New indole-diterpenoids from the algal-associated fungus Aspergillus nidulans

Two new compounds, including a chlorinated indole-diterpenoid 19-hydroxypenitrem A (1) and its dechlorinated derivative 19-hydroxypenitrem E (2), along with two known congeners (3 and 4), were isolated and identified from the cultures of Aspergillus nidulans EN-330, an endophytic fungus obtained from the marine red alga Polysiphonia scopulorum var. villum. Their structures and absolute configurations were assigned on the basis of 1D and 2D NMR and CD experiments. Compounds 1–4 exhibited cytotoxic activity against brine shrimp with LD₅₀ values of 3.2, 4.6, 1.7, and 8.7 μM, respectively. Besides, the chlorinated 19-hydroxypenitrem A (1) showed antimicrobial activity against four human- and aqua-pathogens. Preliminary SAR study revealed that the Cl-substitution at C-6 enhanced the cytotoxic activity against brine shrimp and antimicrobial activity, while the 19-OH substitution suppressed the activity.     

Betaine-homocysteine methyltransferase in the fungus Aspergillus nidulans.

A betaine:homocysteine methyltransferase activity was demonstrated in the cell-free extracts from the fungus $\text{Aspergillus}\text{nidulans}$. Among methionine-requiring mutants which do not grow on homocysteine one class responds to betaine indicating that this compound can serve as a methyl donor in methionine synthesis $\text{in vivo}$. Mutants of the second class which grow only on methionine were shown to have betaine: homocysteine — and methyltetrahydrofolate: homocysteine methyltransferases simultaneously impaired.     

Glycerol uptake mutants of the hyphal fungus Aspergillus nidulans

A new class of glycerol non-utilizing mutants, designated glcC, has been isolated. The glcC gene was mapped in linkage group VI and mutants were found to complement the reference strains glcA1 (linkage group V) and glcB33 (linkage group I) in diploids. The new mutants were unable to grow on glycerol. However, in contrast to the glcA and glcB phenotype these mutants did grow well on dihydroxyacetone and D-galacturonate. By in vivo 13C NMR spectroscopy it was shown that the glcC mutant did not take up glycerol but did take up dihydroxyacetone. The latter substrate was converted intracellularly into glycerol which was then catabolized as normal.     

Repair of alkylation damage in the fungus Aspergillus nidulans

The repair of alkylation damage in Aspergillus nidulans was investigated. We have assayed soluble protein fractions for enzymes known to be involved in the repair of this type of damage in DNA. The presence of a glycosylase activity that can remove 3-methyladenine from DNA was demonstrated, as well as a DNA methyltransferase activity that appears to act against O6-methylguanine. In addition to this approach, a series of mutants were isolated which display increased sensitivity to alkylating agents (sag mutants). 5 such mutants were further characterized, and at least 4 are shown to map to genes which have not previously been characterized. The behaviour of double mutant combinations demonstrates the existence of at least 2 pathways for the repair of alkylation damage. The majority of the sag mutants (sagA1, sagB2, sag4 and sagE5) exhibit an increased sensitivity to a range of alkylating agents, but not to UV light, while sagC3, when irradiated at the germling stage, also shows sensitivity to UV. None of the mutants isolated are defective in either the 3-methyladenine DNA glycosylase activity, or the DNA methyltransferase activity, and the nature of the defects in these strains remains to be determined.     

Mating type and the genetic basis of self-fertility in the model fungus Aspergillus nidulans

Sexual reproduction occurs in two fundamentally different ways: by outcrossing, in which two distinct partners contribute nuclei, or by self-fertilization (selfing), in which both nuclei are derived from the same individual. Selfing is common in flowering plants, fungi, and some animal taxa. We investigated the genetic basis of selfing in the homothallic fungus Aspergillus nidulans. We demonstrate that alpha and high-mobility group domain mating-type (MAT) genes, found in outcrossing species, are both present in the genome of A. nidulans and that their expression is required for normal sexual development and ascospore production. Balanced overexpression of MAT genes suppressed vegetative growth and stimulated sexual differentiation under conditions unfavorable for sex. Sexual reproduction was correlated with significantly increased expression of MAT genes and key genes of a pheromone-response MAP-kinase signaling pathway involved in heterothallic outcrossing. Mutation of a component MAP-kinase mpkB gene resulted in sterility. These results indicate that selfing in A. nidulans involves activation of the same mating pathways characteristic of sex in outcrossing species, i.e., self-fertilization does not bypass requirements for outcrossing sex but instead requires activation of these pathways within a single individual. However, unlike heterothallic species, aspects of pheromone signaling appeared to be independent of MAT control.     

Mitotic recombination accelerates adaptation in the fungus Aspergillus nidulans

Understanding the prevalence of sexual reproduction in eukaryotes is a hard problem. At least two aspects still defy a fully satisfactory explanation, the functional significance of genetic recombination and the great variation among taxa in the relative lengths of the haploid and diploid phases in the sexual cycle. We have performed an experimental study to explore the specific advantages of haploidy or diploidy in the fungus Aspergillus nidulans. Comparing the rate of adaptation to a novel environment between haploid and isogenic diploid strains over 3,000 mitotic generations, we demonstrate that diploid strains, which during the experiment have reverted to haploidy following parasexual recombination, reach the highest fitness. This is due to the accumulation of recessive deleterious mutations in diploid nuclei, some of which show their combined beneficial effect in haploid recombinants. Our findings show the adaptive significance of mitotic recombination combined with flexibility in the timing of ploidy level transition if sign epistasis is an important determinant of fitness.     

Osmotic adjustment in the filamentous fungus Aspergillus nidulans.

