Requirement of spermidine for developmental transitions in Aspergillus nidulans.

Abstract

Deletion of the spermidine synthase gene in the fungus Aspergillus nidulans results in a strain, deltaspdA, which requires spermidine for growth and accumulates putrescine as the sole polyamine. Vegetative growth but not sporulation or sterigmatocystin production is observed when deltaspdA is grown on media supplemented with 0.05-0.10 mM exogenous spermidine. Supplementation of deltaspdA with >/= 0.10 mM spermidine restores sterigmatocystin production and >/= 0.50 mM spermidine produces a phenotype with denser asexual spore production and decreased radial hyphal growth compared with the wild type. DeltaspdA spores germinate in unsupplemented media but germ tube growth ceases after 8 h upon which time the spores swell to approximately three times their normal diameter. Hyphal growth is resumed upon addition of 1.0 mM spermidine. Suppression of a G protein signalling pathway could not force asexual sporulation and sterigmatocystin production in deltaspdA strains grown in media lacking spermidine but could force both processes in deltaspdA strains supplemented with 0.05 mM spermidine. These results show that increasing levels of spermidine are required for the transitions from (i) germ tube to hyphal growth and (ii) hyphal growth to tissue differentiation and secondary metabolism. Suppression of G protein signalling can over-ride the spermidine requirement for the latter but not the former transition.

Full text

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Molecular Microbiology (2002) 46(3), 801–812
© 2002 Blackwell Publishing Ltd
Blackwell Science, LtdOxford, UKMMIMolecular Microbiology 0950-382X Blackwell Science, 200246Original Article Y. Jin et al.Sper-
midine regulates Aspergillus development
Accepted 2 August, 2002. *For correspondence. E-mail npk@
plantpath.wisc.edu; Tel. (+1) 608 262 9795; Fax (+1) 608 263 2626.
Requirement of spermidine for developmental
transitions in Aspergillus nidulans
Yuan Jin,1 Jin Woo Bok,2 Doralinda Guzman-de-Peña3
and Nancy P. Keller2*
1Department of Plant Pathology, Texas A & M University,
College Station, TX 77843-2132, USA.
2Department of Plant Pathology, University of Wisconsin,
1630 Linden Drive, Madison, WI 53706, USA.
3Departamento de Genetica y Biologia Molecular, Centro
de Investigacion y Estudios Avanzados del IPN, Km 9.6
Libramiento Norte, Carretera Irapuato-Leon, Apdo. Postal.
629, Irapuato, Gto., Mexico.
Summary
Deletion of the spermidine synthase gene in the fun-
gus Aspergillus nidulans results in a strain, ∆ ∆ ∆ ∆spdA,
which requires spermidine for growth and accumu-
lates putrescine as the sole polyamine. Vegetative
growth but not sporulation or sterigmatocystin pro-
duction is observed when ∆ ∆ ∆ ∆spdA is grown on media
supplemented with 0.05–0.10 mM exogenous spermi-
dine. Supplementation of ∆ ∆ ∆ ∆spdA with ≥ ≥ ≥ ≥0.10 mM
spermidine restores sterigmatocystin production and
≥ ≥ ≥ ≥0.50 mM spermidine produces a phenotype with
denser asexual spore production and decreased
radial hyphal growth compared with the wild type.
∆ ∆ ∆ ∆spdA spores germinate in unsupplemented media
but germ tube growth ceases after 8 h upon which
time the spores swell to approximately three times
their normal diameter. Hyphal growth is resumed
upon addition of 1.0 mM spermidine. Suppression of
a G protein signalling pathway could not force asex-
ual sporulation and sterigmatocystin production in
∆ ∆ ∆ ∆spdA strains grown in media lacking spermidine but
could force both processes in ∆ ∆ ∆ ∆spdA strains supple-
mented with 0.05 mM spermidine. These results show
that increasing levels of spermidine are required for
the transitions from (i) germ tube to hyphal growth
and (ii) hyphal growth to tissue differentiation and
secondary metabolism. Suppression of G protein sig-
nalling can over-ride the spermidine requirement for
the latter but not the former transition.
Introduction
Polyamines are small aliphatic molecules involved in
cell growth and development in a wide range of organ-
isms (Tabor and Tabor, 1985; Ruiz-Herrera, 1994). The
three most commonly occurring natural polyamines are
putrescine, spermidine and spermine. In fungi, these
compounds have been reported to be important in cell
differentiation processes including sporulation, spore
germination and dimorphic transition (Guevara-Olvera
et al., 1993; Reyna-Lopéz and Ruiz-Herrera, 1993;
Ruiz-Herrera, 1994; Lopez et al., 1997). For example,
polyamine biosynthesis increases during zoospore ger-
mination in Blastocladiella emersonii (Mennucci et al.,
1975). In Sclerotium rolfsii, mycelial growth and sclerotium
germination were positively correlated with increased
putrescine levels, whereas sclerotium formation was
accompanied by a marked increase in spermine content
(Shapira et al., 1989). High levels of cellular polyamine
(putrescine and spermidine) lead to hyphal growth rather
than yeast-like growth in the dimorphic fungus Mucor
racemosus (Inderlied et al., 1980). Disruption of the orni-
thine decarboxylase gene (i.e. odc, required for conver-
sion of ornithine to putrescine) in the corn smut fungus
Ustilago maydis resulted in a failure of the odc mutant
strain to shift from the yeast to mycelial stage (Guevara-
Olvera et al., 1997).
Several studies suggest polyamines are involved in the
regulation of not only sporulation but also mycotoxin bio-
synthesis in Aspergillus species. Aflatoxin (AF) and steri-
gmatocystin (ST) are carcinogenic secondary metabolites
produced by Aspergillus fungi. Treatment of A. nidulans
(ST producer) and A. parasiticus (AF producer) with 1,4-
diamino-2-butanone (DAB), a competitive inhibitor of orni-
thine decarboxylase, repressed both sporulation and myc-
otoxin production. The DAB effects could be counteracted
by adding exogenous putrescine, the ornithine decarbox-
ylase product, to the growth media (Guzman-de-Peña and
Ruiz-Herrera, 1997; Guzman-de-Peña et al., 1998). Fur-
ther studies in A. nidulans showed polyamine depletion
resulted in the failure of brlA, a transcription factor
required for asexual sporulation (Prade and Timberlake,
1993), to be expressed (Guzman-de-Peña et al., 1998).
