The effects of low molecular weight (96.5 KDa) chitosan on the pathogenic yeast Candida albicans were studied. Low concentrations of chitosan, around 2.5 to 10 μg·mL−1 produced (a) an efflux of K+ and stimulation of extracellular acidification, (b) an inhibition of Rb+ uptake, (c) an increased transmembrane potential difference of the cells, and (d) an increased uptake of Ca2+. It is proposed that these effects are due to a decrease of the negative surface charge of the cells resulting from a strong binding of the polymer to the cells. At higher concentrations, besides the efflux of K+, it produced (a) a large efflux of phosphates and material absorbing at 260 nm, (b) a decreased uptake of Ca2+, (c) an inhibition of fermentation and respiration, and (d) the inhibition of growth. The effects depend on the medium used and the amount of cells, but in YPD high concentrations close to 1 mg·mL−1 are required to produce the disruption of the cell membrane, the efflux of protein, and the growth inhibition. Besides the findings at low chitosan concentrations, this work provides an insight of the conditions required for chitosan to act as a fungistatic or antifungal and proposes a method for the permeabilization of yeast cells.
Debaryomyces hansenii was grown in YPD medium without or with 1.0 M NaCl or KCl. Respiration was higher with salt, but decreased if it was present during incubation. However, carbonylcyanide-3-chlorophenylhydrazone (CCCP) markedly increased respiration when salt was present during incubation. Salt also stimulated proton pumping that was partially inhibited by CCCP; this uncoupling of proton pumping may contribute to the increased respiratory rate. The ADP increase produced by CCCP in cells grown in NaCl was similar to that observed in cells incubated with or without salts. The alternative oxidase is not involved. Cells grown with salts showed increased levels of succinate and fumarate, and a decrease in isocitrate and malate. Undetectable levels of citrate and low-glutamate dehydrogenase activity were present only in NaCl cells. Both isocitrate dehydrogenase decreased, and isocitrate lyase and malate synthase increased. Glyoxylate did not increase, indicating an active metabolism of this intermediary. Higher phosphate levels were also found in the cells grown in salt. An activation of the glyoxylate cycle results from the salt stress, as well as an increased respiratory capacity, when cells are grown with salt, and a 'coupling' effect on respiration when incubated in the presence of salt.
Different methods to estimate the plasma membrane potential difference (PMP) of yeast cells with fluorescent monitors were compared. The validity of the methods was tested by the fluorescence difference with or without glucose, and its decrease by the addition of 10 mM KCl. Low CaCl₂ concentrations avoid binding of the dye to the cell surface, and low CCCP concentrations avoid its accumulation by mitochondria. Lower concentrations of Ba²+ produce a similar effect as Ca²+, without producing the fluorescence changes derived from its transport. Fluorescence changes without considering binding of the dyes to the cells and accumulation by mitochondria are overshadowed by their distribution between this organelle and the cytoplasm. Other factors, such as yeast starvation, dye used, parameters of the fluorescence changes, as well as buffers and incubation times were analyzed. An additional approach to measure the actual or relative values of PMP, determining the accumulation of the dye, is presented.
The fermentation and respiration activities of Debaryomyces hansenii were compared with those of Saccharomyces cerevisiae grown to stationary phase with high respiratory activity. It was found that: (a) glucose consumption, fermentation and respiration were lower than for S. cerevisiae; (b) fasting produced a much smaller decrease of respiration; (c) glucose consumed and not transformed to ethanol was higher; (d) in S. cerevisiae, full oxygenation prevented ethanol production but this effect was reversed by CCCP, whereas D. hansenii still showed some ethanol production under aerobiosis, which was moderately increased by CCCP. ATP levels were similar in the two yeasts. Levels of glycolytic intermediaries after glucose addition, and enzyme activities, indicated that the main difference and limiting step to explain the lower fermentation of D. hansenii is phosphofructokinase activity. Respiration and fermentation, which are lower in D. hansenii, compete for the re-oxidation of reduced nicotinamide adenine nucleotides; this competition, in turn, seems to play a role in defining the fermentation rates of the two yeasts. The effect of CCCP on glucose consumption and ethanol production also indicates a role of ADP in both the Pasteur and Crabtree effects in S. cerevisiae but not in D. hansenii. D. hansenii shows an alternative oxidase, which in our experiments did not appear to be coupled to the production of ATP.
Growth of Saccharomyces cerevisiae stopped by maintaining the pH of the medium in a pH-stat at pH 8.0 or 9.0. Studying its main physiological capacities and comparing cells after incubation at pH 6.0 vs. 8.0 or 9.0, we found that (a) fermentation was moderately decreased by high pH and respiration was similar and sensitive to the addition of an uncoupler, (b) ATP and glucose-6-phosphate levels upon glucose addition increased to similar levels and (c) proton pumping and K(+) transport were also not affected; all this indicating that energy mechanisms were preserved. Growth inhibition at high pH was also not due to a significant lower amino acid transport by the cells or incorporation into proteins. The cell cycle stopped at pH 9.0, probably due to an arrest as a result of adjustments needed by the cells to contend with the changes under these conditions, and microarray experiments showed some relevant changes to this response.
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