Correlation between the regulation of sterigmatocystin biosynthesis, assexual and sexual sporulation in Emericella nidulans

Abstract

We analyzed the regulation of sterigmatocystin biosynthesis in wild type and mutant strains of Emericella nidulans (= Aspergillus nidulans). A positive correlation between both asexual and sexual sporulation and synthesis of the mycotoxin was observed. Those conditions which favored sporulation stimulated sterigmatocystin formation, and vice versa. Both processes were stimulated by light in a veA+ genetic background. In contrast, they were inhibited by diaminobutanone, an inhibitor of ornithine decarboxylase. The effect of this inhibitor was partially reverted by putrescine addition. Partial supplementation of specific requirements to auxotrophic mutants allowed normal vegetative growth, but interfered with asexual sporulation and sterigmatocystin biosynthesis. Synthesis of the mycotoxin was neither affected in a brlA mutant or in developmental mutants blocked at later steps in sporulation. As in wild type strain, diaminobutanone inhibited sterigmatocystin biosynthesis and cleisthotecia formation in the brlA mutant, and its effect was reverted by addition of putrescine. The inhibitor also affected the transcription of brlA. Our results indicate that sporulation and the synthesis of sterigmatocystin are co-regulated at a step previous to the brlA execution point.

Full text

Antonie van Leeuwenhoek 73: 199–205, 1998. 199
c
1998 Kluwer Academic Publishers. Printed in the Netherlands.
Correlation between the regulation of sterigmatocystin biosynthesis and
asexual and sexual sporulation in Emericella nidulans
Doralinda Guzm
´
an-de-Pe
˜
na
1
,Jes
´
us Aguirre
2
&Jos
´
e Ruiz-Herrera
1
1
Departamentos de Gen
´
etica y Biolog
´
ia Molecular (Unidad M
´
exico, D.F.) and Ingenier
´
ia Gen
´
etica (Unidad
Irapuato),Centro de Investigaci
´
on y de Estudios Avanzados del IPN. Irapuato, Gto, M
´
exico;
2
Instituto de
Fisiolog
´
ia Celular, Universidad Nacional Aut
´
onoma de M
´
exico. Mexico, D.F.; Departamento de Ingenier
´
ia
Gen
´
etica Centro de Investigaci
´
on y de Estudios Avanzados del IPN Km 9.6 Libramiento Norte, Carretera
Irapuato-Le
´
on. Apdo. Postal 629 Irapuato, Gto. Mexico; Tel: 52-462-39600;Fax: 52 462 39650; E-mail:
dguzm
´
an@irapuato.ira.cinvestav.mx;( author for correspondence)
Received 5 June 1997; accepted 12 December 1997
Key words: Emericella nidulans, Aspergillus nidulans; sterigmatocystin, mycotoxins, sporulation, polyamines,
diaminobutanone,brlA
Abstract
We analyzed the regulation of sterigmatocystinbiosynthesis in wild type and mutant strains of Emericella nidulans
(= Aspergillus nidulans). A positive correlation between both asexual and sexual sporulation and synthesis of the
mycotoxin was observed. Those conditions which favored sporulation stimulated sterigmatocystin formation, and
vice versa. Both processes were stimulated by light in a veA genetic background.In contrast, they were inhibited
by diaminobutanone, an inhibitor of ornithine decarboxylase. The effect of this inhibitor was partially reverted
by putrescine addition. Partial supplementation of specific requirements to auxotrophic mutants allowed normal
vegetative growth, but interfered with asexual sporulation and sterigmatocystin biosynthesis. Synthesis of the
mycotoxin was neither affected in a brlA mutant or in developmental mutants blocked at later steps in sporulation.
As in wild type strain, diaminobutanone inhibited sterigmatocystin biosynthesis and cleisthotecia formation in the
brlA mutant, and its effect was reverted by addition of putrescine. The inhibitor also affected the transcription of
brlA. Our results indicate that sporulation and the synthesis of sterigmatocystin are co-regulated at a step previous
to the brlA execution point.
