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Molecular Microbiology (2009) 72(4), 931946

doi:10.1111/j.1365-2958.2009.06695.x
First published online 29 April 2009

Biosynthesis of the red pigment bikaverin in Fusarium


fujikuroi: genes, their function and regulation
Philipp Wiemann,1,2 Anita Willmann,1
Marcus Straeten,1 Karin Kleigrewe,1,2 Marita Beyer,2
Hans-Ulrich Humpf2 and Bettina Tudzynski1*
1
Institut fr Botanik, Schlossgarten 3, and 2Institut fr
Lebensmittelchemie, Corrensstr. 45, Westflische
Wilhelms-Universitt Mnster, D-48149 Mnster,
Germany.

Summary
Fusarium secondary metabolites are structurally
diverse, have a variety of activities and are generally
poorly understood biosynthetically. The F. fujikuroi
polyketide synthase gene bik1 was previously shown
to be responsible for formation of the mycelial
pigment bikaverin. Here we present the characterization of five genes adjacent to bik1 as encoding a
putative FAD-dependent monooxygenase (bik2), an
O-methyltransferase (bik3), an NmrA-like protein
(bik4), a Zn(II)2Cys6 transcription factor (bik5) and an
MFS transporter (bik6). Deletion of each gene resulted
in total loss or significant reduction of bikaverin
synthesis. Expression studies revealed that all bik
genes are repressed by high amounts of nitrogen in
an AreA-independent manner and are subject to a
time- and pH-dependent regulation. Deletion of the pH
regulatory gene pacC resulted in partial derepression
while complementation with a dominant active allele
resulted in repression of bik genes at acidic ambient
pH. Transcription of all bik genes in strains lacking
bik1, bik2 or bik3 was essentially eliminated, while
transcription of some bik genes was detected in
strains lacking bik4, bik5 or bik6. Thus, bikaverin synthesis is regulated by a complex regulatory network.
Understanding how different factors influence the
synthesis of this model secondary metabolite will aid
understanding secondary metabolism in general.

Introduction
The rice pathogen Fusarium fujikuroi belongs to the Gibberella fujikuroi species complex, which contains 11 difAccepted 4 April, 2009. *For correspondence. E-mail tudzynsb@
uni-muenster.de, bettina.tudzynski@uni-muenster.de; Tel. (+49) 251
83224801; Fax (+49) 251 83221601.

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Journal compilation 2009 Blackwell Publishing Ltd

ferent biologically distinct species (or mating populations)


consisting of 29 closely related Fusarium species. This
entire group is referred to as Fusarium section Liseola
(Nirenberg and ODonnell, 1998; Leslie et al., 2004).
Section Liseola strains have been isolated from a variety
of different host plants and have broad host range
specificity. They may produce one or more of a range
of secondary metabolites, such as gibberellins (GAs)
(reviewed in Tudzynski, 2005), the red pigment bikaverin
and its minor coproduct nor-bikaverin (Balan et al., 1970;
Kjr et al., 1971; Linnemannstns et al., 2002; Bell et al.,
2003), and several mycotoxins such as moniliformin,
beauvericin (Fotso et al., 2002), fumonisins (Proctor
et al., 2004), fusaric acid (Bacon et al., 1996) and fusarin
C (Song et al., 2004). Although the functions of most
secondary metabolites are unknown, it is generally recognized that pigmented materials likely protect fungi from
exposure to environmental stress like UV light (Medentsev et al., 2005). Bikaverin has also been found to affect
a wide range of biological processes. Early work in the
1970s described bikaverin as a fungal vacuolation factor
as it induced morphological changes similar to senescence (Cornforth et al., 1971). It has also been shown to
act as an antibiotic against a variety of organisms, including the protozoon Leishmania brasiliensis, the oomycete
Phytophtora infestans and the nematode Bursaphelenchus xylophilus (Balan et al., 1970; Kwon et al., 2007;
Son et al., 2008). This antigrowth function also extends to
mammalian cell lines as it has been reported to be cytotoxic to Ehrlich ascites carcinoma, lymphoma L5178Y,
sarcoma 37, lymphoadenoma NK/LY, sentinel pancreatic
cancer cell line MIA Pa Ca-2 and HeLa cells (Fuska et al.,
1975; Zhan et al., 2007). These effects are most likely due
to inhibition of ATP synthesis by uncoupling oxidative
phosphorylation (Henderson et al., 1977; Kovc et al.,
1978).
Polyketides constitute the largest and most diverse
group of Fusarium metabolites. Recently, the genomes of
four Fusarium species; F. graminearum, F. oxysporum,
F. solani and F. verticillioides, a close relative of
F. fujikuroi, have been sequenced. Analysis of the
sequence data has revealed that they each contain 1316
polyketide synthase (PKS) genes of which only a few are
functionally conserved between the four of them (Kroken
et al., 2003; Gaffoor et al., 2005; D. Brown, unpubl.

932 P. Wiemann et al.

Fig. 1. Bikaverin biosynthesis in Fusarium


fujikuroi.
A. Structures of nor-bikaverin and bikaverin
produced by the wild-type strain IMI58289.
Structure identification was accomplished as
described in the text. For NMR spectra see
Supporting information.
B. The bikaverin gene cluster in F. fujikuroi
strain IMI58289. Direction of transcription for
each predicted gene is indicted by the arrows.
Intron positions are indicated as white bars.

results). So far, the PKSs for fumonisin (Proctor et al.,


1999), bikaverin (Linnemannstns et al., 2002), aurofusarin (Kim et al., 2005a), fusarin C (Song et al., 2004),
the purple and red perithecial pigments (Graziani et al.,
2004; Proctor et al., 2007), as well as the two adjacent
PKSs for zearalenone (Kim et al., 2005b), have been
functionally characterized. In some cases, e.g. fumonisins
(Proctor et al., 2003) and aurofusarin (Frandsen et al.,
2006), the PKSs are located in clusters of tightly linked
genes all involved in the biosynthesis of the polyketide
product. Little work to date has examined the transcriptional regulation of these gene clusters. For most other
PKSs, the involvement of adjacent genes in metabolite
synthesis needs to be determined.
In the current study, we define the extent of the bikaverin
gene cluster by characterizing five genes located adjacent
to the bikaverin PKS-encoding gene (bik1). The five new
genes encode a putative FAD-dependent monooxygenase
(bik2), an O-methyltransferase (bik3), a putative NmrA-like
transcriptional regulator (bik4), a Zn(II)2Cys6 fungal type
transcription factor (bik5) and an MFS-type transporter
(bik6). Targeted gene disruption of the five new genes
confirmed a role in the synthesis of bikaverin. We also
examined the regulation of all six bik genes and found that
their expression is influenced by nitrogen, pH and time as
well as the presence of the other bik genes, their transcripts
or protein, but most likely not by their chemical products. In
the first case, bik gene expression is strictly repressed by
nitrogen in an AreA-independent manner. Second, we
found that the pH-related transcription factor PacC acts to
repress bik gene expression, and third, bik genes are
subject to a time-dependent regulation. In the last and most
surprising case, we found in single gene deletion-strains
that the expression of the remaining bik genes was significantly downregulated by an unknown mechanism. In addition, we describe the first in vivo production, purification
and structural identification of pre-bikaverin (3,8,10,11tetrahydroxy-1-methyl-12H-benzo[b]xanthen-12-one) as
the first stable intermediate of the bikaverin pathway and
product of the PKS enzyme Bik1.

