Antioxidant Genes for Cells against Atmospheric Pressure Cold Plasma:
Deletion and Overexpression Mutants of Saccharomyces cerevisiae
Ma Ruonan, Feng Hongqing, Li Fangting, Liu Qi, Fang Jing, Zhang Jue
Academy for Advanced Interdisciplinary Studies, Peking University, Beijing,100871, China
Zhu Weidong
Saint Peter’s College, Jersey City, New Jersey,07031, USA
Abstract: Reactive species, and in particular, reactive oxygen species (ROS) produced in atmospheric pressure
cold plasmas are considered important in inducing biological responses [1]. Yet it is not clear what genes in cells
respond (if at all) to these reactive species. In this paper, we study the anti-ROS genes’ protective functions in
eukaryotic cells using S. cerevisiae deletion and overexpression mutants. Four classic anti-ROS genes are
included in the experiment: superoxide dismutases (Sod1 and Sod2), cytoplasmic catalase T (Ctt1), and gamma
glutamylcysteine synthetase (Gsh1). SOD1 and SOD2 catalyze free superoxide radicals to less harmful hydrogen
peroxide (H2O2), and CTT1 catalyzes H2O2 to H2O and O2. GSH1 catalyzes the first step in glutathione (GSH)
biosynthesis, which also plays a role in eliminating H2O2 and lipid peroxides. The deletion, overexpression and
wild-type yeast are exposed to a direct current, atmospheric pressure, cold air plasma mircojet. Their viabilities
after treatment are measured by XTT assay. The intracellular ROS is measured with dichlorodihydrofluorescein
diacetate (DCDHF diacetate) using fluorescence microscopy. The protective roles of antioxidant genes to cold
plasma will be discussed in detail.
Keywords: reactive oxygen species (ROS); Saccharomyces cerevisiae; deletion; overexpression;
antioxidant genes.
1. Introduction
Atmospheric pressure cold plasmas, a kind of
nonthermal plasma, is a source of reactive species
(e.g., OH·, H·, O·, H2O2, and O3), UV radiation,
energetic ions, and charged particles. Recently,
biological application of atmospheric pressure glow
discharges has become a hot topic for research.
Plasmas are considered as a potential mean for
sterilization, such as the sterilization of reusable
medical tools and the decontamination of biological
and chemical warfare agents [2, 3], and for the
removal of bacterial biofilms [4]. It is found that the
sterilants produced by the nonthermal plasma killed
or inactivated a wide range of bacteria, fungi, spores,
and viruses at high concentrations either on the
plastic surface or suspended in saline or culture [5,
6]. Nonthermal plasma is also regarded as effective
for removing infectious proteins from the surfaces of
surgical instruments [7, 8].
According to the published reports, for atmospheric
pressure plasma, chemically reactive species, such as
free radicals, metastable atoms, and molecules,
always play an important role during sterilization [9].
Reactive oxygen species (ROS) are derivatives of
molecular oxygen formed in living cells, which
include hydrogen peroxide, hydroxyl radicals and
superoxide anions. During plasma treatment, large
amounts of ROS are released, resulting in
macromolecular damage and cell death (Fleury et al.,
2002). The damage to cellular components by ROS
is prevented by antioxidant defenses that either
scavenge ROS or repair oxidized molecules.
Catalases in concert with superoxide dismutase form
the first and most important line of antioxidant
defense [10]. Under conditions when ROS
generation prevails over the capacity of antioxidant
defense, cells experience oxidative stress, which
could lead to damages in yeast cells.
Compared with the traditional experiments which
focused on the mechanism of microbial inactivation
by nonthermal plasma from a physical standpoint,
we intended to explain the mechanism of
sterilization from the molecular level. In our
experiments, Saccharomyces cerevisiae, a model
organism for basic studies in cell biology were used,
since the genome of S. cerevisiae has been studied
throughly. Our research has focused on anti-ROS
genes deletion and overexpression mutants treated
with plasma. Four classic anti-ROS genes are
included in the experiment: Sod1 and Sod2 express
superoxide dismutases (SOD) which catalyzes the
dismutation of O-2· to H2O2 and O2 [12] Ctt1 (gene
for cytoplasmic catalase T) expresses Catalase which
decomposes H2O2 into H2O and O2 [13].
