Jpn. J. Protozool. Vol. 41, No. 2. (2008)

153

Original

Difference in sensitivity to amino acid starvation between the wild typeand sir2A-null mutant of Dictyostelium discoideum
Takahiro KATAYAMA1, Takako OKUWA2 and Hiro YASUKAWA1*
1

Graduate School of Science and Engineering, University of Toyama, Toyama 930-8555,
Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa 920-0934, Japan.
2

Key words: Dictyostelium discoideum; Sir2; Sir2A

SUMMARY
Dictyostelium discoideum transits fromgrowth phase to developmental phase when nutrients are depleted. D. discoideum under astarved condition aggregates and forms a cellmass called mound within 12 h and then forms afruiting body. The nutrients critical for the transition are amino acids. Thus, D. discoideum isthought to have a mechanism for sensing aminoacid starvation. We examined in the current study
the function of Sir2A in the initiation of development. The wild type initiated development andformed mounds within 12 h even in the presenceof 1 ~ 3 mg/ml amino acids, while the null mutant did not form mounds within 12 h in the presence of 2 mg/ml amino acid. These results suggest that the null mutant is less sensitive to starvation than is the wild type and that Sir2A plays*Corresponding author
Tel & Fax: +81-76-445-6875
E-mail: hiro@eng.u-toyama.ac.jp
Received: 22 February 2008; Accepted: 24 July 2008.

an important role in the recognition of amino acidstarvation.

INTRODUCTION
The cellular slime mold Dictyostelium discoideum is a soil-dwelling microorganism. D. discoideum cells grow as unicellular free-living
amoebae in the presence of nutrients. Upon starvation, amoebae aggregate and form a cell mass consisting of approximately 105 cells. The cell mass,called a ‘mound’, changes its form to a slug andmigrates as a multicellular organism. Then the slugchanges its form to a fruiting body (Firtel, 1995,1996; Kessin, 2001). The nutrients critical for thetransition from growth phase to developmental
phase are amino acids. Amino acid starvation is
the specific stimulus that initiates the developmental phase of the life cycle of D. discoideum (Marin,1976). D. discoideum is therefore thought to havea mechanism for sensing amino acid starvation.
However, proteins responsible for the mechanism
are not well understood.

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Sensitivity of D. discoideum (sir2A) to amino acid starvation

We have analyzed silent information regulator 2(Sir2) proteins in D. discoideum. Sir2 proteins arean evolutionarily conserved family of NADdependent protein deacetylases (Brachmann et al.,
1995; Imai et al., 2000). By deacetylating histone
and other proteins, Sir2 proteins play importantroles in various biological processes such as gene
expression, apoptosis, metabolism and aging(Blander and Guarente, 2004).
Analysis of genomic databases has revealed that
D. discoideum encodes at least four Sir2 homologs
named Sir2A, Sir2B, Sir2C and Sir2D. Reverse
transcription-PCR (RT-PCR) and whole-mount insitu hybridization (WISH) analyses showed thatthe genes for Sir2A, Sir2C and Sir2D were expressed at high levels in growing cells but at decreased levels in developing cells, whereas thegene encoding Sir2B was expressed in the prestalk-cell region in the developmental phase (Katayamaand Yasukawa, 2008). We have tried to establish
gene disruption mutants and overexpressing mutants to determine the importance of Sir2 proteinsin D. discoideum.
In this report, we show the results obtained
from analysis of a sir2A-disruption mutant. Thismutant exhibits different sensitivity to amino acidstarvation from that of the wild type. It is thoughtthat Sir2A plays an important role in sensing
amino acid starvation.

MATERIALS AND METHODS
Culture conditions
For dish culture, Ax2, an axenic strain of D.
discoideum, was cultured in HL5 liquid medium(Watts and Ashworth, 1970), and the gene disruption mutant was maintained in HL5 supplementedwith 5 mg/l blasticidin S (BS). For shaking culture,cells were grown in HL5 or HL5 containing thedrug with shaking at 150 r.p.m. Cell culture wascarried out at 20°C.

Fruiting body formation
For differentiation on bacterial lawns, cells
grown in medium were collected, washed with abuffer (1.66 g/l NaH2PO4, pH adjusted to 6.1 withKOH), diluted, and spread on SM plates (10 g/lBactopeptone, 1 g/l Yeast extract, 1.9 g/l KH2PO4,1.0 g/l K2HPO4, 0.5 g/l MgSO4, 10 g/l Glucose, 17g/l agar) with Escherichia coli WA802 (metB1
lac3 lacY1 galK2 galT22 supE hsdR2). For differentiation on agar plates, cells grown in medium
were collected, washed with the buffer, and plated
on SM plates or non-nutrient agar plates at a density of 1 x 106 cells/cm2. Amino acid mixture wasadded to the agar plates if necessary. The mixtureof amino acids contained 18 of the 20 naturally
occurring amino acids. Cysteine and tyrosine wereomitted from routine experiments because of low
stability and solubility, respectively. Cells wereincubated at 20°C.
Gene disruption
The construct for disruption of the gene encoding Sir2A was generated as follows. The coding
region was amplified from the Ax2 genome by
PCR using the primers 5’-ATAAAGCTTATGTACGCAGTGAATCCAATTGAA and 5’-AATGAATTCGAACTATTTTGGATATTTACAATATG.
A gene cassette for blasticidin S resistance (a bsrcassette; 1.35-kb in size) (Sutoh, 1993) was inserted into the SpeI site of the Sir2A coding region. The gene disruption construct was introduced
into Ax2 cells by electroporation. Cells in which
the gene was disrupted were selected in HL5 supplemented with blasticidin S. Gene disruption wasconfirmed by PCR and RT-PCR using the primers
5’-GTGCTGGTATCAGTGTTGCTGCTGGTATTC
and 5’-CCTCACGTGCACAATCATTGAATCTT
GATGGC. RT-PCR for rnlA (Ogawa et al., 2000)
was carried out as a control using the primers 5’TTACATTTATTAGACCCGAAACCAAGCG and5’-TTCCCTTTAGACCTATGGACCTTAGCG.
RT-PCR was performed with a One Step RT-PCR
Kit (Takara Bio, Shiga, Japan) using total RNA

Jpn. J. Protozool. Vol. 41, No. 2. (2008)

isolated from cells.

