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A novel gene with antisalt and anticadmium stress activities from a halotolerant marine green alga Chlamydomonas sp. W80

Satoshi Tanaka , Yoshito Suda , Kazunori Ikeda , Masahiro Ono , Hitoshi Miyasaka , Masanori Watanabe , Ken Sasaki , Kazumasa Hirata
DOI: http://dx.doi.org/10.1111/j.1574-6968.2007.00696.x 48-52 First published online: 1 June 2007

Abstract

A novel gene with antistress activities against both salt (NaCl) and cadmium stresses was isolated from the cDNA library of halotolerant green alga Chlamydomonas 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).

Keywords
  • Chlamydomonas
  • salt
  • cadmium
  • antistress gene
  • green alga

Introduction

Microorganisms are attractive resources for antistress genes, as they have a wide variety of tolerance to many environmental stresses, such as high salinity, high temperature and heavy metals (Lowe et al., 1993; Nies, 1999). The halotolerant 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 µM of MV (Miyasaka et al., 2000a, b), while other oxygen-evolving photosynthetic organisms such as higher plants, algae and cyanobacteria usually tolerate only <5 µM 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-, chilling- 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 phytoremediation 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 continuously illuminated by fluorescent lamps at a light intensity of 175 µmol quanta m−2 s−1, with aeration by bubbling at a rate of 200 mL air min−1.

The Luria–Bertani (LB) medium supplemented with 50 µg 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 mL well−1) under a continuous illumination (light intensity: c. 160 µmol 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-carbenicillin 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°C 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 µL 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°C, and the numbers of the colonies were counted.

Screening for anticadmium-stress genes

The λZAPII-cDNA library of C. W80 cells constructed in the previous study (Miyasaka et al., 2000a) was used for screening for anticadmium-stress genes. Briefly, the λZAPII 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°C 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 200 µM 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 µM 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 960 µM, respectively, and these values are c. 60 and 145 times higher than those of C. reinhardtii (IC50=6.5 µM and EC50=6.6 µM). Among the Chlamydomonas species previously reported, Chlamydomonas acidophila shows the highest cadmium tolerance with an IC50 value (for cell growth) of 14.4 µM (Nishikawa & Tominaga, 2001), and C. W80 shows more than twenty times higher cadmium tolerance than C. acidophila does.

1

Cadmium tolerance of Chlamydomonas W80 (C. W80) (▲) in comparison with Chlamydomonas reinhardtii (△). 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 experiments (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.

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 screening method is based on the acquisition of cadmium tolerance 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. Interestingly, 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 5′ untranslated region, a 789 bp of an ORF, and a 364 bp of 3′ 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.

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.

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 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.

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°C on a rotary shaker (150 r.p.m.) in the medium containing 1% (○), 3% (•), 5% (△) and 7% (▲) 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.

View this table:
1

Heavy metal tolerance of CW80Cd404 clone

CdCl2 (mM)
00.20.51
Cd404+++++++++
Control+++++±
CoCl2 (mM)
011.52
Cd404++++++
Control++++++
NiCl2 (mM)
033.54
Cd404+++++±
Control+++++±
CuCl2 (mM)
03.63.84
Cd404+++++±
Control+++++±
  • Approximately 2 × 106 cells were spread onto a LB-carbenicillin plate with various concentration of heavy metals, and cultured at 37°C 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:+++, >1000;++, 100–1000;+, 10–99; ±, 1–9; −, 0.

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), indicating that the function of this gene is not related to antioxidative-stress activity.

In addition, the clone did not show any enhanced stress tolerance against neither freezing (three cycles of freezing at −80°C and thawing at room temperature) nor heat (culture at 48, 50, 52 or 54°C) 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-terminal 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, CARRRTALRERYGIAGTARED, 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 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. Clendennen is also thanked for his editorial revision of this manuscript.

Footnotes

  • Editor: Aharon Oren

References

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