Results
Identification of the bikaverin gene cluster
Previously, we identified and characterized the PKSencoding gene involved in synthesis of bikaverin and
nor-bikaverin (Fig. 1A) as bik1 (formerly pks4) (Linnemannstns et al., 2002). In order to identify new bikaverin
biosynthetic genes, genetic sequence was obtained in
both directions by chromosome walking. Sequence analysis of a 15 kb region upstream of bik1 revealed five new
genes with predicted amino acid sequences that share
significant similarity to previously characterized proteins in
the data bases that are consistent with bikaverin synthesis
(Fig. 1B; Table 1). Three of the genes, bik2, bik3 and bik6,
appear to encode structural proteins involved in synthesis
or transport of bikaverin as they share similarity to FADdependent monooxygenases, O-methyltransferases and
efflux pumps of the major facilitator superfamily (MFS)
respectively. The remaining two genes, bik4 and bik5,
appear to be involved in some aspect of regulation as they
encode proteins with similarity to the NmrA family of regulators (bik4) and to GAL4-like transcription factors carrying
a Zn(II)2Cys6 binuclear cluster DNA-binding domain (bik5)
(Table 1). In F. fujikuroi, a homologue of NmrA from
Aspergillus nidulans, Nmr, was shown to affect nitrogen
regulation by directly binding AreA (Schnig et al., 2008).
In contrast, fungal Zn(II)2Cys6 transcription factors affect
transcription by binding to specific short DNA sequences in
a genes promoter. The predicted functions of the gene
upstream of bik6, an alkaline serine protease, and the gene
downstream from bik1, a putative subtilisin-like peptidase,
are not consistent with bikaverin synthesis and thus are
probably not part of the bikaverin gene cluster.
bik gene expression is co-regulated by nitrogen and pH
We previously showed that bik1 gene transcription and
concomitant bikaverin synthesis is repressed by high
amounts of nitrogen and alkaline pH (Linnemannstns
et al., 2002). Northern analysis indicated that transcripts

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Journal compilation 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 931946

326
749
485

453

489

0
2
5
981
2364
1847
bik4 (AM696287)
bik5 (AM696286)
bik6 (AM696285)

2
1464
bik3 (AM229667)

2
bik2 (AM229668)

1578

IPR001509 NmrA-like protein


IPR001138 Fungal-specific Zn(II)2Cys6 transcription factor
IPR007114 Major facilitator superfamily transporter

Monooxygenase introducing hydroxy groups at C6 and C7 of


pre-bikaverin
O-methyltransferase methylating hydroxy groups at C3 and C8
of pre-bikaverin
Transcription factor enhancer
Positive acting, pathway-specific transcription factor
Efflux pump

2009
Polyketide synthase condensing one acetyl- and eight
malonyl-CoA units to form pre-bikaverin

IPR000794 b-ketoacyl synthase


IPR001031 Thioesterase
IPR001227 Acyl transferase region
IPR006162 Phosphopanthein-binding
IPR002938 Aromatic ring-hydroxylase
IPR003042 FAD-dependent monooxygenase
IPR001077 O-methyltransferase, family 2
3
bik1 (AJ278141)

6270

Domains and motifs


Intron
number
Length
(bp)
Gene name
(accession number)

Table 1. The bikaverin biosynthetic genes and their predicted functions in Fusarium fujikuroi.

Predicted function

Amino
acids

Regulation of bikaverin biosynthesis in F. fujikuroi 933

Fig. 2. Co-regulation of bik cluster genes under different nitrogen


and pH conditions. Wild-type strain IMI58289 was grown in 10% ICI
(glutamine) medium for 3 days before harvesting. The washed
mycelia were then shifted into 0% ICI media or 100% ICI
(glutamine) media, adjusted to pH 4 or pH 8. The mycelia were
harvested 2 h after shifting. The Northern blot was hybridized with
the indicated probes.

corresponding to the five new putative bikaverin genes


described above were almost totally repressed by
glutamine (except for bik4) and at alkaline ambient pH
and highly expressed under nitrogen starvation and acidic
pH conditions (Fig. 2). Transcripts corresponding to the
gene upstream from bik6 and downstream from bik1 were
not detected in any of these conditions (data not shown).
In contrast to the bik genes, the expression of the GA
biosynthesis genes, cps/ks and ggs2, encoding the entcopalyl diphosphate/ent-kaurene synthase CPS/KS and
the geranylgeranyl diphosphate synthase GGS2, respectively, were repressed by nitrogen, but were not affected
by ambient pH (Fig. 2) (Tudzynski and Hlter, 1998).
Targeted gene replacement of the new bik genes and
bikaverin analysis
To examine the effect of each gene deletion on bikaverin
production, we individually deleted all five by exchanging
their coding sequence with a selectable marker gene
(Fig. S1A, and data not shown). As predicted, deletion

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934 P. Wiemann et al.

dards for bikaverin and nor-bikaverin, both compounds


were isolated from cultures of the wild-type by preparative
HPLC and identified by mass spectrometry (MS) and
nuclear magnetic resonance (NMR) spectroscopy. This is
the first publication of 13C-NMR data for nor-bikaverin (see
Supporting information). The visual assessment was then
confirmed by HPLC-DAD, demonstrating that the Dbik1,
Dbik2 and Dbik3 mutants produced no bikaverin and norbikaverin, the Dbik5 mutant produced only a trace
amount, while the Dbik4 and Dbik6 mutants produced
significantly less bikaverin and nor-bikaverin as compared
with the wild-type (Fig. 3). These results are consistent
with our hypothesis that bik1, bik2, bik3 encode enzymes
that catalyse biosynthetic reactions in the bikaverin
pathway while bik4 and bik5 encode proteins that affect
the amount of bikaverin synthesized.
Temporal expression of the bik genes

Fig. 3. Bikaverin production by bik deletion mutants. Single


deletion mutants for bik1, bik2, bik3, bik4, bik5 and bik6 and the
wild-type strain IMI58289 were grown for 10 days in 10% ICI
medium. The pigmentation of cultures was documented by
photography. HPLC-DAD analyses of bikaverin (2) and
nor-bikaverin (1) production was carried out as indicated in the text.

mutants were all significantly affected in their phenotype


when grown in liquid media. Specifically, Dbik1, Dbik2 and
Dbik3 mutants totally lost their ability to produce pigmented mycelia, whereas Dbik4, Dbik5 and Dbik6 mutants
produced significantly less pigment as compared with the
wild-type strain IMI58289 (Fig. 3). In order to obtain stan-

In preliminary experiments, we observed that the bik genes


are weakly expressed at 120 h of incubation in ICI medium
in contrast to the GA genes, which are highly expressed at
this time point. To further explore changes in bik gene
expression over time, we grew the wild-type in 10% ICI
medium with low amounts of nitrogen (0.6 mM NH4NO3)
and examined the expression of all six bik genes and
accumulation of bikaverin over 10 days. Bikaverin production was first observed at 24 h and pigmentation intensified
over the next 2 days. Although expression of all six bik
genes essentially peaked after 48 h, there were differences in overall quantity as well as pattern of expression
(Fig. 4). The expression pattern and transcript level of the
two new predicted structural genes bik2 and bik3 were
almost identical to that observed for bik1. Although bik6
shared a similar expression pattern, it appeared significantly reduced in quantity in comparison. Expression of the
two putative regulatory genes, bik4 and bik5 also peaked
after 48 h, but was detected over the entire 10 days.
We also examined the expression of two GA biosynthesis genes by hybridizing the same filter with probes corresponding to cps/ks and ggs2. The expression of both
genes was first detected at 24 h, albeit significant lower
than most bik genes, and then, in contrast to the bik
genes, expression increased out to 10 days of cultivation
(Fig. 4).
Is AreA involved in nitrogen repression of bikaverin
biosynthesis?
As both the GA and the bik genes are repressed by high
amounts of nitrogen, we expected a similar AreAdependent regulation of bik genes as previously observed
for the GA biosynthetic genes (Tudzynski et al., 1999;
Mihlan et al., 2003). Examination of the promoter regions

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Journal compilation 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 931946

