Analysis of the stress response of yeast Saccharomyces cerevisiae toward pulsed electric field

doi:10.1016/j.elstat.2012.01.003

Journal of Electrostatics

Volume 70, Issue 2, April 2012, Pages 212-216

https://doi.org/10.1016/j.elstat.2012.01.003 Get rights and content

Abstract

This study investigated the stress response of the yeast Saccharomyces cerevisiae toward a pulsed electric field (PEF). Changes in the transcription level of heat stress response gene HSP104 and oxidation stress response genes GSH1, GLR1, SOD1 and SOD2 were analyzed. Comparison of the increases and decreases in the transcript abundance caused by heat and PEF stress showed that these two stresses are different toward yeast. And it was shown that the glutathione-dependent biological defense mechanism against oxidation stress is strongly related to the yeast resistance toward PEF stress.

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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Citation Excerpt :
Liu et al. (2019) came to a similar conclusion by proteomic analysis, they suggest that the PEF treatment could impact the expression of the E. coli proteins and the validity of proteomic results was confirmed by quantitative reverse-transcription polymerase chain reaction data. Additionally, PEF might affect the expression of genes related to oxidation stress response, which leads to microorganisms’ inactivation (Tanino et al., 2012). However, our understanding of the generation mechanism of PEF-induced sub-lethally injured cells is still limited.
The pulsed electric fields (PEF) technology has been widely applied to inactivate spoilage and pathogenic microorganisms in food products because of its conspicuous advantages. However, the inevitable production of sub-lethally injured microorganisms limited its further application and development. In the present study, we investigated the mechanisms underlying the PEF-induced inactivation and sublethal damage of microorganisms to enhance the bactericidal effect of PEF treatments. In our work, Saccharomyces cerevisiae was used as a model organism, and the lethal and sublethal injury of microorganisms induced by PEF was estimated using non-selective and selective media. We found that PEF treatment with 20 kV/cm intensity for 200 or 400 μs, or with 15 kV/cm intensity for 400 μs, caused more sub-lethally injured cells. The PEF with 55 °C treatment produced the highest inactivation rate and the lowest sublethal injury rate. Then two-dimensional electrophoresis with mass spectrometry analysis was conducted to analyze the protein differences of S. cerevisiae under different PEF treatments. Compared with the PEF treatment (20 kV/cm, 200 μs) alone, many proteins changed significantly after PEF combined with 55 °C mild heat treatment, including several heat shock proteins. Finally, the heat shock protein 104 (Hsp104) of S. cerevisiae was knocked out, which led to a decrease in sub-lethally injured cells and an increase in inactivation rate after PEF treatment. Our findings provide critical information for developing more effective PEF-based microbial inactivation approaches in food processing.
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Pulsed electric field (PEF) treatment, an alternative to thermal processing in the food industry, is insufficient to inactivate bacterial spores. Although spores that have been treated in this manner remain alive, specific understanding of their physiological properties is limited. The purpose of this study is to describe the morphology, viability, and germination behavior of Bacillus atrophaeus spores treated with PEF. Our findings indicate that nonlethal PEF treatment results in spore deformation, dipicolinic acid (DPA) leakage, and a shorter and more uniform germination lag time (T_lag), but that there is no change in release duration (ΔT_release), germination ratio, or viability. Based on our findings, we conclude that an intact morphologic state and DPA content are not prerequisites for germination and full viability and that, in contrast to nutrient-induced germination in which initially slowly released DPA triggers subsequent germination events, leaked DPA during PEF treatment does not. Spores that have been subjected to this procedure remain dormant and preserve their full germinability. We found that PEF-treated spores respond to germinants more quickly and with less heterogeneity, possibly because the tiny cracks formed on the spore surface facilitate the germinants' access to the germination receptors situated on the spore's inner membrane. The consensus view that nonlethal PEF has less impact on spores that are still capable of forming CFUs under proper conditions is one-sided. This research advances our understanding of how spores behave following nonlethal PEF treatment and gives information on the topics of nosocomial sterilization, food safety, and public health.
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In our previous research, high voltage prick electrostatic field (HVPEF) was found to have good antibacterial effects against Staphylococcus aureus (S. aureus) on solid surface through destructing cell membranes. In the current study, the mechanism of HVPEF against S. aureus from the aspect of ROS-mediated oxidative stress was investigated. The ROS levels increased remarkably under HVPEF stress, and the addition of ROS scavengers significantly decreased ROS levels and mortality rates of S. aureus, indicating that ROS was a vital mediator in the antibacterial role of HVPEF. HVPEF treatment decreased the superoxide dismutase (SOD) activity and glutathione levels, and induced bacterial peroxidation. Gas chromatography-mass spectrometry (GC-MS) results indicated HVPEF treatment changed the fatty acid composition of the bacterial membranes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), circular dichroism (CD) spectra and flow cytometry results showed that HVPEF treatment induced DNA oxidative damage, destroyed the structure of DNA and proteins, changed the expression of genes related to oxidative stress (sodA, katE, ahpC, G6PD, fabG and fabF) and led to bacteria apoptosis. This study further investigated oxidative stress of S. aureus under HVPEF based on our previous study, aiming to provide a better understanding of the antibacterial mechanism of the electric field.
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Lactic acid bacteria play an important role in functional food and fermentation products for human and animal nutrition, as probiotics, paraprobiotics, postbiotics or high-lactic acid-producing strains in biorefineries. Pulsed electric field (PEF) treatment is gaining recognition in the food industry, but little is known about the effects of PEF treatment on the probiotic characteristics of lactic acid (LA) bacteria or its application for the production of paraprobiotics and postbiotics. Thus, we studied the inactivation kinetics and permeabilization of Lacticaseibacillus rhamnosus and Lacticaseibacillus paracasei as high LA-producing strains with probiotic characteristics by batch and continuous PEF treatment.
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Journal of Electrostatics

Abstract

Highlights

Introduction

Section snippets

Strains and medium

Confirmation of the synthesized first-strand cDNA

Discussion

Acknowledgment

References (21)

Theory of electroporation: a review

Bioelectrochem. Bioenerg.

Physical and chemical modifications of high-voltage pulse sterilization

J. Electrostat.

Thermal and pulsed electric fields pasteurization of apple juice: effects on physicochemical properties and flavor compounds

J. Food Eng.

Pulsed electric field processing of foods: a review

J. Food Prot.

Effect of culture temperature on high-voltage pulse sterilization of Escherichia coli

J. Electrostat.

Inactivation of L. plantarum in a PEF microreactor: the effect of pulse width and temperature on the inactivation

Innovative Food Sci. Emerging Technologies

Electrohydraulic discharge and nonthermal plasma for water treatment

Ind. Eng. Chem. Res.

Killing effect of concentrated pulsed electric field apparatus

Nihon Shokuhin Kougakkkaisi Ronbunshu

A new pulsed electric field microreactor: comparison between the laboratory and microscale

Lab. Chip

Cited by (30)

Reversible electroporation caused by pulsed electric field – Opportunities and challenges for the food sector

Proteomics-based mechanistic study of sub-lethally injured Saccharomyces cerevisiae by pulsed electric fields

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Inhibition mechanism of high voltage prick electrostatic field (HVPEF) on Staphylococcus aureus through ROS-mediated oxidative stress

Pulsed electric field treatment of Lacticaseibacillus rhamnosus and Lacticaseibacillus paracasei, bacteria with probiotic potential

Pulsed electric field-assisted fermentation of Hanseniaspora sp. yeast isolated from Lebanese apples