alcA Promoter Research Articles

Downregulation of the ypdA Gene Encoding an Intermediate of His-Asp Phosphorelay Signaling in Aspergillus nidulans Induces the Same Cellular Effects as the Phenylpyrrole Fungicide Fludioxonil.

Many eukaryotic histidine-to-aspartate (His-Asp) phosphorelay systems consist of three types of signal transducers: a His-kinase (HK), a response regulator (RR), and a histidine-containing phosphotransfer intermediate (HPt). In general, the HPt acts as an intermediate between the HK and the RR and is indispensable for inducing appropriate responses to environmental stresses. In a previous study, we attempted but were unable to obtain deletion mutants of the ypdA gene in order to characterize its function in the filamentous fungus Aspergillus nidulans. In the present study, we constructed the CypdA strain in which ypdA expression is conditionally regulated by the A. nidulans alcA promoter. We constructed CypdA strains with RR gene disruptions (CypdA-sskAΔ, CypdA-srrAΔ, and CypdA-sskAΔsrrAΔ). Suppression of YpdA induced by ypdA downregulation activated the downstream HogA mitogen-activated protein kinase cascade. YpdA suppression caused severe growth defects and abnormal hyphae, with features such as enhanced septation, a decrease in number of nuclei, nuclear fragmentation, and hypertrophy of vacuoles, both regulated in an SskA-dependent manner. Fludioxonil treatment caused the same cellular responses as ypdA suppression. The growth-inhibitory effects of fludioxonil and the lethality caused by ypdA downregulation may be caused by the same or similar mechanisms and to be dependent on both the SskA and SrrA pathways.

Inducible expression of antigens in plants: a study focused on peptides related to multiple sclerosis immunotherapy

Multiple sclerosis (MS) affects 2.3 million patients worldwide with no effective treatments available thus far. Depletion of autoreactive T-cells is considered the basis for immunotherapeutic approaches. For this purpose the peptides BV5S2, BV6S5, and BV13S1 have been identified as candidates for the development of a MS vaccine. Herein, the plant-based simultaneous production of these peptides is described as an effort to generate a new model of MS immunotherapy. A polyprotein comprising the sequence of the target peptides was designed having the picornaviral 2A sequence in between to mediate the release of the individual peptides upon translation. A codon optimized gene was cloned in vectors mediating constitutive (CaMV35S promoter) or inducible (AlcA promoter) expression. No transgenic tobacco plants were recovered from the constitutive vector suggesting toxicity of the target peptides. In contrast, several transformed lines were obtained with the inducible vector. The individual BV5S2, BV6S5, and BV13S1 peptides were detected in transformed lines upon ethanol-mediated induction and a quantitative analysis based on a OVA conjugate carrying the three peptides revealed accumulation levels up to 0.5 μg g-1 FW leaves. The plant-made peptides were able to induce humoral responses in orally immunized mice. This platform will be useful in the development of alternative immunotherapies against MS having low cost and safety as main attributes. Moreover the platform represents an attractive alternative for the expression of antigens having detrimental effects in plants.

Transcriptional analyses of an ethanol inducible promoter in Escherichia coli and tobacco for production of cellulase and green fluorescent protein

Cellulase is a widely used enzyme for saccharification of plant cell walls for the production of biofuels. In our work, we tested an ethanol inducible native alcA promoter from Aspergillus nidulans in Escherichia coli (E. coli) and tobacco. The alcA promoter was fused with a cellulase gene and then the promoter was compared with other widely used promoters, namely, T5 and cauliflower mosaic virus (CaMV) 35S in E. coli. We also tested the activity of alcA promoter in tobacco by transiently expressing a green fluorescent protein gene under the control of alcA promoter. We concluded from our quantitative polymerase chain reaction (qPCR) results that when alcA promoter was expressed along with AlcR transcription factor, the alcA promoter was 11 times more active than the T5 promoter. The qPCR data suggested that there may be a direct correlation between AlcR concentrations and alcA activity, indicating a key role of the activator AlcR in the regulation of the alcA promoter. Our work on inducible promoter system for cellulase production may open new avenues of research in the field of biofuel development from living cells.

