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.     

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.     

Detection of unknown toxic mycotoxins inAspergillus nidulans using a structure-activity approach

Mycotoxins are secondary metabolites of filamentous fungi that can cause various acute and chronic toxic effects in humans. Previous work by Büngeret al. exhibited that the cytotoxicityof Aspergillus nidulans, one of the most frequent toxigenic moulds in composting plants, could not be explained by its content of identified mycotoxins. The presence of additional mycotoxins or other toxic prinpiples was assumed, which may be detected by a structure-activity approach. An HPLC-diode array detector method was used to separate and characterize the components of theA. nidulans-extract within 50 minutes/analysis. Aliquots of the extract were chromatographed and nine 5-minutes-fractions were collected and lyophilized. Rechromatography of aliquots of the residues confirmed the accuracy of the 5-minutes-cuts. The cytotoxicity of these fractions was estimated in three cell lines (A-549, L-929 and Hep-G2) using the neutral red assay (NRU assay). Ethanol/dichloromethane (1:1, v/v) was proven to be a suitable solvent mixture with a low cytotoxicity. HPLC-fractions were dissolved in this mixture prior to the NRU assay. Three 5-minutes-fractions exhibited a strong cytotoxicity in this screening system and will be further analysed to identify the underlying unknown toxic principles. Presented at the 28^th Mykotoxin-Workshop, Bydgoszcz, Poland, May 29–31, 2006, and the 47^th spring meeting of the DGPT, Mainz, Germany, April 4–6, 2006 Financial support: Deutsche Forschungsgemeinschaft (Project MU 1716–2)     

Effects of methyl benzimidazoIe-2-yl carbamate on microtubule and actin cytoskeleton inAspergillus nidulans

Summary The effects of methyl benzimidazole-2-yl carbamate (MBC) on microtubule and actin cytoskeleton were analyzed by indirect immunofluorescence and transmission electron microscopy in a wild-type strain and a benomyl-resistant mutant (benA ₁₀) ofAspergillus nidulans. The treatment of the wild-type strain with sublethal doses of MBC not only caused depolymerization of cytoplasmic microtubules (MTs), but also changed the pattern of actin at the hyphal tips. In the MBC-treated hyphae, the actin fluorescence was concentrated at the very tip region of the hypha, whereas in the control hyphae, the actin fluorescence was weak at the very tip and strong below the tip. The dose of MBC used for the wild-type strain did not depolymerize the MTs or modify the actin organization at the apex in the mutant strain, which confirmed that the change in actin distribution in the wild-type strain was due to the disruption of MTs. In the mutant strain, a seven times higher concentration of MBC than in the wild-type strain was required to depolymerize MTs and to alter the actin organization at the apex. The ultrastructural study of the MBC-treated hyphae revealed that the area containing apical vesicles was larger and the number of microvesicles was higher than in control hyphae. These changes probably resulted from the disassembly of MTs and the reorientation of actin cytoskeleton in MBC-treated apexes and suggested that MTs would organize the actin at the apex, which in turn would restrict the vesicle fusion to a narrow area at the hyphal tip. In treated hyphae of both strains without cytoplasmic MTs, mitotic spindles were detected although in lower number and with slightly modified morphology.Abbreviations DAPI 4,6-diamidino-2-phenylindole - DMSO dimethyl sulfoxide - EM electron microscopy - ER endoplasmic reticulum - IIP indirect immunofluorescence - MBC methyl benzimidazole-2-yl carbamate - MTs microtubules     

An asparaginase of Aspergillus nidulans is subject to oxygen repression in addition to nitrogen metabolite repression

Summary Of five amidohydrolase activities subject to nitrogen metabolite repression in Aspergillus nidulans, l-asparaginase shows clearest evidence of also being subject to repression by atmospheric oxygen. Such oxygen repressibility is only evident under nitrogen metabolite derepressed conditions. Asparaginase levels are also considerably elevated by areA300, an altered function allele of the positive acting wide domain regulatory gene areA mediating nitrogen metabolite repression and are drastically reduced by loss of function mutations in areA. A. nidulans has two l-asparaginase enzymes and it has been shown by the use of appropriate mutants that these regulatory effects are exerted on the expression of that specified by the ahrA gene but probably not that specified by the apnA gene. Present address: (until 25 August, 1988) Department of Genetics, University of Georgia, Athens, GA 30602, USA     

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.     

A translocation associated, loss-of-function mutation in the nitrogen metabolite repression regulatory gene of Aspergillus nidulans can revert intracistronically

Summary The areA ^r -18 mutation is a loss-of-function mutation in areA, the positive acting regulatory gene mediating nitrogen metabolite repression in Aspergillus nidulans. It results from a reciprocal translocation which splits the coding region into 5 and 3 moieties. Surprisingly, we have selected rare intracistronic revertants of areA ^r -18. From crosses heterozygous for areA ^r -18 revertant alleles, duplication-deficiency progeny containing two copies of a substantial portion of chromosome IV but lacking part of chromosome III, including the 5 moiety of areA, have been obtained. For all four revertants analysed genetically, growth properties of these duplication-deficiency strains indicate that the reversion events involve the 3 portion of areA and that the 5 portion of areA is unnecessary for the revertant phenotype. This conclusion was directly confirmed for one revertant using Southern blotting. As all four reversion events involve additional chromosomal rearrangements, they probably fuse functional promoters, ribosome binding sites and in frame initiation codons to the 3 portion of the gene. In the course of characterisation of these mutations, new mapping data for a large region of chromosome IV have been generated, and a new reciprocal translocation activating the cryptic regulatory gene areB, whose product can substitute for that of areA, has been identified.     

