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Anovel genewith antisalt andanticadmiumstress activities fromahalotolerantmarine greenalgaChlamydomonas sp.W80Satoshi Tanaka1, Yoshito Suda2, Kazunori Ikeda3, Masahiro Ono3, Hitoshi Miyasaka1, MasanoriWatanabe4, Ken Sasaki4 & Kazumasa Hirata2
1The Kansai Electric Power Co, Environmental Research Center, Kyoto, Japan; 2Environmental Biotechnology Laboratory, Graduate School of
Pharmaceutical Sciences, Osaka University, Osaka, Japan; 3The General Environmental Technos Co. Ltd, Adzuchicho 1-Chome Chuo-ku, Osaka, Japan;
and 4Graduate School of Engineering, Hiroshima Kokusai Gakuin University, Nakano Aki-ku, Hiroshima, Japan
Correspondence: Hitoshi Miyasaka, The
Kansai Electric Power Co., Environmental
Research Center, Keihanna-Plaza, 1-7
Hikari-dai, Seikacho, Sourakugun, Kyoto
619-0237, Japan. Tel.: 181 774 93 2892;
fax: 181 774 93 2894; e-mail:
Received 22 December 2006; revised 12
February 2007; accepted 13 February 2007.
First published online 28 March 2007.
DOI:10.1111/j.1574-6968.2007.00696.x
Editor: Aharon Oren
Keywords
Chlamydomonas ; salt; cadmium; antistress
gene; green alga.
Abstract
A novel gene with antistress activities against both salt (NaCl) and cadmium
stresses was isolated from the cDNA library of halotolerant green alga Chlamydo-
monas sp. strain W80 by a functional expression screening with Escherichia coli.
The C-terminal region of this protein is responsible for the antistress activity,
because N-terminal truncated clone of this gene retains the antistress activity, and
the C-terminal truncated clone loses the activity. In the C-terminal region, there is
a histidine and aspartic acid-rich domain (HD-rich domain).
Introduction
Microorganisms are attractive resources for antistress genes,
as they have a wide variety of tolerance to many environ-
mental stresses, such as high salinity, high temperature and
heavy metals (Lowe et al., 1993; Nies, 1999). The halotoler-
ant green alga Chlamydomonas W80 (C. W80), isolated in
the coastal area of Wakayama in Japan, shows a surprisingly
high oxidative stress tolerance caused by methyl viologen
(MV), which is reduced by the photosynthetic apparatus
generating highly toxic superoxide (O2�) (Rabinowitch et al.,
1987). Chlamydomonas W80 tolerates up to 200 mM of MV
(Miyasaka et al., 2000a, b), while other oxygen-evolving
photosynthetic organisms such as higher plants, algae and
cyanobacteria usually tolerate only o 5 mM of MV. In the
previous studies, the authors isolated several antistress genes
from this alga by a functional expression screening method
with Escherichia coli and cyanobacterial cells (Miyasaka
et al., 2000a, b; Takeda et al., 2000, 2003; Tanaka et al.,
2001, 2004), and also successfully enhanced the salt-, chil-
ling- and oxidative-stress tolerances of the higher plants by
introducing the antistress gene of C. W80 (Yoshimura et al.,
2004), proving the usefulness of the antistress genes of
C. W80 for plant molecular breeding.
In this study, it was found that C. W80 also has a very
high cadmium-tolerance, and anticadmium-stress genes
were screened by a functional expression screening with
E. coli to isolate some useful genes, which can be applied to
environmental biotechnologies, such as the phytoremedia-
tion of heavy metals.
Materials and methods
Algal and bacterial cultures
Modified Okamoto medium (MOM; pH 8.0) supplemented
with 5 mM NH4Cl (Miyasaka et al., 1998) and modified
Bristol medium (pH 6.0) were used for the halotolerant
C. W80 and fresh water Chlamydomonas reinhardtii (IAM
C-238) cultures, respectively. The algal cultures were con-
tinuously illuminated by fluorescent lamps at a light inten-
sity of 175mmol quanta m�2 s�1, with aeration by bubbling
at a rate of 200 mL air min�1.
