Endo-exonuclease of Aspergillus nidulans

Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5'-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical phi X-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5'-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.     

Tubulins in Aspergillus nidulans

The discovery and characterization of the tubulin superfamily in Aspergillus nidulans is described. Remarkably, the genes that encode alpha-, beta-, and gamma-tubulins were all identified first in A. nidulans. There are two alpha-tubulin genes, tubA and tubB, two beta-tubulin genes, benA and tubC, and one gamma-tubulin gene, mipA. Hyphal tubulin is encoded mainly by the essential genes tubA and benA. TubC is expressed during conidiation and tubB is required for the sexual cycle. Promoter swapping experiments indicate that the alpha-tubulins encoded by tubA and tubB are functionally interchangeable as are the beta-tubulins encoded by benA and tubC. BenA mutations that alter resistance to benzimidazole antimicrotubule agents are clustered and define a putative binding region for these compounds. gamma-Tubulin localizes to the spindle pole body and is essential for mitotic spindle formation. The phenotypes of mipA mutants suggest, moreover, that gamma-tubulin has essential functions in addition to microtubule nucleation.     

Hyphal morphogenesis in Aspergillus nidulans

The formation of hyphae that grow solely by apical extension is a defining feature of filamentous fungi. Hyphal morphogenesis involves several key steps, including the establishment and maintenance of a stable polarity axis, as well as cell division via the deposition of septa. Several filamentous fungi have been employed in attempts to decipher the mechanisms underlying these steps. Amongst these fungi, Aspergillus nidulans has proven to be a particularly valuable model. The genetic tractability of this fungus coupled with the availability of sophisticated post-genomics resources has enabled the identification and characterization of numerous genes involved in hyphal morphogenesis. Here, we summarize current progress towards understanding the function of these genes and the mechanisms involved in polarized hyphal growth and septation in A. nidulans. We also highlight important areas for future investigation.     

Ammonium regulation in Aspergillus nidulans

l-Glutamate uptake, thiourea uptake, and methylammonium uptake and the intracellular ammonium concentration were measured in wild-type and mutant cells of Aspergillus nidulans held in various concentrations of ammonium and urea. The levels of l-glutamate uptake, thiourea uptake, nitrate reductase, and hypoxanthine dehydrogenase activity are determined by the extracellular ammonium concentration. The level of methylammonium uptake is determined by the intracellular ammonium concentration. The uptake and enzyme characteristics of the ammonium-derepressed mutants, meaA8, meaB6, DER3, amrA1, xprD1, and gdhA1, are described. The gdhA mutants lack normal nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase (NADP-GDH) activity and are derepressed with respect to both external and internal ammonium. The other mutant classes are derepressed only with respect to external ammonium. The mutants meaA8, DER3, amrA1, and xprD1 have low levels of one or more of the l-glutamate, thiourea, and methylammonium uptake systems. A model for ammonium regulation in A. nidulans is put forward which suggests: (i) NADP-GDH located in the cell membrane complexes with extracellular ammonium. This first regulatory complex determines the level of l-glutamate uptake, thiourea uptake, nitrate reductase, and xanthine dehydrogenase by repression or inhibition, or both. (ii) NADP-GDH also complexes with intracellular ammonium. This second and different form of regulatory complex determines the level of methylammonium uptake by repression or inhibition, or both.     

Mitochondrial beta-oxidation in Aspergillus nidulans

Beta-oxidation (beta-ox) occurs exclusively in the peroxisomes of Saccharomyces cerevisiae and other yeasts, leading to the supposition that fungi lack mitochondrial beta-ox. Here we present unequivocal evidence that the filamentous fungus Aspergillus nidulans houses both peroxisomal and mitochondrial beta-ox. While growth of a peroxisomal beta-ox disruption mutant (DeltafoxA) was eliminated on a very long-chain fatty acid (C(22:1)), growth was only partially impeded on a long-chain fatty acid (C(18:1)) and was not affected at all on short chain (C4-C6) fatty acids. In contrast, growth of a putative enoyl-CoA hydratase mutant (DeltaechA) was abolished on short-chain and severely restricted on long- and very long-chain fatty acids. Furthermore fatty acids inhibited growth of the DeltaechA mutant but not the DeltafoxA mutant in the presence of an alternate carbon source (lactose). Disruption of echA led to a 28-fold reduction in 2-butenoyl-CoA hydratase activity in a preparation of organelles. EchA was also required for growth on isoleucine and valine. The subcellular localization of the FoxA and EchA proteins was confirmed through the use of red and green fluorescent protein fusions.     

Supersuppressors in Aspergillus nidulans.

Summary Simultaneous reversion of mutations in two different Aspergillus nidulans loci adA and metG was found to be due monogenic suppressor mutations. Prelimirary evidence for the existance of supersuppressors in A. nidulans is presented.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Selection arena in Aspergillus nidulans

The selection arena hypothesis states that overproduction of zygotes--a widespread phenomenon in animals and plants--can be explained as a mechanism of progeny choice. As a similar mechanism, the ascomycetous fungus Aspergillus nidulans may overproduce dikaryotic fruit initials, hereafter called dikaryons. Then, progeny choice might involve selection on which of these dikaryons will thrive to produce thousands of zygotes. These zygotes each produce eight sexual spores which together fill up one fruiting body. In this study, we test the selection arena hypothesis in this homothallic fungus that produces both sexual and asexual spores. We analyzed two mitochondrial and 15 auxotrophic mutations for consequences on sexual and asexual reproduction. We found that many of these mutations confer sexual self-sterility as a pleiotropic effect under conditions of normal asexual spore production. This confirms an important prediction of the selection arena, namely that dikaryons carrying a (slightly) deleterious mutation are not able to proliferate and produce sexual spores. The selection arena ensures that reproductive energy is invested mainly in dikaryons and thus sexual spores of good genetic quality.     

Xylanase production by Aspergillus nidulans

Abstract The effect of phagocyte activation by TNF-α on the ability to trigger a chemiluminescence (CL) response, associated with the release of oxidizing species was evaluated in healthy human mononuclear cells in the presence of Mycobacterium leprae . Recombinant TNF-α (r-TNF-α) increased the CL response of unstimulated M. bovis BCG- and PMA-stimulated cells but did not reverse the M. leprae defective activation of the human phagocyte oxidative burst. M. leprae was less well phagocytosed than M. bovis BCG but phagocytosis of mycobacteria was not altered by addition of r-TNF-α. The failure of activation of oxygen-free radical production might have some relevance to the pathogenesis of leprosy.