Quantitative physiological study of the fast dynamics in the intracellular pH of Saccharomyces cerevisiae in response to glucose and ethanol pulses

The ability of cells to maintain pH homeostasis in response to environmental changes has elicited interest in basic and applied research and has prompted the development of methods for intracellular pH measurements. Many traditional methods provide information at population level and thus the average values of the studied cell physiological phenomena, excluding the fact that cell cultures are very heterogeneous. Single-cell analysis, on the other hand, offers more detailed insight into population variability, thereby facilitating a considerably deeper understanding of cell physiology. Although microscopy methods can address this issue, they suffer from limitations in terms of the small number of individual cells that can be studied and complicated image processing. We developed a noninvasive high-throughput method that employs flow cytometry to analyze large populations of cells that express pHluorin, a genetically encoded ratiometric fluorescent probe that is sensitive to pH. The method described here enables measurement of the intracellular pH of single cells with high sensitivity and speed, which is a clear improvement compared to previously published methods that either require pretreatment of the cells, measure cell populations, or require complex data analysis. The ratios of fluorescence intensities, which correlate to the intracellular pH, are independent of the expression levels of the pH probe, making the use of transiently or extrachromosomally expressed probes possible. We conducted an experiment on the kinetics of the pH homeostasis of Saccharomyces cerevisiae cultures grown to a stationary phase after ethanol or glucose addition and after exposure to weak acid stress and glucose pulse. Minor populations with pH homeostasis behaving differently upon treatments were identified.

show abstract

“…4B). As expected (34), adding 20 g of glucose liter Ϫ1 to the stationary cells increased the pH i to a value Ͼ7 for most of the cells (Fig. 4D).…”

Section: Analysis Of S Cerevisiae Ph By Flow Cytometrysupporting

confidence: 82%

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

Valkonen

Mojžita

Penttilä

et al. 2013

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

“…Because the intracellular pH is close to neutral (19,20), virtually all fumaric acid inside the cells is present as the double-charged form, while the concentration of undissociated acid is negligible. Hence, at a low extracellular pH, the driving force is always from outside the cells to inside and not reversible.…”

Section: Resultsmentioning

confidence: 99%

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

Jamalzadeh

Verheijen

Heijnen

et al. 2012

Appl Environ Microbiol

View full text Add to dashboard Cite

Microbial production of C 4 dicarboxylic acids from renewable resources has gained renewed interest. The yeast Saccharomyces cerevisiae is known as a robust microorganism and is able to grow at low pH, which makes it a suitable candidate for biological production of organic acids. However, a successful metabolic engineering approach for overproduction of organic acids requires an incorporation of a proper exporter to increase the productivity. Moreover, low-pH fermentations, which are desirable for facilitating the downstream processing, may cause back diffusion of the undissociated acid into the cells with simultaneous active export, thereby creating an ATP-dissipating futile cycle. In this work, we have studied the uptake of fumaric acid in S. cerevisiae in carbon-limited chemostat cultures under anaerobic conditions. The effect of the presence of fumaric acid at different pH values (3 to 5) has been investigated in order to obtain more knowledge about possible uptake mechanisms. The experimental results showed that at a cultivation pH of 5.0 and an external fumaric acid concentration of approximately 0.8 mmol · liter ؊1 , the fumaric acid uptake rate was unexpectedly high and could not be explained by diffusion of the undissociated form across the plasma membrane alone. This could indicate the presence of protein-mediated import. At decreasing pH levels, the fumaric acid uptake rate was found to increase asymptotically to a maximum level. Although this observation is in accordance with proteinmediated import, the presence of a metabolic bottleneck for fumaric acid conversion under anaerobic conditions could not be excluded.

show abstract

“…When the capacity of the proton transporters is overloaded, intracellular biochemical processes can further extend cell-buffering capabilities. The buffering capacity of yeast cell-free extract was previously studied by two groups (Kresnowati et al, 2008;Sigler et al, 1981), who stated the amounts of acid or base necessary to change the pH of the cytosolic fluid without the influence of biological processes. We have for the first time developed a method of measuring yeast cell buffering capacity in vivo, by quantifying the short-term effects of weak base addition.…”

Section: Discussionmentioning

confidence: 99%

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Marešová

Hošková

Urbánková

et al. 2010

Yeast

View full text Add to dashboard Cite

AbstractpHluorin is a pH-sensitive variant of green fluorescent protein for measuring intracellular pH (pH in ) in living cells. We constructed a new pHluorin plasmid with the dominant selection marker KanMX. This plasmid allows pH measurements in cells without auxotrophic mutations and/or grown in chemically indefinite media. We observed differing values of pH in for three prototrophic wild-types. The new construct was also used to determine the pH in in strains differing in the activity of the plasma membrane Pma1 H + -ATPase and the influence of glucose on pH in . We describe in detail pHluorin measurements performed in a microplate reader, which require much less hands-on time and much lower cell culture volumes compared to standard cuvettes measurements. We also utilized pHluorin in a new method of measuring the buffering capacity of yeast cell cytosol in vivo, shown to be ca. 52 mM/pH for wild-type yeast and moderately decreased in mutants with affected potassium transport.

show abstract

Quantitative physiological study of the fast dynamics in the intracellular pH of Saccharomyces cerevisiae in response to glucose and ethanol pulses

Cited by 41 publications

References 26 publications

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

Noninvasive High-Throughput Single-Cell Analysis of the Intracellular pH of Saccharomyces cerevisiae by Ratiometric Flow Cytometry

pH-Dependent Uptake of Fumaric Acid in Saccharomyces cerevisiae under Anaerobic Conditions

New applications of pHluorin—measuring intracellular pH of prototrophic yeasts and determining changes in the buffering capacity of strains with affected potassium homeostasis

Contact Info

Product

Resources

About