Transport of L-glucose by Rhodotorula glutinis

The kinetics of L-glucose transport by Rhodotorula glutinis were studied over a 720-fold range of sugar concentrations. Analysis of the saturation isotherm revealed the presence of a one-carrier system for L-glucose in the plasma membrane of Rhodotorula glutinis. This carrier exhibited a km of 3.7 +/- 0.3 mM. D-Ribose was found to be a competitive inhibitor with a Ki of 19 +/- 1 mM. The results suggest that L-glucose is transported by the high-Km, D-ribose carrier. L-Glucose was transported against a concentration gradient and the transport was inhibited by the proton conductor 2,4-dinitrophenol.     

Transport and metabolism of 2-deoxy-D-glucose by Rhodotorula glutinis

2-Deoxy-D-glucose transport by Rhodotorula glutinis is an active process. The intracellular concentration of free deoxyglucose after 15 min incubation of Rhodotorula cells with this sugar was 230 times the extracellular concentration. Although cell extracts at this time contained more 2-deoxy-D-glucose 6-phosphate than deoxyglucose, pulse-labelling experiments demonstrated that deoxyglucose is transported as the free sugar and subsequently phosphorylated. After transport, Rhodotorula cells metabolize deoxyglucose. The major metabolites during 30-90 min incubations were determined to be 2-deoxy-D-glucose 6-phosphate, 2-deoxy-D-glucitol, 2-deoxy-D-gluconate and 2,2'-dideoxy-alpha, alpha'-trehalose. Rhodotorula glutinis also degrades deoxyglucose to CO2. The concentrations of intermediates in this pathway were too low to detect and resolve in extracts of control cells. In 2,4-dinitrophenol-poisoned cells, however, it appears that deoxyglucose degradation is restricted largely to loss of C-1 as CO2 and it was possible to identify 1-deoxy-D-ribulose 5-phosphate as an intermediate presumably arising from metabolism of deoxyglucose by the oxidative portion of the hexose monophosphate pathway.     

Inactivation of Nitrate Reductase of the Yeast, Rhodotorula glutinis

Nitrate reductase (NR) from the yeast, Rhodotorula glutinis var. salinaria was composed of two enzymatic components, diaphorase and terminal nitrate reducing moieties. The enzyme used NADPH as electron donor and FAD as cofactor. The synthesis of nitrate reductase was promoted specifically by nitrate and repressed by ammonium and amino acids. Nitrate reductase from this yeast had an inactive as well as an active form. Inactive enzyme was reactivated by oxidation with ferricyanide in vitro. Hydroxylamine promoted the formation of inactive enzyme in vivo. Ammonium could neither promote the inactivation nor reduce the total level of nitrate reductase activity. Nitrate could protect nitrate reductase from inactivation caused by nitrogen starvation or hydroxylamine.     

Effect of glucose on pyruvate utilization by Rhodotorula glutinis

Summary The utilization of glucose and pyruvate by the yeast Rhodotorula glutinis in a medium containing both carbon sources has been studied. Glucose is readily consumed whereas the uptake of pyruvate is completely blocked by the presence of the sugar.The content of pyruvate kinase and phosphoenolpyruvate carboxykinase in R. glutinis cells growing on glucose plus pyruvate are drastically affected with time by the disappearance of the sugar from the culture medium. After complete exhaustion of glucose, the level of pyruvate kinase drops sharply down to a minimum whereas that of phosphoenolpyruvate carboxykinase rises abruptly up to a maximum.Feeding experiments with labelled compounds show that glucose affects the utilization of the amino acids alanine and aspartate, and conversely that the amino acids influence the utilization of the sugar. Glucose breakdown and its incorporation into polysaccharides is controlled by the amino acids and gluconeogenesis from the amino acids is controlled by the sugar.     

Carotenoid Biosynthesis in Rhodotorula glutinis

It was determined that lycopene could be cyclized directly by Rhodotorula glutinis. It was also shown the the temperature effect (i.e., increased beta-carotene synthesis in response to lower incubation temperatures) in R. glutinis was controlled by changes in enzyme concentration.     

Regulation of phenylalanine biosynthesis in Rhodotorula glutinis.