Overexpression of the laeA gene leads to increased production of cyclopiazonic acid in Aspergillus fumisynnematus

To explore novel bioactive compounds produced via activation of secondary metabolite (SM) gene clusters, we overexpressed an ortholog of laeA, a gene that encodes a global positive regulator of secondary metabolism in Aspergillus fumisynnematus F746. Overexpression of the laeA gene under the alcA promoter resulted in the production of less pigment, shorter conidial head chains, and fewer conidia. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis revealed that SM production in OE::laeA was significantly increased, and included new metabolites that were not detected in the wild type. Among them, a compound named F1 was selected on the basis of its high production levels and antibacterial effects. F1 was purified by column chromatography and preparative TLC and identified as cyclopiazonic acid (CPA) by LC/MS, which had been previously known as mycotoxin. As A. fumisynnematus was not known to produce CPA, these results suggest that overexpression of the laeA gene can be used to explore the synthesis of useful bioactive compounds, even in a fungus for which the genome sequence is unavailable.

A mutation in the converter subdomain of Aspergillus nidulans MyoB blocks constriction of the actomyosin ring in cytokinesis

We have identified a mutant allele of the Aspergillus nidulans homologue of myosin II (myoB; AN4706), which prevents normal septum formation. This is the first reported myosin II mutation in a filamentous fungus. Strains expressing the myoBG843D allele produce mainly aberrant septa at 30°C and are completely aseptate at temperatures above 37°C. Conidium formation is greatly reduced at 30°C and progressively impaired with increasing temperature. Sequencing of the myoBG843D allele identified a point mutation predicted to result in a glycine-to-aspartate amino acid substitution at residue 843 in the myosin II converter domain. This residue is conserved in all fungal, plant, and animal myosin sequences that we have examined. The mutation does not prevent localization of the myoBG843D gene product to contractile rings, but it does block ring constriction. MyoBG843D rings at sites of abortive septation disassemble after an extended period and dissipate into the cytoplasm. During contractile ring formation, both wild type and mutant MyoB::GFP colocalize with actin – an association that begins at the pre-ring “string” stage. Down-regulation of wild-type myoB expression under control of the alcA promoter blocks septation but does not prevent actin from aggregating at putative septation sites – the actin rings, however, do not fully coalesce. Both septation and targeting of MyoB are blocked by disruption of filamentous actin using latrunculin B. We propose a model in which myosin assembly at septation sites depends upon the presence of F-actin, but assembly of the actin component of contractile rings depends upon normal levels of myosin only for the final stages of ring compaction.

The histone acetyltransferase GcnE (GCN5) plays a central role in the regulation of Aspergillus asexual development.

Acetylation of histones is a key regulatory mechanism of gene expression in eukaryotes. GcnE is an acetyltransferase of Aspergillus nidulans involved in the acetylation of histone H3 at lysine 9 and lysine 14. Previous works have demonstrated that deletion of gcnE results in defects in primary and secondary metabolism. Here we unveil the role of GcnE in development and show that a ∆gcnE mutant strain has minor growth defects but is impaired in normal conidiophore development. No signs of conidiation were found after 3 days of incubation, and immature and aberrant conidiophores were found after 1 week of incubation. Centroid linkage clustering and principal component (PC) analysis of transcriptomic data suggest that GcnE occupies a central position in Aspergillus developmental regulation and that it is essential for inducing conidiation genes. GcnE function was found to be required for the acetylation of histone H3K9/K14 at the promoter of the master regulator of conidiation, brlA, as well as at the promoters of the upstream developmental regulators of conidiation flbA, flbB, flbC, and flbD (fluffy genes). However, analysis of the gene expression of brlA and the fluffy genes revealed that the lack of conidiation originated in a complete absence of brlA expression in the ∆gcnE strain. Ectopic induction of brlA from a heterologous alcA promoter did not remediate the conidiation defects in the ∆gcnE strain, suggesting that additional GcnE-mediated mechanisms must operate. Therefore, we conclude that GcnE is the only nonessential histone modifier with a strong role in fungal development found so far.

