The molecular and genetic basis of conidial pigmentation in Aspergillus niger.

A characteristic hallmark of Aspergillus niger is the formation of black conidiospores. We have identified four loci involved in spore pigmentation of A. niger by using a combined genomic and classical complementation approach. First, we characterized a newly isolated color mutant, colA, which lacked pigmentation resulting in white or colorless conidia. Pigmentation of the colA mutant was restored by a gene (An12g03950) which encodes a putative 4'phosphopantetheinyl transferase protein (PptA). 4'Phosphopantetheinyl transferase activity is required for the activation of Polyketide Synthases (PKSs) and/or Non-Ribosomal Peptide Synthases (NRPSs). The loci whose mutation resulted in fawn, olive, and brown color phenotypes were identified by complementation. The fawn phenotype was complemented by a PKS protein (FwnA, An09g05730), the ovlA mutant by An14g05350 (OlvA) and the brnA mutant by An14g05370 (BrnA), the respective homologs of alb1/pksP, ayg1 and abr1 in A. fumigatus. Targeted disruption of the pptA, fwnA, olvA and brnA genes confirmed the complementation results. Disruption of the pptA gene abolished synthesis of all polyketides and non-ribosomal peptides, while the naphtho-γ-pyrone subclass of polyketides were specifically dependent on fwnA, and funalenone on fwnA, olvA and brnA. Thus, secondary metabolite profiling of the color mutants revealed a close relationship between polyketide synthesis and conidial pigmentation in A. niger.

Peroxicretion: a novel secretion pathway in the eukaryotic cell.

BACKGROUND: Enzyme production in microbial cells has been limited to secreted enzymes or intracellular enzymes followed by expensive down stream processing. Extracellular enzymes consists mainly of hydrolases while intracellular enzymes exhibit a much broader diversity. If these intracellular enzymes could be secreted by the cell the potential of industrial applications of enzymes would be enlarged. Therefore a novel secretion pathway for intracellular proteins was developed, using peroxisomes as secretion vesicles. RESULTS: Peroxisomes were decorated with a Golgi derived v-SNARE using a peroxisomal membrane protein as an anchor. This allowed the peroxisomes to fuse with the plasma membrane. Intracellular proteins were transported into the peroxisomes by adding a peroxisomal import signal (SKL tag). The proteins which were imported in the peroxisomes, were released into the extra-cellular space through this artificial secretion pathway which was designated peroxicretion. This concept was supported by electron microscopy studies. CONCLUSION: Our results demonstrate that it is possible to reroute the intracellular trafficking of vesicles by changing the localisation of SNARE molecules, this approach can be used in in vivo biological studies to clarify the different control mechanisms regulating intracellular membrane trafficking. In addition we demonstrate peroxicretion of a diverse set of intracellular proteins. Therefore, we anticipate that the concept of peroxicretion may revolutionize the production of intracellular proteins from fungi and other microbial cells, as well as from mammalian cells.

A novel screening method for cell wall mutants in Aspergillus niger identifies UDP-galactopyranose mutase as an important protein in fungal cell wall biosynthesis.

To identify cell wall biosynthetic genes in filamentous fungi and thus potential targets for the discovery of new antifungals, we developed a novel screening method for cell wall mutants. It is based on our earlier observation that the Aspergillus niger agsA gene, which encodes a putative alpha-glucan synthase, is strongly induced in response to cell wall stress. By placing the agsA promoter region in front of a selectable marker, the acetamidase (amdS) gene of A. nidulans, we reasoned that cell wall mutants with a constitutively active cell wall stress response pathway could be identified by selecting mutants for growth on acetamide as the sole nitrogen source. For the genetic screen, a strain was constructed that contained two reporter genes controlled by the same promoter: the metabolic reporter gene PagsA-amdS and PagsA-H2B-GFP, which encodes a GFP-tagged nuclear protein. The primary screen yielded 161 mutants that were subjected to various cell wall-related secondary screens. Four calcofluor white-hypersensitive, osmotic-remediable thermosensitive mutants were selected for complementation analysis. Three mutants were complemented by the same gene, which encoded a protein with high sequence identity with eukaryotic UDP-galactopyranose mutases (UgmA). Our results indicate that galactofuranose formation is important for fungal cell wall biosynthesis and represents an attractive target for the development of antifungals.

Survival in the presence of antifungals: genome-wide expression profiling of Aspergillus niger in response to sublethal concentrations of caspofungin and fenpropimorph.

How yeast cells respond to cell wall stress is relatively well understood; however, how filamentous fungi cope with cell wall damage is largely unexplored. Here we report the first transcriptome analysis of Aspergillus niger exposed to the antifungal compounds caspofungin, an inhibitor of beta-1,3-glucan synthesis, and fenpropimorph, which inhibits ergosterol synthesis. The presence of sublethal drug concentrations allowed A. niger to adapt to the stress conditions and to continue growth by the establishment of new polarity axes and formation of new germ tubes. By comparing the expression profile between caspofungin-exposed and nonexposed A. niger germlings, we identified a total of 172 responsive genes out of 14,509 open reading frames present on the Affymetrix microarray chips. Among 165 up-regulated genes, mainly genes predicted to function in (i) cell wall assembly and remodeling, (ii) cytoskeletal organization, (iii) signaling, and (iv) oxidative stress response were affected. Fenpropimorph modulated expression of 43 genes, of which 41 showed enhanced expression. Here, genes predicted to function in (i) membrane reconstruction, (ii) lipid signaling, (iii) cell wall remodeling, and (iv) oxidative stress response were identified. Northern analyses of selected genes were used to confirm the microarray analyses. The results further show that expression of the agsA gene encoding an alpha-1,3-glucan synthase is up-regulated by both compounds. Using two PagsA-GFP reporter strains of A. niger and subjecting them to 16 different antifungal compounds, including caspofungin and fenpropimorph, we could show that agsA is specifically activated by compounds interfering directly or indirectly with cell wall biosynthesis.

