Plant Cell Physiol.

45(6): 667–671 (2004)

JSPP © 2004

Rapid Paper

Improvement of Culture Conditions and Evidence for Nuclear

Transformation by Homologous Recombination in a Red Alga,
Cyanidioschyzon merolae 10D
Ayumi Minoda 1, Rei Sakagami 2, Fumi Yagisawa 3, 4, Tsuneyoshi Kuroiwa 3 and Kan Tanaka 1, 5
1
Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Tokyo, 113-0032 Japan
2
School of Life Science, Tokyo University of Pharmacy and Life Science, Hachioji, Tokyo, 192-0392 Japan
3
Department of Life Science, College of Science, Rikkyo (St. Paul’s) University, Nishiikebukuro, Tokyo, 171-8501 Japan
4
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Tokyo, 113-0033 Japan

;
Although the nuclear genome sequence of Cyanidio- genome project aimed at promoting an understanding of plant
schyzon merolae 10D, a unicellular red alga, was recently evolution and molecular biology. In addition, its simple life
determined, DNA transformation technology that is impor- cycle also facilitates laboratory manipulations (Terui et al.
tant as a model plant system has never been available thus 1995, Kuroiwa 1998). Despite its usefulness as a model plant
far. In this study, improved culture conditions resulted in a system, the basic methods for cultivating C. merolae have not
faster growth rate of C. merolae in liquid medium (dou- been investigated sufficiently to take advantage of it. Under
bling time = 9.2 h), and colony formation on gellan gum conventional culture conditions with continuous illumination
plates. Using these conditions, spontaneous mutants (5- (35 µmol photons m–2 s–1) at 40°C with aeration, the doubling
fluoroortic acid resistant) deficient in the UMP synthase time was reported as approximately 32 h in liquid medium.
gene were isolated. The lesions were then restored by intro- And limited use of cultures on solid plate media has been
ducing the wild-type UMP synthase gene into the cells sug- reported for C. merolae 10D (Toda 1996). In this study, we
gesting DNA transformation by homologous recombination. investigated these culture conditions to obtain a faster growth
rate, and examined the possibility for nuclear transformation
Keywords: Cultivation — Cyanidioschyzon merolae — 5-FOA using exogenous DNA and the improved cultivation conditions.
resistance — Homologous recombination — Red algae —
Transformation. Results and Discussion

Abbreviations: 5-FOA, 5-fluoroorotic acid; MA medium, modi- To improve the cultivation conditions, the composition of
fied Allen medium; OMP, orotidine 5′-monophosphate; UMP, uridine the medium was examined. In our laboratory thus far, C. mero-
5′-monophosphate. lae has been grown in Allen’s photoautotrophic medium (Allen
1959), replacing the trace elements with Arnon’s A6 solution
for experimental convenience. Previously, Dr. Seckbach sug-
gested that 2× Allen’s medium; in which all ingredients are
Introduction concentrated two times more than the original Allen’s medium,
supports better cyanidiophycea growth (Seckbach 1994). Thus,
Cyanidioschyzon merolae 10D is a unicellular red alga we examined C. merolae growth in Allen’s media concentrated
that lives in acid hot springs rich in sulfate (pH 1.5, 45°C). This to various strengths. As a result, the doubling time in the dou-
alga contains one nucleus, one mitochondrion and one plastid, ble strength 2× Allen’s medium was shortened from 32 to 24 h,
and is considered one of the most primitive algae by many lines however, increasing concentrations of the ingredients up to
of evidence (Kuroiwa 1998, Nozaki et al. 2003, Matsuzaki et fivefold did not improve this growth rate further. Thus, 2×
al. 2004). A sample of C. merolae 10D was isolated (Toda et al. Allen’s medium was used for the subsequent experiments.
1995) from the hot spring algal collection provided from Dr. When the iron ingredient of the 2× Allen’s medium was
Pinto, Naples University. Because this thermophilic alga does mixed with the other ingredients prior to autoclaving, a signifi-
not possess any rigid cell walls, C. merolae is a suitable mate- cant amount of sediment appeared, which was inconvenient for
rial for studies of biochemistry, structural biology and biotech- the experimental manipulations. To prevent this the iron ingre-
nology. Furthermore, the nuclear (Matsuzaki et al. 2004) and dient was added after autoclaving. Considering the modifica-
the organelle (Ohta et al. 1998, Ohta et al. 2003) genome tions described above, a modified Allen’s autotrophic medium
sequences of C. merolae were recently determined by a nuclear (MA medium) was established for C. merolae growth (Table 1).
5
Corresponding author: E-mail, kntanaka@iam.u-tokyo.ac.jp; Fax, +81-3-5841-8476.

