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A mutant strain of Emericella nidulans MNU 82 was isolated by multistep mutation. The beta-amylase produced by the mutant was able to digest raw starch. It was readily and strongly adsorbed onto raw starch at pH 5.0. The enzyme to starch ratio was 1950 U/g starch. The enzyme showed no correlation between the capacity of raw starch digestion and adsorption of the enzyme.
OBJECTIVE: Chronic granulomatous disease (CGD) is a rare inherited disorder of NADPH oxidase in which phagocytes fail to generate reactive antimicrobial oxidants. Invasive fungal infections are an important cause of morbidity and mortality in CGD patients, with Aspergillus spp. being the most frequent fungal pathogens. We reviewed the reported cases of osteomyelitis in CGD patients due to Aspergillus nidulans and compared them with those due to Aspergillus fumigatus. METHODS: Twenty-four cases of osteomyelitis due to Aspergillus spp. in 22 male CGD patients were found in MEDLINE. RESULTS: Fourteen cases (58%) were due to Aspergillus nidulans and ten cases to Aspergillus fumigatus. No other aspergilli were reported as causes of osteomyelitis. Osteomyelitis due to Aspergillus nidulans was associated with pulmonary infection and involved 'small bones' more frequently than Aspergillus fumigatus osteomyelitis (p=0.032). Half of the CGD patients with Aspergillus nidulans osteomyelitis died compared with none of those with Aspergillus fumigatus osteomyelitis (p=0.019). In both Aspergillus nidulans and Aspergillus fumigatus cases, cure was achieved by prompt antifungal treatment combined with surgery and immunotherapy. CONCLUSION: Aspergillus nidulans causes osteomyelitis in CGD patients relatively frequently compared with Aspergillus fumigatus and may be accompanied by higher mortality. This contrasts with the low frequency with which Aspergillus nidulans causes osteomyelitis in patients with other types of immunodeficiency.
Aspergillus niger grows poorly on acetamide as a nitrogen or carbon source and lacks sequences detectably homologous to the amdS gene encoding the acetamidase of Aspergillus nidulans. We have taken advantage of these observations to develop a transformation system for A. niger using the amdS gene as a dominant heterologous marker for selecting transformants on the basis of acetamide utilization. Transformants varied in their ability to grow on amide media and the number of integrated copies of the amdS plasmid ranged from 1 or 2 to greater than 100. Southern analysis of transformants revealed that the multiple copies were integrated into the chromosome in tandem arrays. This result indicates that transformation of A. niger is more similar to mammalian cells than to yeast. Analysis of enzyme activity levels and RNA levels showed that most of the copies of amdS were expressed. Mitotic stabilities of transformants were found to be high. A transformant containing greater than 100 copies of the amdS gene was impaired in omega-amino acid utilization, a result that has also been found in A. nidulans. Since, in A. nidulans, omega-amino acids induce acetamidase via a characterizied regulatory gene (amdR/intA) this observation implies that titration of an analogous A. niger regulatory gene product by multiple amdS copies has occurred. Additional evidence suggested that the amdS gene is regulated in A. niger. It has also been shown that an unselected plasmid can be co-transformed with the amdS plasmid into A. niger.
Aspergillus nidulans grows by apical extension of multinucleate cells called hyphae that are subdivided by the insertion of crosswalls called septa. Apical cells vary in length and number of nuclei, whereas subapical cells are typically 40 microm long with three to four nuclei. Apical cells have active mitotic cycles, whereas subapical cells are arrested for growth and mitosis until branch formation reinitiates tip growth and nuclear divisions. This multicellular growth pattern requires coordination between localized growth, nuclear division, and septation. We searched a temperature-sensitive mutant collection for strains with conditional defects in growth patterning and identified six mutants (designated hyp for hypercellular). The identified hyp mutations are nonlethal, recessive defects in five unlinked genes (hypA-hypE). Phenotypic analyses showed that these hyp mutants have aberrant patterns of septation and show defects in polarity establishment and tip growth, but they have normal nuclear division cycles and can complete the asexual growth cycle at restrictive temperature. Temperature shift analysis revealed that hypD and hypE play general roles in hyphal morphogenesis, since inactivation of these genes resulted in a general widening of apical and subapical cells. Interestingly, loss of hypA or hypB function lead to a cessation of apical cell growth but activated isotropic growth and mitosis in subapical cells. The inferred functions of hypA and hypB suggest a mechanism for coordinating apical growth, subapical cell arrest, and mitosis in A. nidulans.