Regulation of bikaverin biosynthesis in F. fujikuroi 935

ICI (NH4NO3) medium. At this time point, almost no


expression of bik1, bik4 and bik6 genes was observed in
the Dbik5 mutant, while the expression of bik2 and bik3
was significant and similar to the wild-type (Fig. 6A). In
contrast, in the Dbik4 mutant, almost no expression of
bik1, bik2 and bik3 was observed while bik6 expression
was similar to the wild-type and bik5 expression appeared
reduced compared with the wild-type.
We also studied the expression of the bik genes in the
Dbik1, Dbik2, Dbik3 and Dbik6 mutants. Surprisingly, deletion of bik1 bik2, or bik3 or deletion of both bik2 and bik3
(DDbik2/3 double mutant) led to the strict downregulation
of all remaining cluster genes. In contrast, deletion of bik6
led to the loss of bik1, bik2 and bik3 expression, while bik4
and bik5 expression appeared greater than in the wildtype (Fig. 6A).
Due to the downregulation of the cluster genes by deletion of a structural gene (bik1, bik2 or bik3), it was not
possible to identify any intermediate of the bikaverin
pathway in the Dbik2, Dbik3 or DDbik2/3 mutant
respectively. To examine if an overexpression of bik5
would restore expression of the remaining bik genes, we

Fig. 4. Expression of bik genes over time. Wild-type strain


IMI58289 was grown in 10% ICI (NH4NO3) medium. Samples were
taken after 24, 48, 72, 120 and 240 h. The Northern blot was
hybridized with probes as indicated.

of all bik genes revealed multiple putative AreA binding


sites (Table S1), but only a few promoters contained two
sites located not more than 3040 nt apart from each
other, which is considered optimal for AreA binding
(Marzluf, 1997). In order to determine if AreA positively
regulated the expression of bik genes in a similar way as
the GA genes, we examined their expression in an DareA
mutant. We compared the expression of the bik genes in
the wild-type with that in the DareA mutant over 10 days of
growth in 10% ICI medium with glutamine (0.6 mM) as a
nitrogen source (the DareA mutant does not grow on
NH4NO3). We found that the overall expression of all bik
genes was higher, peaking at 48 h, then in the wild-type.
After this point, transcript levels decreased quickly to
levels lower than the wild-type. This was in contrast to the
dependency of the expression of the GA genes cps/ks
and ggs2 on AreA (Fig. 5).
Transcriptional analysis of bik deletion strains
We studied the expression of all six bik genes in strains
deficient in each individual gene as well as the DDbik2/3
double mutant after 3 days of cultivation in synthetic 10%

Fig. 5. Expression of bik genes in the wild-type compared with


the DareA mutant over time. The wild-type strain IMI58289 and the
DareA mutant were grown in 10% ICI (0.6 mM glutamine) medium.
Samples were taken after 24, 48, 72, 120 and 240 h. The Northern
blot was hybridized with the indicated probes.

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936 P. Wiemann et al.

Fig. 6. Influence of bik gene deletions on


expression of remaining bik genes.
A. The wild-type strain IMI58289, the single
bik gene deletion mutants (Dbik1-Dbik6) and
double mutant (DDbik2/3) were grown for
3 days in 10% ICI (NH4NO3) medium. Each
blot was hybridized with the indicated probes.
B. The wild-type strain IMI58289, the single
bik gene deletion mutants (Dbik2 and Dbik5)
and the bik5 overexpression mutants
(Dbik2+OE::bik5 and WT+OE::bik5) were
grown for 3 days in 10% ICI (NH4NO3)
medium. The Northern blot was hybridized
with the indicated probes.

transformed the Dbik2 mutant with a vector containing


bik5 under control of the constitutive promoter of the
F. fujikuroi glnA gene encoding the glutamine synthetase
(Teichert et al., 2004). Although the Dbik2+OE::bik5
mutant generated significantly more bik5 transcript than
the parent strain, neither bik1 transcription (Fig. 6B) nor
bikaverin or intermediates were detected. To examine if
overexpression of bik5 had any effect on expression of
the other bik genes in the wild-type, we transformed the
same vector also into wild-type strain IMI58289. In this
case, overexpression of bik5 led to a much stronger
expression of bik5 than in the Dbik2 background, suggesting an impact of bik2 deletion on expression of bik5.
Again, increased expression of bik5 did not result in
upregulation of bik1, as compared with the wild-type
(Fig. 6B).

course of this work, through the overexpression of the


F. fujikuroi pks4 (bik1) gene in Escherichia coli (Ma et al.,
2007). Our work is the first identification of pre-bikaverin
production in vivo and supports the hypothesis that it is an
authentic bikaverin intermediate.
In order to further explore the effect bik1 overexpression may have on other bik genes, we also transformed
the individual Dbik2 and Dbik3 mutants with the bik1 overexpression vector, yielding the strains Dbik2+OE::bik1
and Dbik3+OE::bik1 respectively. As observed with the
DDbik2/3 double mutant, transformants only showed
increased bik1 transcription and concomitant prebikaverin accumulation, but no additional transcription of
the other structural gene (e.g. bik3 in the Dbik2+OE::bik1
mutant or bik2 in the Dbik3+OE::bik1 mutant) (Fig. S2).
Addback of bik1 in the Dbik1 mutant

In vivo identification of the first stable intermediate in


the bikaverin pathway
To facilitate the identification of the chemical product of
Bik1, we overexpressed bik1 in the DDbik2/3 double
mutant using the constitutive glnA promoter. This strategy
was employed to overcome the bik1 downregulation
and to allow the chemical intermediate of Bik1 to accumulate to detectable levels for analysis without further
modifications. Transformants (DDbik2/3+OE::bik1) with
the correct integration of the vector displayed a different
color when grown in 10% ICI media (Fig. 7A). Although
transcriptional analysis indicated that the transformants
accumulated significantly more bik1 transcripts, as compared with the wild-type and the parent strain, expression
of bik4, bik5 or bik6 was not restored (Fig. 7B). Analysis of
culture filtrates identified a metabolite with MS and NMR
spectra consistent with the chemical SMA76a, which
we refer to as pre-bikaverin (Fig. 7C and Supporting information). SMA76a was first identified, during the

To examine if the downregulation of the cluster genes by


the deletion of a single bik gene is due to a perturbation of
the chromatin structure through the integration of the
vector constructs, we transformed the Dbik1 mutant with a
vector containing bik1 under the control of the constitutive
glnA promoter. The accumulation of significant red pigmentation in some transformants (Fig. 7A) immediately
suggested that the construct was functioning as intended.
PCR verified that these transformants (bik1 C) contained
the overexpression construct (data not shown). The
expression of all bik genes in the bik1 addback strains
was then examined under different pH conditions. In contrast to the DDbik2/3+OE::bik1 mutants, the complemented Dbik1 transformants accumulated transcripts for
all six bik genes at pH 4. Interestingly, bik1 expression
was significantly higher under both pH conditions as compared with the wild-type. The deregulation at pH 8 is probably due to the exchange of the bik1 promoter for the
pH-independent glnA promoter (Fig. 7B and D).

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Journal compilation 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 931946

Regulation of bikaverin biosynthesis in F. fujikuroi 937

Fig. 7. Identification of pre-bikaverin as the


product of Bik1 and bik gene expressions
after addback of bik1.
A. Photography of cultures of the wild-type
IMI58289, the DDbik2/3, DDbik2/3+OE::bik1,
Dbik1 and bik1 C mutants after 10 days growth
in 10% ICI (NH4NO3) medium, pH 4.
B. Expression of bik genes in the wild-type,
the DDbik2/3 mutant and the bik1
overexpression mutant DDbik2/3+OE::bik1.
The strains were grown for 3 days in 10% ICI
(NH4NO3) medium, pH 4. The Northern blot
was hybridized with probes as indicated. 28S
and 18S rRNA was visualized by EtBr staining
as control.
C. Structure of pre-bikaverin produced by the
bik1 overexpression mutant
DDbik2/3+OE::bik1. Structure identification
was accomplished as described in the text.
For NMR spectra see Supporting information.
D. The wild-type and the bik1 addback strain
bik1 C were grown in 10% ICI (NH4NO3)
medium for 3 days before harvesting. The
washed mycelia were then shifted into 0% ICI
media adjusted to pH 4 or pH 8. The mycelia
were harvested 2 h after shifting. Each
Northern blot was hybridized with the
indicated probes.

