Transitional states in a chemostat culture of Candida utilis

Transient states of the chemostat Candida utilis 1668-3-37 culture were studied when its growth was limited by ethanol and an abrupt acidification of the medium from pH 5.0 to 2.2 was done or when the dilution rate was rapidly changed from D = 0.1 to 0.3 h-1 and back to 0.07 h-1. The pH shock was found to cause stronger oscillations in a number of parameters (the weight of dry biomass, the content of residual ethanol, the content of RNA in the cells) than a change in the dilution rate. In the latter case the population density changed more smoothly than the content of RNA did. DNA content remained at one and the same level in all of the experiments. All of the oscillations were observed only in the first generation after a shock; there upon, the culture remained for a long time (7 to 10 generations) in a very stable state typical of chemostat cultures. The oscillations induced by the unfavourable pH of the medium were compared with those caused by an abrupt change in the dilution rate. The pH shock brought about multiple damping oscillations of the parameters whereas a change in the dilution rate resulted, most often, in a merely one oscillation.     

Effect of cyclic AMP on RNA and protein synthesis in Candida albicans.

Sequence analysis by the automated Edman degradation shows that dopamine $\text{β}$-hydroxylase (dopamine $\text{β}$-monooxygenase; EC 1.14.17.1) from bovine adrenal medulla contains equal amounts of NH₂-terminal alanine and serine residues. The sequence data are in agreement with the proposal that this enzyme consists of two types of polypeptide chains which are identical in the NH₂-terminal ends, except that one of the chains lack the NH₂-terminal tripeptide Ser-Ala-Thr.     

Respiratory oscillations and heat evolution in synchronous cultures of Candida utilis

Synchronous cultures of the budding yeast Candida utilis prepared by continuous-flow size selection showed respiratory oscillations when the energy source was either glucose, acetate or glycerol. The period of the oscillations was about one-third of the cell cycle time (i.e. about 0.5 h). No fluctuations in heat evolution could be detected. In organisms growing with acetate or glycerol, the effects of cyanide, N,N'-dicyclohexylcarbodi-imide and carbonyl cyanide m-chlorophenylhydrazone (maximum inhibition of respiration at respiratory maxima, maximum uncoupling of energy conservation at respiratory minima) suggest that the control mechanism responsible for the oscillations is mitochondrial respiratory control in vivo. The effects of cyanide and N,N'-dicyclohexylcarbodi-imide on the respiration of cultures growing synchronously with glucose were different from those for cultures growing with the non-fermentable substrates; this suggests that the mitochondrial respiratory system interacts with the early reactions of glucose utilization.     

Several parameters of the growth of chemostatic culture of Candida utilis in the presence of optimal and submaximal temperatures

Candida utilis BKM Y-1668 was cultivated in the chemostat (limitation with glycerol) with the rate of flow D from 0.05 to 0.3 hr-1; the economic coefficient Y and Ks were constant at the optimum temperature of growth (30 degrees C). The maximum growth rate was 0.35 hr-1. The content of ATP in the cells and the energy charge of the cell decreased, and the content of ADP and AMP and the activity of phoshohydrolases increased in the cell, with an increase in D from 0.05 to 0.3 hr-1. Small amounts of glycerol and phosphorus were expended for maintaining life without multiplication (m) at 30 degrees C. At the submaximum temperature (40 degrees C), growth of the cells was inhibited, the rate of assimilation of glycerol and phosphorus, and m, increased. The content of ATP in the cells and their energy charge also increased.     

Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.     