Aspergillus nidulans reproduces asexually via uninucleate, haploid spores, which are produced on morphologically differentiated aerial structures, called conidiophores. These consist of four distinct cell types, a foot with a terminally swollen stalk, metulae, phialides and conidiospores. The molecular mechanisms underlying the morphological changes that occur during conidiophore development have been studied by mutant analysis. We have isolated the hym A mutant, in which conidiophore development is affected at the metula stage. In the mutant metulae do not differentiate properly but come to resemble hyphae (hym = hypha-like metulae). In this paper we have analyzed the corresponding gene. It encodes a highly expressed 44 kDa protein which resides in the cytoplasm and has homologues in yeast, plants, fly, worm, fish, mice and man. We constructed hym deletion strains of Saccharomyces cerevisiae and of A. nidulans and found that the gene is essential in S. cerevisiae but is dispensable in the filamentous fungus. A cellular function for the Hym protein has not yet been defined in any organism. To demonstrate functional conservation we constructed a chimeric protein comprised of the N-terminal half of the A. nidulans and the C-terminal half of the mouse homologue MO25. This hybrid protein could fully substitute for HymA function in A. nidulans. In addition, the mouse protein itself partially rescued the hym A mutation in the fungus. HymA is thus highly conserved in evolution and probably serves similar functions. The fact that hym A is required for conidiophore development in A. nidulans suggests that homologous genes in other organisms might also be involved in morphogenesis.
Aspergillus nidulans when supplemented with egg-white protein (1 %) produced considerable amounts of proteases extracellularly. The order of the appearance of the proteolytic enzymes growth on untreated and heat-inactivated egg-white were studied. They showed pH optima of 5, 7.2 and 9 when assayed at 37 degrees C. It is proposed that one of the factors leading to higher cell mass of A. nidulans in the presence of egg-white is the availability of protease-catalyzed hydrolyzed products of egg-white.
Aspergillus nidulans is able to grow on oleic acid as sole carbon source. Characterization of the oleate-induced beta-oxidation pathway showed the presence of the two enzyme activities involved in the first step of this catabolic system: acyl-CoA oxidase and acyl-CoA dehydrogenase. After isopicnic centrifugation in a linear sucrose gradient, microbodies (peroxisomes) housing the beta-oxidation enzymes, isocitrate lyase and catalase were clearly resolved from the mitochondrial fraction, which contained fumarase. Growth on oleic acid was associated with the development of many microbodies that were scattered throughout the cytoplasm of the cells. These microbodies (peroxisomes) were round to elongated, made up 6% of the cytoplasmic volume, and were characterized by the presence of catalase. The beta-oxidation pathway was also induced in acetate-grown cells, although at lower levels; these cells lacked acyl-CoA oxidase activity. Nevertheless, growth on acetate did not cause a massive proliferation of microbodies in A. nidulans.
Aspergillus nidulans has functioned as a model system for the study of fungal genetics since the 1950s. Application of methodologies ranging from Mendelian genetics to the most sophisticated molecular biological techniques have resulted in a detailed understanding of genes and pathways involved in primary metabolism, secondary metabolism and development in A. nidulans. We have taken advantage of this background in developing A. nidulans as a genetic system to study the molecular mechanisms regulating aflatoxin biosynthesis. Aflatoxin, a carcinogenic polyketide, is the product of a lengthy biochemical pathway found in the asexual spp., A. flavus and A. parasiticus. A. nidulans possesses most if not all of this pathway and produces sterigmatocystin, the penultimate precursor of the aflatoxin pathway. We have identified a approximately 60 kb cluster of genes in A. nidulans whose products are involved in sterigmatocystin biosynthesis. This cluster contains at least 20 genes proposed to encode both enzymatic activities and regulatory proteins. Our results have shown that at least some of these genes are functionally conserved between A. nidulans, A. flavus and A. parasiticus, and that they are regulated in similar ways. Further studies of sterigmatocystin regulation in A. nidulans should yield information transferable to studies of (i) secondary metabolism in other filamentous fungi and (ii) aflatoxin regulation in A. flavus and A. parasiticus in particular.
