Hyphal tip ultrastructure ofAspergillus nidulans andAspergillus giganteus and possible implications of woronin bodies close to the hyphal apex of the latter species

Summary The hyphal tip ultrastructure ofAspergillus nidulans andAspergillus giganteus indicates that their apical organization is very similar to that found in other filamentous fungi. Both species have an area immediately behind the hyphal apex free of all large organelles and containing a high concentration of vesicles. InA. giganteus only one size class of vesicle is clearly evident, with a mean diameter of 72 nm. InA. nidulans two size classes of vesicle were found, with mean diameters of 75 nm and 31 nm. A Spitzenkörper is evident inA. nidulans as an area very close to the tip containing only the smaller vesicles. InA. giganteus one or more apparently mature Woronin bodies were found within the first 1 m of some hyphal apices. The possible significance of their presence is discussed.     

A case of chronic necrotizing pulmonary aspergillosis due toAspergillus nidulans

A 55-year old man without immunosuppression clinically showed a coin lesion in the right lower lung on the chest radiographs.Aspergillus nidulans was isolated and identified in both trans-bronchial lung biopsy specimen and resected tissue. The specimens revealed characteristics of chronic necrotizing pulmonary aspergillosis pathologically. Very few reports on cases of pulmonary aspergillosis due toA. nidulans exist, and we were not able to find any reports of similar cases. This case may be the first reported case of chronic necrotizing pulmonary aspergillosis due toA. nidulans.     

Genetical and biochemical aspects of quinate breakdown in the filamentous fungus Aspergillus nidulans

In the ascomycetous fungus Aspergillus nidulans, the expression of two inducible, contiguous or closely linked genes (qutB and qutC) which encode enzymes for quinate breakdown to protocatechuate, appears to be controlled by the product of a tightly linked third gene (qutA). The qut gene cluster locates on chromosome VIII. The catalytic steps required for this conversion are dehydrogenase, dehydroquinase, and dehydratase, and these activities are induced by the presence of quinate in a similar manner. The dehydroquinase enzyme has been purified and shown to be multimeric, consisting of 20–22 identical subunits of approximately 10,000 MW. The enzyme has a pI value of 5.84, a K _m of 5×10^–4 m, and an amino acid composition that lacks tryptophan and cysteine. The enzyme also cross-reacts with rabbit antibodies raised against Neurospora crassa catabolic dehydroquinase.This work was supported in part by European Molecular Biology Organisation grants to J.R.K. and A.R.H. and by National Institutes of Health Grant GM23051 to N.H.G.     

Nitrite toxicity inAspergillus nidulans: Effect of mutation at thenihB gene

ThenihB gene ofAspergillus nidulans was found to confer sensitivity to elevated concentrations of nitrite, compact morphology and absence of conidiation. ThenihB locus was allocated to linkage group II and was recessive in heterozygous diploids. When thenihB1 mutant was grown on a mixture of nitrite plus NH ₄ ⁺ its sensitivity to nitrite was unchanged. A possible role for this gene in nitrite transport and/or the maintenance of membrane integrity is discussed.     

Expression of a synthetic Artemesia annua amorphadiene synthase in Aspergillus nidulans yields altered product distribution

A gene encoding a plant terpene cyclase, Artemisia annua amorpha-4,11-diene synthase (ADS), was expressed in Aspergillus nidulans under control of a strong constitutive promoter, (p)gpdA. The transformants produced only small amounts of amorphadiene, but much larger amounts of similar sesquiterpenes normally produced as minor by-products in planta. In contrast, expression of ADS in Escherichia coli produced almost exclusively amorpha-4,11-diene. These results indicate that the host environment can greatly impact the terpenes produced from terpene synthases.     

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular l-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. α-l-Arabinofuranosidase B, endoarabinase, l-arabinose reductase, l-arabitol dehydrogenase, xylitol dehydrogenase, and l-xylulose reductase were all inducible to varying degrees by l-arabinose and l-arabitol and subject to carbon catabolite repression by d-glucose. With the exception of l-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on d-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of “self” catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.     