Influence of acetate on the growth of Candida utilis in continuous culture

Candida utilis was grown on acetate in chemostat cultures that were, successively, carbon and ammonia-limited (30° C; pH 5.5). With carbon(acetate)-limited cultures, the specific rate of oxygen consumption (q _{O 2}) was not a linear function of the growth rate but was markedly stimulated at the higher dilution rates, thus effecting a marked decrease in the Y _O value. This increased respiration rate, and decreased yield value, correlated closely with a marked increase in the extracellular acetate concentration. Under ammonia-limiting conditions, very low Y _O values were found, generally comparable with those found with carbon-limited cultures growing at the higher dilution rates, but these varied markedly with the extracellular acetate concentration. Thus, when the unused acetate concentration was raised progressively from about 5 g/l to about 21 g/l, the Y _O value decreased non-linearly from 11.4 to 5.8. When the extracellular acetate concentration was further increased to 25 g/l, growth was inhibited and the culture washed out. This relationship between respiration rate and the extracellular concentration of unused acetate was also markedly influenced by the culture pH value. Thus, with a fixed extracellular acetate concentration (16±2g/l) and dilution rate (0.14 h^–1), lowering the culture pH value progressively from 6.9 to 5.1 effected a marked and progressive increase in the respiration rate. Further lowering of the culture pH to 4.8, however, caused a complete collapse of respiration. In contrast to this situation, progressively lowering the pH value of an acetatelimited culture from 6.9 to 4.5 affected only slightly the culture respiration rate, and growth was possible even at a pH value of 2.5. These results are discussed in the context of the possible mechanisms whereby acetate exerts its toxic effect on the growth of C. utilis.     

Simultaneous action of environmental factors on Candida utilis growth in a chemostat culture

The effect of carbon dioxide and carbon-containing metabolites on the growth of Candida utilis was studied under the conditions of phosphate limitation. The limiting factor and the hydrocarbonate form of CO2 were shown to act simultaneously on the yeast growth, decreasing its rate. The threshold concentration of the limiting factor remained unchanged. Carbon-containing metabolites produced a similar action on the chemostat yeast culture. The factors limiting the rate of the yeast growth did not switch over.     

Effect of X rays on protein synthesis and RNA synthesis in adenovirus-infected cells.

Ionizing radiation exerts a deteriorating effect on the rate of protein and RNA synthesis in HeLa cells. This effect is higher in cells infected with adenovirus, thus pointing to a higher radiosensitivity of viral syntheses compared with the cellular ones.     

Batch culture of Candida utilis in a medium deprived of a phosphorous source

Summary Candida utilis var. major NRRL-Y-1084 was grown in a defined medium without a phosphorous (P) source. During the exponential phase, cells divided according to a specific growth rate of 0.32 h^-1, which is lower than the usual rate for a balanced medium (0.4–0.6 h^-1). The relative P content of the biomass decreased from 2.70% to 0.75% over a period of 6 h, including 2 h of cell division arrest. At the end of this period there was another interruption of cell division. After that, multiplication restarted at a considerably lower rate and it deviated slightly from the exponential pattern. The stationary phase began when biomass P content reached 0.4%–0.5%, slowly decreasing afterwards to 0.25–0.20%. Biomass synthesis was less affected than cell division by the relative decrease of endogenous P, the two processes differing partially in their kinetics. Cell lysis started shortly before the stationary phase and affected about 20% of the population by the end of the assay. RNA and P content of the resulting biomass were 2.4% and 0.25% respecitvely, P being mainly incorporated to RNA.The relationship of biomass production to glucose uptake was very low, probably because the marked P deficiency called for an increase in energy consumption for growth and specially for maintenance. Compared with yeasts grown in a balanced medium, 40% increase in glycogen was observed, whereas no mean changes in the content of cell wall carbohydrates (glucan and mannan) and that of true protein were found.Member of the Scientific Researcher's Career of the Consejo Nacional de Investigaciones Cientificas y Técnicas (CONICET). Agrentina     

Effect of elevated temperature on the morphology of a chemostat culture of Candida utilis yeasts and on their content of nucleic acids

The effect of elevated temperature (40 degrees C) on a chemostat culture of Candida utilis was studied at different rates of dilution, D = 0.1 and 0.3 hr-1. The cells in the fermenter being in the stationary state at the optimum temperature of 30 degrees C were gradually washed-out 5 hours after the action of this temperature, and the population consisted of non-divided cells. In the majority of such "double" cells, the nucleus was contained in both the parent and the daughter parts. The content of RNA decreased by 31%, that of DNA, by 20%, and that of protein, by 13% (per 1 mg of biomass).     