Abbreviations: AF aflatoxins; DAB 1,4 diamino-2-butanone; ODC ornithine decarboxylase; ST sterig-
matocystin
Introduction
Aflatoxins (AF) are potent toxic, carcinogenic, and
mutagenic compounds produced by three species of
Aspergillus (Ellis et al. 1991; Kurtzman et al.
1987). They are polyketide derivatives whose synthe-
sis involves the condensation of acetyl and malonyl
groups by an enzymatic complex similar to fatty acid
synthetase, denominated polyketide synthetase (Dut-
ton 1988). Other species such as Emericella nidulans
(=Aspergillus nidulans), accumulate sterigmatocystin
(ST), an AF precursor, also with carcinogenic and
mutagenicproperties. The genesinvolvedintheST/AF
biosynthetic pathways are organized as clusters in dif-
ferent species, several genes of which show a high
degree of homology (Keller et al. 1994; Trail et al.
1995; Brown et al. 1996).
Through manipulation of the environmentalcondi-
tions, the use of the ornithine decarboxylase inhibitor
1,4 diamino-2-butanone (DAB), and the analysis of
asporogenous mutants, we provided evidence that
sporulationandAF synthesisarelinkedprocessesin A.
parasiticus (Guzm
´
an-de-Pe
˜
na & Ruiz-Herrera 1997).
Trail et al. (1995) suggested the possibility that some
of the genes involved in AF synthesis might be linked
to differentiation, because their mutation also affect-

200
Table 1. Emericella nidulans (=Aspergillus nidulans) strains used in this study
Strain Relevant genotype Reference/Source
FGSC26 biA1,veA1 FGSC
1
FGSC4 biA1 FGSC
AJC7.1 biA1, veA1, brlA1 Clutterbuck 1969
GO171 biA1, puA1, veA1, Clutterbuck 1969
SJA17 pabaA1, brlA(-2900 p/l)::lacZ, abaA2, veA1 Aguirre 1993
GO102 biA1, wA5, inoB2, veA1 Clutterbuck 1969
NK002 biA1, pabaA1, yA2 wA3, veA1, trpC801 Mayorga & Timberlake 1990
CRO1 cfwA2, pabaA1, yA2, veA1 Aguirre et al. 1993
AJC22 biA1, veA1, wetA6 Clutterbuck 1969
TJA22 biA1, brlA(-2900 p/l)::lacZ, metG1, veA1 Aguirre 1993
CRO5 wA1, argB2, pyroA, veA1 Clutterbuck 1969
1
Fungal Genetics Stock Center, Dept. of Microbiology, University of Kansas Medical Center,
Kansas City, KS.
ed sclerotial morphology and the synthesis of spore
pigments. These observations agree with data accu-
mulated during the study of Streptomyces,wherean
associationbetweentheonsetofsecondarymetabolism
and sporulation has been established (Hopwood1988;
Bibb 1996). The existence of mutants deficient in
both processes indicates common elements of genetic
control (Champnes & Chater 1994). In Streptomyces
griseus an autoregulatory factor (A-factor) that links
secondarymetabolismandcelldifferentiationhasbeen
identified. Mutants of S. griseus deficient in A-factor
lose simultaneously the ability to produce secondary
metabolites and to form spores (Hara & Beppu 1982).
A-factorbindstoaspecificprotein,leadingtoderepres-
sion of gene(s) important for secondary metabolism
and sporulation (Miyake et al. 1989; 1990).
To analyze further the relationship between sec-
ondary metabolism and sporulation in Aspergillus we
have chosen E.nidulans as a model for two main rea-
sons:first, this fungusisableto produceST, an AFpre-
cursor, and second, several well characterized devel-
opmental mutants are available, providing important
tools to analyze both processes.
Materials and methods
Strains and culture conditions
The strains used in this study are described in Table 1.
