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

Abstract

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

In eukaryotes, the mitogen-activated protein (MAP) kinase signaling transduction pathways convey a variety of exterior information to nuclear targets to regulate cell growth and differentiation (1, 2, 18, 19). In Saccharomyces cerevisiae, FUS3 is a MAP kinase that regulates mating. Homologs of FUS3 have also been characterized in 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 with biosynthesis of natural products (10). Although a regulatory role for MAP kinases in fungal morphogenesis has been established (22, 27, 34, 41, 42), only one study of a MAP kinase (homologous to S. cerevisiae SLT2 in Fusarium graminearum) affecting toxin production has been reported previously (21). The possible role of MAP kinases in fungal secondary metabolism remains obscure, and the implications of FUS3 homologs for natural product biosynthesis have not been investigated. Aspergillus nidulans is a model filamentous fungus used to study regulation of development and secondary metabolism (10, 44). We recently reported that a mutation in mpkB, encoding the FUS3 putative homolog in A. nidulans, blocked sexual development (34). A. nidulans is also known to generate diverse natural products, including the mycotoxin sterigmatocystin (ST), penicillin (PN), and the antitumor compound terrequinone A (10, 24, 39, 44). In this study, we investigated the role of mpkB in the biosynthesis of secondary metabolites. This is the first study reporting the role of Aspergillus MAP kinase signaling pathways in the regulation of fungal natural product biosynthesis.

Phylogenetic analysis.

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

Growth conditions.

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

TABLE 1.

A. nidulans strains used in this study

Strain	Pertinent genotype	Source or reference
TN02A7	pyrG89 argB2 pyroA4 ΔnkuA::argBariboB2a	Gift from Berl Oakley
TNK7.3.6	ΔmpkB::pyrGAFbargB2 pyroA4 riboB2 ΔnkuA::argB	35
TNK7.6.7	argB2 pyroA4 riboB2 ΔnkuA::argB	35
TDB1.1	ΔmpkB::pyrGAFargB2 pyroA4 mpkB::pyroA riboB2 ΔnkuA::argB	35

^aThe nkuA::argB mutation greatly reduces the frequency of nonhomologous integration of transforming DNA fragments (32a, 47).

^bThe pyrG marker gene is from Aspergillus fumigatus.

Mycotoxin analysis.

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

PN analysis.

The culture conditions and bioassay used to quantify PN production were 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.

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 virus reverse 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 primers shown in Table 2.

TABLE 2.

Primers used in qRT-PCR analysis

Primer	Sequence
actinF	5′-ATGGAAGAGGAAGTTGCTGCTCTCGTTATCGACAATGGTTC-3′
actinR	5′-CAATGGAGGGGAAGACGGCACGGG-3′
stcE-F	5′-GATCGAAGTCCGATCCCGCCGAC-3′
stcE-R	5′-GTGGATCTTGCGCACCAGATAGCAGG-3′
stcU-F	5′-CATGTCAAGGACGTTACGCCAGATGAATTCGACCGAGTATTTCGGGTC-3′
stcU-R	5′-GCGGCACACTCATCCACCTGCTCATC-3′
aflR-F	5′-ATGGAGCCCCCAGCGATCAGCCAG-3′
aflR-R	5′-TTGGTGATGGTGCTGTCTTTGGCTGCTCAAC-3′
ipnA-F	5′-TCCCTACCCCGAGGCTGCTATCAAGACG-3′
ipnA-R	5′-CATTTCACCCGATGGATGGGCGCTTT-3′
acvA-F	5′-GACAAGGACAGACCGTGATGCAGGAGA-3′
acvA-R	5′-CCCGACGCAGCCTTAGCGAACAAGAC-3′
aatA-F	5′-GCTGCGCATGGCCCTCGAAAGTAC-3′
aatA-R	5′-GCCTTCCGGCCCACATGATCGAAGAC-3′
tdiA-F	5′-CCGATGCCTGGAGTGCGAATGCG-3′
tdiA-R	5′-TCTGCGCCTGCTCGAGAGCAGCATC-3′
tdiB-F	5′-GCTACCTGCACACGAGCAGCAACA-3′
tdiB-R	5′-GCGCTCTCAAAGTTCCGCTCAGCG-3′
laeA-F	5′-CATGAGCCCTATGTATAGCAACAATTCCGAGCGAAACCAG-3′
laeA-R	5′-ACCTCGATCGCCCAGATACCAGTTCCAC-3′