FEMS Microbiol Lett 271 (2007) 48–52c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
The Luria–Bertani (LB) medium supplemented with
50 mg mL�1 of carbenicillin was used for E. coli (SOLR strain,
Stratagene, La Jolla CA) cultures. The bacterial cell growth
was monitored by measuring the OD600 nm of cultures.
For the cadmium stress-tolerance experiments, the algal
cells were cultured in a 12-well plate (2 mLwell�1) under a
continuous illumination (light intensity: c. 160mmol
quanta m�2 s�1). To avoid the precipitation of the cadmium
ion, the potassium phosphate (K2HPO4 and KH2PO4; total
5 mM) in the MOM was replaced with 5 mM disodium
glycerophosphate and 5 mM HEPES (pH 8.0). The cells were
inoculated into a fresh medium at a low cell density
(OD680 nm = 0.05, c. 4� 105 cells mL�1) for the growth-
inhibition experiments, and at a high cell density
(OD680 nm = 1.0, c. 8� 106 cells mL�1) for the cell-toxicity
experiments, respectively. Cell growth and viability were
monitored by measuring the OD680 nm values of the cultures.
For the bacterial heavy metal-tolerance experiments,
overnight cultures were started by inoculating LB-carbeni-
cillin liquid medium with a single E. coli colony. On the
following day, the cultures were diluted to an OD600 nm of
0.05 with a fresh LB-carbenicillin medium, and cultured at
37 1C on a rotary shaker (150 r.p.m.) until the OD600 nm
value became c. 0.3. Then the E. coli cells were diluted to an
OD600 nm of 0.05 with a fresh LB-carbenicillin medium, and
50 mL cultures (c. 2� 106 cells) were plated onto an
LB-carbenicillin plate (90 mm diameter) containing various
concentrations of heavy metals, and cultured for 3 days at
37 1C, and the numbers of the colonies were counted.
Screening for anticadmium-stress genes
The lZAPII-cDNA library of C. W80 cells constructed in the
previous study (Miyasaka et al., 2000a) was used for screen-
ing for anticadmium-stress genes. Briefly, the lZAPII cDNA
library was mass excised into phagemid DNA, and the host
E. coli cells carrying the mass-excised phagemid DNA were
plated onto the selection plate with a high concentration
(1 mM) of CdCl2. The plates were incubated at 37 1C for
2 days and the cadmium-tolerant bacterial colonies were
isolated.
Results and discussion
The halotolerant green alga C. W80 shows a surprisingly high
oxidative stress-tolerance caused by MV (up to 200mM of
MV) (Miyasaka et al., 2000a). Because it is well known that a
high concentration of cadmium also causes severe oxidative
stress (Yoshida et al., 2003; Mendoza-Cozatl et al., 2005;
Watanabe & Suzuki, 2002), it was expected that this alga
might also have a high stress tolerance against cadmium, and
examined the cadmium tolerance of C. W80. As a reference
strain the C. reinhardtii, a fresh water strain, which is widely
used as a model photosynthetic microorganism, was chosen,
and the cadmium tolerance of these two algal strains was
compared. The cadmium tolerance of the algal cells was
examined in two terms: growth inhibition and cell toxicity
(cell bleaching). As was expected, C. W80 cells show a very
high tolerance to cadmium chloride up to 500 mM for cell
growth (Fig. 1a) and 5 mM for cell bleaching (Fig. 1b). The
50% inhibitory concentration (IC50) value for growth, and
the effective concentration for 50% (EC50) value for cell
toxicity for C. W80 cells are 390 and 960mM, respectively,
and these values are c. 60 and 145 times higher than those of
C. reinhardtii (IC50 = 6.5 mM and EC50 = 6.6 mM). Among
0 0.001 0.01 0.1 10
50
100