Aspergillus nidulans asparaginase activity may be assayed conductimetrically. The method is based on the increase of conductivity which is due to the production of ammonia and/or aspartate in a reaction mixture containing A. nidulans cell-free extract and asparagine or aspartate hydroxamate. This conductivity is linear with time and enzyme concentration and it follows Michaelis kinetics. Conductimetric activity was not detectable in mutants lacking asparaginase activity.
Aspergillus fumigatus produces conidia that are highly dispersable and resistant to degradation. We have sought to analyze these properties by studying the rodlets which form the outer spore coat protein. Degenerate primers based on hydrophobins in other fungi were applied to genomic DNA from A. fumigatus in PCR. A product of this reaction with similarity to an Aspergillus nidulans gene as judged by Southern hybridization was chosen for further study. Cloning and sequencing revealed a gene with two introns which encodes a protein of 159 amino acids. Structural characteristics consistent with those of other fungal hydrophobin genes, especially conserved cysteine residues, are present. The expression of the gene is limited to the developmental stages in which maturing conidiophores are present. This A. fumigatus gene, HYP1, was used to transform a mutant strain of A. nidulans that lacks rodlets. Transformants with a single copy of HYP1 expressed a rodlet layer on their conidia as observed by freeze-fracture electron microscopy.
Aspergillus nidulans synthesizes penicillins as secondary metabolites when grown under certain culture conditions. Broths containing carbon (C) sources that give rise to carbon catabolite repression (CCR) support a much lower antibiotic yield than broths with non-repressing C sources. Steady-state levels of the isopenicillin N synthetase (IPNS) gene transcript are considerably reduced in mycelia grown with repressing C sources and are depressed in mycelia grown with sugars which do not cause CCR, indicating that penicillin biosynthesis is regulated by CCR through transcriptional control of structural genes. CCR is sufficient to explain the temporal window of expression of the IPNS gene during the growth cycle since (i) the transcript becomes derepressed as soon as the concentration of a repressing C source drops to non-repressing levels and (ii) derepressing C sources sustain derepressed IPNS transcription at all tested moments of the growth cycle. Several tested hypofunctional mutations in creA (the negatively acting regulatory gene which mediates CCR in A. nidulans) do not cause full derepression of IPNS transcript in the presence of a repressing C source. The slight degree of IPNS derepression caused by some creAd (derepressed) alleles parallels the strength of the mutation (as determined by the morphological effect they elicit).
Aspergillus nidulans is one of the model ascomycete fungi. Transposition events have never been described in this organism. We have determined that this organism has at least 13 copies of a Fot1-related element. These copies are transcribed, non-methylated and polymorphic in various wild isolates. In spite of this, we have failed to isolate transposon insertions when the resident niaD gene is used as a transposon trap. This contrasts with the situation described previously in Fusarium oxysporum. We show that two elements of F. oxysporum, Fot1 and impala, transpose efficiently in A. nidulans. We have developed the impala system by tagging it with the yA gene. This permits the visual detection of the transposon by the colour of the conidiospores. We demonstrate that no endogenous transposase of A. nidulans is able to act in trans on a defective impala element, whereas its own transposase driven by two different promoters is able to mobilize this element. The frequency of excision of these modified elements is between 10(-4) and 10(-5). Loss of the transposable element occurs in about 10% of all excision events. In the remaining 90%, the transposon seems to be integrated at random positions in the genome. The availability of mitochondrially inherited mutations has allowed us to demonstrate that hybrid dysgenesis is apparently absent in A. nidulans. The development of this system opens the way to investigating the mechanism underlying the paucity of transposition events leading to visible phenotypes. It should allow us to develop efficient gene-tagging tools, useful in this and other fungi.