The pH-dependent regulation of bik genes: is PacC


involved?
A major component of the pH regulatory system in filamentous fungi is PacC, a 72 kDa Cys2His2 zinc finger transcription factor. Seminal work in A. nidulans showed that PacC
undergoes two proteolytic processing steps in response to
alkaline ambient pH. The first, signalling protease cleavage of PacC removes a processing-inhibitory C-terminal
domain, making its truncated PacC product accessible to a
second processing protease, yielding a 27 kDa truncated
active PacC (reviewed in Pealva et al., 2008).
Although the bik genes are clearly repressed at alkaline
pH (Fig. 2), the expression of bik1 at alkaline pH, when its
natural promoter was exchanged for the constitutive glnA
promoter, suggests that alkaline pH directly affects bik1
transcription (Fig. 7D). The findings of three potential
PacC binding motifs (5-GCCARG-3; Espeso et al., 1997)
in the promoter region of bik1 and none in the glnA promoter region (Table S1) suggested that PacC may be
involved, albeit in a non-typical manner. To examine
whether PacC might play a role in bik gene regulation

despite the opposite pH optimum, we cloned and deleted


the pacC gene (Fig. S1B). The F. fujikuroi pacC gene
encodes a 618-amino-acid protein (accession AJ514259),
which shares 94% identity with the PacCs from
both F. verticillioides (AY216461) and F. oxysporum
(AY125958). As expected, F. fujikuroi DpacC strains
behaved similarly to DpacC mutants described for the
other Fusarium (Caracuel et al., 2003a,b; Flaherty et al.,
2003), and displayed a reduced growth rate on synthetic
medium (SM) buffered to pH 4 and pH 6. On SM buffered
to pH 8, there was severely restricted growth of the
mutant (Fig. 8A), indicating that PacC is required for
growth in alkaline pH media. We also created pacC 246
gain-of-function mutants by complementing the DpacC
mutant with a truncated version of the gene, refered to as
pacC 246, which yields a 246 aa (27 kDa) active PacC
protein. As expected, the transformants examined were
restored in their ability to grow on alkaline media, but
showed a severe growth defect at acidic pH (Fig. 8A).
Growth experiments with the DpacC strains in liquid
culture (10% ICI medium, pH 4) led to the production of
significantly more bikaverin than in the wild-type. In

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938 P. Wiemann et al.

Fig. 8. Influence of PacC on growth,


pigmentation and bik gene expressions.
A. Comparison of growth of pacC mutants
(deletion: DpacC and constitutive: pacC246)
to the wild-type strain IMI58289 on synthetic
media (SM) adjusted to pH 4, pH 6 or pH 8
after 5 days.
B. Photography of cultures of the wild-type
strain IMI58289, the DpacC and pacC246
mutants after 3 days growth in 10% ICI
(NH4NO3) medium, pH 4.
C. Expression of bik1 in the wild-type, and the
DpacC and pacC246 mutants after 3 days
growth in 10% ICI (NH4NO3) medium, pH 4.
D. The wild-type, DpacC mutant and bik1
complementation strains (bik1 C and
bik1 CDPacProm) were grown in 10% ICI
(NH4NO3) medium for 3 days before
harvesting. The washed mycelia were then
shifted into 0% ICI media adjusted to pH 4 or
pH 8. The mycelia were harvested 2 h after
shifting. The Northern blot was hybridized with
the indicated probes as indicated.
E. The wild-type, DpacC and Dbik5 mutants
as well as the DDpacC/bik5 double knock-out
mutant were grown in 10% ICI (NH4NO3)
medium for 3 days before harvesting. The
washed mycelia were then shifted into 0% ICI
media, adjusted to pH 4 or pH 8. The mycelia
were harvested 2 h after shifting. Each
Northern blot was hybridized with the
indicated probes.

contrast, the pacC 246 mutants produced only small


amounts of bikaverin (Fig. 8B). Northern analysis of all six
bik genes in both strains were consistent with the level of
pigmentation observed. All six bik genes were significantly
expressed in the DpacC mutant, while, in contrast, they
were minimally expressed, as compared with the wildtype, in the pacC 246 mutant (Fig. 8C).
To examine the role of PacC in bik gene expression
more closely, we constructed a vector carrying the entire
bik1 gene under control of its natural promoter in which all
three potential PacC binding motifs (5-GCCARG-3) were
mutated or deleted. The vector was transformed into the
Dbik1 mutant yielding bik1 CDPacCProm mutants. The
expression of bik1 in the wild-type, the DpacC and Dbik1
mutants as well as in the bik1 addback strains (bik1 C and
bik1 CDPacCProm) was then examined under different pH

conditions. Interestingly, bik1 expression was more significant even at acidic pH in the DpacC and the
bik1 CDPacCProm strain, as compared with the wild-type
(Fig. 8D). At an alkaline pH no transcription of bik1 was
detected in the wild-type, whereas significant transcription
was detected in the pacC knock-out mutant and the bik1
addback strain bik1 C. In the bik1 CDPacCProm mutant an
even higher expression level has been obtained than in
the wild-type under acidic conditions (Fig. 8D).
To investigate whether the activating effect of the pacC
deletion on bik1 expression is more significant than the
downregulating effect of the bik5 deletion, we constructed
pacC and bik5 double deletion mutants (DDpacC/bik5)
and tested bik1 expression at different pH conditions.
Interestingly, bik1 expression in the double mutant
resembled the signal intensity of the bik5 single deletion

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Journal compilation 2009 Blackwell Publishing Ltd, Molecular Microbiology, 72, 931946

Regulation of bikaverin biosynthesis in F. fujikuroi 939

strain at acidic and alkaline pH conditions, indicating that


PacC is not involved in interdependent regulation of bik
gene expression (Fig. 8E).

Discussion
Many filamentous fungi produce red, green, bluish green or
black polyketide-derived pigments, which have been
studied extensively because of their taxonomic value, biological activity and in vivo function. Their often toxic nature
suggested to early researchers that they quelled competition and thus were antibiotics. Work since has shown that
some pigments play a protective role against environmental stresses such as irradiation and oxidation while others
contribute to virulence (reviewed in Duran et al., 2002). In
the genus Fusarium, numerous pigments have been characterized, including aurofusarin in F. graminearum
and F. culmorum, rubrofusarin in F. culmorum, bikaverin
in F. fujikuroi, F. verticillioides and F. oxysporum, fusarubin
in F. solani and two different perithecial pigments in
F. graminearum, F. verticillioides and F. solani (reviewed in
Medentsev and Akimenko, 1998; Duran et al., 2002;
Proctor et al., 2007). All of the known PKSs required for
fungal pigment production have a similar multidomain
organization and belong to the non-reducing class of PKSs
(Kroken et al., 2003; D. Brown, pers. comm.).
Previously, we identified and characterized the
F. fujikuroi PKS-encoding gene (bik1) that is essential for
bikaverin synthesis (Linnemannstns et al., 2002). In this
study, we define the extent of the bikaverin gene cluster.
We identified and characterized the five genes adjacent to
bik1, referred to as bik2bik6, and show, by gene deletion
and analysis of their predicted proteins, that they are
involved in either modifying the Bik1 product (bik2 and
bik3), transport (bik6) or regulation (bik4 and bik5). Gene
deletion and chemical analysis of extracts showed that
the two new structural genes bik2 and bik3 were absolutely required for bikaverin synthesis. Based on the similarity of Bik2 to FAD-dependent monooxygenases, we
propose that this protein is responsible for the oxidation of
C6 and C7 of pre-bikaverin. The similarity of Bik3 to
O-methyltransferases suggests that this protein is
involved in methylating the C3 and C8 hydroxyl groups
(Table 1). In contrast, Dbik6 mutants synthesize some
bikaverin. The similarity of Bik6 to MFS-type transporters
suggests that this protein transports bikaverin (and/or
pathway intermediates) across a membrane. Using the
transporter classification database (TCDB; http://www.
tcdb.org; Saier and Ren, 2006), Bik6 belongs in the DHA1
(Drug:H+ Antiporter-1) family based on the presence of 12
predicted transmembrane spanning domains and four
conserved motifs. The likely H+-antiporter nature of Bik6
parallels the acidic pH optimum observed for bikaverin
production. At acidic pH, H+ ions predominate in the