667

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 668 Cyanidioschyzon merolae as a model plant system

Table 1 Modified Allen’s (MA) autotrophic medium ml–1, Kanto Kagaku, Japan), nalidixic acid (10 µg ml–1, Itoh et
al. 1997), novobiocin (100 µg ml–1), and 5-fluoroorotic acid
(NH4)2SO4 2.62 g liter–1 (5-FOA) (800 µg ml–1) effectively prevented growth, while
KH2PO4 0.54 g liter–1 ampicillin (1 mg ml–1), kanamycin (1 mg ml–1), spectinomycin
MgSO4·7H2O 0.5 g liter–1 (1 mg ml–1), bleomycin (20 µg ml–1), and vancomycin (1 mg
CaCl2·2H2O 0.14 g liter–1 ml–1) had no effect on growth. The lack of growth inhibition by
a
FeCl3 (EDTA·2Na) 0.016 g liter–1 (0.028 g liter–1) kanamycin and bleomycin was consistent with the results of
b
Trace elements (2× A6 solution) Yagisawa et al. (2004). In other organisms such as yeast (Sac-
Adjust the pH to 2.5 with H2SO4 charomyces cerevisiae), 5-FOA is used to select mutants defi-
a
250× Fe solution (filtered sterilization) is 7 g liter–1 EDTA·2Na and cient in orotidine 5′-monophosphate (OMP) decarboxylase
4 g liter–1 FeCl3. because 5-FOA is converted to a highly toxic compound, 5-
b
A6 solution is 2.85 g liter–1 H2BO3, 1.8 g liter–1 MnCl2·4H2O, 0.105 g fluorouracil, by this enzyme (Boek et al. 1987). Conversely,
liter–1 ZnCl2, 0.39 g liter–1 Na2MoO2·2H2O, 0.04 g liter–1 CoCl2·6H2O,
0.043 g liter–1 CuCl2. OMP decarboxylase-deficient mutants show auxotrophy for
250× Fe solution was sterilized by filtration and added to the medium uracil, and the structural gene for this enzyme (URA3 in S. cer-
after the addition of all other ingredients and completion of autoclaving. evisiae) can be used as a selectable marker for genetic transfor-
mation (Boek et al. 1987, Peck et al. 2000). Taking advantage
When the liquid culture was performed under higher light con- of this, a gene in C. merolae encoding OMP decarboxylase was
ditions (90 µmol photons m–2 s–1) and bubbled with 5% (v/v) identified based on its nuclear genome sequence (Matsuzaki et
CO2 at 40°C, doubling time was shortened further, down to al. 2004). In C. merolae, OMP decarboxylase was found to be
9.2 h (Fig. 1A). However, cells grown under illumination with expressed as a fused protein with orotidine-5′-phosphoribosyl-
200 µmol photons m–2 s–1 showed a decrease in the growth rate transferase, which is encoded by URA5 in S. cerevisiae, as in
and a yellowish color, suggesting they were suffering from high Arabidopsis thaliana and Homo sapiens (Nasr et al. 1994). Its
light stress at this light intensity. Under the growth conditions corresponding gene was named URA5.3 in C. merolae. The
tested [35 µmol photons m–2 s–1, 0.04% (v/v) CO2, 40°C], C. inhibitory concentration of 5-FOA was roughly the same as in
merolae formed colonies on the MA-0.4% gellan gum plates S. cerevisiae; basically no colonies emerged when 107 cells
(Fig. 1B); no colony formation has previously been observed were spread on MA-0.4% gellan gum plates containing 800 µg
on plates based on the original Allen’s medium. The addition ml–1 5-FOA (data not shown). To obtain spontaneous 5-FOA-
of 0.4% glycerol to the medium also improved the plate and resistant mutants, 9×107 cells were spread on nine MA-0.4%
liquid culture growth of C. merolae (data not shown), indicat- gellan gum plates containing 800 µg ml –1 5-FOA and 500 µg
ing that there are further possibilities for improving culture ml–1 uracil. Eight independent clones were isolated after 2
conditions. months of plating, and designated M1–M8, respectively. 5-
The construction of a nuclear transformation system is FOA resistance and uracil auxotrophy was a stable phenotype
important for making C. merolae a model experimental organ- as shown in Fig. 2A and B. The entire DNA of three clones,
ism. For this purpose, it is essential to establish selectable M1, M3 and M4, was isolated, and the nucleotide sequences of
marker genes and selection conditions. To find potentially use- the DNA regions around the URA5.3 gene were directly deter-
ful drugs for marker selection, various available antibiotics and mined after the PCR amplification using combinations of the
drugs generally used for genetic selection were tested, and their primers listed in Table 2. As a result, we found that a dA7 tract
effect on growth was examined using MA plates. It was in the mid-portion of the URA5.3 open reading frame (462
revealed that chloramphenicol (100 µg ml–1), imazapyr (500 µg amino acids) had mutated to a dA8 tract as a result of a frame

Fig. 1 C. merolae growth. (A) Cell growth under

70 µmol photons m–2 s–1 at 40°C with bubbling of 5%
CO2 (v/v). Values are representative of two independent
experiments. (B) Formation of single C. merolae colonies
on plates. Cells were streaked onto the MA-0.4% gellan
gum plates using a toothpick, and incubated for 10 d
before the photograph was taken.

