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[Studies on the cell wall of yeast of the genus Rhodotorula. X. Isolation and purification of cell-wall glycoproteins from Rhodotorula rubra (author's transl)].

Following ethylene diamine treatment of the cell wall of Rhodotorula rubra, a water soluble fraction has been isolated. This fraction can be resolved into three glycoproteins and one protein. The major part is a glycoprotein, purified to homogeneity which has a molecular weight of 64 000. The glyco-part of this protein contains mannose, glucose and one residue of glucosamine. After pronase treatment, the presence of an ""Asparaginyl-N-acetylglucosamine" linkage is established by the existence of one aspartic acid residue and one glucosamine residue. After permethylation, the initial data give some evidence that the branching points in the molecule were the carbon atoms 3 and (or) 6 of the mannose moiety and that some glucose moieties are bound to the non-reducing terminal end.

Acetylglucosamine

[Taxonomical investigations in sexual strains of Rhodotorula glutinis (Fres.) Harrison, Rh. aurantiaca (Saito) Lodder and Rh. rubra (Demme) Lodder (author's transl)].

Several strains of Rhodotorula were crossed with the strains IFO 0559a and IFO 0880alpha of Rhodotorula glutinis. We found nine new sexual Rhodotorula strains, belongong to the species Rh. glutinis, Rh. aurantiaca and Rh. rubra. 22 combinations, whose physiological and biochemical qualities were found out, followed from crossings of all sexual strains. The cell walls of some selected mycel combinations contained a pore in the center. Resting spores and life cycles of all combinations correspond to Rhodosporidium toruloides Banno. Results and taxonomical problems are being discussed.

Carbohydrate Metabolism

Rhodotorula fungaemia: a life-threatening complication of indwelling central venous catheters.

A 30-year-old woman receiving total parenteral nutrition via an indwelling central venous catheter for an intestinal motility disorder developed fever, tachycardia, tachypnea, and hypotension. Multiple blood cultures drawn through the catheter prior to these events, as well as a peripheral blood culture obtained earlier, grew the red yeast Rhodotorula rubra. The patient was critically ill for over one month but eventually recovered with therapy including the systemic antifungal agents amphotericin B and flucytosine and removal of the catheter. Although Rhodotorula has generally been regarded as having low pathogenicity, this case emphasizes the serious nature of Rhodotorula sepsis and suggests the need for both systemic antifungal therapy and removal of a colonized indwelling catheter.

Adult

A kinetic analysis of D-xylose transport in Rhodotorula glutinis.

The kinetics of D-xylose transport were studied in Rhodotorula glutinis. Analysis of the saturation isotherm revealed the presence of at least two carriers for D-xylose in the Rhodotorula plasma membrane. These two carriers exhibited Km values differing by more than an order of magnitude. The low-Km carrier was repressed in rapidly growing cells and derepressed by starvation of the cells. Several hexoses were observed to inhibit D-xylose transport. In the studies reported here, the inhibitions produced by D-galactose and 2-deoxy-D-glucose were examined in some detail in order to define the interactions of these sugars with the D-xylose carriers. 2-Deoxy-D-glucose competitively inhibited both of the D-xylose carriers. In contrast, only the low-Km carrier was competitively inhibited by D-galactose.

Biological Transport, Active

Meningitis caused by Rhodotorula.

A case of rhodotorula meningitis in a 21 year old male compromised with acute lymphoblastic leukemia is reported. The yeast was identified as Rhodotorula rubra (Demme) Lodder.

Adult

Endocarditis caused by Rhodotorula successfully treated with 5-fluorocytosine.

A 7-year-old boy suffering from aortic regurgitation and mitral stenosis and regurgitation was admitted with endocarditis caused by Rhodotorula pilimanae and was treated successfully with orally administered antifungal agent, namely 5-fluorocytosine (5-FC). A dose of 100 mg per kg body weight, divided into four equal parts, was prescribed. After a prolonged febrile period his temperature dropped to normal on the fourth day of 5-FC therapy. Review of the published reports disclosed few cases of endocarditis due to Rhodotorula spp. and this case seems to be the first treated with 5-FC. Follow-up in one year, after discharge from the hospital, revealed no evidence of relapse.

