures, in which metabolism was respiro-fermentative, contained small numbers of large, branched mitochondria. These changes did not significantly affect the fraction of the cellular volume occupied by the mitochondria. Similar differences in mitochondrial morphology were observed in glucose-limited chemostat cultures. In aerobic chemostat cultures, glucose metabolism was strictly respiratory and cells contained a large number of small mitochondria. Anaerobic, fermentative chemostat cultivation resulted in the large, branched mitochondrial structures also seen in glucose-grown batch cultures. Upon aeration of a previously anaerobic chemostat culture, the maximum respiratory capacity increased from 10 to 70 µmole.min^–1.g weight^–1 within 10 h. This transition resulted in drastic changes of mitochondrial number, morphology and, consequently, mitochondrial surface area. These changes continued for several hours after the respiratory capacity had reached its maximum. Cyanide-insensitive oxygen consumption contributed ca. 50% of the total respiratory capacity in anaerobic cultures, but was virtually absent in aerobic cultures. The response of aerobic cultures to oxygen deprivation was qualitatively the reverse of the response of anaerobic cultures to aeration. The results indicate that mitochondrial morphology inS. cerevisiae is closely linked to the metabolic activity of this yeast: conditions that result in repression of respiratory enzymes generally lead to the mitochondrial morphology observed in anaerobically grown, fermenting cells.     

Isolation and characterization of pcsB, the gene for a polyene carboxamide synthase that tailors pimaricin into AB-400

From cell-free extracts of Streptomyces RGU5.3, a tailoring activity of pimaricin, leading to the biosynthesis of its natural carboxamide derivative AB-400, was recently identified. The two polyene macrolides, pimaricin and AB-400, were produced in almost equal quantities and can be detected in the fermentation broth of the producer strain. This report concerns the isolation and partial characterization of the gene, polyene carboxamide synthase (pcsB), responsible for the bioconversion. The gene encoded an asparagine synthase-like protein, belonging to the type II glutamine amidotransferase family, and was named pcsB. The fermentation broth of a recombinant strain carrying the engineered pcsB gene under the control of the inducible tipA promoter within an integrative vector produces the carboxamide AB-400 as the main polyene macrolide.