The permeability of the yeast cells (Saccharomyces cerevisiae) to lipophilic tetraphenylphosphonium cations (TPP(+) ) after their treatment with single square-shaped strong electric field pulses was analyzed. Pulsed electric fields (PEF) with durations from 5 to 150 µs and strengths from 0 to 10 kV/cm were applied to a standard electroporation cuvette filled with the appropriate buffer. The TPP(+) absorption process was analyzed using an ion selective microelectrode (ISE) and the plasma membrane permeability was determined by measurements obtained using a calcein blue dye release assay. The viability of the yeast and the inactivation of the cells were determined using the optical absorbance method. The experimental data taken after yeasts were treated with PEF and incubated for 3 min showed an increased uptake of TPP(+) by the yeast. This process can be controlled by setting the amplitude and pulse duration of the applied PEF. The kinetics of the TPP(+) absorption process is described using the second order absolute rate equation. It was concluded that the changes of the charge on the yeast cell wall, which is the main barrier for TPP(+) , is due to the poration of the plasma membrane. The applicability of the TPP(+) absorption measurements for the analysis of yeast cells electroporation process is also discussed.
An investigation of the yeast cell resealing process was performed by studying the absorption of the tetraphenylphosphonium (TPP+) ion by the yeast Saccharomyces cerevisiae. It was shown that the main barrier for the uptake of such TPP+ ions is the cell wall. An increased rate of TPP+ absorption after treatment of such cells with a pulsed electric field (PEF) was observed only in intact cells, but not in spheroplasts. The investigation of the uptake of TPP+ in PEF treated cells exposed to TPP+ for different time intervals also showed the dependence of the absorption rate on the PEF strength. The modelling of the TPP+ uptake recovery has also shown that the characteristic decay time of the non-equilibrium (PEF induced) pores was approximately a few tens of seconds and this did not depend on the PEF strength. A further investigation of such cell membrane recovery process using a florescent SYTOX Green nucleic acid stain dye also showed that such membrane resealing takes place over a time that is like that occurring in the cell wall. It was thus concluded that the similar characteristic lifetimes of the non-equilibrium pores in the cell wall and membrane after exposure to PEF indicate a strong coupling between these parts of the cell.
+ effect on the cell wall of the yeast Saccharomyces cerevisiae as probed by FT-IR spectroscopy IntroductionThe capacity of metal cations to induce competence of yeast and bacterial cells to exogenous DNA is the basis of biological methods in biotechnology and molecular genetics [1][2][3]. The most commonly used, efficient protocol, for genetic transformation of Saccharomyces cerevisiae is treatment with alkali metal ions. Li + ions are the most effective of all the cations tested, and the transformation efficiency is comparable to levels obtained by the protoplast method [4]. Li + ions enhance the transformation of intact cells, but no effect is observed on transformation of protoplasts, implying that Li + ions facilitate DNA passage through the cell wall [5]. The mechanism underlying S. cerevisiae transformation includes DNA attachment and penetration through the cell wall, although how DNA passes through the cell wall is not yet clear [6][7][8]. Cell wall density, thickness and structure are factors of major importance during penetration of exogenous molecules into the cell. However, despite the extensive use of Li + ions in yeast transformation protocols, the influence of these cations on the structure of the yeast cell wall has not been widely investigated and, therefore, remains unclear. The influence of Li + ions on the cell surface topography of intact S. cerevisiae cells was observed by atomic force microscopy and it was found that the surface of Li + treated yeast cells became much rougher [9].The S. cerevisiae cell wall is composed of three major components: β-glucans, chitin, and mannoproteins. Glucose residues are linked to other glucose molecules through β(1→3) and β(1→6) linkages and to N-acetylglucosamine via β(1→4) bonds [10,11]. S. cerevisiae mannan has a linear α(1→6)-linked Keywords: FT-IR spectroscopy • Lithium • Saccharomyces cerevisiae • TransformationAbstract: The effect of Li + ions as a transformation inducing agent on the yeast cell wall has been studied. Two Saccharomyces cerevisiae strains, p63-DC5 with a native cell wall, and strain XCY42-30D(mnn1) which contains structural changes in the mannan-protein complex, were used. Fourier transform infrared (FT-IR) spectroscopy has been used for the characterization of the yeast strains and for determination of the effect of lithium cations on the cell wall. A comparison of the carbohydrate absorption band positions in the 970-1185 cm -1 range, of Na + and Li + treated yeast cells has been estimated. Absorption band positions of the cell wall carbohydrates of p63-DC5 were not influenced by the studied ions. On the contrary, the treatment of XCY42-30D(mnn1) cells with Li + ions shifted glucan band positions, implying that the cell wall structure of strain XCY42-30D(mnn1) is more sensitive to Li + ion treatment. backbone with side chains of α(1→2)-and α(1→3)-linked mannose units. In S. cerevisiae, mannoproteins contribute to the regulation of cell wall porosity, and therefore control both the exit of secretary proteins and the entrance of ma...
The electrical field-induced changes of the yeast Saccharomyces cerevisiae cells permeabilization to tetraphenylphosphonium (TPP+) ions were studied using square-shaped, nanosecond duration high power electrical pulses. It was obtained that pulses having durations ranging from 10 ns to 60 ns, and generating electric field strengths up to 190 kV/cm significantly (up to 65 times) increase the absorption rate of TPP+ ions without any detectible influence on the yeast cell viability. The modelling of the TPP+ absorption process using a second order rate equation demonstrates that depending on the duration of the pulses, yeast cell clusters of different sizes are homogeniously permeabilized. It was concluded, that nanosecond pulse-induced permeabilization can be applied to increase the operational speed of whole cell biosensors.
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