SummaryWe 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 g g g g -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.

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.

The reticulocyte 15-lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12) is implicated in oxidative damage to reticulocyte mitochondria before their elimination by degradation during maturation to the erythrocyte. A proportion of the 15-lipoxygenase sediments with the mitochondrial-rich stromal fraction of density-gradient-fractionated rabbit reticulocytes suggesting a physical association with mitochondria before their elimination. Ca2+ promotes binding of reticulocyte 15-lipoxygenase to isolated rat liver and reticulocyte mitochondria and 15-lipoxygenase-mediated lipid peroxidation of mitochondrial lipids and free linoleic acid. Association of reticulocyte 15-lipoxygenase with isolated mitochondria is not simply a consequence of Ca(2+)-induced swelling, but implies that Ca2+ mediates translocation of soluble lipoxygenase to mitochondrial membranes. Therefore, Ca2+ may have an important physiological role in the regulation of 15-lipoxygenase-mediated targeting of reticulocyte mitochondria for degradation.

The presence, but not expression, of homologs of three structural genes and a regulatory gene necessary for aflatoxin biosynthesis inAspergillus parasiticus and A. flavus was shown for A. oryzae and A. sojae. Homologs of the regulatory gene aflR were cloned and sequenced fromA. oryzae and A. sojae.