Strain Improvement by Overexpression of the laeA Gene in Monascus pilosus for the Production of Monascus-Fermented Rice

Monascus species have been used to produce fermented rice called Monascus-fermented rice (MFR). To improve a Monascus strain via activation of secondary metabolite (SM) gene clusters for use in the production of MFR, we overexpressed an ortholog of the laeA gene, which encodes a global positive regulator of secondary metabolism under the control of the strong heterologous Aspergillus nidulans alcA promoter in Monascus pilosus. The OE::laeA transformant produced more SMs, including those not detected under uninduced conditions. MFR produced using the M. pilosus OE::laeA strain contained 4 times more monacolin K, a cholesterol-lowering agent, than MFR produced using the wild-type strain. In addition, pigment production was remarkably increased, and the antioxidant activity was increased as well. The results from this study suggest that Monascus species, which are important industrial fermentative fungi in Asia, can be improved for the production of functional foods by overexpressing the laeA gene.

Genetic and structural validation of Aspergillus fumigatus UDP‐N‐acetylglucosamine pyrophosphorylase as an antifungal target

The sugar nucleotide UDP-N-acetylglucosamine (UDP-GlcNAc) is an essential metabolite in both prokaryotes and eukaryotes. In fungi, it is the precursor for the synthesis of chitin, an essential component of the fungal cell wall. UDP-N-acetylglucosamine pyrophosphorylase (UAP) is the final enzyme in eukaryotic UDP-GlcNAc biosynthesis, converting UTP and N-acetylglucosamine-1-phosphate (GlcNAc-1P) to UDP-GlcNAc. As such, this enzyme may provide an attractive target against pathogenic fungi. Here, we demonstrate that the fungal pathogen Aspergillus fumigatus possesses an active UAP (AfUAP1) that shows selectivity for GlcNAc-1P as the phosphosugar substrate. A conditional mutant, constructed by replacing the native promoter of the A. fumigatus uap1 gene with the Aspergillus nidulans alcA promoter, revealed that uap1 is essential for cell survival and important for cell wall synthesis and morphogenesis. The crystal structure of AfUAP1 was determined and revealed exploitable differences in the active site compared with the human enzyme. Thus AfUAP1 could represent a novel antifungal target and this work will assist the future discovery of small molecule inhibitors against this enzyme.

Functional Analysis of the α-1,3-Glucan Synthase Genes agsA and agsB in Aspergillus nidulans: AgsB Is the Major α-1,3-Glucan Synthase in This Fungus

Although α-1,3-glucan is one of the major cell wall polysaccharides in filamentous fungi, the physiological roles of α-1,3-glucan remain unclear. The model fungus Aspergillus nidulans possesses two α-1,3-glucan synthase (AGS) genes, agsA and agsB. For functional analysis of these genes, we constructed several mutant strains in A. nidulans: agsA disruption, agsB disruption, and double-disruption strains. We also constructed several CagsB strains in which agsB expression was controlled by the inducible alcA promoter, with or without the agsA-disrupting mutation. The agsA disruption strains did not show markedly different phenotypes from those of the wild-type strain. The agsB disruption strains formed dispersed hyphal cells under liquid culture conditions, regardless of the agsA genetic background. Dispersed hyphal cells were also observed in liquid culture of the CagsB strains when agsB expression was repressed, whereas these strains grew normally in plate culture even under the agsB-repressed conditions. Fractionation of the cell wall based on the alkali solubility of its components, quantification of sugars, and 13C-NMR spectroscopic analysis revealed that α-1,3-glucan was the main component of the alkali-soluble fraction in the wild-type and agsA disruption strains, but almost no α-1,3-glucan was found in the alkali-soluble fraction derived from either the agsB disruption strain or the CagsB strain under the agsB-repressed conditions, regardless of the agsA genetic background. Taken together, our data demonstrate that the two AGS genes are dispensable in A. nidulans, but that AgsB is required for normal growth characteristics under liquid culture conditions and is the major AGS in this species.