Our BLAST search and phylogenetic analysis revealed an identity of 60% and a similarity of 78% between A. nidulans MpkB and S. cerevisiae FUS3 (see Fig. S1A and B in the supplemental material). The phylogenetic tree of FUS3 homologs revealed that A. nidulans MpkB 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 fails to develop sexual structures (34). Previous studies have shown that some developmental genes also regulate mycotoxin production (10, 44). In this study we evaluated the effect of the mpkB mutation on ST biosynthesis in A. nidulans. Our TLC analysis revealed that the mpkB mutant strain produced low levels of ST compared with the levels produced by the control strains over time (Fig. 1A). At 20 h ST had clearly accumulated in the control strains, while only trace amounts of ST were observed in the mutant strain under the experimental conditions assayed. In this study we also evaluated the effect of the mpkB mutation on the ST transcriptional regulator gene, aflR (11, 43, 45), as well as the expression of two structural genes, stcE and stcU (8), as indicators of cluster activation (Fig. 1B). qRT-PCR analysis of aflR, stcU, and stcE expression showed a drastic reduction in transcription levels (Fig. 1B). The wild-type phenotype for both gene expression levels and ST production was almost fully restored in the complemented strain.

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, ΔmpkB 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 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 other metabolites that were produced at lower levels in the mpkB mutant than in the control strains. This suggests that mpkB could have a broader effect (direct or indirect) on multiple metabolic pathways (Fig. 1A). For this reason we looked at the possible effect of the mpkB mutation on PN biosynthesis. The mpkB mutation resulted in a drastic decrease in PN biosynthesis (which was approximately 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 in a decrease in the transcription of the analyzed genes (Fig. 2C). It is known that the expression of acvA is the rate-limiting step in PN biosynthesis (17). In our study acvA transcription was most affected by the mpkB mutation (>50% decrease). Alteration of PN gene expression, particularly in the case of acvA, could cause the reduction in PN production observed in the mpkB mutant (Fig. 2B).

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, ΔmpkB 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 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 the expression of tdiA and tdiB, which are required for terrequinone A biosynthesis (5, 39). Our experiments revealed that the mpkB mutant showed a dramatic reduction in the expression of tdiB and a slight reduction in the expression of tdiA (Fig. 3).

FIG. 3.

Expression levels of the terrequinone A biosynthetic genes, tdiA and tdiB. The relative expression levels were calculated using the 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, characterized in detail in S. cerevisiae, FUS3 kinase activates Ste12. Activated Ste12 is able to bind and induce the expression of pheromone-responsive genes (13). We found a putative ste12/steA binding site in the promoter of the A. nidulans hapE gene (position −408). Expression of PN biosynthesis enzyme genes is regulated by HAP-like complexes (3, 6). It is possible that mpkB-dependent steA regulation of PN gene expression could be at least in part mediated by the HAP complex. Additionally, we found another putative ste12/steA binding site directly in the divergently oriented and shared acvA-ipnA promoter (position −343 with respect to the acvA translation start site).

Interestingly, our study indicated that mpkB affects the expression of laeA (Fig. 1B and 2C). The latter gene encodes a putative methyltransferase known to regulate secondary metabolic gene clusters in Aspergillus (4, 25, 35), including ST, PN, and terrequinone A gene clusters. These findings suggest that the effect of mpkB on the transcription of genes involved in secondary metabolism could be at least in part influenced through the regulation of laeA transcription. In conclusion, this study demonstrated that the FUS3-like signaling pathway in A. nidulans not only regulates morphological differentiation in response to environmental stimuli but also modulates the biosynthesis of different natural products, adapting to environmental variations. Due to the high level of conservation among FUS3 homologs, it is likely that this signaling pathway could also control secondary metabolism in other fungal species.