A subtraction library was prepared from cultures of Aspergillus niger that had or had not been exposed to dithiothreitol (DTT), in order to identify genes involved in the unfolded protein response (UPR) or in the response to reductive stress. A large fraction of the clones in the library (40%) encoded two putative methyltransferases (MTs) whose function has yet to be determined. Other stress-responsive genes included a homologue of the Mn2+ -containing superoxide dismutase gene (sodB) and a number of genes predicted to code for products that function in protein turnover and in intra- and extracellular transport of molecules. Transcriptional microarray analysis was carried out with a group of 15 genes, comprising 11 from the cDNA library, two genes linked to the putative MT genes but not represented in the library, and two UPR control genes (bipA and pdiA). Eleven of the 15 genes were inducible with DTT. This was either reflected by the presence of transcripts in cells subjected to DTT stress compared to absence under control conditions, or by an induction ratio of between 1.4 and 8.0 in cases where transcripts were already detectable under control conditions. The MT genes were among the four most highly induced. None of the genes, apart from bipA and pdiA, showed significant induction in response to other stresses that are known to induce the UPR in fungi. We conclude that DTT alone does not provide for specific induction of UPR genes and that other stress conditions must also be examined.

ABSTRACT The growth of the filamentous fungus Aspergillus niger , a common food spoilage organism, is inhibited by the weak acid preservative sorbic acid ( trans-trans -2,4-hexadienoic acid). Conidia inoculated at 10 5 /ml of medium showed a sorbic acid MIC of 4.5 mM at pH 4.0, whereas the MIC for the amount of mycelia at 24 h developed from the same spore inoculum was threefold lower. The MIC for conidia and, to a lesser extent, mycelia was shown to be dependent on the inoculum size. A. niger is capable of degrading sorbic acid, and this ability has consequences for food preservation strategies. The mechanism of action of sorbic acid was investigated using 31 P nuclear magnetic resonance (NMR) spectroscopy. We show that a rapid decline in cytosolic pH (pH cyt ) by more than 1 pH unit and a depression of vacuolar pH (pH vac ) in A. niger occurs in the presence of sorbic acid. The pH gradient over the vacuole completely collapsed as a result of the decline in pH cyt . NMR spectra also revealed that sorbic acid (3.0 mM at pH 4.0) caused intracellular ATP pools and levels of sugar-phosphomonoesters and -phosphodiesters of A. niger mycelia to decrease dramatically, and they did not recover. The disruption of pH homeostasis by sorbic acid at concentrations below the MIC could account for the delay in spore germination and retardation of the onset of subsequent mycelial growth.

