Analysis of the stress response of yeast Saccharomyces cerevisiae toward pulsed electric field
Highlights
► The stress response of the yeast toward a pulsed electric field was investigated. ► No heat stress was detected in the PEF-treated yeast cells in our study. ► The PEF treatment induced the expression of the oxidation stress response genes. ► The glutathione played an important role in the stress resistance toward PEF.
Introduction
A pulsed electric field (PEF) with nanoseconds of rising time and microseconds of half bandwidth could be generated by a brief application of voltage. The direct-current and alternate-current voltage applied to water generates a large amount of heat which results from ohmic heating. However, PEF characterized by this extremely brief application of voltage does not generate a large amount of heat. Application of PEF to microorganisms has been investigated as a liquid inactivation technology. When a microorganism in a liquid is exposed to a strong PEF, electrical compression and fenestration of the cell membrane could occur [1]. Fenestration of the cell membrane could lead to irreversible destruction; the cell would die with the release of cytoplasmic components. From these characteristics, PEF has been attracting attention as a non-thermal inactivation technology based on a physical mechanism without chemicals [2].
To increase the inactivation efficiency, the improvement of reactor components, especially the shape and material of the electrode has been widely investigated [3], [4], [5], [6], [7], [8]. Many PEF inactivation studies under various conditions focused on high-voltage generation technology, applied voltage, power supply frequency and conductivity of the solution [4], [9], [10], [11], [12], [13]. These reports provided additional knowledge regarding the inactivation of microorganisms by PEF treatment and encouraged technical improvements. Although the effect of PEF inactivation seems to be the destruction of the cell membrane, local and instant heat generation could not be ignored. If the heat generated by PEF treatment affects the inactivation of the cell, biological heat stress should be induced. However, the biological responses of the cells toward PEF were still unclear, and attempt to improve the PEF inactivation efficiency from the biological approach was also not investigated.
This study investigated the stress response of the yeast Saccharomyces cerevisiae (viz., one of the most widely researched microorganisms concerning stress responses) toward PEF by analyzing the changes in the transcript abundance of several genes. The genes that respond to the heat and oxidation stresses were selected as the target genes. After the analysis of the changes in transcript abundance, inactivation investigations using the stress-response-inhibited yeast by gene knockout and chemical inhibition of the metabolic pathway were also carried out.
Section snippets
Strains and medium
S. cerevisiae BY4742 (MATalpha, leu2Δ0, ura3Δ0, his3Δ1, lys2Δ0) was used for the analysis of the various stress responses of yeast. A GSH1 (Z449376) knockout strain of S. cerevisiae BY4742 (MATalpha, leu2Δ0, ura3Δ0, his3Δ1, lys2Δ0, gsh1Δ::KanMX) was purchased from Open Biosystems, Inc. (Clone ID:17097). This strain was designated as BY4742gsh1Δ in this study and used for the analysis. These yeasts were cultivated in YPD medium (0.5% (w/v) yeast extract, 1% peptone and 0.9% glucose). For solid
Confirmation of the synthesized first-strand cDNA
The quantitative difference in the first-strand cDNAs synthesized from the total RNAs of the heat and PEF-treated cells was confirmed by partially amplifying the GLK1 gene, which encodes one of the constitutive glycolytic enzymes, by PCR. Fig. 2 shows the results of the agarose gel electrophoresis using the amplified DNAs (lane 1–4). No differences in the DNA band strength were observed. This result indicated that first-strand cDNAs were successfully synthesized without any quantitative
Discussion
This study investigated the stress response of the yeast S. cerevisiae toward PEF by focusing on heat and oxidation stress response genes. The first-strand cDNAs were reverse-transcribed from the extracted total RNAs of the heat- and PEF-treated yeast cells and target genes were partially amplified from the first-strand cDNA. Increases and decreases in the transcript abundances of the target genes as stress responses were qualitatively analyzed by image analysis of those amplified DNAs
Acknowledgment
This research was supported by a Grand-in-Aid for Scientific Research (C) from the Japan Society for the Promotion of Science (22510082).
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