The concomitant effect of polyamine deprivation on brlA
expression, sporulation and ST production is reminis-
cent of a conserved G protein-mediated growth pathway
that links asexual development to ST/AF production in

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802Y. Jin et al.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
Aspergillus spp. (Hicks et al., 1997). Aspergillus mutants
containing an activated α subunit of a heterotrimeric G
protein, FadA (Yu et al., 1996a), exhibit a purely vegetative
growth phenotype with no sporulation or ST/AF biosyn-
thesis (Hicks et al., 1997). Both sporulation and ST/AF
production require that this FadA signalling pathway be
inactivated and this requires FlbA, a RGS domain protein
which is postulated to enhance the endogenous GTPase
activity of FadA (Lee and Adams, 1994; Yu et al., 1996a).
Both FadA activated mutants and FlbA loss-of-function
mutants cannot express brlA or aflR, a transcription factor
required for ST/AF biosynthesis (Yu et al., 1996b).
Guzman-de-Peña and Ruiz-Herrera (1997) therefore pro-
posed that polyamine effects on sporulation and ST/AF
production might be mediated in part through the FadA/
FlbA signalling pathway.
Various arguments have been proposed suggesting a
role for polyamines in the transduction of extracellular
messages. Polyamines can act as an intracellular signal
enhancing Ca2+ influx across the plasma membrane and
Ca2+ efflux from the mitochondria in mouse heart, liver and
kidney (Koenig et al., 1983). Polyamines also have been
found to stimulate Gα protein GTPase activity in calf brain
(Bueb et al., 1991; Bueb et al., 1992), mitogen-activated
kinase activity in human breast epithelial cells and in
mouse 10T1/2 fibroblasts and serine/threonine CK2 activ-
ity in chicken COS cells (Kubota et al., 1997; Leroy et al.,
1997; Manni et al., 1997). Furthermore, polyamines have
been implicated in depressing cAMP synthesis in bacteria
(Escherichia coli; Wright and Boyle, 1982) and fungi (Mag-
naporthe grisea and Mucor spp.; Orlowski, 1995; Choi
et al., 1997).
Our goals in this study were, first, to investigate a role
for polyamines in both A. nidulans spore development and
ST production at a genetic level by analysing a spermidine
synthase mutant, and, second, to explore a possible rela-
tionship between polyamine metabolism and the FadA/
FlbA signal transduction pathway in this fungus.
Results
Analysis of the A. nidulans spdA gene
Cosmid pW30B01 was identified by hybridization to an A.
nidulans EST with homology to spermidine synthases.
Sequencing of a 5.2 kb XhoI fragment from cosmid
pW30B01 revealed an internal open reading frame (ORF)
of 1488 bp encoding a putative 291-amino-acid protein
interrupted by four introns, 159, 86, 107 and 257 bp in
length (data not shown). This ORF was named spdA and
encoded the putative spermidine synthase of A. nidulans.
The deduced amino acid sequence not only showed high
identity with spermidine synthases of Saccharomyces cer-
evisiae (Hamasaki-Katagiri et al., 1997) and Schizosac-
charomyces pombe (Barrel et al., 1995) (∼ 63%), but also
shared significant identity with spermidine synthases of
bacteria and mammals (c. 24%).
Disruption of A. nidulans spdA gene
Transformation of A. nidulans strain PW1 with the spdA
disruption construct pYJ3 yielded 250 transformants. Six-
teen of these 250 transformants grew well and sporulated
on media supplemented with 1.0 mM spermidine but grew
poorly and did not sporulate on 0.1 mM spermidine
media. Genomic DNA was isolated from these 16 putative
spdA mutants, digested with XhoI and probed with the
5.2 kb XhoI pYJ2 fragment containing the entire spdA
locus. Strain TYJ3.119 had the 4.4 and 2.4 kb fragments
predicted if the SphI–SacI fragment of spdA was replaced
by argB. spdA disruption was confirmed by EcoRV diges-
tion and polymerase chain reaction (PCR) amplification
(data not shown).
For further examination of the function of different
polyamines in cells of A. nidulans, we constructed a puA,
spdA double mutant strain TYJ3.174 (puA2, ∆spdA) by
transforming RYJ6 with pYJ3 using the same transforma-
tion procedures as above. Mutants at the puA locus of A.
nidulans are crippled in the ability to synthesize putrescine
because they are deficient in ornithine decarboxylase
activity (Stevens, 1975). puA2, which was created by
mutagenesis with nitrous acid by C. Herman, is allelic to
puA1 isolated by Sneath (1955).
Analysis of polyamines in Aspergillus mutants
Table 1 shows the chemically unconjugated polyamine
composition of wild-type TPK1.1 and TYJ3.119 (∆spdA).
The wild type accumulated no putrescine but increasing
levels of spermidine were accumulated as the spermidine
Table 1. Chemically unconjugated polyamine accumulation in wild-
type and ∆spdA strains of Aspergillus nidulans.
Strain
Spermidine
added (mM)
Concentration
(µM mg−1 protein)
Wet weight
(mg)a
Putrescinea
Spermidinea
TPK1.1
(wt)
0.0
0.1
0.5
1.0
3.0
0.0
0.1
0.5
1.0
3.0
0.0
0.0
0.0
0.0
0.0
3.8
1.8
1.1
5.2
5.8
0.96
1.93
0.55
6.80
14.02
0.0
0.0
0.0
0.0
0.12
163
170
220
206
267
45
41
49
147
147
TYJ3.119
(DspdA)
a. Numbers are the average of two replications. Wet weight reflected
weight of mycelium or, in the case of TYJ3.119 at 0–0.5 mM spermi-
dine, germlings.

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Spermidine regulates Aspergillus development 803
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
concentration increased in the media. TYJ3.119 accumu-
lated putrescine but no spermidine (with the exception
of cells incubated with 3.0 mM spermidine). FGSC682
(puA2) is a leaky mutant accumulating some putrescine
and the polyamine content of the double mutant TYJ3.174
(puA2, ∆spdA) was very similar to TYJ3.119 with accumu-
lation of putrescine (data not shown).
Spermidine is essential for A. nidulans growth
and development
We tested the growth of the spdA mutant TYJ3.119 on
putrescine, spermidine and spermine containing media
(Table 2 and data not shown). TYJ3.119 was unable to
grow on any concentration of putrescine and grew only
poorly on high concentrations of spermine (data not
shown). In contrast, weak hyphal growth of this mutant
was observed at spermidine concentrations of 0.05 and
0.1 mM. One, 3 and 6 mM spermidine allowed some
radial growth (Table 2). Substantial conidial production
required spermidine concentrations ≥0.5 mM (Table 2).