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 Cyanidioschyzon merolae as a model plant system 669

Fig. 2 Analysis of spontaneous 5-FOA-resistant C. merolae mutants. (A) Sensitivity to 5-FOA of the wild type and M3 and M4 mutants grown
on MA-0.4% gellan gum plates containing 800 µg ml–1 5-FOA and 500 µg ml–1 uracil. The three spots represent reproducibility. (B) Effects of
uracil on 5-FOA-resistant mutant growth. The M4 mutant was grown in the presence (crosses) or absence (open circles) of 2.5 mg ml–1 uracil. The
wild type was also grown in the absence of uracil as a control (closed squares). (C) The domain structure of UMP synthase (URA5.3) in C. mero-
lae, whose amino terminal and carboxy terminal domains correspond to orotidine-5′-phosphoribosyltransferase and OMP decarboxylase, respec-
tively. The grey area represents the catalytic site of OMP decarboxylase while the black area indicates the changed amino acid stretch of the
URA5.3 protein as a result of a frameshift mutation in M3 and M4. The structure of the mutated region was also shown in detail. The nucleotide
sequence of the URA5.3 gene has been deposited in the DDBJ/EMBL/GenBank databases under Accession No. AB164641, and the depicted
nucleotide sequence of the wild-type gene corresponds to positions 3,602 to 3,655.

Table 2 List of primers

Primer name Sequence 5′-Position 3′-Position a
ClaI-URA5.3 5′-CCATCGATGGGAACTGAGGGGCGAACGC-3′ 2012 2029
URA5.3-SpeI 5′-GGACTAGTCCTGGCATGCGCAGAACGCG-3′ 5170 5151
primer 1 5′-GCGAGGTAGGTGCTAGTTTG-3′ 2411 2430
primer 2 5′-CGAGGATACGAGACAGCAAC-3′ 3030 3011
primer 3 5′-TACGGTGCAGAACACCGAAC-3′ 2834 2853
primer 4 5′-GTTCAAATTCCGCAGCGTCC-3′ 3448 3429
primer 5 5′-TCACACAAGGCTCTCGTTGC-3′ 3225 3244
primer 6 5′-CCTGCGCTGAACTGTGCAAC-3′ 3949 3830
primer 7 5′-TGCGGCAGACGTACAATCGA-3′ 3632 3651
primer 8 5′-TTGCTGGCTCTTCACTCCCG-3′ 4251 4232
primer 9 5′-GCGAAGCGTTTCCGTTCTGT-3′ 4041 4060
primer 10 5′-CGACACTAGAGCGATTCCCT-3′ 4670 4651
a
The 5′ and 3′ positions of the primers represent the nucleotide positions in the database entry, AB164641.

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 670 Cyanidioschyzon merolae as a model plant system