medium facilitating the transport of H+ ions into the cells in


exchange for bikaverin. The production of a metabolite
despite the loss of a cluster transporter type gene has
previously been observed. In F. verticillioides, deletion of
fum19, encoding a putative ABC transporter located in the
fumonisin gene cluster, did not affected the overall quantity of fumonisins produced (Proctor et al., 2003).
Our studies examining the regulation of bikaverin production proved to be very interesting. Northern analysis of
all six genes over time and in the DareA and DpacC
mutants showed that they are regulated in a similar
manner and is consistent with previous genome-wide
microarray studies (Brown et al., 2008). We assumed,
based on the similarity of bik4 and bik5 to previously
characterized transcriptional regulators, that they play a
role in regulating the transcription of the other cluster
genes. While Zn(II)2Cys6 transcription factors (Bik5) have
been shown to be involved in the regulation of numerous
fungal metabolites, the role or mode of action of NmrA-like
proteins (Bik4) in regulation is not yet known. Interestingly,
a gene encoding an NmrA-like protein is also present in
the Claviceps purpurea alkaloid gene cluster (N. Lorenz
and P. Tudzynski, pers. comm.). Based on the dramatic
reduction of bikaverin production by the Dbik4 and Dbik5
mutants (5 and 100 respectively), we propose that both
play an important, but not absolute role in bikaverin
synthesis. There is precedence in Fusarium for the production of a secondary metabolite in mutants lacking
putative regulatory genes located within the biosynthetic
gene cluster. Some fum gene transcripts as well as some
fumonisin was produced in Dfum21 mutants lacking the
fumonisin-specific Zn(II)2Cys6 transcription factor Fum21
(Brown et al., 2007). We are currently examining how and
why bik2 and bik3 are significantly transcribed in the Dbik5
strain while bik1, bik4 and bik6 transcription is essentially
eliminated.
Responding to nutrient and environmental signals:
nitrogen and pH
The synthesis of many secondary metabolites is regulated by environmental conditions, such as carbon and
nitrogen sources, pH and light (Keller et al., 1997). In
F. fujikuroi, both GA and bikaverin synthesis are
repressed by high nitrogen concentrations (Linnemannstns et al., 2002; Mihlan et al., 2003; Schnig
et al., 2008). As the GA genes are directly regulated by
binding the general transcription factor AreA (Mihlan et al.,
2003), we expected a similar process to be the case for
the bik genes. However, in contrast to the direct and major
role of AreA for GA gene expression, the bik genes seems
to be only indirectly affected by AreA. We found that the
presence of AreA is not essential for high expression
levels of the bikaverin biosynthesis genes despite the

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940 P. Wiemann et al.

presence of some potential GATA sequence elements in


the promoters of the bik genes. Evidence for an indirect
involvement of AreA centres around the observation that,
at 120 h of incubation, the expression of bik1, bik2 and
bik3 is already completely downregulated in the DareA
mutant in contrast to the wild-type.
In previous work, we were not aware of the temporal
manner of the expression of bik genes and ascribed
contrasting roles for AreA in bikaverin synthesis (Linnemannstns et al., 2002; Teichert et al., 2004; Schnig
et al., 2008). Thus, samples for genome-wide microarray
studies were taken from the wild-type and DareA mutant
at a time point of 120 h, leading to identification of bik
genes as AreA target genes (Schnig et al., 2008). In
the current study, we monitored expression over 10 days
and clearly demonstrate that the mechanism of the
strong nitrogen repression of bik genes differs from that
of the AreA-dependent GA gene expression. The possibility that AreA indirectly affects bik gene expression by
regulating the expression of the putative pathwayspecific transcription factor Bik5 can be excluded
because its expression is not at all affected in the DareA
mutant (see Fig. 5).
Regulation of gene expression by ambient pH is mediated via the Cys2His2 zinc finger transcription factor PacC
(Tilburn et al., 1995; Pealva et al., 2008). A direct role for
PacC in activating genes preferentially expressed at alkaline pH has been demonstrated for several genes encoding extracellular enzymes (Caddick et al., 1986), proteins
involved in cell wall biosynthesis (Fonzi, 1999), and in
production of secondary metabolites such as penicillin
and cephalosporin (Espeso et al., 1993; Schmitt et al.,
2001; Brakhage et al., 2004). A role for PacC as a repressor at alkaline pH has been demonstrated for the
g-aminobutyrate (GABA) permease-encoding gene in
A. nidulans (Espeso and Arst, 2000) and for the fumonisin
biosynthetic gene fum1 encoding a PKS involved at an
early step in the formation of fumonisins in F. verticillioides
(Flaherty et al., 2003). Previously, we showed that bik1
is only expressed at acidic pH (Linnemannstns et al.,
2002). In this study, we demonstrated that the five new bik
genes are co-regulated with bik1 and that they are all
strongly repressed at neutral and alkaline pH. The presence of PacC recognition sequences (5-GCCARG-3) in
the promoters of three of the bik genes (3 in the bik1
promoter and 1 each in the bik4 and bik5 promoters)
supported a role for PacC in bikaverin regulation. Our
finding that the DpacC mutant was even more pigmented
at acidic pH further supports our hypothesis, suggesting
that PacC acts as a repressor of bik gene expression and
bikaverin formation, similar to fumonisin production in
F. verticillioides (Flaherty et al., 2003). To confirm the
repressing effect of PacC, we complemented the DpacC
mutant with a dominant activating pacC 246 allele. In con-

trast to the DpacC mutant, transformants carrying the


pacC 246 allele were significantly reduced in pigment production and expression of the bik genes (Fig. 8B and C).
Furthermore, we found that either deletion of pacC, substitution of the natural bik1 promoter for a constitutive
promoter without PacC recognition sites or specific
mutation/deletion of these motifs in the natural bik1 promoter led to a significant bik1 transcription signal under
alkaline pH conditions (Fig. 7D and Fig. 8D). In addition,
the expression of bik genes was much stronger under
acidic conditions, suggesting that PacC acts as repressor
under all pH conditions (Fig. 8C). Interestingly, the
repressed bik1 expression in a DDpacC/bik5 double
knock-out mutant supports our theory that Bik5 is essential for bik1 expression and suggests an independent
mode of action from PacC (Fig. 8E).
Deletion of single bik genes affects the expression of
the other cluster genes
Surprisingly, the loss of any one of the biosynthetic genes
bik1, bik2 or bik3 fully abolished the expression of the
other bik genes except for bik5 which was downregulated.
In addition, the loss of bik4 and bik6 resulted in downregulation of bik1, bik2 and bik3. We explored three possibilities that could account for these observations. First,
the deletion of any cluster DNA may interfere with general
gene cluster regulation by perturbing chromatin structure.
Second, gene transcription may be influenced by a negative feedback process related to either other cluster gene
transcripts or by bikaverin (or pathway intermediates).
Finally, the transcription factor PacC might be involved in
feedback regulation of bik gene expression.
To address the first possibility, we complemented the
bik1 mutant with a plasmid carrying the bik1 gene fused
to the strong glnA promoter. The ectopic integration of
this construct led to the accumulation of bik1 transcripts,
restored the expression of all the other bik genes and,
most importantly, the production of bikaverin (see
Fig. 7). Therefore, changes in chromatin structure by
integrating replacement cassettes into the cluster locus
can be excluded as a reason for the transcriptional
interdependence.
The theory of a negative feedback process involving
pathway intermediates was first suggested in Cercospora
nicotianae to influence cercosporin gene transcription
(Chen et al., 2007). However, this possibility is very
unlikely for bik gene regulation as overexpression of bik1
in either single knock-out mutants (Dbik2 or Dbik3)
restored bik1 transcription, as expected, but not transcription of the remaining bik genes, respectively (Fig. S2),
despite the accumulation of high amounts of prebikaverin. In fact, we propose that the loss of bik gene
expression observed in the single gene knock-outs is