Interestingly, although radial growth was less than wild
type, conidia production per unit area was significantly
higher in TYJ3.119 at
≥0.5 mM.
The phenotype of increased asexual spore production
and decreased radial growth of TYJ3.119 at high spermi-
dine supplementation was unexpected. We further exam-
ined this phenotype by examining the puA2 mutants.
TPK1.1 (wild-type alleles), FGSC682 (puA2), TYJ3.119
(∆spdA) and TYJ3.174 (puA2, ∆spdA) were grown on
minimal medium plates containing 0.0, 0.05 mM or
1.0 mM spermidine (Table 3 and data not shown).
FGSC682 showed some growth on 0.0 and 0.05 mM
spermidine media and was completely restored to wild-
type vegetative growth and sporulation in media amended
with 1.0 mM spermidine. Colony growth of TYJ3.119 was
more vigorous than TYJ3.174 on minimal medium con-
taining either 0.05 mM or 1.0 mM spermidine although
both lagged behind TPK1.1 and FGSC682. Both
TYJ3.119 and TYJ3.174 produced more conidia per unit
spermidine concentrations
area than either wild type or FGSC682 when grown on
1.0 mM spermidine. Considering the leaky nature of the
puA2 allele, these data suggest that the observed pheno-
type was largely dependent on the ∆spdA allele.
As other studies described a relationship between asex-
ual and sexual spore production in A. nidulans (Champe
et al., 1987; Calvo et al., 1999) where an increase in pro-
duction of one type of spore is associated with a decrease
of production of the other spore, we examined ascospore
production at day 10 in WIM126 (wild type) and RYJ4
(∆spdA) in YGT medium supplemented with 1.0 mM sper-
midine, an amount of spermidine yielding significantly
more conidia in ∆spdA than in wild type. As expected,
WIM126 produced a lawn of cleistothecia (the sexual body
containing ascospores) where a 1-cm core contained 104−
105 ascospores (Calvo et al., 1999). Although RYJ4 pro-
duced the same number of cleistothecia, they were barren
(data not shown).
Conidiophore initiation but not conidia formation is
delayed in A. nidulans polyamine mutants
Because all three polyamine requiring strains FGSC682,
Table 2. Effects of different concentrations of spermidine on the
growth and conidiation of an Aspergillus nidulans wild-type and a
spermidine synthase mutant after 72 h incubation.
Spermidine
concentration (mM)
Diameter (cm) Spores 107 cm−2
TPK1.1
wt
TYJ3.119
(∆spdA)
TPK1.1
wt
TYJ3.119
(∆spdA)
0.00
0.05
0.10
0.50
1.00
3.00
6.00
7.12a
7.21
7.16
7.30
6.90
7.28
6.65
0.00
0.39
0.47
3.34
5.00
5.94
5.71
5.01
5.04
4.44
4.17
3.96
5.19
4.98
0.00
0.00
0.43
7.83
6.57
7.89
8.25
a. Values are means of three replicates. Means were analysed by
t-test (TPK1.1 versus TJY3.119) for each concentration of spermidine
at P = 0.05. The ∆spdA strain was significantly different from the wild
type at every concentration of spermidine.
Table 3. Diameters and spore densities of Aspergillus nidulans strains after 6 days growth on minimal medium.
Strains
Spermidine
0 mM 0.05 mM1.0 mM
Diameter (cm)Spores cm−2
Diameter (cm) Spores cm−2
Diameter (cm) Spores cm−2
TPK1.1 (wt)
FGSC682 (puA2)
TYJ3.119 (∆spdA)
TYJ3.174 (puA2, DspdA)
LSD0.05b
6.47a
0.40
0.00
0.00
0.1869
6.0 × 107
0
0
0
0.9494
6.52
1.12
0.57
0.00
0.1759
5.0 × 107
2.0 × 105
7.02
7.10
5.13
3.93
0.5244
5.54 × 107
5.48 × 107
8.26 × 107
8.62 × 107
0.9375 × 107
0
0
0.4747 × 105
a. Values are means of three replicates.
b. Means were analysed using Fisher’s protected least significant difference at P = 0.05.

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804Y. Jin et al.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
TYJ3.119 and TYJ3.174 required higher levels of spermi-
dine supplementation for asexual sporulation than for
vegetative growth, it appeared that a specific level of
spermidine is required for activation of A. nidulans conid-
iation, the process of asexual spore development. To fur-
ther examine the nature of spermidine requirement in
conidiation, we grew FGSC682, TYJ3.119, TYJ3.174 and
wild-type strain TPK1.1 on 1.0 mM spermidine plates and
recorded the timing of conidiophore initiation and conidia
formatioo. Colonies were point inoculated and data only
taken from a centre core so the fungal material vas of the
same approximate age. Conidiophore development was
delayed in all three polyamine mutants (Table 4) with
increasing delays in this order: puA2 > ∆spdA > puA2;
∆spdA. However, once conidiophore differentiation was
initiated, the time required for conidia formation (4 h) was
identical in all four isolates.
Effects of polyamine starvation on spore germination of
A. nidulans
Although the ∆spdA strains did not grow on media without
spermidine, they remained alive for at least 3 days on this
media. When transferred to medium supplemented with
1.0 mM spermidine after 3 days, growth and conidiation
resumed in these strains. In an effort to determine the
developmental step at which spermidine starvation
affected the growth process of A. nidulans, we grew
TYJ3.119 (∆spdA) and TYJ3.174 (puA2; ∆spdA) and the
wild-type strain TPK1.1 in minimal shake cultures contain-
ing 0, 0.05, or 1.0 mM spermidine for 24 h at 37°C. Asex-
ual spore germination of both polyamine requiring strains
was delayed by ∼ 2 h (a spore was considered germinated
when the germ tube was the same diameter as the spore,
Table 5). However, the per cent germination was not
affected.
Microscopic examination of these strains showed that
enlarged conidia formed in the two polyamine requiring
strains grown in 0.0 and 0.05 mM spermidine media
(Fig. 1D–I and data not shown). These cells were not
present when TYJ3.119 and TYJ3.174 were grown in
1.0 mM spermidine (Fig. 1J–L and data not shown). The
diameter of the enlarged conidia ranged from 8.2 to
13.0 µm whereas wild-type conidia average ∼ 3 µm. In
medium lacking spermidine, germ tube extension of most
of the ∆spdA conidia typically slowed at 8–10 h and then
stopped shortly thereafter with no further cell division.
At the same time germ tube growth slowed (∼ 8 h), the
germinating spores began to swell. At 12 h, the enlarged
conidia were obviously distinct from other spores and
mycelia. The growth and differentiation of the enlarged
conidia could be restored by adding 1.0 mM exogenous
spermidine to the culture.