clones. After 1 month, a number of colonies formed only on the

plates where pURA5.3-KS had been electroporated in every
case, suggesting nuclear transformation by the exogenous DNA
(Fig. 3A). The transformation frequency differed from time to
time, but typically more than 100 colonies appeared by the
introduction of 0.5 µg pURA5.3-KS plasmid DNA.
To analyze whether these clones appeared due to nuclear
transformation, three uracil-prototrophic clones were collected
from each transformation protocol, and their total DNAs were
isolated. The results of Southern hybridization with a URA5.3
probe indicated only one copy of the URA5.3 gene in the
Fig. 3 Nuclear transformation of C. merolae. (A) pURA5.3-KS con-
taining the wild-type URA5.3 gene introduced into C. merolae cells by nuclear genome (Fig. 3B), showing that random integration of
electroporation, and plated on MA-0.4% gellan gum plates. An exam- the transformed plasmid into the nuclear genome did not occur.
ple of the Effectene Transfection Reagent results is shown (right) Sequencing analysis of the URA5.3 region of these five clones
together with a negative control with the vector plasmid (left). (B) confirmed that the frameshift mutation found in the M4 mutant
Southern hybridization analysis of the transformants. The total DNAs was reverted to the wild-type sequence (data not shown). Thus,
were isolated from the wild type (lane 1), and the M4 mutant (lane 2)
and representative transformants (lanes 3–5) were digested with PstI
transformation of the nuclear genome by homologous recombi-
and hybridized with the URA5.3 probe. Each transformant was nation was thought to have occurred in this study. The recent
obtained by the introduction of pURA5.3-KS (lane 3) using Effectene isolation of cycloheximide-resistant mutants could also facili-
Transfection Reagent (lane 4) or SuperFect Transfection Reagent (lane tate the establishment of gene targeting technology in the
5). Lane M is the molecular weight marker. nuclear genome of C. merolae (Yagisawa et al. 2004).
The unicellular green alga Chlamydomonas is known to
shift after the 259th amino acid and the production of a trun- be a good model organism for studies of cell and molecular
cated gene product (261 amino acids, Fig. 2C). Due to this biology (Harris 1998). However, gene targeting of its nuclear
mutation, the carboxy terminal domain corresponding to OMP genome by homologous recombination has never been
carboxylase activity was truncated, consistent with the 5-FOA- achieved. Homologous recombination of the nuclear genome of
resistant and uracil-auxotrophic phenotype of the M3 and M4 plants has so far only been reported in the moss Phys-
mutants. Since the same mutation was found in two independ- comitrella patens (Schaefer 2002). Therefore gene-targeting
ent isolates, this dA-tract appears to be a hot spot for slippage technology using the unicellular C. merolae system will help
mutations during DNA replication (Seki et al. 1999). We could solve many problems with regards to plant as well as basic
not find any mutated bases in the URA5.3 gene of the M1 eukaryotic biology.
strain, indicating an unknown mutation at another locus.
Next, using the isolated M4 mutant as a recipient, the pos- Materials and Methods
sibility of nuclear transformation was examined. A DNA frag-
DNA sequencing was performed with the CEQ™ 2000XL DNA
ment encompassing the wild-type URA5.3 gene was PCR analysis system (BECKMAN COULTER, CA, U.S.A.) and CEQ™
amplified from the chromosome DNA, and cloned on a plasmid DTCS-Quick Start Kit (BECKMAN COULTER, CA, U.S.A.). To con-
vector to make pURA5.3-KS. To introduce DNA into C. mero- struct pURA5.3-KS, 3,158 bp DNA fragment containing the wild-type
lae, 1.5 ml cultures were harvested at the mid-exponential URA5.3 gene was PCR amplified from chromosomal DNA with a
primer set, ClaI-URA5.3 and URA5.3-SpeI (see Table 2), and cloned
phase (A750 = 0.3–0.4), and concentrated to 40 µl by centrifuga-
into the ClaI–SpeI sites of pBluescriptKS (Stratagene, CA, U.S.A.).
tion. The cell suspension containing 3–4×108 cells was mixed The identity of the cloned sequence containing the chromosomal copy
with 1 µl of pURA5.3-KS (0.5 µg µl–1), and electroporated at a was confirmed by sequencing. Electroporation was performed with
field strength of 2 kV m–2 s–1. In addition to this, 4 µg DNA Electro-gene-transfer-equipment GTE-10 equipped with Extended
was treated with Effectene Transfection Reagent (QIAGEN, capacitance unit ECU-1 (Shimadzu, Japan). Southern hybridization
analysis was performed as described previously (Oguchi et al. 1999).
Hilden, Germany) or SuperFect Transfection Reagent (QIAGEN,
The URA5.3 probe (625 bp) was designed to detect the 3-kbp PstI
Hilden, Germany) as described by the supplier, mixed with the fragment derived from the chromosomal URA5.3 copy, and larger frag-
40 µl of the cell suspension, and electroporated at a field ments were expected to be detected in case of random plasmid integra-
strength of 2.5 kV m–2 s–1. The vector plasmid, pBluescriptKS, tion. The DIG-labeled probe was prepared by PCR amplification using
was also electroporated using the same conditions as the con- primers 5 and 6 shown in Table 2.
trols. After electroporation, cell suspensions were inoculated
Acknowledgments
with 2 ml MA medium, and incubated overnight in the dark at
40°C with shaking. The cells were spread on MA-0.4% gellan We would like to thank Drs. Osami Misumi and Haruko Kuroiwa
gum plates, and incubated under light conditions with 35 µmol for helpful discussions and suggestions. This study was supported by a
photons m–2 s–1 at 40°C to allow selection of uracil prototrophic Grant-in-Aid for Scientific Research on Priority Area “Genome Biol-

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 Cyanidioschyzon merolae as a model plant system 671

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Oguchi, K., Liu, H., Tamura, K. and Takahashi, H. (1999) Molecular cloning
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(Received March 11, 2004; Accepted April 6, 2004)

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