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Regulation of bikaverin biosynthesis in F. fujikuroi 941

likely due to negative feedback regulation related to the


loss of other cluster gene transcripts or gene products.
Addback of bik1 in Dbik1 strains led to transcriptional
restoration of all bik genes, but did not affect other bik
gene expression in Dbik2, Dbik3 and DDbik2/3 strains
(Fig. 7 and Fig. S2). Our observation that bik5 overexpression in bik2 strains did not enhance bik1 expression
supports this theory and indicates that Bik5 is not involved
in this feedback regulation (Fig. 6B).
To address the question if PacC might be involved in
feedback regulation, we generated double knock-out
mutants of bik5 and pacC (DDpacC/bik5). However, the
deletion of pacC in the Dbik5 background did not result in
any upregulation of bik1, demonstrating that the feedback
regulation of early cluster genes does not require PacC
(Fig. 8E).
It is important to note that the significant upregulation of
bik6 in the bik4 mutant, and bik4 and bik5 in the
bik6 mutant suggested that additional transcriptional interdependence of some genes by an as yet unknown
mechanism exists (Fig. 6A). It is possible that the characterization of these additional processes may shed light on
how the deletion of simple biosynthetic genes such as
bik1, bik2 or bik3 would have the significant impact on
transcription that we observe.

GA biosynthetic genes, the bikaverin genes are


repressed by nitrogen. However, in contrast to the GA
genes, AreA is not essential for the expression of the
bikaverin genes, suggesting a different mechanism of
nitrogen regulation. Second, we demonstrate that PacC
represses the expression of bik genes at alkaline and
even at acidic pH conditions. This is in contrast to
several other secondary metabolite genes from Aspergillus spp., which are preferentially expressed at alkaline
pH, and are repressed by PacC under acidic conditions.
Third, studying the transcript levels of bik genes over a
10-day period showed that the expression of bik genes
is time-dependent, having a maximum at 4872 h of cultivation. Forth, expression of some genes is dependent
on the presence of other cluster genes or their protein
products and likely involves a form of feedback inhibition. We found that chromatin restructuring, chemical
intermediates, Bik5 and PacC do not play a role in this
regulatory mechanism.
Understanding all of the factors that regulate polyketide
biosynthesis is a critical step towards controlling their
synthesis. Bikaverin biosynthesis is a worthwhile model
system for better understanding fungal secondary
metabolism, and the information we learn will be directly
applicable to other economically valuable materials.

First in vivo production of a bikaverin intermediate

Experimental procedures

An initial goal of our programme was to structurally characterize bikaverin pathway intermediates. Unfortunately,
the lack of bik1 transcription in the knock-out strains precluded this possibility. We overcame this problem by overexpressing bik1 under the control of the glnA promoter in
the DDbik2/3 mutant. This led to the isolation and characterization of pre-bikaverin as the product of the PKS and
the first intermediate of the pathway. NMR and MS analysis (see Supporting information) revealed that the structure of this compound is identical to SMA76a, which was
identified in cultures of E. coli expressing bik1 (Ma et al.,
2007). In addition, we characterized the structure of norbikaverin, which we isolated from extracts of the wild-type.
The significant accumulation of this material (33% relative
to bikaverin) suggests that the methylation of the C3
hydroxyl group is not absolutely required for function or
export. The possible existence of strains of F. fujikuroi that
produce different ratios of nor-bikaverin/bikaverin would
support the possibility that these two compounds function
differently.
In summary, we identified and characterized five new
genes, adjacent to bik1, as required for bikaverin synthesis. Targeted gene replacement of each led to the
loss or drastic reduction of bikaverin production. We
show that bik gene expression is complicated and regulated by several different principles. First, similar to the

Fungal strains and culture conditions


The wild-type strain F. fujikuroi IMI58289 (Commonwealth
Mycological Institute, Kew, UK) was used as the parent strain
for all knock-out experiments. The DareA mutant (Tudzynski
et al., 1999) and the Dbik1 (Dpks4) mutant (Linnemannstns
et al., 2002) have been described. For all cultures, F. fujikuroi
was pre-incubated for 48 h in 300 ml Erlenmeyer flasks with
100 ml Darken medium (DVK) (Darken et al., 1959) on a
rotary shaker. ICI (Imperial Chemical Industries Ltd, UK)
media (Geissman et al., 1996) containing 0.6 mM NH4NO3
(10% ICI medium) or 6 mM NH4NO3 (100% ICI medium) was
inoculated with 0.5 ml of the DVK starter culture. For experiments with the DareA mutant, glutamine was used as nitrogen source instead of (NH4)2SO4 (DVK) or NH4NO3 (ICI
medium) respectively. For protoplasting, 0.5 ml of the starter
culture was transferred into Erlenmeyer flasks with 100 ml
complete medium (CM) (Pontecorvo et al., 1953) and incubated at 28C on a rotary shaker at 190 r.p.m. for 18 h. For
analysis of bikaverin and intermediates, the fungus was
grown for 10 days at 28C on a rotary shaker (190 r.p.m.) in
10% ICI. For RNA isolation, the fungal strains were grown in
10% ICI medium with NH4NO3 or with glutamine for 1, 2, 3, 5
or 10 days on a rotary shaker at 28C. For shift experiments,
the mycelia were grown for 3 days in 10% ICI medium and
then transferred into ICI medium without nitrogen (0% ICI)
or 6 mM NH4NO3 (100% ICI) media adjusted to pH 4 or
pH 8. Mycelia were harvested after 2 h. For plate assays, SM
(Caracuel et al., 2003b) adjusted to different pH values was
used.

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942 P. Wiemann et al.