Administration, Oral

Engineering of xylose metabolic pathways in Rhodotorula toruloides for sustainable biomanufacturing.

The oleaginous yeast Rhodotorula toruloides is a promising microbial cell factory for the sustainable production of biofuels and value-added chemicals from renewable carbon sources. Unlike the conventional yeast Saccharomyces cerevisiae, R. toruloides can naturally metabolize xylose, the second most abundant sugar in lignocellulosic hydrolysates. However, its native xylose metabolism is inefficient, characterized by slow xylose uptake and accumulation of D-arabitol. Moreover, despite its phenotype, research on the enzymes involved in xylose metabolism has yet to reach a consensus. Therefore, this review provides a comprehensive analysis of the non-canonical xylose metabolism in R. toruloides, focusing on the properties of key enzymes involved in xylose metabolism. Native xylose reductase and xylitol dehydrogenase exhibit broad substrate promiscuity compared to their counterparts in the xylose-fermenting Scheffersomyces stipitis. Additionally, the absence of xylulokinase expression under xylose-utilizing conditions redirects metabolism toward D-arabitol accumulation. Consequently, D-arabitol dehydrogenases and ribulokinase play essential roles in the xylose metabolism of R. toruloides. These findings highlight the fundamental differences between R. toruloides xylose metabolism and the oxidoreductase pathways observed in other xylose-fermenting yeast, providing insights for metabolic engineering strategies to improve xylose utilization and enhance bioconversion of cellulosic hydrolysates to different bioproducts by R. toruloides.

Xylose

[Transport and utilization of alditols in the yeast Rhodotorula gracilis glutinis (author's transl)].

Although alditols reached the interior of the cells of Rhodotorula gracilis by means of the constitutive monosaccharide carrier, a second carrier of pentitols was found to be inducible. Ribitol and L-arabinitol, entering the cells by the constitutive carrier, induced a single catabolic enzyme system, including a carrier of pentitols. This induction depended on de novo protein synthesis. In addition to ribitol and L-arabinitol, the induced catabolic enzyme system was also specific for xylitol and D-arabinitol; these two alditols were not inducers. Hexitols, erythritol and pentoses were not transported or catabolized by the induced system. The induced carrier had a lower half-saturation constant, KT, and higher maximal velocity, V, than the constitutive carrier of penitols. Kinetic studies of induced transport indicated, that this was an active process which appeared to be coupled with the simultaneous uptake of H+-ions (H+-substrate symport). The induced transport of pentitols was strongly inhibited by monosaccharides, although not competitively. The enzyme synthesis induced by ribitol or L-arabinitol was repressed by monosaccharides.

Biological Transport, Active

Monosaccharide transport systems in the yeast Rhodotorula glutinis.

By using D-glucose, D-xylose, D-galactose and D-fructose in the strictly aerobic yeast Rhodotorula glutinis and by comparing the half-saturation constants with inhibition constants the yeast was shown to possess a single common system for D-xylose and D-galactose (Km's and Ki's all between 0.5 and 1.1 mM) but another distinct transport system for D-fructose. The transport of D-glucose has a special position in that glucose blocks apparently allotopically all the other systems observed although it uses at least one of them for its own transport. The different character of D-glucose uptake is underlined by its relative independence of pH (its "Km" is completely pH-insensitive) in contrast with all other sugars. At low concentrations, all sugars show mutual positive cooperativity in uptake, suggesting at least two transport sites plus possibly a modifier site on the carrier.

Binding Sites

Regulation of ornithine transcarbamylase in Rhodotorula glutinis.

The regulation of ornithine transcarbamylase (OTC) of Rhodotorula glutinis has been studied, by growing the yeasts in different carbon and nitrogen sources and estimating the enzyme level in crude yeasts extracts. The results show a nutritional repression of OTC by arginine, when added to the culture media as carbon, nitrogen or carbon and nitrogen sources. On the other hand ornithine does not exert any effect in the same experimental conditions.

Amino Acids

Oxygen pressure, fatty acid composition and ergosterol level in Rhodotorula gracilis.