They were kept in minimal medium or in silica gel at
4 C. To analyze the synthesis of ST, different culture
media were utilized: potato dextrose agar (PDA, Dif-
co); a medium containing 1.5% agar, 2% yeast extract
and 1% sucrose (YES, Davis et al. 1967); and 1.5%
agar-containing minimal-nitrate media (M-AN, K
¨
afer
1977), supplemented as required. Amino acids, vita-
mins, and putrescine or/and 1,4 diamino-2-butanone
(DAB) were added to the media as filter sterilized
solutions. To analyze the effect of light, the fungus
was inoculated in the middle of YES agar plates. A set
of plates were coveredwith black paper and cardboard
and incubated at 28 C in a dark room whereasanother
set was placed in an illuminated room (40 W/m
2
/seg)
at 28 C also. After 60 h of incubation, spores were
countedand STextracted.Dataareexpressedperplate,
and in some experiments they are also expressed per
mg of dry weight.
Sterigmatocystin determination
Sterigmatocystin (ST) was extracted with acetone and
chloroform (Keller et al. 1994). The concentration of
ST in the extracts was determined by HPLC in a C
18
-
reverse phase column with a solvent system of deion-
ized water and methanol with the following elution
program: from 0 to 5 min, 60% methanol; and from
5 to 20 min, 70% methanol. The absolute levels of
ST varied in different experiments, but the reported
differences observed among the different samples or
treatments were reproducible. All experiments were
repeated at least twice with duplicate or triplicate sam-
ples.
Miscellaneous
Determination of growth as dry weight, and spore
numbers were performed as described (Guzman-de-

201
Table 2. Sterigmatocystin production by morphological mutants of E. nidulans in YES
medium
Mutant Relevant genotype Spores ( 10
7
) Sterigmatocystin ( g)
N
1
W
2
N
1
W
2
SJA-17 abaA2 0 0
3
0582.1
GO-102 wA5 0 1.2 0.3 0 25 8.5
AJC-22 wetA6
4
100 0.8 nd
5
930 7.0 nd
NK002 wA3, yA2 0 14.0 2.4 9 0.4 95 5.0
CRO5 wA1 0
3
0.4
6
84 2.0 80 0.3
CRO1 yA2, cfwA2 1 0.5 13.0 1.5 0 0
AJC7.1 brlA1 0
3
nd 463
6
nd
YES agar plates were incubated during 5 days at 28 C. Average from triplicates SEM
values
1
No additional supplementation of auxotrophic requirements
2
With additional supplementation of auxotrophic requirements
3
Cleistothecia were formed
4
The phenotype is not expressed at the temperature used
5
Not determined
6
Average of duplicate samples.
Pe
˜
na & Ruiz-Herrera 1997). -Galactosidase activity
was measured according to Miller (1972). Protein was
measured by Bradford’s method (1976). PDA, yeast
extract, and agar were obtained from Difco Laborato-
ries (Detroit, MI); amino acids, vitamins, putrescine,
DAB, O-nitrophenyl- -D-galactopyranoside, and ST
standard were purchased from Sigma Chemical Co.
(St. Louis, MO). HPLC gradesolventswere from Mer-
ck M
´
exico. Other reagents were of the highest purity
available.
Results
Co-regulation of asexual sporulation and ST
biosynthesis by light
Mooney & Yager (1990) described that wild type E.
nidulans (veA ) displays a period of photosensitivity
for sporulation. Since most strains of E. nidulans are
veA , and thereforeinsensitiveto lightfor sporulation,
we utilized E. nidulans FGSC 4 strain (veA ) to deter-
minethe effectof illuminationonSTbiosynthesis. The
strainwasincubatedonplatesofYESmediumat28 C
for 60 h, either under dark or illuminated conditions.
At the end of the incubation period, ST was extracted
and quantified, and spores were counted. Striking dif-
ferences were observed. Mycelium grown in darkness
was fluffy, and producedonly a small number of white
spores: 1 10
7
per plate, and a low amount of ST:
37 g per plate; as compared with mycelium grown in
the presence of light, which produced abundant green
spores: 110 10
7
, and a larger amount of ST: 257 g
per plate.