Bacterial strains and plasmids


Escherichia coli strain Top10 (Invitrogen, Groningen, the
Netherlands) was used for plasmid propagation. F. fujikuroi
bik genes or gene fragments were cloned into the vector
pUC19 (Fermentas, Germany). Each gene deletion vector
was created essentially by cloning the two ~0.8 kb DNA fragment flanking each side of the target gene into a vector
containing the hygromycin (pGPC1; Desjardins et al., 1992)
or nourseothricin (pNR1; Malonek et al. 2004) resistance
markers such that the resistance gene was flanked by the
Fusarium DNA. All DNA fragments were amplified by PCR
using the primers indicated (Table S2) and cloned into the
pCR2.1-TOPO vector (Invitrogen) and sequenced. Many of
the primers introduced restriction sites (underlined), which
were used in subsequent cloning steps.
The bik2 deletion vector was created by cloning the 0.8 kb
5 flank (primers MO-GR1-SacI and MO-GR2-KpnI) and the
0.9 kb 3 flank (primers MO-MT-GR3-XbaI and MO-MT-GR4HindIII) into pGPC1 creating pDbik2. The bik3 deletion vector
was created by cloning a 0.9 kb SacI/KpnI fragment (primers
MT-GR1-SacI and MT-GR2-KpnI) and a 0.8 kb HindIII/XbaI
fragment (primers MT-GR3-XbaI and MT-GR4-HindIII) into
pGPC1 to give pDbik3. For the bik2/3 double knock-out, the
KpnI/SacI fragment of vector pDbik3 was exchanged for the
HindIII/XbaI fragment of pDbik2 revealing vector pDbik2/bik3.
The bik4, bik5 and bik6 deletion vectors included the
nourseothricin resistance cassette. The bik4 deletion vector
was created by cloning the 0.71 kb 5 flank (primers LF4F and
LF4R) and the 0.75 kb 3 flank (primers RF4F and RF4R) into
pNR1 yielding pDbik4. The bik5 deletion vector was created
by cloning the 0.75 kb 5 flank (primers LF5F and LF5R) and
the 1.3 kb 3 flank (primers RF5F and RF5R) into pNR1
yielding pDbik5. The bik6 deletion vector was created by
cloning a 0.79 kb 5 flank (primers LF6F and LF6R) and a
0.69 kb 3 flank (primers RF6F and RF6R) into pNR1 yielding
pDbik6.
The pacC deletion vector was created by cloning 1 kb 5
flank (primers PACC-GR1 and PACC-GR2) and 1.1 kb 3
flank (primers PACC-GR3 and PACC-GR4) into pGPC1 creating pDpacC. The constitutive on pacC mutant gene vector
was creating by cloning a 1.9 kb SacI/EcoRI amplicon
(primers PACC-GR1 and PACC-246-R1) and a 1 kb EcoRI/
XbaI amplicon (primers PACC-Ter-F1 and PACC-GR3) simultaneously into SacI/XbaI linearized pNR1 yielding pPacC246.
The 1.9 kb fragment included 1 kb upstream of the predicted
pacC start codon (e.g. the pacC promoter) and 896 bp of
predicted coding sequence. A stop codon was introduced
after residue Val246 by the primer PACC-246-R1. The 1 kb
amplicon was entirely downstream of the original predicted
pacC stop codon and was predicted to include the natural
terminator sequence.
The bik1 overexpression vector pglnAprom::bik1-1 was constructed by replacing the endogenous bik1 promoter with the
glnA promoter (Teichert et al., 2004) in plasmid pPKS4 (Linnemannstns et al., 2002). The glnA promoter was first
amplified using primers glnA-prom-KpnI and glnA-prom-SphI.
For construction of the bik1 complementation vector
pbik1DPacCProm two amplicons of the endogenous bik1 promoter were created using primers introducing mutations
(Table S2) at putative PacC binding regions. A 0.33 kb SphI/

ClaI amplicon (primers bik1-DP-SphI-F1 and bik1-DP-R1)


and a 0.44 kb KpnI/SphI amplicon (primers bik1-DP-F2 and
bik1-DP-SphI-R2) were cloned simultaneously into pPKS4
that was previously KpnI/ClaI digested, thereby releasing the
endogenous bik1 promoter. The bik1 promoter replacement
vector pglnAprom::bik1-2 was constructed by cloning the 1 kb
N-terminal portion of bik1(primers bik1-F1-SalI and bik1-R1ApaI) adjacent to the glnA promoter in pUCH-N-glnA. pUCHN-glnA was created by first cloning the nourseothricin
resistance cassette from vector pNR1 into the XbaI/HindIII
sites of pUCH2-8 (Alexander et al., 1999) creating pUCH28-N. pUCH2-8-N was then digested with HindIII and SalI
(thereby releasing the 2.5 kb hygromycin cassette) and
ligated to the HindIII- and SalI-digested 1 kb glnA promoter
fragment (primers GS-prom-HindIII and GS-prom-SalI)
creating pUCH-N-glnA. The bik5 overexpression vector
pglnAProm::bik5 was constructed by cloning the bik5 gene
(primers bik5-F-SalI and bik5-R-XhoI) adjacent to the glnA
promoter in pUCH2-8-N.

Screening of genomic library for bik1 and pacC


About 40 000 phages of the F. fujikuroi IMI58289 genomic
library (Malonek et al., 2004) were plated with E. coli strain
Xl1-blue MRF and screened by plaque hybridization essentially as described (Sambrook et al., 1989). Plaque lifts (Gene
Screen nylon membranes, DuPont, Germany) were hybridized with a [32P]dCTP-labelled 1.5 kb PCR fragment of the
F. fujikuroi bik1 and a 1 kb PCR fragment of the F. fujikuroi
pacC genes. Hybridization and washing steps were performed as described (Teichert et al., 2004). Putative positive
phages were purified in a second screening round. Phage
DNA was isolated as described by Sambrook et al. (1989)
and used for restriction analysis and subcloning.

Standard molecular methods


Lyophilized mycelium was ground into a fine powder with a
mortar and pestle and dispersed (in the case of DNA for use
in PCR) in extraction buffer as described by Cenis (1992).
DNA for Southern hybridization experiments was prepared
following the protocol of Doyle and Doyle (1990). Plasmid
DNA was extracted using the Genomed plasmid extraction kit
(Genomed, Germany). For Southern blot analysis, genomic,
plasmid or phage DNA was digested with the indicated
restriction enzymes (Fermentas, Germany), fractionated in
1% (w/v) agarose gels, and transferred to nylon N+ membranes (Amersham, Germany) by downward blotting
(Ausubel et al., 1987). 32P-labelled probes were prepared
using the random oligomer-primer method (Sambrook et al.,
1989). Membranes were hybridized in 5 Denhardts solution
containing 5% dextran sulphate and washed at the same
temperature used for hybridization in 2 SSPE (1 SSPE is
0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA, pH 7.7),
0.1% SDS and 1 SSPE, 0.1% SDS. Total F. fujikuroi RNA
was isolated using the RNAgents total RNA isolation kit
(Promega, Germany). Samples of 15 mg of total RNA were
transferred to Hybond-N+ membranes after electrophoresis
on a 1% (w/v) agarose gel containing formaldehyde, according to Sambrook et al. (1989). Northern blot hybridizations

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Regulation of bikaverin biosynthesis in F. fujikuroi 943

were accomplished by the method of Church and Gilbert


(1984). For cDNA synthesis 1 mg of total RNA was taken
using the oligo(dT)1218 primer and the SuperScript II
reverse transcriptase (Invitrogen) according to the manufacturers instructions.
PCR reactions contained 25 ng DNA, 5 pmol of each
primer, 200 nM concentrations of desoxynucleotide triphosphates, and 1 unit of BioThermTMDNA polymerase (GeneCraft
GmbH, Ldinghausen, Germany). The reactions started with
4 min at 94C, followed by 35 cycles of 1 min at 94C, 1 min
at 5665C, 1 min at 70C, and a final 10 min at 70C. PCR
products were cloned into pCR2.1-TOPO (Invitrogen, Groningen, the Netherlands).
Subclones were sequenced on both strands with a LI-COR
4000 instrument (MWG, Munich, Germany) according the
manufacturers directions using standard plasmid-specific
primers and sequence-specific primers obtained from MWG
Biotech (Munich, Germany). DNA and protein sequence
alignments were done with DNA STAR (Madison, WI, USA).
Sequence homology searches were performed using the
NCBI database server. Protein homology was based on
BlastX searches (Altschul et al., 1990).