SpdA mRNA levels do not vary during
A. nidulans conidiation
As described above, spdA and its product are required for
vegetative growth as well as for activation of conidiophore
formation. To determine if the expression of spdA is devel-
opmentally regulated, spdA transcript was examined in
wild-type strain FGSC26 during conidiophore formation.
As shown in Fig. 2, a 1.1 kb mRNA corresponding to spdA
is present and the levels remained relatively constant at
all time points examined. This suggests spermidine levels
Table 4. Time of conidiophore formation and conidiation of Aspergil-
lus nidulans strains grown on minimal medium containing 1.0 mM
spermidine.
Strains
Time of conidiophore
formationa (h)
Time of
conidiationa (h)
TPK1.1 (wt)
FGSC682 (puA2)
TYJ3.119 (∆spdA)
TYJ3.174 (puA2, ∆spdA)
16b
20
26
30
20
24
30
34
a. Hours post incubation in 37°C.
b. Data based on counts of 100 conidiphores or conidia.
Table 5. Effect of spermidine starvation on spore germination of Aspergillus nidulans strains.
Spermidine
Strain
TPK1.1 (wt) TYJ3.119 (∆spdA) TYJ3.174 (puA2, ∆spdA)
00.05 mM 1.0 mM0 0.05 mM1.0 mM0 0.05 mM1.0 mM
Time (h)
2
4
6
8
10
1a
3
0
4
0
7
0
1
0
2
0
0
0
1
1
0
1
1
66
93
92
68
96
97
69
91
93
12
56
92
10
59
94
14
53
96
14
58
91
12
56
89
12
56
91
a. Per cent of germinated spores, 100 spores were counted for each test.

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Spermidine regulates Aspergillus development
examined ST production and stc gene transcription in
TYJ3.119 as these processes are jointly repressed by
polyamine inhibitors (Guzman-de-Peña et al., 1998).
Figure 3 shows that aflR and stcU are not expressed in
the ∆spdA strain unless the media is supplemented with
≥0.10 mM spermidine. stcU, formerly called verA (Keller
et al., 1994), encodes a highly expressed biosynthetic
gene required for ST synthesis. Furthermore, compared
with wild type, the expression of stcU was delayed in
TYJ3.119 in amended medium.
ST production followed a concomitant course as stcU
expression. TLC data clearly show that, while 0.10 mM
and 1.0 mM spermidine allowed the ∆spdA strain to pro-
duce ST, no ST was produced in lower concentrations of
spermidine. Also when ST was produced in TYJ3.119, it
was delayed compared with wild type.
805
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
are not transcriptionally regulated, at least at this level of
detection. This transcript was not detected in the spdA
disrupted strain TYJ3.119 (data not shown).
Polyamines control sterigmatocystin production in
A. nidulans
Having observed the extreme affects that spdA disruption
has on both vegetative growth and conidiation, we next
Complementation of ∆spdA with a functional copy of the
gene restores wild-type phenotype
To confirm that the characteristics described above are
solely due to the deletion of spdA, we transformed the
∆spdA strain RJY13 with a functional copy of spdA. All
transformants yielded wild-type growth. Two transfor-
mants, TJW28, in which the wild-type spdA was intro-
duced at the trpC locus, and TJW32, in which the
wild-type spdA was integrated ectopically, were further
analysed. There were no significant difference in colony
diameter, conidia or ascospsore production between wild-
type RYJ12 and the transformants TJW28 or TJW32
(Table 6 and data not shown). Polyamine analyses of
TJW28 and TJW32 showed that they produced similar
levels of spermidine and ST as wild type (data not shown).
Thus, the ∆spdA phenotype appeared to be a direct result
of loss of SpdA activity.
Fig. 1. Spermidine starvation causes formation of enlarged conidia.
Strains TPK1.1 (A–C; wild type), TYJ3.119 (D–L; ∆spdA) and
TYJ3.174 (puA2; ∆spdA) were grown in shake minimal medium
containing 0.0 mM spermidine (A–F), 0.05 mM spermidine (G–I) or
1.0 mM spermidine (J–L). Micrographs shown were taken at 8, 12
and 16 h post inoculation time points. As TYJ3.119 and TYJ3.174
gave identical results, only TYJ3.119 micrographs are shown. The
scale bar, shown in A, is 16.0 µm.
Fig. 2. Northern analysis of the spdA mRNA. Wild-type strain
FGSC26 was grown in shake minimal medium for 20 h at 37°C and
then transferred onto agar plates containing the same medium and
incubated at 37°C for the times indicated. Total RNA was isolated
from FGSC26 at the time of the mycelial transfer (0 h), −6 h and
−3 h before the transfer and 2, 4, 6, 8 h after the transfer and then
hybridized with a 0.7 kb spdA specific cDNA probe. The lower panel
shows equal loading of total RNA as evaluated by ethidium bromide
staining.
Fig. 3. Spermidine is required for sterigmatocystin biosynthesis.
RNA and ST from TPK1.1 (wild type) and TYJ3.119 (∆spdA) were
extracted from cultures 0, 12, 24 and 48 h after shifting from 1.0 mM
spermidine shake media to shake media containing 0, 0.01, 0.05,
0.10 or 1.0 mM spermidine. A 0.75 kb SstII–SmaI fragment from
plasmid pRB7 (Yu et al., 1996b) was used as a stcU-specific probe;
a 1.2 kb aflR fragment from plasmid pAHK25 (Yu et al., 1996b) was
used as an aflR-specific probe. Loading of total RNA (20 µg lane−1)
was evaluated by ethidium bromide staining; ST was separated by
TLC and the sample labelled Std is the ST standard (Sigma).

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806Y. Jin et al.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
Requirement of spermidine for suppression of a G protein
signalling pathway
The loss of asexual sporulation and ST production in the
∆spdA strains is not unique to this genetic lesion. Mutants
in genes (e.g. fluG and fadA) involved in a G protein
signalling pathway in A. nidulans are also defective in
sporulation, stc gene expression and ST production
(Hicks et al., 1997). However, sporulation and/or stc gene
expression and ST production can be restored in these
mutants by overexpressing flbA under the control of the
threonine-inducible promoter, alcA (Hicks et al., 1997; K.
Shimizu, J. K. Hicks and N. P. Keller, unpublished data).
Overexpression of flbA results in decreased activity of
FadA – and presumably other Gα proteins – through
facilitation of endogenous GTPase activity. We were inter-
ested to ask if overexpressing flbA in a ∆spdA background
could restore conidiation and ST production in the sper-
midine mutant.