Sequence, organization and expression of the core histone genes of Aspergillus nidulans

Summary The core histone gene family ofAspergillus nidulans was characterized. The H2A, H2B and H3 genes are unique in theA. nidulans genome. In contrast there are two H4 genes, H4.1 and H4.2. As previously reported for the H2A gene (May and Morris 1987) introns also interrupt the other core histone genes. The H2B gene, like the H2A gene, is interrupted by three introns, the H3 and H4.1 gene are each interrupted by two introns and the H4.2 gene contains one intron. The position of the single intron in H4.2 is the same as that the first intron of the H4.1 gene. The H2A and H2B genes are arranged as a gene pair separated by approximately 600 by and are divergently transcribed. The H3 and H4.1 genes are similarly arranged and are separated by approximately 800 bp. The H4.2 gene is not closely linked to either the H2A-H2B or H3-H4.1 gene pairs. Using pulse field gel electrophoresis an electrophoretic karyotype was established forA. nidulans. This karyotype was used to assign the H3–H4.1 gene pair and the H4.2 gene to linkage group VIII and the H2A–H2B gene pair to either linkage group III or VI. The abundance of each of the histone messenger RNAs was determined to be cell cycle regulated but the abundance of the H4.2 mRNA appears to be regulated differently from the others.     

Allocation of random amplified polymorphic DNA markers and enzyme activities toAspergillus nidulans andAspergillus tetrazonus chromosomes

Chromosome-substituted haploid segregants of anA. nidulans × A. tetrazonus somatic hybrid were used to allocate several random amplified polymorphic DNA and isoenzyme markers to parental chromosomes. Twenty-six amplified DNA fragments, and nine isoenzyme activities, including lactate dehydrogenase, superoxide dismutase, and arylesterase isoenzymes were assigned to chromosomes. Chromosome-specific markers were found for eachA. nidulans andA. tetrazonus chromosome. These markers could be used to saturate the genetic map ofA. nidulans. The formation of two secondary metabolites was also assigned to chromosomes III and VIII. Attempts were made to allocate extracellular enzyme activities to parental chromosomes, mostly without success, possibly because multiple enzyme forms located on different chromosomes could be responsible for the production of an enzyme activity.     

Isozyme polymorphism of endo-β-1,4-glucanase inAspergillus nidulans

An electrophoretic survey of the natural populations ofAspergillus nidulans, theA. nidulans group, and various species belonging to the genusAspergillus from diverse geographical areas of India was carried out to determine the isozyme polymorphism of endoglucanase. The data revealed the presence of three forms of endoglucanase designated EG I, EG II, and EG III. In some isolates, EG I and EG II were present separately; in others, instead of two separate bands, one thick band was detected, which was designated EG I. In natural isolates ofA. nidulans and theA. nidulans group, EG III was detected in most, but not all, isolates, while EG I and EG II were always present. However, in various other species of the genusAspergillus, EG II was totally lacking. In all the populations at the EG I and EG II region, seven electrophoretic variants each were detected, and at the EG III region four variants were seen. The data suggest that there may be two structural genes for endoglucanase, one coding for proteins in the EG I/EG II zone and another for protein in the EG III zone.This research work was supported by the Council of Scientific and Industrial Research (CSIR), New Delhi.     

Cloning and molecular characterisation of the amdR controlled gatA gene of Aspergillus nidulans

Summary The gamma-amino-n-butyrate transaminase gene (gatA) of Aspergillus nidulans is one of several genes under positive control by the regulatory gene amdR (also called intA). The gatA gene has been cloned from a cosmid library by complementation of a gatA mutation. The sequence of a 2.6 kb genomic fragment containing gatA has been determined. An open reading frame of 1497 bp within this sequences is interrupted by three putative introns and predicts a protein of 55 kDa. Northern analysis confirms control of gatA RNA levels by amdR and also indicates that gatA is not strongly regulated by areA-mediated nitrogen metabolite repression. A. nidulans transformants containing multiple copies of a plasmid carrying an 88 bp fragment from the 5 untranscribed region of gatA grew poorly on substrates whose utilisation is dependent on genes controlled by amdR. This indicated titration of limiting amounts of the amdR gene product by this 88 bp fragment. Comparison of this sequence with the 5 region of the coregulated gene, amdS, reveals probable sites of action for the amdR protein.     