Aspergillus nidulans galactofuranose biosynthesis affects antifungal drug sensitivity

The cell wall is essential for fungal survival in natural environments. Many fungal wall carbohydrates are absent from humans, so they are a promising source of antifungal drug targets. Galactofuranose (Galf) is a sugar that decorates certain carbohydrates and lipids. It comprises about 5% of the Aspergillus fumigatus cell wall, and may play a role in systemic aspergillosis. We are studying Aspergillus wall formation in the tractable model system, A. nidulans. Previously we showed single-gene deletions of three sequential A. nidulans Galf biosynthesis proteins each caused similar hyphal morphogenesis defects and 500-fold reduced colony growth and sporulation. Here, we generated ugeA, ugmA and ugtA strains controlled by the alcA(p) or niiA(p) regulatable promoters. For repression and expression, alcA(p)-regulated strains were grown on complete medium with glucose or threonine, whereas niiA(p)-regulated strains were grown on minimal medium with ammonium or nitrate. Expression was assessed by qPCR and colony phenotype. The alcA(p) and niiA(p) strains produced similar effects: colonies resembling wild type for gene expression, and resembling deletion strains for gene repression. Galf immunolocalization using the L10 monoclonal antibody showed that ugmA deletion and repression phenotypes correlated with loss of hyphal wall Galf. None of the gene manipulations affected itraconazole sensitivity, as expected. Deletion of any of ugmA, ugeA, ugtA, their repression by alcA(p) or niiA(p), OR, ugmA overexpression by alcA(p), increased sensitivity to Caspofungin. Strains with alcA(p)-mediated overexpression of ugeA and ugtA had lower caspofungin sensitivity. Galf appears to play an important role in A. nidulans growth and vigor.

Molecular genetic analysis reveals that a nonribosomal peptide synthetase-like (NRPS-like) gene in Aspergillus nidulans is responsible for microperfuranone biosynthesis

Genome sequencing of Aspergillus species including Aspergillus nidulans has revealed that there are far more secondary metabolite biosynthetic gene clusters than secondary metabolites isolated from these organisms. This implies that these organisms can produce additional secondary metabolites, which have not yet been elucidated. The A. nidulans genome contains 12 nonribosomal peptide synthetase (NRPS), one hybrid polyketide synthase/NRPS, and 14 NRPS-like genes. The only NRPS-like gene in A. nidulans with a known product is tdiA, which is involved in terrequinone A biosynthesis. To attempt to identify the products of these NRPS-like genes, we replaced the native promoters of the NRPS-like genes with the inducible alcohol dehydrogenase (alcA) promoter. Our results demonstrated that induction of the single NRPS-like gene AN3396.4 led to the enhanced production of microperfuranone. Furthermore, heterologous expression of AN3396.4 in Aspergillus niger confirmed that only one NRPS-like gene, AN3396.4, is necessary for the production of microperfuranone.

Myosin‐II is Necessary for Septum Formation in Aspergillus nidulans

Cytokinesis in filamentous fungi involves the progressive inward growth of the cell wall and plasma membrane, resulting in a series of functionally connected multinucleate compartments, delimited by crosswalls termed septa. The process of septation, despite intensive study, is still incompletely understood. By screening chemically mutagenized strains of the model fungus Aspergillus nidulans, we have identified several isolates with impaired septum formation, which complementation studies using previously‐identified Aspergillus nidulans “sep” mutations show to be mutations in novel loci. Here we report that the mutation in one of these strains (RCH‐2) occurs in the gene that encodes the fungal homologue of mammalian myosin II (MyoB, AN4706). Sequencing of the MyoB allele in the RCH‐2 mutant reveals a point mutation that is predicted to result in a glycine‐to‐aspartate amino acid substitution at residue 843 in the myosin‐II converter domain. This residue is conserved in all fungal, plant, and animal myosin‐II sequences that we have examined. Down‐regulation of wild‐type MyoB expression under control of the A. nidulans AlcA promoter replicates the septation‐minus phenotype of the RCH‐2 mutation. A GFP‐tagged version of MyoB localizes to constricting septal rings in a manner consistent with the protein's involvement in the process of septation.