150
Concentration of cadmium (mM)
Gro
wth
(%
)
0 0.001 0.01 0.1 1 100
50
100
Concentration of cadmium (mM)
Via
bilit
y (%
)
(a)
(b)
Fig. 1. Cadmium tolerance of Chlamydomonas W80 (C. W80) (m) in
comparison with Chlamydomonas reinhardtii (W). The algal cells were
cultured in a medium containing various concentrations of cadmium
chloride. The initial cell densities are c. 4� 105 and 8�106 cells mL�1, for
the growth inhibition experiments (a) and for the cell toxicity experi-
ments (b), respectively. The cells were cultured for 72 h (for the cell
growth experiments) and 7 days (for the cell toxicity experiments),
respectively, and the cell growth and viability were monitored by
measuring the OD680 nm values of the cultures. The final cell densities
(cells mL�1) of control (no cadmium) cultures are c. 3.2� 106 (C. W80)
and 3.6�106 (C. reinhardtii) for the growth inhibition experiments, and
1.5�107 (C. W80) and 4.7� 106 (C. reinhardtii) for the cell toxicity
experiments, respectively. Values are shown as % of the OD680 nm values
of control cultures at the end of culture, and are the means� SE for three
cultures.
FEMS Microbiol Lett 271 (2007) 48–52 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
49Antistress genes from marine Chlamydomonas
the Chlamydomonas species previously reported, Chlamydo-
monas acidophila shows the highest cadmium tolerance with
an IC50 value (for cell growth) of 14.4 mM (Nishikawa &
Tominaga, 2001), and C. W80 shows more than twenty
times higher cadmium tolerance than C. acidophila does.
Given this very high cadmium stress tolerance of C. W80,
anticadmium-stress genes were tried to be isolated by a
functional expression screening. The principle of the screen-
ing method is based on the acquisition of cadmium toler-
ance of the E. coli cells carrying the algal gene, and with this
method the authors successfully isolated several antisalt and
antioxidative stress genes (Miyasaka et al., 2000a, b; Takeda
et al., 2000, 2003; Tanaka et al., 2001, 2004). The cDNA
library of C. W80 was screened for anticadmium-stress
genes using LB-carbenicillin plates with 1 mM cadmium
chloride, and 12 candidate clones were isolated. Interest-
ingly, the DNA sequence of one clone (clone no.
CW80Cd404, DDBJ Accession No. AB243758) was found
to be identical to that of a previously isolated clone (clone
no. CW80Na58, DDBJ Accession No. AB009142), which was
isolated as an antisalt-stress gene with unknown function
(Miyasaka et al., 2000a). The cDNA insert of CW80Cd404
clone is 1284 bp in length consisting of a 108 bp of 50
untranslated region, a 789 bp of an ORF, and a 364 bp of 30
untranslated region with a 23 bp of poly(A) tail. The ORF,
encoding a 236-amino acid polypeptide with a calculated
molecular mass of 28 782 Da, was found to be located in the
proper reading frame of the pBluescript SK(� ) expression
vector. The deduced amino acid sequence of the
CW80Cd404 and CW80Na60 clones are shown in Fig. 2. In
the previous study, the authors also isolated an N-terminal
truncated clone of the same gene as an antisalt-stress gene
(clone no. CW80Na60); the start position of this clone is
indicated in Fig. 2 by an arrow. Both DNA and the deduced
amino acid sequence showed no significant homology to the
previously found sequences in the database, including the
C. reinhardtii genome database (ChlamyDB: http://
www.chlamy.org/chlamydb.html). The DNA sequence data
of the other 11 clones were also deposited in the DDBJ DNA
Database with accession numbers of AB186738 through
AB186748.