Cells of Rhodotorula gracilis grown for 6 hrs at 2 mm Hg O2 pressure when compared with cells grown for 6 hrs at 140 mm Hg, show: a) a large decrease in the level of ergosterol, b) a significant increase in the level of oleic acid and a decrease in the levels of linoleic and linolenic acids, both in fatty acid fraction of the phospholipids and in the free fatty acids and neutral fat fractions. The results suggest that the dehydrogenation of oleic acid to linoleic acid is preferentially inhibited at low O2 pressure. The possibility is considered that these changes of lipid metabolism might be causally related with decrease of the growth rate observed at low O2 pressure.

Ergosterol

Some properties of the adenosine triphosphatase systems of two yeast species, Saccharomyces cerevisiae and Rhodotorula glutinis.

1. Total ATPase levels were determined in homogenate fractions of baker's yeast, Saccharomyces cerevisiae K and Rhodotorula glutinis. The maximum ATPase activities in 8000 X g supernatant of the three yeast strains were 6.0, 1.9, and 2.2 mmol Pih-1 (gDS)-1, respectively; the activities in the sediment were somewhat higher. Exponential cells of S. cerevisiae K and R. glutinis exhibited higher ATPase levels than did the stationary cells. 2. The total ATPase activity in both yeast species showed a maximum at ph 6.8 a minimum at pH 7.2, and another broader masimum around pH 8.0. 3. No significant NaK-ATPase activity was detected in baker's yeast, in either the exponential or the stationary cells of R. glutinis, and in exponential S. cerevisiae K cells in the pH range of 6.0-9.3. 4. Stationary cells of S. cerevisiae K exhibited, at pH 7.0-8.5, A Na,K-ATPase activity attaining 9% of total ATPase level. 5.3 X 10(-3) M phenylmethyl sulphonyl fluoride had no effect on the total ATPase level in S. cerevisiae and inhibited the activity in R. glutinis by 25%; it did not bring forth any Na,K-ATPase activity apart from that found in its absence. 6. 1.5 M urea lowered the ATPase activity in R. glutinis by 68% but had no effect on S. cerevisiae cells. 10(-5) M dicyclohexylcarbodiimide suppressed the ATPase activity in S. cerevisiae and R. glutinis by 74 and 79%, respectively. Neither agent revealed and additional Na,K-ATPase activity. 7. The comparison of Na,K-ATPase activities with data on K+ fluxes across the yeast plasma membrane suggested that even with the lower flux values the Na,K-ATPase, even if present, would account for a mere 40% of transported ions. The results imply that the active ion transport in yeasts is energized by mechanisms other than the Na,K-ATPase.

Adenosine Triphosphatases

Temperature dependence of the energy-linked monosaccharide transport across the cell membrane of Rhodotorula gracilis.

The temperature dependence of the active monosaccharide transport across the cell membrane of the yeast Rhodotorula gracilis has been studied between 0 and 55 degrees C with D-xylose as the transported substrate: (i) Between 0 and 10 degrees C there is virtually no transport. (ii) The initial velocity of transport increases exponentially from 15 to 30 degrees C (deltaE equal to 32 plus or minus 2 kcal/mol). (iii) At 30 degrees C a sharp "break" occurs in the Arrhenius plot and with increasing temperature the transport becomes inactivated, with a positive slope of the corresponding straight line ("deltaE equal to minus 15 kcal/mol"). (iv) In the temperature range of 50-55 degrees C, both the transport and the metabolic activity cease. In order to account for the abrupt changes of the membrane permeability, we attempted to ascribe them to phase transitions in the membrane structure: the first one, between 10 and 15 degrees C, to the crystalline: liquid-crystalline phase change; the second one, around 30 degrees C, to a change from highly ordered (low entropy) to less ordered (high entropy) membrane structure. Whereas the former phase transition is reversible, the latter appears to be irreversible. Arrhenius plots of the cell respiration exhibit a "break" at 30 degrees C, as well. However, at higher temperatures there is no thermal inactivation of the respiratory activity. The importance of a proper organization of the cell membrane constituents for the efficient transport function is discussed.

Biological Transport, Active

Cytological and chemical changes in cell walls of Rhodotorula gracilis. III. Characteristics and life cycle of the yeast.