Sporulation and ST production in different mutant
backgrounds
Three groups of mutants were used in this study:
those affected in genes essential for sporulation,
such as brlA, abaA,andwetA;
those affected in non essential genes involved in
sporulation, such as wA and yA;and
auxotrophic mutants, such as the putrescine-
requiring mutant strain GO171, which lacks
ornithine decarboxylase activity.
Among the first group of mutants, the brlA1 mutant,
contains a null allele of the brlA regulator gene (Clut-
terbuck 1969). This mutation blocks the expression
of many genes that are specifically triggered during
asexual sporulation, whereas sexual sporulation is not
affected. The brlA1 mutant produced high amounts of
ST (Table 2) and abundant cleisthotecia.
The other mutants analyzed affected in abaA and
wetA, as well as those of the second group affected in
wA and yA genes, carried different auxotrophic mark-
ers. When these mutants were grown in complex YES
medium without addition of the total supplementation
of their requirements, vegetative growth was rough-
ly equivalent to wild type, but most of the mutant
strains: SJA17, GO102, NK002, and CRO1, did not
sporulate, nor produced ST. Mutants AJC22 which
requires biotin, and CRO5 were the exceptions to this
behavior. BothproducedST, however,thefirstonepro-

202
Figure 1. Putrescine is required for normal sporulation of an E. nidulans mutant lacking ODC activity. A. PDA plate inoculated with mutant
strain GO171 and incubated for 72 h. B. As above but PDA medium contained 0.1 mM putrescine. C. As above but PDA medium contained
1 mM putrescine.
Table 3. Putrescine is required for sporulation and sterigmatocystin biosynthesis
in E. nidulans GO171 mutant lacking ODC activity
Putrescine Growth Spores Sterigmatocystin
added (mM) (mg D.W.
1
)(10
7
)(g) (ng/mgD.W.)
0 126 16 1.5
2
0.8 4.5 1.7 35.7
0.1 96 11.0 620.0
3
1.5 50.0 1.4 520.0
1.0 108 2.5 1500.0
3
0.7 54.0 0.3 500.0
White spores obtained in non-supplemented PDA (1.8 10
6
) were inoculated
on PDA plates containing different additional concentrations of putrescine, and
incubated at 28 C for 72h. Average from triplicates SEM values
1
Dry weight
2
White spores
3
Green spores.
ducedabundantconidiain the absenceof addedbiotin,
whereas the second one produced abundant cleisthote-
cia (Table 2). When total supplementation of nutri-
ents were added to the media, asexual sporulation and
ST synthesis occurred in strains GO102, NK002 and
CRO5. Strain SJA17 did not form conidia, but pro-
duced abundant cleistothecia, whereas strain CRO1,
did not produce ST under any of the tested conditions
(Table 2).
The effect of polyamine auxotrophy was ana-
lyzed in the putrescine-requiringmutant strain GO171
(puA1) whose phenotype is expressed only when the
polyamine pool is exhausted. This was achieved by
growing the strain for about three cycles in unsup-
plemented PDA. At this stage, scant white spores
were produced. These were inoculated on plates of
PDA containing different additional concentrations of
putrescine (0, 0.1, or 1.0 mM). After four days spore
numbers and ST were determined. In the absence of
additional putrescine, abundant vegetative mycelium
wasobtained,probablybecausethe PDA medium con-
tains limiting amounts of polyamines, but it produced
only scant white spores (Fig.1), and very low amounts
of ST (Table 3). On the other hand, in the presence
of the additional supplement of putrescine, the mutant
produced large numbers of normal green spores (Fig-
ure 1), and high amounts of ST (Table 3).
Inhibition of ornithine decarboxylase reduces
sporulation and ST production
The effect of DAB, a competitive inhibitor of
ornithine decarboxylase, was analyzed using E. nidu-
lansFGCS26and the brlA1mutant underconditionsin
which wild type strain FGCS26 sporulated abundantly
and produced ST. When 5mM DAB was added to the
medium, ca. 50% inhibition of growth, a total inhibi-
tion of sporulation and an almost complete inhibition
of ST synthesis occurred (Table 4A). Similar behavior
was observed when the brlA1 mutant was used. Aeri-

203
Table 4. Effect of 1,4 diamino-2-butanone on sporulation and sterigmatocystin
synthesis in E. nidulans
Strain DAB Growth Spores Sterigmatocystin
(mM) (mg D.W.
