Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

The Aspergillus nidulans putative mitogen-activated proteinkinase encoded by mpkB has a role in natural product biosynthesis.An mpkB mutant exhibited a decrease in sterigmatocystin geneexpression and low mycotoxin levels. The mutation also affectedthe expression of genes involved in penicillin and terrequinoneA synthesis. mpkB was necessary for normal expression of laeA,which has been found to regulate secondary metabolism gene clusters.

INTRODUCTION

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INTRODUCTION

In eukaryotes, the mitogen-activated protein (MAP) kinase signalingtransduction pathways convey a variety of exterior informationto nuclear targets to regulate cell growth and differentiation(1, 2, 18, 19). In Saccharomyces cerevisiae, FUS3 is a MAP kinasethat regulates mating. Homologs of FUS3 have also been characterizedin other filamentous fungi (12, 14, 16, 22, 26, 27, 29, 30,31, 32, 36, 37, 40, 41, 42, 46).

Cell differentiation or development is often associated withbiosynthesis of natural products (10). Although a regulatoryrole for MAP kinases in fungal morphogenesis has been established(22, 27, 34, 41, 42), only one study of a MAP kinase (homologousto S. cerevisiae SLT2 in Fusarium graminearum) affecting toxinproduction has been reported previously (21). The possible roleof MAP kinases in fungal secondary metabolism remains obscure,and the implications of FUS3 homologs for natural product biosynthesishave not been investigated. Aspergillus nidulans is a modelfilamentous fungus used to study regulation of development andsecondary metabolism (10, 44). We recently reported that a mutationin mpkB, encoding the FUS3 putative homolog in A. nidulans,blocked sexual development (34). A. nidulans is also known togenerate diverse natural products, including the mycotoxin sterigmatocystin(ST), penicillin (PN), and the antitumor compound terrequinoneA (10, 24, 39, 44). In this study, we investigated the roleof mpkB in the biosynthesis of secondary metabolites. This isthe first study reporting the role of Aspergillus MAP kinasesignaling pathways in the regulation of fungal natural productbiosynthesis.

Phylogenetic analysis.

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Phylogenetic analysis.

Protein sequence alignment and phylogenetic analysis were performedusing CLUSTAL W. A phylogenetic tree was visualized using TREEVIEW(33).

Growth conditions.

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Growth conditions.

The strains used are listed in Table 1. Conidia (10⁶ spores/ml)were inoculated into 500-ml flasks containing 200 ml liquidGMM (9) plus supplements (23) and incubated at 37°C at 300rpm for 18 h. Approximately 3 g of filtered mycelium from eachstrain was spread on solid GMM and allowed to grow in the darkat 37°C. At 8, 20, and 30 h after the shift, mycelial sampleswere collected for ST analysis and mRNA analysis of ST genes.The same culture conditions were also used to analyze tdiA andtdiB expression.

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TABLE 1. A. nidulans strains used in this study

Mycotoxin analysis.

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Mycotoxin analysis.

ST extraction was carried out as described by Hesseltine etal. (20), with some modifications. Twenty milligrams of driedmycelia was ground and mixed with 1 ml of methanol-4% NaCl (55:45,vol/vol). After 20 min of incubation at room temperature, mixtureswere centrifuged, and the supernatant was extracted with chloroform.Thin-layer chromatography (TLC) analysis was performed as previouslydescribed (24).

PN analysis.

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PN analysis.

The culture conditions and bioassay used to quantify PN productionwere the same as those previously described by Brakhage et al.(7); Bacillus calidolactis C953 (a gift from Geoffrey Turner)was used as the test organism.

qRT-PCR analysis.

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qRT-PCR analysis.

RNA extraction was carried out as previously described (38).Four micrograms of total RNA was treated with DNase I RQI (Promega)and reverse transcribed using Moloney murine leukemia virusreverse transcriptase (Promega). Quantitative reverse transcription-PCR(qRT-PCR) was performed with an Mx3000P thermocycler (Stratagene),using SYBR green JumpStart Taq Ready Mix (Sigma) and the primersshown in Table 2.

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TABLE 2. Primers used in qRT-PCR analysis

Our BLAST search and phylogenetic analysis revealed an identityof 60% and a similarity of 78% between A. nidulans MpkB andS. cerevisiae FUS3 (see Fig. S1A and B in the supplemental material).The phylogenetic tree of FUS3 homologs revealed that A. nidulansMpkB grouped with other homologs from the genus Aspergillus(see Fig. S1B in the supplemental material).

We recently reported that an mpkB mutant of A. nidulans failsto develop sexual structures (34). Previous studies have shownthat some developmental genes also regulate mycotoxin production(10, 44). In this study we evaluated the effect of the mpkBmutation on ST biosynthesis in A. nidulans. Our TLC analysisrevealed that the mpkB mutant strain produced low levels ofST compared with the levels produced by the control strainsover time (Fig. 1A). At 20 h ST had clearly accumulated in thecontrol strains, while only trace amounts of ST were observedin the mutant strain under the experimental conditions assayed.In this study we also evaluated the effect of the mpkB mutationon the ST transcriptional regulator gene, aflR (11, 43, 45),as well as the expression of two structural genes, stcE andstcU (8), as indicators of cluster activation (Fig. 1B). qRT-PCRanalysis of aflR, stcU, and stcE expression showed a drasticreduction in transcription levels (Fig. 1B). The wild-type phenotypefor both gene expression levels and ST production was almostfully restored in the complemented strain.

