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.     

Properties of deoxyribonuclease 4 from Aspergillus nidulans

Deoxyribonuclease 4 from Aspergillus nidulans was purified to over 70% homogeneity. It contains a polypeptide of Mr about 30000, and behaves as a dimer, but with some evidence of dissociation on gel filtration and ultracentrifugation. The pH optimum is 7-9. Activity is supported by metal ions in the order (Mn2+ + Ca2+) greater than Mn2+ approximately equal to (Mg2+ + Ca2+) much greater than Mg2+. Mn2+ is optimal at 10-20 mM. DNAase 4 strongly prefers native DNA, for which the Km is about 0.5 mM, and on which it acts as an endonuclease. The specific activity is about 2000 mumol of nucleotide made acid-soluble in 30 min at 37 degrees C per mg of protein. Action on denatured DNA, which has a lower optimal Mn2+ concentration and a different time course from its action on native DNA, may be due to partial renaturation of the DNA used. It has no action on RNA. With native DNA the enzyme gave mainly, or entirely, double-strand cleavages by a single-hit mechanism, with either Mn2+ or Mg2+. The enzyme has no strongly preferred sequences. Action stops, or becomes very slow, when 50-60% acid-solubility has been reached. In a near-limit digest, mononucleotides were absent, dinucleotides to at least heptanucleotides occurred in similar weight yields, there was an excess of chains of 10-11 (or rather longer) and a rapid decline at greater lengths. Products have 3'-OH, 5'-P termini. The products and kinetics can be understood in terms of the enzyme's causing non-staggered double-strand cleavages randomly in DNA but subject to a requirement for at least two base pairs at one side of the cleavage site and at least 10 (or perhaps rather more) base pairs on the other side of the site.     

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.     

Cryptic Aspergillus nidulans antimicrobials

Secondary metabolite (SM) production by fungi is hypothesized to provide some fitness attribute for the producing organisms. However, most SM clusters are "silent" when fungi are grown in traditional laboratory settings, and it is difficult to ascertain any function or activity of these SM cluster products. Recently, the creation of a chromatin remodeling mutant in Aspergillus nidulans induced activation of several cryptic SM gene clusters. Systematic testing of nine purified metabolites from this mutant identified an emodin derivate with efficacy against both human fungal pathogens (inhibiting both spore germination and hyphal growth) and several bacteria. The ability of catalase to diminish this antimicrobial activity implicates reactive oxygen species generation, specifically, the generation of hydrogen peroxide, as the mechanism of emodin hydroxyl activity.     

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.     

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.     

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.     

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.     

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.