The phenotype and genotype of six strains of the genus Rhodotorula Harrison and of one strain of the genus Cryptococcus Phaff et Fell, with anomalous thickening of cell walls were investigated. The present studies showed that the strains investigated represent different stages of the life cycles of the genus Rhodosporidium Banno. The anomalous thickening of the cell walls can be explained by extreme conditions resulting in the formation of surviving forms (teliospores, chlamydospores).

Bacterial Proteins

Temperature dependence of the apparent affinity and the maximum velocity of the membrane-bound monosaccharide transport system in the yeast Rhodotorula gracilis.

Analysis of the temperature dependence of the monosaccharide transport system in the yeast Rhodotorula gracilis (ATCC 26194, CBS 6681), as tested with D-xylose, revealed that the apparent affinity of the transport system, measured as the reciprocal of the half-saturation constant KT, increased when transport velocity was stimulated by temperature (15--30 degrees C) and decreased when the rate of uptake was reduced at temperatures aboce 30 degrees C. Breaks in Arrhenius plots were accompanied by corresponding breaks in van't Hoff plots. Whereas untreated cells exhibited in the van't Hoff plot a discontinuity at 28--30 degrees C this was not observed in heat-treated cells (at either 37 or 45 degrees C). In heat-treated cells the maximum transport velocity was always lower and the apparent affinity higher than in untreated cells at the same temperature; the optimum temperature for both transport velocity and apparent affinity was shifted to higher values. The data are interpreted in terms of a reversible phase transition of membrane lipids effecting an irreversible alteration of membrane structure. The temperature-induced reversible alkalinization of unbuffered yeast suspensions supports this interpretation.

Biological Transport, Active

The effect of nystatin on active transport in Rhodotorula glutinis (gracilis) is restricted to the plasma membrane.

The effect of the polyene antibiotic nystatin on the energy-requiring transport of substrates in the obligatory aerobic yeast Rhodotorula glutinis (gracilis) is restricted to its interaction with the plasma membrane, whereas the energy metabolism in mitochondria remains unaffected by the antibiotic. A method for preparation of the mitochondrial fraction from this yeast is described.

Adenosine Triphosphate

Purification and properties of the NAD(P)H:nitrate reductase of the yeast Rhodotorula glutinis.

The assimilatory nitrate reductase from the yeast Rhodotorula glutinus has been purified 740-fold, its different catalytic activities have been characterized and some inhibitors studied. The purified enzyme (150 units per mg protein) contains a cytochrome of the b-557 type. An S20,w of 7.9 S was found by the use of sucrose density gradient centrifugation, and a Stokes radius of 7.05 nm was determined by gel filtration. From these values, a molecular weight of 230 000 was estimated for the native enzyme. The purified preparation consisted of two electrophoretically distinguishable proteins, both of which exhibited nitrate reductase activity. The species with the higher electrophoretic mobility which represented the great majority of the total nitrate reductase gave a positive stain for heme and was shown to be composed of subunits with a molecular weight of about 118 000. Thus the molecule contains two subunits of the same size.

Azides

Adaptation of the mitochondrial systems of Rhodotorula gracilis to low oxygen pressure.

The obligate aerobic yeast, Rhodotorula gracilis, was grown in a liquid minimal medium at 1 mm Hg partial pressure of oxygen, conditions under which growth (measured as the increase in total protein) is reduced to 30% of the maximum rate. A significant increase in the ratio between mitochondrial oxidative enzymes and total protein occurs rapidly under these conditions. A concurrent increase in the ratio area of mitochondria/area of cytoplasm was also observed. The relative increase in mitochondrial enzymes, oxidase activities and mitochondrial membranes is due to the inhibition affecting the increase in cytoplasmic structures more significantly than the increase in mitochondrial structures at low pO2. The difference between mitochondrial and cytoplasmic syntheses cannot be ascribed to changes in the availability of ATP but it might rest with some other oxygen-utilising process (pyrimidine redox coenzymes, synthesis of sterols). The experimental conditions studied appear to offer a valuable tool for the investigation of the relationships between mitochondrial and cytoplasmic structures.

Aerobiosis