Aspergillus nidulans produces an extracellular invertase when incubated in the presence of sucrose and about half of the activity produced was found to be associated with the mycelium. Sixty percent of this mycelial invertase could be solubilised by simple mechanical disruption. Among the agents tested for solubilisation of invertase, proteinase K and dithiothreitol were the most effective.
A cell surface glycoprotein (Bm86) from cells of the digestive tract of the cattle tick Boophilus microplus, which has been shown to elicit a protective immunological response in vaccinated cattle, was expressed and secreted in the filamentous fungi Aspergillus nidulans and Aspergillus niger by using the fungal amdS promoter system. The cloned gene coded for the Bm86 secretory signal and all of the Bm86 mature polypeptide except for the hydrophobic carboxy-terminal segment. High levels of Bm86 mRNA were detected in the transformed cells. Bm86 polypeptide was secreted from the cells in a soluble form and it was glycosylated, probably to a similar extent to the native glycoprotein. The recombinant product had an apparent molecular mass of 83 to 87 kilodaltons, whereas that predicted from the amino acid sequence was 69 kilodaltons. The Bm86 was expressed at levels of up to 1.8 mg/liter, or approximately 6% of secreted protein under the growth conditions used. No intracellular Bm86 was detected. A general relationship was observed between transformants containing a high number of copies of the expression plasmid and high expression levels.
Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.
Aspergillus nidulans produces acetamidase and formamidase enzymes. The acetamidase is produced in reduced amounts during growth on glucose, whereas the formamidase is not greatly affected. Mutations in a gene, amdT, which affect glucose repression of amidases are described. One of these, amdT102, causes the acetamidase to be no longer subject to glucose repression and also affects ability to synthesize formamidase. The other, amdT19, results in both the formamidase and the acetamidase being subject to abnormally strong glucose repression, and also in increased maximal acetamidase activities. The dominance relationships at the amdT locus have been investigated. It is suggested that the amdT gene may play a positive role in controlling amidase synthesis.
Aspergillus nidulans WG312 strain secreted lipase activity when cultured in liquid media with olive oil as carbon source. Highest lipase productivity was found when the mycelium was grown at 30 degrees C in a rich medium. The new enzyme was purified to homogeneity from the extracellular culture of A. nidulans by phenyl-Sepharose chromatography and affinity binding on linolenic acid-agarose. The lipase was monomeric with an apparent M(r) of 29 kDa and a pI of 4.85 and showed no glycosylation. Kinetic of enzyme activity versus substrate concentration showed a typical lipase behavior, with K(M) and K(cat) values of 0.28 mM and 494 s(-)(1) and 0.30 mM and 320 s(-)(1) for the isotropic solution and for the turbid emulsion, respectively. All glycerides assayed were hydrolyzed efficiently by the enzyme, but this showed preference toward esters of short- and middle-chain fatty acids. The optimum temperature and pH for the lipolytic activity were 40 degrees C and 6.5, with high activity in the range 0-20 degrees C and reduced thermal stability.
Aspergillus nidulans has three molybdoenzymes, nitrate reductase, purine hydroxylase I and purine hydroxylase II. These three enzymes share a molybdenum-containing cofactor whose synthesis requires the integrity of five loci, designated cnxABC, cnxE, cnxF, cnxG and cnxH. Here we report the existence of a sixth locus, designated cnxJ, which might be involved inthe regulation of cofactor levels. When grown in the presence, but not in the absence, of tungstate or methylammonium, strains carrying cnxJ1 or cnxJ2 have reduced molybdoenzyme levels as judged both from growth properties and enzyme determinations. A new cryosensitive cnxC- allele is also reported. Its phenotype at 37 degrees C (but not 25 degrees C) shows some similarities to that of the two cnxJ- alleles. A structural role for the cnxC (or cnxABC) product in the cofactor is tentatively suggested.