The phenylalanine biosynthetic pathway in the yeast Rhodotorula glutinis was examined, and the following results were obtained. (i) 3-Deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase in crude extracts was partially inhibited by tyrosine, tryptophan, or phenylalanine. In the presence of all three aromatic amino acids an additive pattern of enzyme inhibition was observed, suggesting the existence of three differentially regulated species of DAHP synthase. Two distinctly regulated isozymes inhibited by tyrosine or tryptophan and designated DAHP synthase-Tyr and DAHP synthase-Trp, respectively, were resolved by DEAE-Sephacel chromatography, along with a third labile activity inhibited by phenylalanine tentatively identified as DAHP synthase-Phe. The tyrosine and tryptophan isozymes were relatively stable and were inhibited 80 and 90% by 50 microM of the respective amino acids. DAHP synthase-Phe, however, proved to be an extremely labile activity, thereby preventing any detailed regulatory studies on the partially purified enzyme. (ii) Two species of chorismate mutase, designated CMI and CMII, were resolved in the same chromatographic step. The activity of CMI was inhibited by tyrosine and stimulated by tryptophan, whereas CMII appeared to be unregulated. (iii) Single species of prephenate dehydratase and phenylpyruvate aminotransferase were observed. Interestingly, the branch-point enzyme prephenate dehydratase was not inhibited by phenylalanine or affected by tyrosine, tryptophan, or both. (iv) The only site for control of phenylalanine biosynthesis appeared to be DAHP synthase-Phe. This is apparently sufficient since a spontaneous mutant, designated FP9, resistant to the growth-inhibitory phenylalanine analog p-fluorophenylalanine contained a feedback-resistant DAHP synthase-Phe and cross-fed a phenylalanine auxotroph of Bacillus subtilis.     

Regulation of phenylalanine ammonia lyase in Rhodotorula glutinis.

In the red yeast Rhodotorula glutinis, phenylalanine ammonia lyase (PAL) was induced 10-fold during carbon starvation even in the absence of exogenous phenylalanine, although maximal induction occurred when phenylalanine was the nitrogen (40-fold) or carbon (100-fold) source. Apparent regulatory mutations that affected the expression of PAL were isolated by selecting mutants resistant to the analog p-fluoro-D,L-phenylalanine (PFP). One such mutant, designated FP1, could use phenylalanine as a nitrogen source but not as a carbon source. Similarly, FP1 failed to utilize intermediates of the phenylalanine degradative pathway, namely, benzoate, p-hydroxybenzoate, or 3,4-dihydroxybenzoate, as carbon sources. Although the PFP-resistant mutant contained a low level of PAL, no increase was found when it was grown with phenylalanine as the nitrogen source. A derivative of FP1, FP1a, was isolated that simultaneously regained an inducible PAL and the ability to use phenylalanine, benzoate, p-hydroxybenzoate, and 3,4-dihydroxybenzoate as carbon sources. In addition, when p-hydroxybenzoate was the carbon source, PAL was induced in the mutant FP1a but not in the PFP-sensitive parental strain. We propose that the mutation to PFP resistance occurred in a regulatory gene that controls the entire phenylalanine degradative pathway. Secondary mutations at this locus, as found in strain FP1a, not only restored expression of this pathway, but also altered the induction of PAL by metabolites of this pathway.     

Uptake of disaccharides by the aerobic yeast Rhodotorula glutinis

Sucrose (and raffinose), trehalose, maltose, cellobiose, and lactose were examined for their transport into Rhodotorula glutinis. Melibiose and lactose were found not to be transported at all. Sucrose, raffinose and trehalose are split by periplasmic hydrolases prior to the penetration of their monosaccharide components into cells, the hydrolysis being the rate-limiting factor for the uptake process. Maltose and cellobiose appear to use specific uptake systems. Experiments with protoplasts of Rhodotorula glutinis support the conclusions that sucrose and trehalose are not consumed in the absence of exoenzymes.     

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.     