To confirm the antistress activity of the clones
CW80Dd404 (and CW80Na58), and CW80Na60 against
cadmium and high concentration of NaCl, the authors
isolated the plasmids of these clones, back-transformed the
E. coli cells with the isolated plasmids and examined the
stress tolerance of the transformant E. coli cells. Figure 3
0 MSADAEKQSLLATGVPAHAAGDAPKVAPRE
31 WRHRWYAILGDCSAPDVVSCLLAWKLPFVA
61 WAWNQNRALGMSFWRELLRFAVIVVGFVVA
91 THVAYCGVMMAMCPEIHDRDGASVDGGPGM
121 MRKLLHMHQHHSHHHDDDSTDDSTDSHDHG
151 MWGEDGPHGIPRECVARVAPAYVAITGVFL
181 ALAVYMTLFFARRRTALRERYGIAGTARED
211 CLLYAFCTPCALAQETRTLIHEQVHDGIWY
241 GALPGVAPPAATVAAPAPQKMAV
CW80Na60
DFU614 domain
HD-rich domain
Fig. 2. Deduced amino acid sequences of Chlamydomonas W80 scsr
gene. The start position of the N-terminal truncated clone (CW80Na60)
is shown by an arrow. The HD-rich domain and the domain with a
homology to DFU614 domain are underlined.
201000
1
2
3
Time (h)Time (h)20100
Time (h)
0
1
2
3
201000
1
2
3
Gro
wth
(O
D60
0 nm
)
Gro
wth
(O
D60
0 nm
)
Gro
wth
(O
D60
0 nm
)
(a) (b) (c)
Fig. 3. NaCl salt stress tolerance of CW80Cd404 and CW80Na60 clones. The Escherichia coli cells of the pBluescript vector control (a), clone
CW80Cd404 (b) and clone CW80Na60 (c) were cultured at 37 1C on a rotary shaker (150 r.p.m.) in the medium containing 1% (�), 3% (�), 5% (W) and
7% (m) of NaCl. One percent is the standard NaCl concentration in LB medium. The cell growth was monitored by measuring the OD600 nm values of the
cultures. Values are means� SE for three cultures.
FEMS Microbiol Lett 271 (2007) 48–52c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
50 S. Tanaka et al.
shows the growth of the clones of CW80Cd404, CW80Na60
(N-terminal truncated clone) and vector control in the LB
medium containing 1%, 3%, 5% and 7% NaCl. The growth
of the control cells was much suppressed in the 3% NaCl,
and only a slight growth was observed in the 5% NaCl
medium. On the other hand, the CW80Cd404 and
CW80Na60 clones showed a much better growth in the 3%
NaCl medium compared with the control, and these clones
retained the growth even in the 5% NaCl medium. The
cadmium tolerance of the CW80Cd404 clone is shown in
Table 1. In addition to cadmium, the tolerance of this clone to
other heavy metals, cobalt, nickel and copper was also
examined. The CW80Cd404 clone showed an enhanced
tolerance to cadmium, but did not show any significant
difference in the tolerance to other heavy metals. The
CW80Na60 (N-terminal truncated) clone also showed an
enhanced tolerance to cadmium, and not to other heavy
metals (data not shown). These results indicate that the
antistress activity of CW80Cd404 clone is specific to NaCl salt
stress and cadmium stress, and the authors designated this
gene as C. W80 scsr (salt and cadmium stress related) gene.
Because both a high salt concentration and cadmium
stresses cause oxidative stress, the acquisition of both salt
and cadmium stress tolerance of the CW80Cd404 clone is
supposed to be potentially due to the antioxidative-stress
activity of this gene. In the previous studies, it was observed
that the E. coli cells carrying ascorbate peroxidase (APX) or
glutathione peroxidase (GPX) genes of C. W80 showed an
enhanced tolerance against MV (Miyasaka et al., 2000a;
Takeda et al., 2000; Tanaka et al., 2004), thus it was expected
that the clones of CW80Cd404, CW80Na58 and CW80Na60
could also show an enhanced MV tolerance, if the coded
protein had an antioxidative-stress activity. All the clones,
however, did not show any enhanced tolerance against MV
compared with the vector control (data not shown), indicat-
ing that the function of this gene is not related to anti-
oxidative-stress activity.