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FIG. 1. TLC analysis of ST (A) and qRT-PCR analysis of expression of the stcU, stcE, aflR, and laeA genes (B). Mycelial samples were harvested for ST extraction, and mRNA was analyzed at 8, 20, and 30 h after a shift onto GMM plates. The tested strains were wild-type strain TN02A7, ${Delta}$ mpkB mutant TNK7.3.6, isogenic transformation control strain TNK7.6.7 (trans. cont.), and complementation strain TDB1.1 (comp.). std, ST standard. The relative expression levels were calculated using the Formula method (28), and all values were normalized to expression of the A. nidulans actin gene. The error bars indicate the ranges for three replicates. The dashed arrows indicate additional unknown metabolites whose production was affected by the mpkB mutation.

Our TLC analysis also indicated a different profile for othermetabolites that were produced at lower levels in the mpkB mutantthan in the control strains. This suggests that mpkB could havea broader effect (direct or indirect) on multiple metabolicpathways (Fig. 1A). For this reason we looked at the possibleeffect of the mpkB mutation on PN biosynthesis. The mpkB mutationresulted in a drastic decrease in PN biosynthesis (which wasapproximately sevenfold less than that of controls) (Fig. 2).Next, we analyzed the expression levels of the PN genes, acvA,ipnA, and aatA. We found that the mpkB mutation resulted ina decrease in the transcription of the analyzed genes (Fig.2C). It is known that the expression of acvA is the rate-limitingstep in PN biosynthesis (17). In our study acvA transcriptionwas most affected by the mpkB mutation (>50% decrease). Alterationof PN gene expression, particularly in the case of acvA, couldcause the reduction in PN production observed in the mpkB mutant(Fig. 2B).

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FIG. 2. PN bioassays and PN gene expression. (A) Effect of fungal extracts on the growth of B. calidolactis C953. Spots a, c, e, and g contained extracts from wild-type strain TN02A7, ${Delta}$ mpkB mutant TNK7.3.6, isogenic transformation control strain TNK7.6.7 (trans. cont.), and complementation strain TDB1.1 (comp.), respectively. Spots b, d, f, and h contained to the same extracts mixed with 5 U of penicillase. (B) PN production, expressed in micrograms per milliliter of culture supernatant. Commercial PN G (Sigma) was used as the standard. (C) Expression levels of the PN biosynthesis genes acvA, ipnA, aatA, and laeA. The relative expression levels were calculated using the Formula method (28), and all values were normalized to the expression of the A. nidulans actin gene. The error bars indicate the ranges for three replicates.

We also investigated the effect of the mpkB mutation on theexpression of tdiA and tdiB, which are required for terrequinoneA biosynthesis (5, 39). Our experiments revealed that the mpkBmutant showed a dramatic reduction in the expression of tdiBand a slight reduction in the expression of tdiA (Fig. 3).

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FIG. 3. Expression levels of the terrequinone A biosynthetic genes, tdiA and tdiB. The relative expression levels were calculated using the Formula method (28), and all values were normalized to the expression of the A. nidulans actin gene. The error bars indicate the ranges for three replicates. trans. cont., transformation control strain; comp., complementation strain.

In the conserved pheromone response MAP kinase pathway, characterizedin detail in S. cerevisiae, FUS3 kinase activates Ste12. ActivatedSte12 is able to bind and induce the expression of pheromone-responsivegenes (13). We found a putative ste12/steA binding site in thepromoter of the A. nidulans hapE gene (position –408).Expression of PN biosynthesis enzyme genes is regulated by HAP-likecomplexes (3, 6). It is possible that mpkB-dependent steA regulationof PN gene expression could be at least in part mediated bythe HAP complex. Additionally, we found another putative ste12/steAbinding site directly in the divergently oriented and sharedacvA-ipnA promoter (position –343 with respect to theacvA translation start site).

Interestingly, our study indicated that mpkB affects the expressionof laeA (Fig. 1B and 2C). The latter gene encodes a putativemethyltransferase known to regulate secondary metabolic geneclusters in Aspergillus (4, 25, 35), including ST, PN, and terrequinoneA gene clusters. These findings suggest that the effect of mpkBon the transcription of genes involved in secondary metabolismcould be at least in part influenced through the regulationof laeA transcription. In conclusion, this study demonstratedthat the FUS3-like signaling pathway in A. nidulans not onlyregulates morphological differentiation in response to environmentalstimuli but also modulates the biosynthesis of different naturalproducts, adapting to environmental variations. Due to the highlevel of conservation among FUS3 homologs, it is likely thatthis signaling pathway could also control secondary metabolismin other fungal species.

ACKNOWLEDGMENTS

This study was funded by Northern Illinois University.

FOOTNOTES

* Corresponding author. Mailing address: Department of Biological Sciences, Northern Illinois University, DeKalb, IL 60115. Phone: (815) 753-0451. Fax: (815) 753-0461. E-mail: amcalvo{at}niu.edu

Published ahead of print on 31 March 2008.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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