In the fungus Aspergillus nidulans, the assimilatory conversion of nitrate to ammonia is dependent on the activation function of NirA, a transcription factor belonging to the binuclear Zn2-C6 cluster family. We have previously shown that sequence-specific binding of the activator to a target sequence of nitrate regulated genes in vivo depends on the presence of intracellular nitrate and on the function of a GATA factor (AreA) responsible for chromatin remodelling on the promoter. Nitrate induction promotes NirA binding whereas addition of repressing ammonia leads to gradual dissociation of the activator from its binding site. Here we show that a functional NirA-GFP protein is excluded from the nucleus under no-nitrate conditions and that addition of the inducer results in rapid and complete translocation of the fusion protein into the nucleus. Using a chromatin enzyme-accessibility assay we further show that the physiologically relevant NirA binding site 2 is uncovered already under no-nitrate conditions which suggests that exclusion of NirA from the nucleus is the main factor determining its DNA binding and transcriptional activity. Interestingly, under conditions of induction plus repression, gradual dissociation of NirA from the binding site coincides with repositioning of the relevant nucleosome –1 but the activator is not transported back to the cytosol. Simultaneous over-expresssion of NirA-GFP and the nitrate transporter crnA generates a constitutive nuclear localization signal for NirA and a novel supressor of areAfor nitrate utilization. These data suggest that the nitrate transporter is involved in the generation of the signal and that high levels of the pathway-specific activator can replace the chromatin and activation function of the GATA factor. FUNCTIONAL GENOMICS IN USTILAGO MAYDIS: IDENTIFICATION OF AN IRON UPTAKE GENE CLUSTER WITH A HIGH AFFINITY IRON PERMEASE AFFECTING PATHOGENIC DEVELOPMENT Heiko Eichhorn, Franziska Lessing, Jörg Kämper, Philip Müller and Regine Kahmann Max Planck Institute for terrestrial Microbiology Karl von Frisch Strasse, 35043 Marburg, Germany Phone +49 6421 178 511 Fax +49 6421 178 509 eichhorn@staff.uni-marburg.de, lessingska@web.de, kaemper@staff.uni-marburg.de, mueller4@staff.uni-marburg.de, kahmann@staff.uni-marburg.de Ustilago maydis is the causative agent of corn smut disease. In recent years, it has been demonstrated that a tightly regulated cyclic AMP signalling cascade is necessary for pathogenic development. In this study we have performed a transcriptome analysis using whole genome microarrays (Affymetrix) to identify target genes of the PKA catalytic subunit Adr1. A set of 400 genes was found to be differentially regulated. In this set were ten genes with a putative function in iron uptake clustered to three chromosomal regions. The cluster contains the known genes sid1 and sid2, involved in siderophore biosynthesis plus 8 new genes, designated fer1-8. We have investigated the expression of these genes in mutants affected in cAMP signalling and show that all genes are repressed by iron. Two genes were analysed in more detail. A new nonribosomal peptide synthetase, fer3, was shown to be required for the biosynthesis of ferrichrome A, while the high affinity ferric permease, fer2, is a critical a virulence factor in Ustilago maydis. Side 124-235 26/03/04 15:03 Side 137

We describe a new endoplasmic reticulum (ER)-associated stress response in the filamentous fungus Aspergillus niger. The inhibition of protein folding within the ER leads to cellular responses known collectively as the unfolded protein response (UPR) and we show that the selective transcriptional downregulation of the gene encoding glucoamylase, a major secreted protein, but not two non-secreted proteins, is an additional consequence of ER stress. The transcriptional downregulation effect is shown by nuclear run-on studies to be at the level of transcription, rather than mRNA stability, and is found to be mediated through the promoter of glaA in a region more than 1 kb upstream of the translational start. The inhibition of protein folding in the ER can be induced in a variety of ways. We examined the effects of dithiothreitol (DTT), a reducing agent that causes the formation of unfolded proteins. Although a general downregulation of transcription was seen with DTT treatment, we show that selective downregulation was observed with the glaA gene compared with genes encoding the non-secreted proteins gamma-actin and glyceraldehyde 3'-phosphate dehydrogenase. The DTT-treated fungal cells also showed evidence for the induction of the UPR because expression of bipA and pdiA, encoding an ER-resident chaperone and foldase, respectively, are upregulated and splicing of hacA, the gene encoding the transcription factor responsible for induction of the UPR, occurs allowing the production of an active HacA protein. As a preliminary attempt to investigate if the transcriptional downregulation effect was mediated through HacA (i.e. part of the UPR), we examined ER stress induced through antisense technology to lower the level of PDI in the ER of A. niger. Although the transcription of glaA was attenuated in that strain of A. niger, UPR was not evident, suggesting that the transcriptional downregulation mechanism is controlled differently from the UPR.

Genome sequence data has recently become available for certain Aspergillus species. We consider the transition from genomics to a post-genomic era in Aspergillus, describing resources and methodologies available to underpin research efforts. Advances in our understanding of the fundamental biology of the Aspergilli, together with applications within the biotechnology and medical fields, are anticipated.

A gene encoding a putative pyruvate decarboxylase (EC 4.1.1.1) was isolated from a genomic library of the filamentous fungus Aspergillus parasiticus strain SU-1. The deduced amino acid sequence showed 37% homology to PDC1 from Saccharomyces cerevisiae. Although A. parasiticus has an obligate growth requirement for oxygen, it produced ethanol in shake flask cultures indicating a response to anoxic conditions mediated by pyruvate decarboxylase.