Gsh1(gene for gamma-glutamylcysteine synthetase )
expresses GSH1 which catalyzes the first step in
glutathione(GSH) biosynthesis, also plays a role in
eliminating H2O2 and lipid peroxides [14].
In our previous experiment, we also observed that
the oxidative stress pathway of Eukaryotic cells
Saccharomyces cerevisiae could lead to their
hypersensitivity to plasma treatment [1]. In summary,
we may draw a conclusion that ROS produced by
PMJ in water contributes significantly to the damage
of yeast cells.
2. Materials and Methods
A. Experimental Setup and Apparatus
The plasma device used in this study comprised of
two copper tubes as electrodes separated by a
ceramic tube. The device was driven by a direct
current negative-polarity high-voltage power supply
(Matsuada AU5R120) through a 5 kΩ ballast resistor.
Detailed schematic diagram of the device as well as
the electrical circuit can be found in references [15,
16]. Premixed helium and oxygen was used as the
working gas. The samples were treated with PMJ for
1–3 min. Triplicate cell samples were performed for
each time point. Untreated cells were used as
controls.
respective open reading frame (ORF) with a G418
resistant gene kanMX4 [15] and inoculated in yeast
YPAD medium with antibiotics G418. Before the
plasma treatment, all mutant strains were confirmed
by polymerase chain reaction to make certain of the
correct gene deletion or modification. The
overexpression mutants were constructed through a
recombinant plasmid transforming the wild-type
strain BY4741.The strains were selected on SCminus L-histidine plates. The plasmid-bearing yeast
strains were grown in SC-minus L-histidine medium.
C. Survival Assays
Survival evaluation of each strain to plasma was
performed using XTT assay, an alternative way to
colony forming units counting to evaluate cell
viability [16]. The stains were cultured to the
exponential growth phase, and harvested at a
concentration of 2.5X107 cells/ ml. Then 1 ml cells
were resuspended in 5 ml autoclaved deionized
water and treated with PMJ for 0 min, 1 min, 2 min
and 3 min, respectively. The 1min, 2min and 3min
data of each strain were normalized to that of their
respective nontreated samples to evaluate the
survival ratios of the strains.
D.ROS Scavenger Assays
The wild-type strain were cultured to the exponential
growth phase, and washed with autoclaved
deionized water. Then the cells were centrifuged at
1000 rpm for 5min and resuspended with three
different ROS scavengers(0.75 mol/l Mannitol , 0.25
mol/l L-His and SOD.) which were used as •OH ,
1O2 and ·O2- quenchers in the PMJ-water system,
respectively. After exposure to PMJ for 1-3 minutes,
survival rate was obtained by using XTT assay.
B. Strains and and Culture Conditions
E. Assays of Intracellular ROS
S. cerevisiae is a well studied biological model for
research on eukaryotic cells. In this experiment,
three groups of S. cerevisiae were employed: wildtype, single antioxidant gene (Sod1, Sod2, Ctt1,
Gsh1) deletion and overexpression mutants. Wildtype strain BY4741 (MAT a his3Δ1 leu2Δ0 ura3Δ0
met15Δ0; C.B. Brachmann, et al., 1998) were
maintained routinely in YPAD. The single gene
deletion mutants were constructed by replacing the
Intracellular ROS was measured with the probe 2’,
7’-dichlorofluorescein diacetate (DCFH-DA) [17,
18]. The cells were harvested after exposure to
plasma, and washed with phosphate buffer saline
(PBS, pH 7.4, 0.01M), then incubated at 30 ◦C for 1
h in PBS containing 10-μM DCFH-DA. After
incubation, the cells were observed with
fluorescence microscopy. In all cases two or three
different fields were observed, containing 300-400
cells.
3. Results
Single gene deletion strains have a higher
sensitivity to PMJ treatment than wild-type
strain.