To test this, near-isogenic strains TPK1.3, RYJ2
(both wild-type spdA) and TYJ3.35 (∆spdA), containing
an inducible flbA gene, were examined for conidiophore
development and ST production in 0.0, 0.05 or 0.10 mM
spermidine. As expected, TPK1.3 and RYJ2 produced
conidiophores (Fig. 4A–C and data not shown) and ST
(Fig. 5) at all spermidine concentrations when grown in
flbA inducing conditions. In contrast, flbA overexpression
did not restore development and ST production in TYJ3.35
grown in media lacking spermidine (Fig. 4D–F and data
not shown). However, conidiophore development and ST
production were restored in TYJ3.35 when the strain was
grown in 0.05 and 0.10 mM spermidine (Fig. 4G–I and
Fig. 5 and data not shown). Conidiophore vesicles and
sterigmata were observed by 12 h after flbA induction in
TYJ3.35 in 0.05 spermidine minimal medium (Fig. 4H)
and complete conidiophores were observed by 24 h post
induction (Fig. 4I). This was the same time frame for
development in wild types TPK1.3 and RYJ2.
mRNA analysis confirmed visual and TLC results.
Figure 5 shows that brlA and stcU had accumulated in
TYJ3.35 after 12 h post induction in 0.05 mM spermidine
but that these transcripts were not present in this strain
when grown in media without spermidine. As expected,
both brlA and stcU were expressed in TPK1.3 and RYJ2
in media without spermidine. Interestingly, alcA expres-
sion in TYJ3.35 required no addition of spermidine.
Discussion
The absolute requirement for polyamines in growth and
development in eukaryotic cells has driven the interest in
understanding the actual molecular functions of the
polyamines in biological processes. The broad physiolog-
Table 6. Wild-type spdA allele rescues the ∆spdA phenotype.
Strains
0 mM Spermidine1.0 mM Spermidine
Diameter (cm)Spores cm−2
Diameter (cm)Spores cm−2
RYJ12 (wt)
RYJ11 (DspdA)
TJW 28 (DspdA; spdA::trpC)
TJW 32 (DspdA; spdA::trpC)
LSD 0.05b
7.425a
0.000
7.700
7.800
0.1316
9.10 × 107
0.0
9.00 × 107
9.06 × 107
0.5299 × 107
7.825
4.225
7.950
8.000
0.7342
8.50 × 107
17.5 × 107
8.39 × 107
8.30 × 1 07
0.8563 × 1 07
a. Values are means of three replicates. Diameters and spore densities of Aspergillus nidulans strains were calculated after 6 days growth on
minimal medium.
b. Means were analysed by using Fisher’s protected least significant differences at P = 0.05.
Fig. 4. Overexpression of flbA causes conidiophore development in
the ∆spdA mutant. TPK1.3 (A–C: alcA(p)::flbA) and TYJ3.35 (D–L:
alcA(p)::flbA; ∆spdA) were grown for 18 h in alcA(p) repressing shake
medium (glucose) containing 0 mM (A–F), 0.05 mM (G–I) or 1.0 mM
spermidine (J–L) and then shifted to alcA(p)-inducing shake medium
(threonine) with the same spermidine concentrations. Cultures were
observed and photographed at the time of the shift (0 h; A, D, G and
J), 12 h (B, E, H and K) and 24 h (C, F, I and L) after alcA(p) induction.
The scale bar in A is 16.0 µm and all other panels are the same scale.
V, vesicle; S, sterigmata; C, conidia.

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Spermidine regulates Aspergillus development807
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
ical effects of polyamines suggest that these compounds
may have profound intracellular regulatory properties.
Although spermidine has been found to be the major
polyamine in most fungi (Tabor and Tabor, 1985; Walter
et al., 1997), the exact role of spermidine and its impor-
tance in fungal growth and development remains unclear.
In our studies, we cloned and characterized the A. nidu-
lans spdA gene that encodes spermidine synthase, the
enzyme required to produce spermidine. Our results dem-
onstrate that in the filamentous fungus A. nidulans, sper-
midine is an essential polyamine for viability and suggest
that certain threshold levels of spermidine are required for
specific A. nidulans developmental processes.
Aspergillus nidulans follows a well-defined programme
of development. Wild-type conidia placed on solid medium
initially germinate, produce germ tubes which branch to
produce vegetative hyphae followed by aerial hyphae and
then conidiophores. When grown in liquid medium the
conidia germinate, giving rise to germ tubes that branch
to produce mycelia but not conidiophores. Our results
indicate that spermidine plays a significant role in regulat-
ing these developmental transitions in this organism. At
different stages of development, the level of polyamines
determines the progression through to the next stage of
development. We propose that several intracellular
spermidine threshold levels exist. One level is that con-
centration (∼ 0.05 mM exogenous spermidine) required for
development to proceed past germ tube formation and
another level is that concentration (greater than 0.05 mM
exogenous spermidine) required for development to pro-
ceed past vegetative hyphal growth to conidiophore and
ST production. Results from the overexpression of flbA
also indicate a requirement for a minimal amount of sper-
midine to allow FlbA activation of asexual sporulation and
ST biosynthesis to proceed. Polyamine threshold levels
were also observed for U. maydis (Guevara-Olvera et al.,
1997). When putrescine concentrations lower than
0.5 mM were employed, the U. maydis odc null mutant
grew at a normal rate but was unable to engage in the
yeast-to-mycelium dimorphic transition. Reversion to nor-
mal dimorphic phenotype required higher concentrations
of putrescine or spermidine (Guevara-Olvera et al., 1997).
Not all Aspergillus developmental processes were sen-
sitive to spermidine availability. The rate of conidial germi-
nation, mycelial formation and conidiophore initiation in
the ∆spdA strains are delayed by spermidine starvation,
while the time of conidial development following conidio-
phore formation, per cent conidial germination and per-
cent germ tube emergence were the same as those of
wild type (Tables 5 and 6). This result is consistent with
previous observations that conidial germination and
appressorial formation of Uromyces viciae-fabae (Reiz
et al., 1995) and Magnaporthe grisea (Choi et al., 1997)
are unaffected when treated with DFMO whereas somatic
hypha growth, conidiophore formation and conidiation
were affected. Apparently polyamines differentially affect
various steps in fungal development.