Fungal transformations
Preparation of protoplasts from F. fujikuroi mycelium was
carried out as described (Tudzynski et al., 1999). Approximately 107 protoplasts of strains IMI58289 were transformed
with 10 mg of the replacement cassettes of the vectors
pDbik2, pDbik3, pDDbik2/bik3, pDbik4, pDbik5, pDbik6 and
pDpacC respectively. To generate the DDpacC/bik5 double
mutant 107 protoplasts of the Dbik5 mutant were transformed
with 10 mg of the replacement cassettes of the vector
pDpacC. For gene replacement, transformed protoplasts
were regenerated at 28C in a complete regeneration agar
(0.7 M sucrose, 0.05% yeast extract, 0.1% casaminoacids)
containing 120 mg ml-1 hygromycin B (for pDbik2, pDbik3,
pDDbik2/bik3 and pDpacC) (Calbiochem, Germany),
100 mg ml-1 nourseothricin (Werner-Bioagents, Germany) (for
pDbik4pDbik6) or both antibiotics (for selection of DDpacC/
bik5 double mutants) for 67 days.
For complementation of bik1, the protoplasts from the
Dbik1 mutant were transformed with 10 mg of each plasmid
pglnAProm::bik1-1 and pNR1 or pbik1DPacCProm and pNR1
respectively. For pacC complementation with a truncated
gene copy and overexpression experiments, the protoplasts
generated from the appropriate deletion mutants or the wildtype were transformed with 10 mg of the plasmids pPacC246,
pglnAProm::bik1-2 and pglnAProm::bik5 respectively. For gene
complementation and overexpression, transformed protoplasts were regenerated at 28C in a complete regeneration
agar, as described above, containing 120 mg ml-1 hygromycin
B and 100 mg ml-1 nourseothricin, or only 100 mg ml-1
nourseothricin in case of selection for WT+OE::bik5 mutants.
Single conidial cultures were established from hygromycin
B and/or nourseothricin resistant transformants and used for
DNA isolation and Southern blot analysis.
The homologues integration events of replacement fragments of vectors pDbik2, pDbik3, pDDbik2/bik3 and pDpacC,
carrying the hygromycin resistance cassette, were verified
using two primer pairs targeting the replaced coding region

(WTgeneF/WTgeneR) and two primer pairs located within


the hygromycin resistence cassette and outside the replacement fragment (dLFgene/pUCH-P and dRFgene/pUCH-T).
The homologues integration events of replacement
fragments of vectors pDbik4, pDbik5, pDbik6, carrying
the nourseothricin resistance cassette, were verified using
two primer pairs targeting the replaced coding region
(WTgeneF/WTgeneR) and two primer pairs located within
the hygromycin resistence cassette and outside the replacement fragment (dLFgene/pLOF-oliP and dRFgene/Tub-T).
Nourseothricin/hygromycin-resistant transformants (bik1 C
and bik1 CDPacCProm) generated by transformation of the Dbik1
mutant with pglnAProm::bik1-1 and pNR1 or pbik1PacCProm and
pNR1, respectively, were screened for integration by PCR
using primers bik1-DP-F2 and bik1-dR. Nourseothricin/
hygromycin-resistant transformants (pacC246) generated by
transformation of the DpacC mutant were analysed for integration by PCR using primers PACC-GR1 and PACC-GR4.
Nourseothricin/hygromycin-resistant transformants (DDbik2/
3+OE::bik1, Dbik2+OE::bik1 and Dbik3+OE::bik1) generated
by transformation of the Dbik2, Dbik3 or DDbik2/3 double
mutant, respectively, with pglnAProm::bik1-2 were screened for
homologous integration at the bik1 locus by PCR using
primers glnA-prom and bik1-dR. Nourseothricin/hygromycinresistant transformants (Dbik2+OE::bik5) and nourseothricinresistant transformants (WT+OE::bik5) generated by
transformation of the Dbik2 mutant and the wild-type respectively, with the plasmid pglnAProm::bik5, were analysed for
integration using primers glnA-prom and bik5-R-XhoI.

Isolation of bikaverin, nor-bikaverin and pre-bikaverin


To obtain standards for structure elucidation 10 100 ml
culture fluid of 10-day-old cultures of the wild-type (bikaverin
and nor-bikaverin) or of the DDbik2/3+OE::bik1 mutant (prebikaverin) were each extracted three times with 300 ml ethyl
acetate acidified with 1 ml of 25% HCl. After solvent evaporation the residue was dissolved in acetonitrile (ACN)/1%
formic acid (50 : 50, v/v) and separated on a 300 19 mm
i.d., 7 mm Symmetry PrepTM C8 Column (Waters, Eschborn,
Germany), using a binary gradient delivered by a Varian Pro
Star M-210 pump with 0.1% formic acid as solvent A and ACN
as solvent B. The flow rate was 15 ml min-1 and the HPLC
was programmed as follows. For bikaverin and nor-bikaverin:
0 min, 30% solvent B; 5 min, 80% solvent B; 10 min, 80%
solvent B; 12 min, 100% solvent B. For pre-bikaverin: 0 min,
30% solvent B, 10 min, 85% solvent B, 11 min, 85% solvent
B; 12 min, 100% solvent B. After each HPLC run, the column
was washed with 100% solvent B and equilibrated for 10 min
at the starting conditions. For injection, a rheodyne 7752i
six-port valve with a 2 ml sample loop was used. Bikaverin
(yield: 5.7 mg) and nor-bikaverin (yield: 3.5 mg) were collected after peak detection using a Varian Pro Star 325
UV-Vis Detector set at 510 nm and pre-bikaverin (yield:
2.1 mg) was detected at 431 nm.

HPLC-DAD
Culture fluid (100 ml) of 10-day-old cultures of the wild-type
and the mutants, Dbik1Dbik6, was each extracted three

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944 P. Wiemann et al.

times with 300 ml of ethyl acetate acidified with 1 ml of 25%


HCl. After solvent evaporation, the residue was dissolved in
ACN/1% formic acid (50 : 50, v/v) and separated on a
250 4.6 mm i.d., 5 mm Gemini 5 m C6-Phenyl 110 A
Column (Phenomenex, Aschaffenburg, Germany), using a
binary gradient delivered by a Shimadzu LC-10AT pump
with 1% formic acid as solvent A and ACN as solvent B. The
HPLC was programmed as follows: isocratic step at 10%
solvent B for 1 min followed by a linear gradient to 100%
solvent B in 26 min. After each HPLC run, the column was
washed with 100% solvent B and equilibrated for 10 min at
the starting conditions. The flow rate was 1 ml min-1.
Bikaverin and nor-bikaverin were detected using a Shimadzu SPD-M20A prominence Diode Array Detector (DAD)
set at 510 nm.

High-resolution ESI-MS/MS
High-resolution MS data were measured on a Bruker MicroTOF (Bruker Daltronics, Bremen, Germany) MS with flow
injection. Calibration was achieved by sodium formiat cluster.
The resolution of the MS was Rfwhm = 10000 (full width at half
maximum).
Electrospray ionization (ESI) mass spectra and production spectra were acquired on an API 4000 QTrap MS
(Applied Biosystems, Darmstadt, Germany) with direct flow
infusion. For electrospray ionization, the ion voltage was set
at -4500 V in the negative mode and at 5500 V in the positive mode. Nitrogen served as curtain gas (20 psi); the
declustering potential, being the accelerating current from
atmospheric pressure into high vacuum, was set at -50 V in
the negative mode and 50 V in the positive mode. The
MS/MS parameters were dependent on the substances,
detecting the fragmentation of the [M+H]+ or [M-H]- molecular ions into specific product ions after collision with nitrogen
(4.5 10-5 Torr). The collision energies are given at the
respective compounds.

NMR spectroscopy
The 1H-, 13C- and 2-D-NMR spectra were acquired on a
Bruker DPX-400 (Bruker BioSpin, Rheinstetten, Germany) or
on a Unity plus (Varian, Palo Alto, CA) NMR spectrometer.
Signals are reported in parts per million referenced to CDCl3/
TFA (1:1, v/v) or DMSO-d6 respectively. For structural elucidation and NMR signal assignment 2-D-NMR experiments,
such as gradient-selected correlated spectroscopy, heteronuclear multiple quantum correlation and heteronuclear multiple bond correlation, were performed. Pulse programs for
the experiments were taken from the (Bruker) software
library.

Acknowledgements
This research was supported by the Deutsche Forschungsgemeinschaft (DFG) (Tu1245/7). Philipp Wiemann was
holder of a research fellowship of the Graduiertenkolleg 1409
funded by the Deutsche Forschungsgemeinschaft (DFG). We
thank Sabine Huber for excellent technical support.

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