Survival Ratio (%)
The survival ratio of different S. cerevisiae strains
following plasma treatment is shown in
Fig.1.Compared with the single gene deletion, wildtype strain has a higher survival rate. Three of the
antioxidant related deletion strains (gsh1Δ, ctt1Δ,
and sod2 Δ) were hypersensitive to plasma, with the
survival ratios less than 10% after the treatment of
three minutes while the control of wild-type was
approximately 60%. These results clearly indicate
that these anti-ROS genes are involved in yeast’s
response to plasma stress. The absence of those
genes may result in defective defending ROS so that
the cells have an impaired ability to survive during
plasma treatment.
1min
2min
3min
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
fluorescence of all treated samples (Fig2-b,Fig2-c)
was significantly higher than the control (Fig2-a).
According to these results, it indicated that
intracellular prevention and interception processes
for oxidants are not completely effective; products
of oxidative damage are continuously formed in low
yields and hence may accumulate to cause the cells
suffering oxidative stress [20]. Yeast cells have a
range of responses to ROS that depends on the dose.
At very low doses, the cells can adapt to become
more resistant to subsequent lethal exposure [19]. At
higher doses, when the extent of oxidative stress
exceeds the capacity of the cell defense systems,
oxygen toxicity may result through oxidating cell
membrane, proteins, and DNA, eventually leading to
cell death.
(a)
(b)
(c)
Figure2. Fluorescence microscopy images of intracellular ROS
produced by PMJ in Saccharomyces cerevisiae cells.The wild
type suspended in liquid was treated with He/O2 (2%) PMJ for
(a) 0 mins, (b) 4 minutes,(c) 8 minutes (ROS are stained green).
Funding
WILD
GSH1
CTT1
SOD2
Figure. 1. Survival rate of four types of Saccharomyces
cerevisiae (They are wild, Gsh1 gene deletion, Ctt1 gene
deletion and Sod2 gene deletion , respectively.) treated with a
He/O2 (2%) PMJ in water. All data are from three independent
experiments.
Oxidative Stress was induced in S. Cerevisiae
after treatment with PMJ.
Intracellular ROS fluorescence (Fig.2) showed that
oxidative stress existed in wild-type of S. cerevisiae
cells after plasma discharge. For cells that were
subjected to intracellular ROS assay directly
following plasma exposure, the intracellular ROS
This research is supported by National Natural
Science Foundation of China (30970131) and
Beijing Natural Science Foundation (7102149) to Dr.
Wei Liu, and is also sponsored by Bioelectrics Inc.
(U.S.A.), MST Program of International Science and
Technology
Cooperation
(under
Grant
#
2009DFB30370: ‘‘Cold Plasma induced biological
effect and its clinical application studies’’) and
National
Basic
Research
Program
(No.
2007CB935602)
References
[1] Hongqing Feng, Ruixue Wang, Peng Sun,
Haiyan Wu, Qi Liu, Jing Fang, Weidong Zhu,
Fangting Li and Jue Zhang, “A study of eukaryotic
response mechanisms to atmospheric pressure cold
plasma by using Saccharomyces cerevisiae single
gene mutants,” Appl. Phys. Lett., 97, 131501 (2010)
[2] M. Laroussi, J. P. Richardson, and F. C. Dobbs,
“Effects of nonequilibrium atmospheric pressure
plasmas on the heterotrophic pathways of bacteria
and on their cell morphology,” Appl. Phys. Lett., vol.
81, no. 4, pp. 772–774, Jul. 2002.
[3] T. C. Montie, K. W. Kimberly, and J. R. Roth,
“An overview of research using the one atmosphere
uniform glow discharge plasma (OAUGDP) for
sterilization of surfaces and materials,” IEEE Trans.
Plasma Sci., vol. 28, no. 1, pp. 41–50, Feb. 2000.
[4] N. Abramzon, J. C. Joaquin, J. Bray, and G.
Brelles-Mariño, “Biofilm destruction by RF highpressure cold plasma jet,” IEEE Trans. Plasma Sci.,
vol. 34, no. 4, pp. 1304–1309, Aug. 2006.
[5] R. A. Venezia, M. Orrico, E. Houston, S. M. Yin,
and Y. Y. Naumova, “Lethal activity of nonthermal
plasma sterilization against microorganisms,”Infect.
Control Hosp. Epidemiol., vol. 29, no. 5, pp. 430–
436, May 2008.
[6] H. Yu, Z. L. Xiu, C. S. Ren, J. L. Zhang, D. Z.