Effect of RNA synthesis inhibitors on insulin-induced protein synthesis by 3T3 cells

1. The increased protein synthesis of quiescent 3T3 cells in response to insulin was separated into three distinct phases based on their response to various inhibitors of RNA synthesis. 2. The first increase in protein synthesis was insensitive to the inhibitors used, and probably resulted from activation of existing protein synthesizing mechanism. 3. The second phase was sensitive to a varying extent to alpha-amanitin and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole, implying the need for new mRNA synthesis as well as the production of new ribosomes indicated by its further sensitivity to low concentration (10 ng/ml) of Actinomycin D. 4. The final phase was insensitive to inhibitors of new ribosome formation, but still depended on new mRNA. alpha-difluoromethylornithine, an inhibitor of de novo polyamine synthesis, partly inhibited the insulin induced stimulation of protein synthesis.     

Growth and physiology of Candida utilis NCYC 321 in potassium-limited chemostat culture

When grown in a defined simple salts medium, plus vitamins, Candida utilis displayed an absolute requirement for potassium. But the potassium content of this yeast was exceedingly variable and, with aerobic chemostat cultures (grown at a dilution rate of 0.1 h^-1; 30° C; pH 5.5), was low (< 0.2%, w/w) when they were potassium-limited and high (> 2%, w/2) when glucose-limited. With potassium-limited cultures, the cell-bound potassium content also varied markedly with growth rate, though hardly at all with glucose-limited cultures; magnesium- and phosphate-limited cultures gave intermediate responses.Changes in cell-bound potassium content correlated only weakly with changes in the cellular contents of magnesium, phosphate and RNA, but strongly with changes in both the Y_glucose and Y_O values, indicating an involvement of potassium in the generation of energy by oxidative phosphorylation reactions and/or the utilization of this energy for growth processes.Studies with isolated mitochondria revealed that potassium-limited organisms had a changed content of cytochrome b relative to cytochrome a, and lacked coupling at either site 2 or site 3 of the respiratory chain.These results are discussed in relation to the reported functions of potassium in the growth of micro-organisms, and the organizational differences between prokaryotic and eukaryotic cells.     

Effect of iron and EDTA on ethyl acetate accumulation in Candida utilis

Summary Production of economically-recoverable products from dilute sugar or ethanol is of practical importance. Conversion of glucose to ethyl acetate by Candida utilis was inhibited by FeCl₃ supplementation as low as 10 uM. EDTA added at the onset of growth on glucose relieved such an inhibition and also caused faster and greater amounts of accumulation of the ester. Addition of EDTA during conversion of ethanol to ethyl acetate showed little effect. EDTA may affect cell permeability and/or oxidative metabolism. For continual ethyl acetate production iron limitation must be maintained during as well as before ethanol utilization.EDTA = Ethylenediaminetetraacetic acid.     

RNA turnover and protein synthesis in fish cells

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from ³H-phenylalanine and ³H-uridine incorporation, respectively, and expressed as % · day⁻¹ and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (∼175 μg RNA · mg⁻¹ cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 ± 1.8)>RTP (25.1 ± 1.7)>CHSE 214 (17.6 ± 1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised · μg⁻¹ RNA · day⁻¹, corresponded to the lowest RNA half-life, 75.4 ± 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5–6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis. Accepted: 12 November 1999     

Effect of alpha-isopropylmalate on the synthesis of RNA and protein in Neurospora

Summary The leu-3/-IPM (-isopropylmalate) regulatory system, previously shown to control several genes of leucine, isoleucine, valine, and histidine biosynthesis, appears likely to be involved also in the regulation of overall RNA and protein synthesis in Neurospora. Upon addition of -IPM the synthesis of all major species of stable RNA was found to be transiently inhibited by approximately 50%. A similar reduction was observed in overall protein synthesis. The inhibition was dependent in both cases on a functional leu-3 gene product, in conformance with previously established patterns of -IPM dependent gene regulation. The overt resemblance of the phenomenon described here to the stringent response of bacteria is noted but neither the mechanism of inhibition nor the precise role of -IPM in the process has been established.