Genetic analysis of the Arabidopsis protein kinases MAP3Kε1 and MAP3Kε2 indicates roles in cell expansion and embryo development

MAP3Kε1 and MAP3Kε2 are a pair of Arabidopsis thaliana genes that encode protein kinases related to cdc7p from Saccharomyces cerevisiae. We have previously shown that the map3kε1;map3kε2 double-mutant combination causes pollen lethality. In this study, we have used an ethanol-inducible promoter construct to rescue this lethal phenotype and create map3kε1-/-;map3kε2-/- double-mutant plants in order to examine the function of these genes in the sporophyte. These rescued double-mutant plants carry a yellow fluorescent protein (YFP)-MAP3Kε1 transgene under the control of the alcohol-inducible AlcA promoter from Aspergillus nidulans. The double-mutant plants were significantly smaller and had shorter roots than wild-type when grown in the absence of ethanol treatment. Microscopic analysis indicated that cell elongation was reduced in the roots of the double-mutant plants and cell expansion was reduced in rosette leaves. Treatment with ethanol to induce expression of YFP-MAP3Kε1 largely rescued the leaf phenotypes. The double-mutant combination also caused embryos to arrest in the early stages of development. Through the use of YFP reporter constructs we determined that MAP3Kε1 and MAP3Kε2 are expressed during embryo development, and also in root tissue. Our results indicate that MAP3Kε1 and MAP3Kε2 have roles outside of pollen development and that these genes affect several aspects of sporophyte development.

Localization and function of calmodulin in live-cells of Aspergillus nidulans

Calmodulin (CaM) is a small, eukaryotic protein that reversibly binds Ca 2+. Study of CaM localization in genetically tractable organisms has yielded many insights into CaM function. Here, we described the dynamic localization of Aspergillus nidulans CaM (AnCaM) in live-cells by using recombination strains with homologous, single cross-over insertions at the target gene which placed the GFP fused copy under the inducible alcA promoter and the RFP–CaM integration under the native cam promoter. We found that the localization of CaM fusion was quite dynamic throughout the hypha and was concentrated to the active growing sites during germination, hyphal growth, cytokinesis and conidiation. The depletion of CaM by alcA promoter repression induced the explicit abnormalities of germlings with the swollen germ tubes. In addition, the position of highly concentrated GFP–CaM in the extreme apex seemed to determine the hyphal orientation. These data collectively suggest that CaM is constantly required for new hyphal growth. In contrast to this constant accumulation at the apex, GFP–CaM was only transiently localized at septum sites during cytokinesis. Notably, depletion of CaM caused the defect of septation with a completely blocked septum formation indicating that the transient CaM accumulation at the septum site is essential for septation. Moreover, the normal localization of CaM at a hyphal tip required the presence of the functional actin cytoskeleton and the motor protein KipA, which is indispensable for positioning Spitzenkörper. This is the first report of CaM localization and function in live-cells by the site-specific homologous integration in filamentous fungi.

Dynein Light Intermediate Chain in Aspergillus nidulans Is Essential for the Interaction between Heavy and Intermediate Chains