The respiratory system of Rhodotorula glutinis. II. Mechanism of inhibitor tolerant respiration

The mechanism of inhibitor-tolerant respiration in Rhodotorula glutinis was studied. This inhibitor-tolerant respiratory pathway was not due to the presence of an excess of cytochrome c oxidase, nor to the operation of an inhibitor-resistant cytochrome c oxidase. Carotenoids do not appear to be involved in this respiratory chain pathway; data are also presented which show that the inhibitors penetrate into the cell. Although the initial rate of oxygen uptake by intact cells was not inhibited in the presence of cyanide or antimycin A, in the presence of these inhibitors the rate of oxygen uptake decreased significantly when the oxygen concentration fell below 100 mum. This change in rate of oxygen uptake as a function of pO(2), suggests that a respiratory chain with a low affinity for oxygen operates in the presence of inhibitors. The characteristics of this alternate pathway are described.     

Synthesis of intracellular and extracellular metabolites by Rhodotorula glutinis

A close relationship has been established between the production of extracellular and intracellular lipids and polysaccharides by Rhodotorula glutinis. Their synthesis depended on the pH of the medium. The composition and dynamics of polyol content in the cells, and the presence of acids typical of exolipids in the composition of intracellular lipids under certain conditions, suggest that the components of extracellular lipids are synthesized within the yeast cell.     

Carbohydrate assimilation by clinical and environmental Rhodotorula glutinis strains

This study was carried to determine the carbohydrate assimilation patterns of Rhodotorula strains isolated from clinical and environmental specimens. We have tested the commercial system ID 32C (bioMerieux, France) on 80 different strains of Rhodotorula glutinis: 47 strains from clinical samples and 33 strains from environmental samples. The assimilation percentages obtained in our study for galactose, cellobiose, gluconate and sorbose were lower than those showed in the identification table of the method. However, the assimilation percentages for mannitol and esculin were higher. According to our results, we conclude that the numerical profiles and the identification software of the commercial system present limitations for the characterization of some R. glutinis strains.     

Transport und Umsatz von Polyalkoholen bei der Hefe Rhodotorula gracilis (glutinis)

Zusammenfassung Die Polyalkohole Sorbitol, d-Mannitol, Ribitol, Xylitol, d-Arabitol, l-Arabitol und Erythritol werden von der obligat aeroben Hefe Rhodotorula gracilis über einen beweglichen Träger in der Zellmembran aufgenommen. Der Transportmechanismus ist aktiv, das erreichte Akkumulationsverhältnis ist jedoch bei allen Polyalkoholen erheblich geringer als bei Monosacchariden. Es nimmt, wie auch bei Monosacchariden, mit steigender Außenkonzentration ab, sogar auf Werte kleiner als 1.Kinetische Daten weisen darauf hin, daß das Trägersystem für Polyalkohole identisch ist mit dem für Monosaccharide, jedoch für Polyalkohole eine wesentlich geringere Affinität und maximale Geschwindigkeit aufweist. Aufgrund des hohen Affinitätsunterschiedes wird die Polyalkoholaufnahme in der Anwesenheit von Monosacchariden unterbunden.Die aufgenommenen Polyalkohole werden im Zellinneren nicht umgesetzt; eine Ausnahme stellen Ribitol und l-Arabitol dar, in deren Anwesenheit ein Abbausystem für Pentitole induziert wird.

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Transport and utilization of alditols in the yeast Rhodotorula gracilis (glutinis)I. Constitutive transport of alditols

The obligate aerobic yeast Rhodotorula gracilis was found to take up the alditols d-glucitol, d-mannitol, ribitol, xylitol, d-arabinitol, l-arabinitol and erythritol by means of a constitutive mobile membrane carrier. This uptake involved active transport, that is, it was dependent on the supply of metabolic energy, leading to the accumulation of alditols inside the cells. The accumulation ratio (intracellular concentration to extracellular concentration, S_i/S_o) was much lower for alditols than for monosaccharides. As for monosaccharides, this ratio decreased with increasing extracellular concentration, even to values below 1.The kinetic data showed that the carrier system for alditols was identical to that for monosaccharides, though it had a much lower affinity and maximum velocity for alditols. Hence the uptake of alditols was blocked in the presence of monosaccharides.Only ribitol and l-arabinitol were catabolized following enzyme induction. The other alditols were not broken down.