RESULTS AND DISCUSSION
Establishment of a sir2A-null mutant
We investigated the importance of Sir2A inD. discoideum by transforming Ax2 with a knockout construct of the gene that was disrupted by absr cassette (Fig. 1A). The cells were cultured in
HL5 supplemented with BS, and genomic DNA
extracted from a selected clone was used as a template for PCR. Comparison of the sizes of the PCR
products indicated that the gene in the BS-resistantstrain was disrupted by insertion of the 1.35-kb
fragment (Fig. 1B). RT-PCR using total RNA as atemplate also indicated that sir2A was expressed in
the wild-type cells but not in the BS-resistant

155strain (Fig. 1C). These results indicate that thesir2A gene in the strain tested was disrupted byinsertion of the bsr cassette.
We established two independent clones. Since
they exhibited the same phenotype, we show inthis report the experimental results obtained fromone of these clones. The sir2A-null mutant wasmaintained stably in liquid medium. The growthrate of the mutant was almost the same as that of
the wild type (data not shown), indicating that
Sir2A is not essential for growth in D. discoideum.
Development of null cells
D. discoideum amoebae were spread on SM
plates with bacteria, incubated, and photographed(Fig. 2A). The wild-type amoebae formed plaqueson the bacterial lawns. Inside the plaques, where
the bacteria were depleted, D. discoideum formed

Fig. 1. Establishment of the sir2A-null mutant.
(A) Construct used for gene disruption. Black boxes indicate exons. The bsr cassette (1.35 kb) was inserted into theSpeI site in the sir2A coding region. Arrowheads indicate the primers used in PCR and RT-PCR, which were carried out
to confirm gene disruption. (B) PCR analysis. (C) RT-PCR analysis. Amplification of rnlA was performed as a control.
In (B) and (C), lane 1 contains products from Ax2, and lane 2 contains products from the sir2A-null mutant.

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Sensitivity of D. discoideum (sir2A) to amino acid starvation

Fig. 2. Morphological phenotype of Ax2 and the sir2A-null mutant. (A) D. discoideum cells were axenically grown inHL5, harvested, diluted, and spread on SM plates with E. coli. (B) Amoebae cultured in HL5 were collected, washed,and plated on plain agar plates. Mounds and fruiting bodies formed by the D. discoideum cells were photographed at theindicated times after incubation. The bars indicate 0.5 mm.

fruiting bodies. The sir2A-null mutant also formed
plaques but did not aggregate. We confirmed thatthe null mutant did not aggregate on SM plates
without bacteria (data not shown). This observation eliminates the possibility that bacterial load ora bacterial metabolite inhibits the development of
the null mutant.
We then examined whether the null mutant
could develop on a non-nutrient agar plate. Logphase cells were harvested from a shaking culture
and plated on plain agar plates. Both the wild-type
cells and null-mutant cells initiated development
and formed mounds within 12 h and completed
fruiting body formation within 24 h (Fig. 2B).
These results indicate that a factor(s) in the SM
plate inhibited the initiation of development of themutant.
Sensitivity to amino acid starvation
Amino acids are the only nutrients that inhibit
the initiation of development. Under a starved condition, D. discoideum usually initiates the develop-mental process, aggregates and forms mounds
within 12 h. We determined in the next experiments the difference in sensitivity to amino acidstarvation between the wild type and the null mutant. D. discoideum cells were plated on agar containing various concentrations of amino acids, incubated, and photographed (Fig. 3). The wild-type
cells aggregated and formed mounds normally on
the agar plates even in the presence of 1 ~ 3 mg/ml
amino acids. No aggregate was observed at 12 hon the agar containing more than 4 mg/ml aminoacids.
The null cells formed mounds within 12 h on
the agar plates containing 1 mg/ml amino acids.
On the agar containing more than 2 mg/ml aminoacids, no aggregate was observed 12 h after thestarvation. We obtained almost the same resultsfrom three sets of experiments.
Our results suggest that the wild-type cells
recognize amino acid starvation at 3 mg/ml,
whereas the sir2A-null cells do so when the concentration of amino acids falls to 1 mg/ml. Thus, itJpn. J. Protozool. Vol. 41, No. 2. (2008)

157

Fig. 3. Development of Ax2 and the sir2A-null mutant in the presence of amino acids. Amoebae grown in liquid medium
were collected, washed, and plated on agar plates containing various concentrations of amino acids. Multicellular structures formed on the plates were photographed at 12 h after the plating. The bars indicate 0.5 mm.

appears that null cells are less sensitive to starvation than are wild-type cells.
In summary, the current results indicate that
Sir2A is dispensable for growth but plays an important role in the sensing of amino acid starvation. Further analysis to determine the function ofSir2A in the transition from growth phase to developmental phase is in progress.

ACKNOWLEDGMENT
The analysis of D. discoideum Sir2 proteins
was started by the late Dr. Shoji Ohkuma. We succeeded in the analysis and published the results.
This paper is dedicated to the memory of Dr. ShojiOhkuma.

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