Cytoplasmic dynein is a complex containing heavy chains (HCs), intermediate chains (ICs), light intermediate chains (LICs), and light chains (LCs). The HCs are responsible for motor activity. The ICs at the tail region of the motor interact with dynactin, which is essential for dynein function. However, functions of other subunits and how they contribute to the assembly of the core complex are not clearly defined. Here, we analyzed in the filamentous fungus Aspergillus nidulans functions of the only LIC and two LCs, RobA (Roadblock/LC7) and TctexA (Tctex1) in dynein-mediated nuclear distribution (nud). Whereas the deletion mutant of tctexA did not exhibit an apparent nud mutant phenotype, the deletion mutant of robA exhibited a nud phenotype at an elevated temperature, which is similar to the previously characterized nudG (LC8) deletion mutant. Remarkably, in contrast to the single mutants, the robA and nudG double deletion mutant exhibits a severe nud phenotype at various temperatures. Thus, functions of these two LC classes overlap to some extent, but the presence of both becomes important under specific conditions. The single LIC, however, is essential for dynein function in nuclear distribution. This is evidenced by the identification of the nudN gene as the LIC coding gene, and by the nud phenotype exhibited by the LIC down-regulating mutant, alcA-LIC. Without a functional LIC, the HC-IC association is significantly weakened, and the HCs could no longer accumulate at the microtubule plus end. Thus, the LIC is essential for the assembly of the core complex of dynein in Aspergillus.

Study of the essentiality of the Aspergillus fumigatus triA gene, encoding RNA triphosphatase, using the heterokaryon rescue technique and the conditional gene expression driven by the alcA and niiA promoters

The identification of essential genes represents a critical step in the discovery of novel therapeutic targets in Aspergillus fumigatus. Structural analyses of the Saccharomyces cerevisiae RNA triphosphatase pointed out this enzyme as an attractive therapeutic target for fungal infections. In addition, demonstration of the essentiality of the S. cerevisiae RNA triphosphatase encoding gene enhanced the value of this potential therapeutic target. Nevertheless, consideration of a fungal RNA triphosphatase as an ideal therapeutic target needs confirmation of the essentiality of the respective gene in a fungal pathogen. In this work, we analyzed the essentiality of the A. fumigatus triA gene, encoding RNA triphosphatase, by conditional gene expression and heterokaryon deletion. Using the conditional gene expression driven by the alcA promoter ( alcA P), we found that TriA depletion causes morphological abnormalities that result in a very strong growth inhibition. Nevertheless, since a strict terminal phenotype was not observed, the essentiality of the triA gene could not be ensured. Accordingly, the essentiality of this gene was analyzed by the heterokaryon rescue technique. Results obtained unequivocally demonstrated the essentiality of the A. fumigatus triA gene, indicating the suitability of the RNA triphosphatase as an ideal therapeutic target to treat A. fumigatus infections. Besides, a second conditional gene expression system, based on the niiA promoter ( niiA P), was utilized in this work. Although the niiA P-mediated repression of triA was less severe than that driven by the alcA P, a strong growth inhibition was also found in niiA P- triA strains. Finally, E-tests performed to determine whether triA down-regulated cells became more sensitive to antifungals suggest a synergic effect between amphotericin B and another antifungal inhibiting the A. fumigatus RNA triphosphatase activity.

An ethanol inducible alc system for regulating gene expression in Beauveria bassiana

Entomopathogenic fungi have been investigated as biocontrol agents for controlling of several insect pests in agricultural practice. Current studies mostly focus on improving the virulence and speed of kill by insight in the mechanism of fungal pathogenesis. Investigations on the molecular basis of virulence of these fungi, in some cases, involved in controlling gene expression in time and space, thus appropriate inducible promoter or conditional expression system are demanded. To establish an eligible system for precise temporal control of exogenous gene expression in the important entomopathogenic fungus Beauveria bassiana, the alc regulon system based on AlcR transcription factor and alcA promoter from the ascomycete fungus Aspergillus nidulans was introduced into B. bassiana. After induction, the alc system responded to ethanol sensitively and initiated the expression of the report gene, β-glucuronidase (gus), strongly. Maximal expression occurred at 16 h after the treatment of 0.5% ethanol and the induction level of gus reached 2/3 of that controlled by a constitutive promoter PgpdA. In addition, the induction in B. bassiana was repressed by glucose. With low basal level and rapid induction, this system demonstrated as an effective tool for controlling the expression of target genes in both fundamental research and agriculture for entomopathogenic fungi.