1
)(10
7
)(g) ( g/mg D.W.)
A. FGSC-26
0 188 0.7 10 0.5 210 3.5 1.11
5903.5 0 4 0.3 0.04
B. AJC7.1(brlA1)
0 210 3.5 0
2
272 13.5 1.29
5 105 4.0 0 8 0.7 0.07
For strain FGSC-26, 8 h-grown germlings (4.4 10
6
) were inoculated on plates of
YES agar medium. For strain brlA1, a mycelial suspension was prepared in a sterile
solution of 0.1% Triton X-100 in distilled water and inoculated on YES agar plates.
Cultures were incubated for 72 h at 28 C. Average from triplicates +/- SEM values.
1
Dry weight
2
Cleistothecia were formed.
Table 5. Reversion of DAB effect by putrescine
Strain DAB Putrescine Growth Spores Sterigmatocystin
(mM) (mM) (mg D.W.
1
)(10
7
)(g) ( g/mgD.W.)
A. FGSC26
0 0 196 6.0 2.9 0.8 130.5 1.5 0.665
5 0 208 4.0 0 3.9 1.0 0.018
0 20 182 6.5 7.0 1.3 50.0 4.0 0.274
5 20 172 6.0 3.1 0.5 21.8 1.5 0.126
B. AJC7.1 (brlA1)
0 0 229 9.5 0 463.0 41 2.020
5 0 115 4.2 0 0 0
5 10 211 6.0 0 389.0 32 1.840
5 20 214 2.5 0 389.0 14 1.810
For strain FGSC26, 8 h-grown germlings (4.4 10
6
) were inoculated on YES agar plates. For
strain brlA1, a mycelial suspension was prepared in a sterile solution of 0.1% Triton X-100 in
distilled water, and inoculated on YES agar plates. Cultures were incubated for 72 h at 28 C.
Average from triplicate samples.
1
Dry weight.
al growth, cleistothecia formation, and ST production
wereabundantin controls,andminimalin the presence
of DAB (Figure 2; Table 4B).
When E. nidulans was grown in the presence of
DAB and putrescine (the product of ornithine decar-
boxylase), the effect of DAB was partially revert-
ed. Thus, the fungus sporulated, and produced ST
(Table5A),althoughinloweramountsthanthecontrol.
This partial reversion of the effect of DAB on myco-
toxin biosynthesis and sporulation was also observed
in A. parasiticus (Guzm
´
an-de-Pe
˜
na & Ruiz-Herrera
1997). Reversion of the DAB effect by putrescine was
also observed in the brlA1 mutant (Table 5B). In this
mutant it was not possible to measure the effect on
sporulation, but growth was similar under all tested
conditions .
Determination of the effect of DAB on the expression
of brlA
We analyzedtheeffectof DABon the expressionof the
brl(A/B)::LacZreportergenefusioncontainedinstrain
TJA22 (Aguirre 1993). Strain TJA22 was grown as
described by Skromne et al. (1995), and transferred to
minimal medium lacking carbon and nitrogen sources
for 24hto induce brlAexpression. The promoteractiv-
ity of brlA was reduced by about 80% in the presence
of 5 mm DAB: 52 Units F -galactosidase per mg pro-
tein in the drug-treated culture vs. 294 in the control in
one experiment, and 145 vs 623 Units in a second one.

204
Figure 2. DAB effect on the formation of aerial mycelium in the
AJC7.1brlA1 mutant. YES agar plates without (A) or with 5 mm
DAB (B) were inoculated and incubated for 72 h, and the edge of
the colonies were photographed.
Discussion
The results obtained in this study indicate that both
asexual and sexual sporulation and mycotoxin forma-
tion are linked phenomena in Aspergilli and suggest
that, as in Streptomyces (Bibb 1996; Miyake et al.