Aspergillus nidulans was shown to be xerotolerant, with optimal radial growth on basal medium amended with 0.5 M NaCl (osmotic potential [psi s] of medium, -3 MPa), 50% optimal growth on medium amended with 1.6 M NaCl (psi s of medium, -8.7 MPa), and little growth on medium amended with 3.4 M NaCl (psi s of medium, -21 MPa). The intracellular content of soluble carbohydrates and of selected cations was measured after growth on basal medium, on this medium osmotically amended with NaCl, KCl, glucose, or glycerol, and also after hyperosmotic and hypoosmotic transfer. The results implicate glycerol and erythritol as the major osmoregulatory solutes. They both accumulated during growth on osmotically amended media, as well as after hyperosmotic transfer, except on glycerol-amended media, in which erythritol did not accumulate. Furthermore, they both decreased in amount after hypoosmotic transfer. With the exception of glycerol, the extracellular osmotic solute did not accumulate intracellularly when mycelium was grown in osmotically amended media, but it accumulated after hyperosmotic transfer. It was concluded that the extracellular solute usually plays only a transient role in osmotic adaptation. The intracellular content of soluble carbohydrates and cations measured could reasonably account for the intracellular osmotic potential of mycelium growing on osmotically amended media.     

Farnesol-induced cell death in the filamentous fungus Aspergillus nidulans

FOH (farnesol), a non-sterol isoprenoid produced by dephosphorylation of farnesyl pyrophosphate, has been shown to inhibit proliferation and induce apoptosis. We have been using Aspergillus nidulans and FOH as a model system and cell death stimulus, respectively, aiming to understand by which means filamentous fungi are driven towards cell death. Here, we review some of our findings about FOH-induced cell death in A. nidulans.     

Identification and characterization of genes required for hyphal morphogenesis in the filamentous fungus Aspergillus nidulans

In the filamentous fungus Aspergillus nidulans, germination of an asexual conidiospore results in the formation of a hyphal cell. A key feature of spore germination is the switch from isotropic spore expansion to polarized apical growth. Here, temperature-sensitive mutations are used to characterize the roles of five genes (sepA, hypA, podB-podD) in the establishment and maintenance of hyphal polarity. Evidence that suggests that the hypA, podB, and sepA genes are required for multiple aspects of hyphal morphogenesis is presented. Notably, podB and sepA are needed for organization of the cytoskeleton at sites of polarized growth. In contrast, podC and podD encode proteins that appear to be specifically required for the establishment of hyphal polarity during spore germination. The role of sepA and the pod genes in controlling the spatial pattern of polarized morphogenesis in germinating spores is also described. Results obtained from these experiments indicate that the normal pattern of germ-tube emergence is dependent upon the integrity of the actin cytoskeleton.     

Chromosome-specific recombinant DNA libraries from the fungus Aspergillus nidulans.

Development of physical genomic maps is facilitated by identification of overlapping recombinant DNA clones containing long chromosomal DNA inserts. To simplify the analysis required to determine which clones in a genomic library overlap one another, we partitioned Aspergillus nidulans cosmid libraries into chromosome-specific subcollections. The eight A. nidulans chromosomes were resolved by pulsed field gel electrophoresis and hybridized to filter replicas of cosmid libraries. The subcollections obtained appeared to be representative of the chromosomes based on the correspondence between subcollection size and chromosome length. A sufficient number of clones was obtained in each chromosome-specific subcollection to predict the overlap and assembly of individual clones into a limited number of contiguous regions. This approach should be applicable to many organisms whose genomes can be resolved by pulsed field gel electrophoresis.     

Lactose and D-galactose catabolism in the filamentous fungus Aspergillus nidulans

The disaccharide lactose is a byproduct of cheese production accumulating to amounts of 800,000 tons per year worldwide, of which 15% is used as a carbon source for various microbial fermentations. Nevertheless, little is known about the regulation of its metabolism in filamentous fungi. Lactose is metabolized slowly, and some important fungi such as A. niger cannot use it at all. A more detailed knowledge on the rate-limiting steps would be helpful to improve its industrial application. We have chosen A. nidulans as an object for investigating how lactose and galactose metabolism are regulated because it has long become a model system for biochemical and genetic research on fungi, and mutants in the lactose-metabolizing pathway of A. nidulans are available. In this paper, we will review the contributions of our research group achieved on this field.     

Molecular and physiological characterization of the NAD-dependent glycerol 3-phosphate dehydrogenase in the filamentous fungus Aspergillus nidulans

In filamentous fungi, glycerol biosynthesis has been proposed to play an important role during conidiospore germination and in response to a hyperosmotic shock, but little is known about the genes involved. Here, we report on the characterization of the major Aspergillus nidulans glycerol 3-phosphate dehydrogenase (G3PDH)-encoding gene, gfdA. G3PDH is responsible for the conversion of dihydroxyacetone phosphate (DHAP) into glycerol 3-phosphate (G3P), which is subsequently converted into glycerol by an as yet uncharacterized phosphatase. Inactivation of gfdA does not abolish glycerol biosynthesis, showing that the other pathway from DHAP, via dihydroxyacetone (DHA), to glycerol is also functional in A. nidulans. The gfdA null mutant displays reduced G3P levels and an osmoremediable growth defect on various carbon sources except glycerol. This growth defect is associated with an abnormal hyphal morphology that is reminiscent of a cell wall defect. Furthermore, the growth defect at low osmolarity is enhanced in the presence of the chitin-interacting agent calcofluor and the membrane-destabilizing agent sodium dodecyl sulphate (SDS). As inactivation of gfdA has no impact on phospholipid biosynthesis or glycolytic intermediates levels, as might be expected from reduced G3P levels, a previously unsuspected link between G3P and cell wall integrity is proposed to occur in filamentous fungi.