Activity of quinones from teak (Tectona grandis) on fungal cell wall stress.

Teak ( Tectona grandis L.f., Verbenaceae) sawdust extract inhibited the growth of Aspergillus niger. Centrifugal partition chromatography was used to isolate the active compounds. By (1)H-NMR the active compounds were identified as deoxylapachol and tectoquinone. Two A. niger transgenic strains which show induction of 1,3 -alpha-D-glucan synthase were used as a cell wall damage model. The result showed that deoxylapachol from T. grandis extract induced fungal cell wall stress.

The Aspergillus niger MADS-box transcription factor RlmA is required for cell wall reinforcement in response to cell wall stress.

In Aspergillus niger, the genes coding for glutamine:fructose-6-phosphate amidotransferase (gfaA) and alpha-1,3-glucan synthase (agsA) are induced in response to cell wall stress. In silico analysis of the promoter region of the two genes revealed the presence of putative DNA binding sites for transcription factors involved in stress responses, including sites identical to the Saccharomyces cerevisiae Rlm1p and Msn2p/Msn4p transcription factors. Promoter analysis indicated that the induction of the agsA gene in response to cell wall stress is fully dependent on a putative Rlm1p binding site in its promoter region. Database searches revealed the presence of S. cerevisiae Rlm1p homologues in most filamentous fungi examined, including A. niger. Deletion of the RLM1 homologue, named rlmA in A. niger, completely eliminated the induction of agsA and resulted in a twofold reduced induction of gfaA during Calcofluor White-induced cell wall stress. The rise in cell wall chitin in the presence of Calcofluor White was also affected in the rlmA deletion strain. In addition, the deletion strain was more sensitive towards cell wall stress agents. Our results indicate that A. niger responds to cell wall stress by transcriptional activation of cell wall reinforcing genes including agsA and gfaA through an Rlm1p-like transcription factor. We propose that such a cell wall salvage mechanism is wide spread in filamentous fungi.

Characterisation of CwpA, a putative glycosylphosphatidylinositol-anchored cell wall mannoprotein in the filamentous fungus Aspergillus niger.

Glycosylphosphatidylinositol (GPI)-anchored proteins in fungi are found at the cell surface, either as plasma membrane proteins (GPI-PMPs) or attached by a remnant of the GPI-anchor to the cell wall (GPI-CWPs). GPI-CWPs can be extracted from the cell wall by treatment with hydrofluoric acid (HF), which cleaves the phosphodiester bond that is present in the remnant of the GPI-anchor. The filamentous fungus Aspergillus niger contains at least seven HF-extractable cell wall mannoproteins. One gene encoding an HF-extractable cell wall mannoprotein, cwpA, was cloned and further characterised. The protein sequence of CwpA indicated the presence of two hydrophobic signal sequences both at the N-terminus and C-terminus of the protein, for entering the ER and the addition of a GPI-anchor, respectively. A CwpA-specific antiserum was raised and in combination with fractionation experiments, we show that this protein was abundantly present as an HF-extractable protein in the cell wall of A. niger.

Expression of agsA, one of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger, is induced in response to cell wall stress.

1,3-alpha-D-Glucan is an important component of the cell wall of filamentous fungi. We have identified a family of five 1,3-alpha-D-glucan synthase-encoding genes in Aspergillus niger. The agsA gene was sequenced and the predicted protein sequence indicated that the overall domain structure of 1,3-alpha-D-glucan synthases is conserved in fungi. Using RT-PCR and Northern blot analysis, we found that expression of the agsA gene and to a lesser extent also of agsE were induced in the presence of the cell wall stress-inducing compounds such as Calcofluor White (CFW), SDS, and caspofungin. Loss of agsA function did not result in an apparent phenotype under normal growth conditions but rendered the cells more sensitive to CFW. The induction of 1,3-alpha-D-glucan synthase-encoding genes in response to cell wall stress was not limited to A. niger, but was also observed in Penicillium chrysogenum. We propose that this response to cell wall stress commonly occurs in filamentous fungi.

The cell wall stress response in Aspergillus niger involves increased expression of the glutamine : fructose-6-phosphate amidotransferase-encoding gene (gfaA) and increased deposition of chitin in the cell wall.

Perturbation of cell wall synthesis in Saccharomyces cerevisiae, either by mutations in cell wall synthesis-related genes or by adding compounds that interfere with normal cell wall assembly, triggers a compensatory response to ensure cell wall integrity. This response includes an increase in chitin levels in the cell wall. Here it is shown that Aspergillus niger also responds to cell wall stress by increasing chitin levels. The increased chitin level in the cell wall was accompanied by increased transcription of gfaA, encoding the glutamine : fructose-6-phosphate amidotransferase enzyme, which is responsible for the first and a rate-limiting step in chitin synthesis. Cloning and disruption of the gfaA gene in A. niger showed that it was an essential gene, but that addition of glucosamine to the growth medium could rescue the deletion strain. When the plant-pathogenic fungus Fusarium oxysporum and food spoilage fungus Penicillium chrysogenum were subjected to cell wall stress, the transcript level of their gfa gene increased as well. These observations suggest that cell wall stress in fungi may generally lead to activation of the chitin biosynthetic pathway.