1990; Horinouchi & Beppu 1992), the onset of sec-
ondarymetabolismand differentiationhave a common
regulation.
We have shown that light affects both processes,
asexualsporulationandSTbiosynthesis.InA.nidulans
thevelvetgene (veA)hasbeenimplicatedin the regula-
tion of conidiation. Mutationsaffecting this gene abol-
ish light dependence of conidiation (Mooney & Yager
1990). Here we have confirmed that sporulation of a
veA strainisabnormalinthedark,withtheproduction
of a low number of white conidia, whereas abundant
green conidia were formed when cultures were illu-
minated. The synthesis of ST was also increased by
illumination. Mooney & Yager (1990) observed that
transcription of brlA did not occur unless the organism
was exposed to light during a critical period. These
observations, and our results suggest that light switch-
es on a process which is common to both sporulation
and toxin formation.
The relationshipexistingbetweenST synthesisand
sporulation is supported by the data obtained with
auxotrophic mutants. We observed that only under
conditions where conidia and/or cleistothecia forma-
tion occurred (by addition of an excess of the specific
growth requirements), did ST biosynthesis take place
in significant amounts. The onlyexceptionwas mutant
strain CRO1, which sporulated abundantly, but did not
produce ST under any culture condition used in this
study. This mutant is affected in the cfwA gene which
has been assigned to chromosome I closely linked to
creA and pyroB(Aguirreet al. 1993, unpublished), and
whose function has not been determined. This gene
seems to play an important role in asexual and sexual
sporulation(Aguirreet al. 1993),and as shownhere, in
ST synthesis. The fact that the cfwA2 mutant does not
synthesize the conidium pigments, which are also of
polyketide nature (Mayorga & Timberlake 1992) sug-
gests that a common precursor for both, pigments and
ST is not produced in the mutant. It is also important
to point out that the results obtained with the mutants
abaA, wetA, wA and yA affected in sporulation genes
acting after brlA, indicate that they are not involved in
the regulation of ST synthesis. This and the fact that
a brlA null mutant is able to produce ST, suggest that
the common step in conidiation and ST biosynthesis
operates upstream of the brlA point of execution.
Theresultsobtainedwiththepolyamineauxotroph-
ic mutant (GO171), and the ornithine decarboxylase
inhibitor DAB (Stevens et al. 1977), may be consid-
ered apart from the general effects of the auxotroph-
ic requirements described above. Previous results
obtained using different fungi have demonstrated that
previous to any differentiative step, including sporula-
tion, there occur elevations in the levels of ornithine
decarboxylase and polyamines, and that DAB addi-
tion prevents both, accumulation of polyamines and
differentiation(reviewed by Ruiz-Herrera 1994). In A.
parasiticus, DABinhibitedsporulationandAFbiosyn-
thesis (Guzman-de-Pe
˜
na& Ruiz-Herrera 1997). In the
present study we observed that polyamine deprivation
and DAB addition, inhibited sporulation and ST syn-
thesis in E. nidulans. Both effects were reverted by
putrescine. These observations suggest an important
role for polyamines in the differentiative processes of
E. nidulans, and in the biosynthesis of ST. Interesting-
ly, the asporogenous mutant brlA1 behaved similarly
to wild type with regards to its sensitivity to DAB.

205
Formation of aerial mycelium, sexual sporulation, and
ST biosynthesis were all blocked by the drug. These
results confirm the idea that the link between these
events occurs before the brlA execution point. This is
supported by the observation that expression of brlA
measured by means of a reporter gene was inhibitedin
the presence of DAB.
In summary, our results a) confirm that both asex-
ual and sexual sporulation and polyketide mycotoxin
biosynthesis are correlated processes in Aspergilli and
their perfect stages; b) suggest that their common reg-
ulator operates before brlA expression, and c) indicate
that genes downstream of brlA play no role in the reg-
ulation of ST biosynthesis inE. nidulans.
Acknowledgements
This work was partially supported by the Consejo
Nacionalde Ciencia y Tecnolog
´
ia(CONACYT),Mex-
ico.
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