Neither vegetative growth nor conidiation of the ∆spdA
strains, either TYJ3.119 (∆spdA) or TYJ3.174 (puA2,
∆spdA), could be returned to wild-type phenotype by
exogenous spermidine (Tables 2 and 3). Interestingly, the
∆spdA mutant had greater asexual spore densities than
wild type but could not be compensated for ascospore
production when grown on 1 mM spermidine. The alter-
ation in asexual to sexual spore production in the sup-
plemented ∆spdA strain was reminiscent of Aspergillus
response to sporogenic fatty acids (Champe et al., 1987;
Calvo et al., 1999). These developmental aberrations
could be a reflection of an altered spermidine to
putrescine ratio as alterations in this ratio affect mycelial
growth versus conidiation in A. flavus (Khurana et al.,
1996) and developmental switches in other fungi (Shapira
et al., 1989; Guevara-Olvera et al., 1997). Exogenous
Fig. 5. Activation of brlA and stcU by overexpression of flbA in the
∆spdA strain requires 0.05 mM spermidine.
A. Strains grown in 0.05 mM spermidine.
B. Strains grown in 0.0 mM spermidine. Total RNA from RYJ2
(alcA(p)::flbA), TPK1.3 (alcA(p)::flbA) and TYJ3.35 (alcA(p)::flbA;
∆spdA) was isolated from cultures 0, 6, 12 and 24 h after shifting from
glucose shake medium to alcA-inducing shake medium. A 1.9 kb
SalI–HindII fragment from pJA139 was used as a brlA-specific probe;
a 0.75 kb SstII–SmaI fragment from plasmid pRB7 (Yu et al., 1996b)
was used as an stcU-specific probe; a 2.5 kb EcoRI fragment from
pBN30 was used as an flbA-specific probe; plasmid pJA1 (Adams
and Timberlake, 1990) was used as an alcA-specific probe. Equal
loading of total RNA (20 µg lane−1) was evaluated by ethidium bro-
mide staining. ST was separated by TLC and the sample labelled Std
is the ST standard (Sigma).

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808Y. Jin et al.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
spermidine is known to reduce the rate of ornithine decar-
boxylase synthesis and increase the rate of ornithine
decarboxylase protein degradation in many organisms
including fungi (Barnett et al., 1988; Gupta et al., 2001).
Perhaps the exogenous spermidine led to a perturbation
in putrescine production that when coupled with the
∆spdA allele altered polyamine ratios in a manner leading
to alterations in production of spore type.
Another interesting morphological variation of the
∆spdA and ∆spdA; puA2 strains was the formation of
enlarged conidia when these mutants were grown in liquid
shake medium (Figs 1 and 4). We found that these
enlarged conidia originated from spores after they had
germinated. The appearance of these cells was similar to
swollen spores produced by an A. nidulans rasA mutant,
rasAG17A, where development is arrested (Som and
Kolaparthi, 1994). When stained with DAPI, both the
rasAG17A and the ∆spdA germlings showed multiple nuclei
in the conidium and inhibition of cell division (Som and
Kolaparthi, 1994 and data not shown), a condition indic-
ative of cell cycle arrest. Spermidine may be important in
cell division as it has been shown to remediate spermine-
induced cell cycle arrest in Chlamydomonas (Theiss et al.,
2002). It is possible that spermidine is important in cell
cycle progression in A. nidulans as cell division was
resumed in the ∆spdA strains upon addition of 1.0 mM
spermidine.
As described in the introduction, polyamines affect
many proteins in signal transduction pathways (Wright
and Boyle, 1982; Bueb et al., 1991; 1992; Orlowski, 1995;
Choi et al., 1997; Kubota et al., 1997; Leroy et al., 1997),
including G α proteins. Although we did not test activity of
signalling proteins or concentration of second messen-
gers in our study, the results presented in this communi-
cation indicate that a relationship exists between G protein
signalling control and polyamine control of sporulation and
ST biosynthesis. Overexpression of flbA, leading to the
suppression of G protein activity (Lee and Adams, 1994;
Hicks et al., 1997), could force asexual spore and ST
production in the ∆spdA strain TYJ3.35 but this was
dependent on the concentration of spermidine in the
medium. ST production and asexual development could
not be forced in TYJ3.35 grown in medium lacking sper-
midine. Yet development and ST production was forced in
TYJ3.35 grown in a spermidine concentration (0.05 mM)
that did not support sporulation and ST biosynthesis in
the ∆spdA strains containing a wild-type flbA allele. It
appears that a certain level of spermidine – less than
required for ST and conidiophore development – is
required for normal function of the FlbA/FadA signalling
pathway. Moreover, microscopic observations of TYJ3.35
in 0.05 mM spermidine showed that the conidiophores
emerged from matured mycelia and not the enlarged
conidia (Fig. 4). This suggests that conidiophores cannot
arise from germ tube or spore tissue and that some level
of cellular maturity, in part controlled by spermidine levels,
is required before suppression of the G protein pathway
can instigate asexual development. Our interest is to con-
tinue to characterize the requirements of polyamines in
Aspergillus development and further explore the interac-
tion of polyamines and signal transduction processes in
this genus. Further knowledge of this interaction could be
fruitful in developing means to control fungal sporulation
and mycotoxin biosynthesis.
Experimental procedures
Fungal strains and growth conditions
Table 7 lists all fungal strains used in this study. Some strains
are not discussed in text but used for sexual crosses to obtain
the strains of interest. Sexual crosses of A. nidulans strains
were conducted according to Pontecorvo et al. (1953). All
strains were maintained as silica stocks and/or glycerol
stocks and were grown at 37°C on minimal medium (Cove,
1976) amended with appropriate supplements (Hicks et al.,
1997).
Cloning of the A. nidulans spdA gene
Examination of expressed sequence tag (EST) sequences
from the A. nidulans genome project (http://www.genome.ou.
edu/fungal.html) identified an EST clone, e4a02a1, that
showed homology to spermidine synthases. Using primers
designed from e4a02a1, a DNA fragment was amplified from
A. nidulans genomic DNA. This fragment was sequenced to
confirm spermidine synthase identity and then used to probe
an A. nidulans cosmid library. One cosmid located on chro-
mosome VIII, pW30B01, was identified. The spdA containing
cosmid pW30B01 was digested with XhoI and probed by
hybridization with the labelled spdA PCR product. A 5.2 kb
DNA fragment which hybridized to the spdA probe was
subcloned in pYJ1 to form pYJ2. pYJ1 is a modified pBlue-
script KS vector in which the SacI and SmaI sites were
eliminated.