In addition, the clone did not show any enhanced stress
tolerance against neither freezing (three cycles of freezing
at � 80 1C and thawing at room temperature) nor heat
(culture at 48, 50, 52 or 54 1C) stresses (data not shown).
Thus the effect of the protein is specific to salt and
cadmium.
The antistress activity of the N-terminal truncated clone
CW80Na60 indicates that the C-terminal region of this
protein is responsible for the antistress activity. To confirm
this point, a C-terminal truncated CW80Cd404 clone was
also generated by changing the codon CAG coding the
glutamine (amino acid no. 129 in Fig. 2) to TAG stop codon
by site-directed mutagenesis, and examined if the N-term-
inal region also has any antistress activity. We found that the
N-terminal region did not show any antistress activity in
E. coli cells (data not shown), thus the antistress activity
exists in the C-terminal region.
In the C-terminal region, there is a distinctive domain
with much histidine and aspartic acid (HD-rich domain),
these two amino acids are known to have metal-binding
properties, and potentially involved in the heavy metal stress
tolerance of this protein. In the C-terminal region, a
conserved domain of unknown function named DUF614
was also found. DUF614 domain of C. W80 was found by an
NCBI conserved domain search (Marchler-Bauer & Bryant,
2004) with an e-value of 9� 10�7. DUF614 has been
found in Arabidopsis thaliana (Accession No. AAD49981),
Lycopersicon pennellii (AAF74287), Oryza sativa
(BAB08185), C. reinhardtii in the early zygote stage
(AAF60168), human placenta (Q9NZF1), and mouse
(BAB24360), but the function and physiological role of this
domain is unknown. The detailed deletion experiments in
E. coli cells to determine the contribution of HD-rich and
DUF614 domains to antistress function are in progress.
The expression of C. W80 scsr gene in response to NaCl,
cadmium and MV stresses with a Western blotting analysis
was also examined, using a polyclonal antibody raised
against synthetic polypeptides, CARRRTALRERYGIAG-
TARED, designed from the deduced amino acid sequence
of C. W80 scsr gene, and found that the SCSR protein is
constitutively expressed, and the expression is not enhanced
Table 1. Heavy metal tolerance of CW80Cd404 clone
CdCl2 (mM)
0 0.2 0.5 1
Cd404 111 111 11 1
Control 111 11 � �
CoCl2 (mM)
0 1 1.5 2
Cd404 111 11 1 �Control 111 11 1 �
NiCl2 (mM)
0 3 3.5 4
Cd404 111 11 � �Control 111 11 � �
CuCl2 (mM)
0 3.6 3.8 4
Cd404 111 11 � �Control 111 11 � �
Approximately 2�106 cells were spread onto a LB-carbenicillin plate
with various concentration of heavy metals, and cultured at 37 1C for 3
days. Control is the E. coli cells with pBluescript vector. The experiments
were repeated more than three times with triplicate plates. Colony
numbers:111, 4 1000;11, 100–1000;1, 10–99; � , 1–9; � , 0.
FEMS Microbiol Lett 271 (2007) 48–52 c� 2007 Federation of European Microbiological SocietiesPublished by Blackwell Publishing Ltd. All rights reserved
51Antistress genes from marine Chlamydomonas
by these stresses (data not shown). Thus, it is not certain if
the function of the scsr gene is directly related to antistress
activity in C. W80 cells, although the expression of this gene
in E. coli cells resulted in acquisition of enhanced tolerance
both against NaCl salt and cadmium stresses. Knock-down
or knock-out experiments of the scsr gene in C. W80 cells
using the RNAi method are required in the future to
determine the exact physiological function of this gene in
C. W80, and RNAi methodology for C. W80 cells is under
development.
Acknowledgement
Dr H. Akiyama of Toray Research Center Inc. is thanked for
his help with Western blotting experiments. Dr G. Clenden-
nen is also thanked for his editorial revision of this
manuscript.
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