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doi:10.1111/j.1365-2958.2009.06695.x
First published online 29 April 2009
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.
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
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
2009
Polyketide synthase condensing one acetyl- and eight
malonyl-CoA units to form pre-bikaverin
6270
Table 1. The bikaverin biosynthetic genes and their predicted functions in Fusarium fujikuroi.
Predicted function
Amino
acids
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
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
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 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
HPLC-DAD
Culture fluid (100 ml) of 10-day-old cultures of the wild-type
and the mutants, Dbik1Dbik6, was each extracted three
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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