DNA sequence analysis
The 37.3 kb cosmid pW30B01 was sequenced and is
available on the OU-ACGT website (Lewis et al., 1998). The
sequence of pYJ2 was compared to the nucleotide sequence
of pW30B01, and the size of the spdA gene was determined
by comparing with the spermidine synthase genes of Sac-
charomyces cerevisiae and Schizosaccharomyces pombe.
The deduced amino acid sequence of SpdA was determined
by analysis of the translational products of genomic and
cDNA clones, using the CLUSTALX software system from
NCBI. The GenBank number of this sequence is AY050641.
Construction of the A. nidulans spdA vectors
Plasmids were generated using standard techniques. pYJ2
was digested with SphI and SacI to remove the 0.2 kb inter-

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Spermidine regulates Aspergillus development 809
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
nal SphI–SacI fragment of the spdA ORF. The 0.2 kb frag-
ment was replaced by the 1.8 kb argB fragment obtained by
digesting pJY3 with SmaI. pJY3 was created by moving the
1.8 kb SmaI argB fragment from pJYArgB2 (Shimizu and
Keller, 2001) into pK19 (Pridmore, 1987). The resulting plas-
mid pYJ3, the spdA disruption construct, was made by
blunt-end ligating of the SphI–SacI digested pYJ2 and the
1.8 kb argB fragment. pYJ26-4, containing the spdA gene in
a 1/2 trpC vector (to direct the gene to the trpC site), was
created by ligating the 5.2 kb XhoI spdA containing fragment
from pYJ2 into XhoI-digested pSH96 (Wieser and Adams,
1995).
Fungal transformation procedures spdA disruption strains
TYJ3.119, TYJ3.35 and TYJ3.174 were generated by the
following steps. First, pYJ3 was digested with PvuI and the
obtained linear DNA fragment was utilized to transform
the A. nidulans strain PW1 to create TYJ3.119, strain RYJ2
to create TYJ3.35 and strain RYJ6 to create TYJ3.174. RYJ2
was generated by crossing RBN138 to PW1. RYJ6 was
generated by crossing FGSC682 to PW1. Transformation,
extraction of DNA from transformants and wild-type DNA,
restriction enzyme digestion, gel electrophoresis, Southern
blotting and hybridization were performed using standard
methods (Miller et al., 1985; Sambrook et al., 1989). To facil-
itate isolation of the spdA disruptants, all the transformants
were tested on two types of minimal medium plates, one
supplemented with 1.0 mM spermidine and one with 0.1 mM
spermidine.
Complementation of ∆spdA strains was achieved by trans-
forming them with a wild-type copy of the spdA gene. First
TYJ3.119 was crossed to RJH26 to create RYJ13. RYJ13
was co-transformed with pYJ2 (containing the wild-type spdA
gene) and pTA11 (containing the entire trpC on a 4.4 kb XhoI
fragment cloned into pIC19H, Marsh et al., 1984) to produce
TJW32. RYJ13 was also transformed with pYJ26-4 (a plas-
mid containing the wild-type spdA gene ligated to the half
trpC gene) to produce TJW28.
Polyamine analysis of Aspergillus strains
Polyamines were calculated for A. nidulans wild-type TPK1.1,
the polyamine requiring strains FGSC682, TYJ3.119 and
TYJ3.174, and the two complemented strains TJW28 and
TJW32. All strains were grown on solid minimal medium
supplemented with 0.0, 0.1, 0.5, 1.0 or 3.0 mM spermidine.
Each test was repeated twice.
Spores for inoculation were obtained from 1.0 mM spermi-
dine plates. Four hundred microlitres of 2.3 × 109 spores
ml−1 were spread on a 0.45 µ millipore membrane placed on
top of the solid media, two plates per strain. Plates were
incubated 19 h at 37°C. From each plate, one half of the
mycelium was used for dry weight and protein determination
and the other half was used for polyamine analysis using wet
mycelium.
Polyamine extraction followed that of San-Blas et al.
(1996). Mycelium was filtered and washed twice with distilled
water. Then samples were extracted with 2 ml of 6% perchlo-
ric acid for 3 h at room temperature and centrifuged at
1500 g. The supernatant was placed in glass tubes and air
dried in an oven at 80°C. The product was resuspended in
1 ml of 6% perchloric acid and filtered through a 0.45 µ mil-
lipore membrane. Chemically unconjugated polyamines were
determined by adding 1 ml of 2 M NaOH and 10 µl of benzoyl
chloride to 0.5 ml of the perchloric acid sample. The mixture
was shaken vigorously and incubated at room temperature
for 10 min. The reaction was stopped with 2 ml of saturated
NaCl. This mixture was extracted with 4 ml of diethylether.
The ether phase was recovered, evaporated in a water
bath until dry and 200 µl of HPLC-grade methanol was
added to each tube to recover the benzoylated derivatives.
Samples were filtered through a C18 column (QC 498
Alltech) and then analysed by HPLC using an ODS-C18
(4.6 mm × 150 mm) column. The solvent system was iso-
cratic 50% methanol–water. The column temperature was
35°C and UV detector was fixed at 254 nm. Benzoylated
Table 7. Aspergillus nidulans strains used in this study.
Fungal strains GenotypeSource
FGSC26
FGSC682
PW1
RBN138
RJH26
RYJ2
RYJ4
RYJ6
RYJ11
RYJ12
RYJ13
TJW28
TJW32
TPK1.1
TPK1.3
TYJ3.35
TYJ3.119
TYJ3.174
WIM126
biA1; veA1
wA3, mauB4, puA2; veA1
biA1; argB2; methG1; veA1
wA3; pyroA4; veA1, alcA(p)::flbA::trpC
biA1; argB2; ∆stcE::argB; veA1
biA1; wA3; argB2; methG1; veA1, alcA(p)::flbA::trpC
pabaA1, yA2; argB2; spdA::argB
biA1; wA3, puA2; argB2; methG1
biA1; argB2; ∆stcE::argB, ∆spdA::argB, veA1
biA1; argB2; ∆stcE::argB, veA1
biA1; argB2; ∆stcE::argB, ∆spdA::argB, veA1, trpC801
biA1; argB2; ∆stcE::argB, ∆spdA::argB, veA1, spdA::trpC
biA1; argB2; ∆stcE::argB, ∆spdA::argB; spdA, trpC; veA1, trpC801
biA1; methG1; veA1
biA1; wA3; argB2; methG1; veA1, alcA(p)::flbA::trpC
biA1; wA3; argB2; methG1; ∆spdA::argB, veA1, alcA(p)::flbA::trpC
biA1; argB2; methG1; ∆spdA::argB, veA1
biA1; wA3, puA2; argB2; methG1; ∆spdA::argB, veA1
pabaA1, yA2
FGSCa
FGSC
P. Weglenski
Lee and Adams (1994)
J. Hicks
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
This study
L. Yager
a. Fungal Genetics Stock Center.