Wang, Y. N. Wang, and T. C. Ma, “Inactivation of
yeast by dielectric barrier discharge (DBD) plasma
in helium at atmospheric pressure,” IEEE Trans.
Plasma Sci., vol. 33, no. 4, pp. 145–1409, Aug. 2005.
[7] O. Kylián, H. Rauscher, D. Gilliland, F.
Brétagnol, and F. Rossi, “Removal of model
proteins by means of low-pressure inductively
coupled plasma discharge,” J. Phys. D, Appl. Phys.,
vol. 41, no. 9, pp. 5201.1–5201.8, Mar. 2008.
[8] X. T. Deng, J. J. Shi, H. L. Chen, and M. G.
Kong, “Protein destruction by atmospheric pressure
glow discharges,” Appl. Phys. Lett., vol. 90, no. 1,
pp. 3903.1–3903.3, Jan. 2007.
[9] M. K. Boudam, M. Moisan, B. Saoudi, C.
Popovici, N. Gherardi, and F. Massines, “Bacterial
spore inactivation by atmospheric-pressure plasmas
in the presence or absence of UV photons as
obtained with the same gas mixture,” J. Phys. D,
Appl. Phys., vol. 39, no. 16, pp. 3494–507, Aug.
2006.
[10] D. J. Jamieson, “Oxidative stress responses of
the yeast Saccharomyces cerevisiae,” Yeast, vol. 14,
no. 16, pp. 1511–1527, Dec. 1998. J. M. McCord
and I. Fridovich, “Superoxide dismutase. An
enzymic function of erythrocuprein,” J. Biol. Chem.,
vol. 244, no. 22, pp. 6049–6055, Nov. 1969.
[11] H. Z. Chae, S. J. Chung, and S. G. Rhee,
“Thioredoxin-dependent peroxide reductase from
yeast,” J. Biol. Chem., vol. 269, no. 44, pp. 27 670–
27 678, Nov. 1984.
[12] R. D. Carratore, C. D. Croce, M. Simili, E.
Taccini, M. Scavuzzo, and S. Sbrana, “Cell cycle
and morphological alterations as indicative of
apoptosis promoted by UV irradiation in S.
cerevisiae,” Mutation Res./Genetic Toxicol. Environ.
Mutagenesis, vol. 513, no. 1/2, pp. 183–191, Jan.
2002.
[13] H. Feng, P. Sun, Y. Chai, G. Tong, J. Zhang,
W. Zhu, J. Fang, IEEE Trans. Plasma Sci, 2009, 37,
121.
[14] J. F. Kolb, A. A H. Mohamed, R. O. Price, R. J.
Swanson, A. Bowman, R. L. Chiavarini, M. Stacey,
K. H. Schoenbach, Appl. Phys. Lett, 2008, 92,
241501.
[15] Saccharomyces Genome Deletion Project,
http://wwwsequence.
stanford.edu/group/yeast
deletion project/deletions3.html.
[16] D. M. Kuhn, T. George, J. Chandra, P. K.
Mukherjee, and M. A. Ghannoum, Antimicrob.
Agents Chemother. 46, 1773 (2002).
[17] C. Favre, P. S. Aguilar, and M. C. Carrillo,
“Oxidative stress and chronological aging in
glycogen-phosphorylase-deleted
yeast,”
Free
Radical Biol. Med., vol. 45, no. 10, pp. 1446–1456,
Nov. 2008.
[18] H. Zhu, G. L. Bannenberg, P. Moldeus, and H.
G. Shertzer, “Oxidation pathways for the
intracellular probe 2 ’, 7 ’-dichlorofluorescein,”
Arch. Toxicol., vol. 68, no. 9, pp. 582–587, Sep.
1994.
[19] G. G. Perrone, S. X. Tan, and I. W. Dawes,
“Reactive oxygen species and yeast apoptosis,”
Biochim. Biophys. Acta, vol. 1783, no. 7, pp. 1354–
1368, Jul. 2008.
[20] H. Sies, “Oxidative stress: Oxidants and
antioxidants,” Exp. Physiol., vol. 82, no. 2, pp. 291–
295, Mar. 1997.