Demonstration of an altered phenylalanyl-tRNA synthetase in an analogue-resistant mutant of Aspergillus nidulans

Summary We have isolated and characterized a new class of p-fluorophenylalanine (FPA)-resistant mutant in Aspergillus nidulans using a phenA strain as the wild type, by optimizing the conditions of growth. All four spontaneous mutants selected on a medium containing FPA were found to be recessive to their wild-type alleles in heterozygous diploids. Complementation analyses and linkage data showed that they were allelic and mapped at a single locus (fpaU) in the facA-riboD interval on the right arm of linkage group V. Partial purification and characterization of Phe-tRNA synthetase from wild-type and mutant strains revealed that the mutant enzyme had a greatly reduced ability to activate the analogue. It is suggested that mutation in the fpaU gene brings about a structural alteration in Phe-tRNA synthetase.Abbreviations FPA DL-p-fluorophenylalanine - phenA auxotroph of phenylalanine - Phe-tRNA synthetase phenylalanyl-transfer ribonucleic acid synthetase Current address: Department of Biological Sciences (M/C 066) The University of Illinois at Chicago, Box 4348, Chicago, IL 60680, USA     

Ultrastructural analysis of conidiophore development in the fungusAspergillus nidulans using freeze-substitution

Summary Conidiation inAspergillus nidulans can be divided conveniently into five morphologically distinct stages. These are development of the conidiophore stalk, formation of the conidiophore vesicle, differentiation of metulae, differentiation of phialides, and production of conidia. The results presented here demonstrate that freeze-substitution fixation greatly facilitates the study of most of these stages. Ultrastructural features of vesicles, mitochondria, microtubules and nuclei were more easily resolved in freeze-substituted samples than in chemically fixed samples. In addition, certain structures and events simply not visible in chemically fixed samples were found routinely in freeze-substituted samples. Examples include Golgi bodies and multivesicular bodies and mitotic divisions associated with various stages of conidiation.Abbreviations C conidium - CI conidium initial - CV conidiophore vesicle - FC foot cell - GB Golgi body - M mitochondrion - ME metula - MT microtubule - MVB multivesicular body - N nucleus - PM plasma membrane - P phialide - RER rough endoplasmic reticulum - S spindle apparatus - SPB spindle pole body - V vacuole - W fungal wall - WB Woronin body     

An intragenic map of the brlA locus of Aspergillus nidulans.

Summary We have constructed an intragenic map for the Aspergillus nidulans brlA gene, mutants in which are distinguishable by visual criteria only. Most of the leaky phenotype mutants map near the right (3) end. The gene shows distinct recombinational polarity consistent with recombination initiation at the promoter (centromereproximal) end of the gene. brlA12 and brlA20 mutants gave abnormal DNA restriction patterns consistent with the III; VIII and VI; VIII translocations, respectively, determined by haploidization.     

Localisation of several chromosome I genes of Aspergillus nidulans: implications for mitotic recombination

Summary Another laboratory previously reported that the vast majority of mitotic recombinants in chromosome I disomics of Aspergillus nidulans arise from double exchange events involving the centromeric region and a far distal, possibly telomeric, region. This conclusion was based on the assumption that the camC gene is located in a position far distal to the centromere on the left arm of chromosome. I. As a left arm location for camC distal to the centromere was possibly in conflict with mapping data obtained in the context of an unrelated project, camC was partially mapped along with three other previously unlocated chromosome I genes, davA, ornD and uapA. The data presented here indicate that camC is located in a position far distal to the centromere but on the right arm of chromosome I, a conclusion also supported by the previous data. The positioning of uapA and camC in far distal locations on the right arm of chromosome I indicates the existence of a vast, otherwise nearly unmapped region on this chromosome arm.     