Effects of Neurospora nuclease halo (nuh) mutants on secretion of two phosphate-repressible alkaline deoxyribonucleases

Various recently isolated nuh mutants of Neurospora crassa (i.e., mutants which show reduced nuclease haloes on DNA-sorbose plates flooded with HCl) were mapped in several new genes or gene clusters and checked for effects on DNA repair and nuclease secretion. Some of them were found to be sensitive to MMS (methylmethane sulfonate) and sterile in meiosis. Release of nuclease activities into filtrates of liquid cultures was analyzed by DEAE-Sepharose chromatography. In the wild type, three alkaline deoxyribonuclease activities (A, B, and C) can be separated after growth in sorbose minimal media [Fraser, M. J. (1979). Nucleic Acids Res. 6: 231]. When strains were grown in phosphate-free DNA sucrose media, high (200-fold derepressed) DNase levels were found, and crude dialyzed filtrates could be chromatographed. Only two peaks were found, namely, those of DNase A, a Ca²⁺-dependent strand-nonspecific endonuclease, and DNase B, a ss-DNA-specific Mg²⁺-dependent exonuclease. Of the nuh mutants analyzed by one or both of these methods, many resembled the wild type. A few showed poor derepression, since their sorbose filtrates were normal, while profiles from DNA media lacked all peaks. These grew variably in liquid media with organic phosphates and probably produced suppressors, as was regularly found for nuc-2. Other mutants, which lacked specific peaks, gave the same results with both methods. One of these, nuh-7, produced no peaks at all but secreted unusually high amounts of protein.This investigation was supported by Operating Grant A2564 from the National Science and Engineering Research Council of Canada.     

Unusual evolutionary conservation of 5S rRNA pseudogenes inAspergillus nidulans: Similarity of the DNA sequence associated with the pseudogenes with the mouse immunoglobulin switch region

Summary AllAspergillus nidulans 5S rRNA pseudogenes known so far are the result of integration of an approx. 0.2-kbp-long DNA sequence into the 5S rRNA genes. This sequence, called block C, is present in at least five copies in theA. nidulans genome and seems to be associated either with 5S rRNA genes or pseudogenes. In contrast to the 78% sequence conservation of the C-block in pseudogenes, the truncated 5 halves of the pseudogenes are very highly conserved (96.9–100%). We postulate that the 5S rRNA pseudogenes are still a subject of concerted evolution. The C-block sequence shows similarity to the switch region of the mouse heavy chain immunoglobulin gene. A characteristic motif GGGTGAG is repeated several times in both sequences; the sequence conservation is 63%.     

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.     

SuppressorssuaC 109 andsuaA 101 ofAspergillus nidulans alter the ribosomal phenotypein vitro

A new homologous, cell-free system for protein synthesis has been devised for use with ribosomes and elongation factors fromAspergillus nidulans. Ribosome preparations from strains with either the suaAlO1 orsuaCl09 mutations have a higher misreading ratio (non-cognate:cognate amino acid incorporation) in the presence of hygromycin than controls. They can be classed as fidelity mutants. These results also prove that the mutations must be in genes coding for ribosomal proteins or enzymes which modify ribosomal proteins post-translationally. Alternatively, the genes could code for translation factors.     

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.     

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.     

A mutation affecting amdS expression in Aspergillus nidulans contains a triplication of a cis-acting regulatory sequence

Summary In Aspergillus nidulans expression of the acetamidase structural gene, amdS, is under the control of at least four regulatory genes including the trans-acting amdA regulatory gene. A cis-acting mutation (amdI66) consisting of an 18 by duplication in the 5 region of the amdS gene results in very high levels of acetamidase activity but only in strains carrying semi-dominant mutations in the amdA gene. In selecting for increased amdS expression in an amdI66 amdA ^– strain, an A. nidulans strain with a mutation in the 5 region of the amdS gene was isolated. The nucleotide sequence was determined of the region containing the mutation, designated amdI666. The mutant strain carries three tandem copies of the 18 by sequence that is duplicated in the amdI66 mutation. Thus, from a strain carrying a duplication of an apparent regulatory protein binding site with little effect on gene expression, a strain has been derived that carries a triplication of the site with consequent major effects on regulation. The multiple copies of regulatory sites present in many genes may have been generated by a similar mechanism.     

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.     

Characterization of phiA, a gene essential for phialide development in Aspergillus nidulans

We have previously identified genes and proteins involved in the fungal response to the Streptomyces-produced antibiotics, bafilomycin B1 and concanamycin A, known inhibitors of V-ATPases. Using mRNA differential display we identified an Aspergillus nidulans gene with 30-fold up-regulated expression in the presence of bafilomycin. This gene, here denoted phiA, and its gene product, were further characterized by targeted gene disruption and immunohistochemistry. Phenotypically, the phiA mutation resulted in reduced growth and severely reduced sporulation. The abnormality could be traced to the phialides, which divided several times instead of forming a single flask-shaped cell. The importance of phiA for phialide and conidium development was supported by immunohistochemistry experiments that showed the protein to be mainly present in these two cell types. Attempts to relate phiA to inhibition of V-ATPases did not result in unambiguous conclusions, but suggest the possibility that changed expression of phiA is correlated with growth arrest caused by inhibited V-ATPases.     

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.     

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.