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810Y. Jin et al.
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
putrescine, spermidine and spermine were used as external
standards. Polyamine concentrations were calculated from a
standard curve and expressed as µmol mg−1 protein.
Thin-layer chromatography analysis
Sterigmatocystin production of A. nidulans wild-type strains
and polyamine-deficient strains were examined by TLC anal-
ysis. Sterigmatocystin was extracted from either shake
cultures or stationary cultures using published procedures
(Keller et al., 1994; Hicks et al., 1997). For solid media, a cork
borer with diameter of 1.1 cm was used to collect samples at
the centres of these plates. Each sample was homogenized
and mixed well with 2 ml of double distilled H2O. Then 2 ml
of CHCl3 was added and agitated with a vortex. After centrif-
ugation for 5 min at 1000 r.p.m., the separated organic phase
was transferred to an Eppendorf tube. All samples were dried
down and resuspended in 100 µl of CHCl3. Twenty-five µl of
each extract was spotted on a TLC plate and separated in
toluene–ethyl acetate–formic acid (40 : 60 : 0.5). The TLC
plates were sprayed with aluminium chloride to enhance ST
fluorescence upon exposure to longwave (365 nm) UV light
(Stalk and Rodricks, 1971).
For cultures grown in liquid media, mycelia were lyophilized
and then weighed. Lyophilized mycelium (0.02 g) of each
sample was ground into fine powder. Four hundred microli-
tres of acetone was added to the powder, mixed well with a
vortex and centrifuged for 10 min at 7000 r.p.m. Superna-
tants (350 µl) were transferred to new tubes and 30 µl of each
extract was separated on a TLC plate for ST analysis.
Time course study of spdA mRNA during
fungal development
Total RNA of A. nidulans strains was extracted with Trizol
(Gibco BRL). Mycelium was collected, lyophilized in liquid
nitrogen, and pulverized, then total RNA was extracted fol-
lowing the manufacturers recommended procedure.
Wild-type strains, FGSC26 and RAMC22, as well as the
spdA disruption strain, TYJ3.119, were inoculated at a den-
sity of 105 spores ml−1 in liquid minimal medium containing
appropriate supplements, and shaken at 300 r.p.m. at 37°C
for 20 h. Mycelia were harvested on miracloth, and trans-
ferred onto Fisher P8 filter paper disks placed on 1.2% agar
minimal medium and incubated at 37°C. Samples were taken
at the time of the mycelia transfer (0 h) and −6, −3, 2, 4, 6,
8 h before or after transfer for microscopic observation and
RNA analysis.
Morphological development of polyamine mutant strains
Three microlitres of 105 spores ml−1 of A. nidulans wild-type
strain TPK1.1 and the spdA disrupted strains TYJ3.119
and TYJ3.174 were inoculated in the centres of plates
supplemented with one of the following concentrations of
polyamines: 0.0 polyamine; 0.10, 1.0 or 3.0 mM spermidine;
0.10, 1.0 or 3.0 mM putrescine; 0.10, 1.0 or 3.0 mM sper-
mine. The diameters of colonies were measured and conidia
(the asexual spore of A. nidulans) were counted after 72 h
incubation at 37°C. Tests were performed in triplicates and
analysed by standard t-tests.
For microscopic observations, A. nidulans polyamine
requiring strains FGSC682, TYJ3.119, TYJ3.174 and wild-
type strain TPK1.1 were inoculated in minimal medium con-
taining 0, 0.05 or 1.0 mM spermidine at 106 spores ml−1 and
shaken for 24 h at 300 r.p.m. at 37°C. Samples were exam-
ined at 2 h intervals. Germination rate was determined by
counting 100 conidia. Pictures of fungal development were
taken at 8, 12 and 16 h after inoculation.
For TPK1.3 and TYJ3.35 (strains overexpressing flbA in the
presence of threonine), microscopic observations were made
at 0, 12 and 24 h after these strains were transferred to
threonine media (for details, see Overexpression of flbA in
the A. nidulans spdA mutant strain).
Effect of polyamine starvation on differentiation and ST
production of A. nidulans
Three µl of 105 spores ml−1 spores of A. nidulans wild-type
TPK1.1 and polyamine requiring strains FGSC682, TYJ3.119
and TYJ3.174 were inoculated on the centres of minimal
medium agar plates containing 0.0, 0.05, 0.10 or 1.0 mM
spermidine. The diameters of colonies were measured and
conidia were counted after 6 days (144 h) incubation at 37°C.
Tests were performed in triplicate and analysed by standard
t-tests.
Overexpression of flbA in the A. nidulans spdA
mutant strain
TYJ3.35 was constructed by transformation of RYJ2 with
pYJ3, whereas TPK1.3 was generated by transformation of
RYJ2 with the argB-containing plasmid pPK1. Spore suspen-
sions of both strains were filtered through miracloth to remove
conidiophores and mycelium. Then 2 × 106 spores ml−1 of
each strain was inoculated in 500 ml of minimal medium (1%
glucose as carbon source) containing 0.0, 0.05 or 1.0 mM
spermidine and shaken at 300 r.p.m. at 37°C for 18 h. Myce-
lium was collected onto miracloth, washed once with minimal
medium lacking glucose, divided into equal parts, transferred
to flasks containing 100 ml of minimal medium with 100 mM
L-threonine as the sole carbon source or 1% glucose as the
sole carbon source and shaken for 48 h at 37°C. The same
concentration of spermidine was included in the transfer
media. For medium with L-threonine as sole carbon, samples
were harvested for RNA analysis at the time of the medium
shift (0 h) and 6, 12 and 24 h after the shift. For glucose
medium, samples were harvested for RNA analysis at time
of the medium shift (0 h) and 6, 12, 24, 48 and 72 h after the
shift. Sterigmatocystin was also examined in these samples
as described above.
Acknowledgements
Yuan Jin and Jin Woo Bok contributed equally to this manu-
script. This research was partly funded by USAID under the
Peanut CRSP Grant LAG-G-00-96-90013-00 and by NSF
Grant MCB-9874646 to N.P.K. Centro de Investigacion y
Estudios Avanzados del IPN funded DG. We acknowledge

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Spermidine regulates Aspergillus development811
© 2002 Blackwell Publishing Ltd, Molecular Microbiology, 46, 801–812
technical help and useful discussions with our colleagues Drs
Daren Brown and Doris Kupfer.
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