Tolerance to higher temperature byAspergillus nidulans and its possible implication in biological control of wilt disease of pigeon-pea

Summary During the course of studies on the ecology ofFusarium udum Butler, the incitant of wilt disease of pigeon-pea (Cajanus cajan (L.) Millsp.),Aspergillus nidulans was found to tolerate higher temperatures of summer, and other species includingF. udum were suppressed in field soil. The population ofA. nidulans increased in the soil incubated at 40±2°C at pH6 and 7 while the population ofF. udum was highly suppressed. The wilt disease of pigeon-pea was significantly suppressed at 38±2°C in the soil having a mixture of the inocula ofF. udum andA. nidulans whereas at lower temperature (25±2°C) no significant impact ofA. nidulans on the disease was found. On the basis of this study an integrated use of higher temperature, alkaline pH andA. nidulans has been suggested for biological control of wilt disease of pigeon-pea.     

Purification and biochemical characterization of a broad substrate specificity thermostable alkaline protease from Aspergillus nidulans

Aspergillus nidulans PW1 produces an extracellular carboxylesterase activity that acts on several lipid esters when cultured in liquid media containing olive oil as a carbon source. The enzyme was purified by gel filtration and ion exchange chromatography. It has an apparent MW and pI of 37 kDa and 4.5, respectively. The enzyme efficiently hydrolyzed all assayed glycerides, but showed preference toward short- and medium-length chain fatty acid esters. Maximum activity was obtained at pH 8.5 at 40°C. The enzyme retained activity after incubation at pHs ranging from 8 to11 for 12 h at 37°C and 6 to 8 for 24 h at 37°C. It retained 80% of its activity after incubation at 30 to 70°C for 30 min and lost 50% of its activity after incubation for 15 min at 80°C. Noticeable activation of the enzyme is observed when Fe²⁺ ion is present at a concentration of 1 mM. Inhibition of the enzyme is observed in the presence of Cu²⁺, Fe³⁺, Hg²⁺, and Zn²⁺ ions. Even though the enzyme showed strong carboxylesterase activity, the deduced N-terminal amino acid sequence of the purified protein corresponded to the protease encoded by prtA gene.     

Comparison of four media for the isolation ofAspergillus flavus group fungi

Four agar media used to isolate aflatoxin producing fungi were compared for utility in isolating fungi in theAspergillus flavus group from agricultural soils collected in 15 fields and four states in the southern United States. The four media wereAspergillus flavus andparasiticus Agar (AFPA, 14), the rose bengal agar described by Bell and Crawford (BCRB; 3), a modified rose bengal agar (M-RB), and Czapek's-Dox Agar supplemented with the antibiotics in BC-RB (CZ-RB). M-RB was the most useful for studying the population biology of this group because it permitted both identification of the greatest number ofA. flavus group strains and growth of the fewest competing fungi. M-RB supported an average of 12% moreA. flavus group colonies than the original rose bengal medium while reducing the number of mucorales colonies and the number of total fungi by 99% and 70%, respectively. M-RB was successfully employed to isolate all three aflatoxin producing species,A. flavus, A. parasiticus andA. nomius, and both the S and L strains ofA. flavus. M-RB is a defined medium without complex nitrogen and carbon sources (e.g. peptone and yeast extract) present in BC-RB. M-RB should be useful for studies on the population biology of theA. flavus group.Abbreviations M-RB Modified Rose Bengal Agar - CZ-RB Czapeks Rose Bengal Agar - BC-RB Bell and Crawford's Rose Bengal Agar - AFPA Aspergillus flavus andparasiticus agar     

Transformation of Aspergillus nidulans with a cloned, oligomycin-resistant ATP synthase subunit 9 gene

Summary An allele (oliC31) of the A. nidulans oliC gene has been cloned using homology with the equivalent gene from N. crassa. OliC31 codes for an oligomycin-resistant, triethyltin-hypersensitive form of subunit 9 of the mitochondrial ATP synthase complex. Direct selection for oligomycin-resistance was possible following transformation of A. nidulans with the oliC31 gene. The phenotypes of transformants cultured in the presence of oligomycin were indicative of the position of integration of the transforming plasmid within the genome. Subsequent recombination events involving the integrated oliC31 gene were also apparent from altered levels of resistance to oligomycin or triethyltin. This gene should prove useful as a marker for transformation of strains lacking auxotrophic lesions and in gene replacement or disruption experiments.