The novel membrane protein SccA affects cell wall integrity in Aspergillus nidulans

This research describes a novel gene designated SccA, which affects cell wall integrity in the filamentous fungus Aspergillus nidulans. Plasmid‐borne extra copies of SccA can suppress the calC2 mutation in the A. nidulans orthologue of protein kinase C (PkcA), which results in hypersensitivity to the chitin‐binding agent Calcofluor White (CFW). In filamentous fungi, as in yeasts, hypersensitivity to CFW correlates with defects in cell wall integrity. The hypothetical translated product of SccA is an ST‐rich 271‐aa protein (42% S/T) of 27.4 kDa mass (unprocessed), with a cleavable N‐terminal ER‐targeting domain and a probable internal membrane anchor. No known homologies have been identified in the sequence databases. A SccA‐GFP hybrid localizes to the plasma membrane of vegetative hyphae and is especially enriched in septa. When SccA is placed under the control of the regulatable AlcA promoter, vegetative growth is normal under both high expression and low expression conditions (growth on glycerol or glucose containing medium, respectively), but asexual sporulation is suppressed during growth on glycerol. Taking into consideration the protein's cell surface location and its influence on the function of PkcA, we hypothesize that SccA plays a role in signal transduction as part of a cell wall integrity pathway.

GDP‐mannose transporter contributes to polarity establishment in the filamentous fungus Aspergillus nidulans

GDP‐mannose transporters (Gmt) carry nucleotide sugars from the cytosol across the Golgi apparatus membrane in various eukaryotic organisms including plants and a variety of fungi. Some fungal species such as the yeast Saccharomyces cerevisiae express only one Gmt, while others including A. nidulans express paralogues (GmtA and GmtB) whose individual roles have not been elucidated. As in S. cerevisiae GmtA is localized within the Golgi apparatus which in A. nidulans consists of widely separated cisternae, in contrast to the stacked cisternae of animals and plants. Here we show that GmtB localization is congruent with GmtA in mature hyphae. Real‐time reverse transcriptase PCR shows a gradual decrease in GmtA expression during the first 12 hours of germination while GmtB expression remains high and unchanged during this period. Depletion of GmtB by the glucose repressible alcA promoter causes abnormalities in polarized growth, a developmental event that is initiated during the early hours of germination. Taken together these observations suggest that GmtB plays an important role in establishment of polarized growth. Although GmtA and GmtB reside within the same Golgi compartments they appear to perform independent functions at different developmental stages.Funded by Research Corporation, the Merck/AAAS Undergraduate Science Research Program and NSF RUI

A Gene Cluster Containing Two Fungal Polyketide Synthases Encodes the Biosynthetic Pathway for a Polyketide, Asperfuranone, in Aspergillus nidulans

The genome sequencing of Aspergillus species including A. nidulans reveals that the products of many of the secondary metabolism pathways in these fungi have not been elucidated. Our examination of the 27 polyketide synthases (PKS) in A. nidulans revealed that one highly reduced PKS (HR-PKS, AN1034.3) and one nonreduced PKS (NR-PKS, AN1036.3) are located next to each other in the genome. Since no known A. nidulans secondary metabolites could be produced by two PKS enzymes, we hypothesized that this cryptic gene cluster produces an unknown natural product. Indeed after numerous attempts we found that the products from this cluster could not be detected under normal laboratory culture conditions in wild type strains. Closer examination of the gene cluster revealed a gene with high homology to a citrinin biosynthesis transcriptional activator (CtnR, 32% identity/47% similarity), a fungal transcription activator located next to the two PKSs. We replaced the promoter of the transcription activator with the inducible alcA promoter, which enabled the production of a novel polyketide that we have named asperfuranone. A series of gene deletions has allowed us to confirm that the two PKSs together with five additional genes comprise the asperfuranone biosynthetic pathway and leads us to propose a biosynthetic pathway for asperfuranone. Our results confirm and substantiate the potential to discover novel compounds even from a well-studied fungus by using a genomic mining approach.