Utilization of betaine aldehyde by choline (chol-) mutants of Neurospora crassa

Summary The addition of betaine aldehyde to solid and liquid minimal media supported the growth of two choline-requiring mutants of Neurospora crassa (chol-1 and chol-2). The results were interpreted as evidence for the occurrence of the enzyme choline dehydrogenase in Neurospora crassa.     

Excretion of pyruvate in nickel toxicity in wild type and Ni2+ resistant mutants ofNeurospora crassa

The parent wild strainNeurospora crassa Em 5297a and three Ni²⁺ resistantNeurospora crassa mutants have been shown to excrete pyruvate into the culture medium in Ni²⁺ and Co²⁺ toxicities. Ni²⁺ has a more pronounced effect in this regard. The excretion is progressive with growth inhibition and is abolished by Mg²⁺ in all strains and by Fe³⁺ partially in the Em strain but not inNeurospora crassa Ni^R1. Pyruvate, citrate and malate supplementation reverse growth inhibition caused by excess Ni²⁺, but with concomitant suppression of Ni²⁺ accumulation. It is suggested that one of the features of Ni²⁺ toxicity inNeurospora crassa is a derangement in carbohydrate metabolism at step(s) beyond pyruvate and that this is possibly due to decreased invivo activity of Mg²⁺ dependent processes     

Caracterisation morphologique,chimique et serologique d'un mutant albinos de Neurospora crassa shear et dodge isole de semences de mache (Valerianella olitoria (L.) Poll.)

Résumé Un mutant albinos (sans carotènes) a deNeurospora crassa Shear &Dodge a été isolé de semences de mâche (Valerianella olitoria L.) sur le marché à Genève. Son appartenance à l'espècecrassa est démontrée sur les plans morphologique et sexuel, et il est désignéN. crassa al-Chg a. Il se distingue du type sauvage et d'un mutant albinos (al-2 15.300 a) étudié près du lieu de vente des semences, par la température optimum de croissance. Des tests sérologiques (immunoélectrophorèse) et des séparations de protéines sur gel d'acrylamide ont mis en évidence le degré beaucoup plus élevé de corrélation entre les mutants albinos (al-Chg et al-2) et le type sauvage deN. crassa qu'avec celui deN. sitophila. La valeur taxonomique de ces deux techniques est discutée, de même que l'origine éventuelle du mutant isolé.

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An albino mutant (without carotenes) ofNeurospora crassa Shear &Dodge has been isolated from seeds ofValerianella olitoria L. on sale in Geneva, Switzerland. Its taxonomic position was demonstrated morphologically and sexually. It is described asN. crassa al-Chg a. By its optimal growth temperature it differs from the wild type and from an albino mutant (al-2 15.300 a) studied near the selling place of the seeds. Serological tests (immunoelectrophoresis) and separation of proteins on acrylamide gel have made conspicuous the considerably higher degree of relationship existing between thealbino mutants (al-Chg and al-2) and the wild type ofN. crassa than between these mutants and the wild type ofN. sitophila. The taxonomic value of these methods and the possible origin of the isolated mutant are discussed.

     

Formation and regeneration of protoplasts of wild-typeNeurospora crassa

Trichoderma reesei was grown using purified cell walls ofNeurospora crassa as a primary source of carbon. The resulting culture medium contained an undefined mixture ofN. crassa cell-wall digesting enzymes. Protoplasts (cell lacking wall) were formed when youngN. crassa hyphae were treated withTrichoderma mixture. The vast majority of protoplasts resynthesized cell-wall material when washed free of cell-wall digesting enzyme; of these, about 40% regenerated a mycelium.     

Regulation of methionine biosythesis in Neurospora crassa.

The regulation of serine hydroxymethyltransferase, methylenetetrahydrofolate reductase, and methyltetrahydropteroylpolyglutamate:homocysteine methyltransferase was investigated in Neurospora crassa. Adding choline to the medium decreased the specific activity of these enzymes. Methionine potentiated the choline effect, but, when added alone, was without effect. Neither choline, methionine, nor S-adenosylmethionine appears to be the immediate corepressor of synthesis of these enzymes.Several nonallelic mutants were examined for the enzymes of methionine methyl group synthesis. The formate-requiring mutant for lacks serine hydroxymethyltransferase. The methionine-requiring mutants me-1 and me-8 lack, respectively, the reductase and the methyltransferase. The methionine-requiring mutants me-1, me-6 (folate polyglutamate synthetase deficient) and me-8 were found to have significantly higher serine hydroxymethyltransferase specific activities than did the wild-type strain.     

Characterization of a mutation that causes overproduction of inositol in Neurospora crassa

Summary Slow-growing (inl ^+/-) spontaneous mutants have been isolated from an inositol requiring (inl) strain of Neurospora crassa that produces defective myo-inositol-1-phosphate synthase (MIPS), the enzyme responsible for the production of inositol-1-phosphate from glucose-6-phosphate. The defective enzyme has some residual activity. In the inl ^+/- strain the synthesis of the defective enzyme is enhanced, which enables the strain to grow slowly on minimal medium. The mutation (opi1) responsible for the partial inositol independence segregates independently from the inositol locus, and suppresses the inositolless character by overproduction of defective MIPS. opi1 acting upon the wild type (inl ⁺) allele increases MIPS production and causes inositol excretion.     

An enzymic method for the determination of chitin and chitosan in fungal cell walls

The free and N-acetyl glucosamine contents, serving as a measure of the amounts of chitosan and chitin respectively, were determined in the chitinase hydrolysates of the cell wall of a wild strain ofNeurospora crassa. Chitinase, obtained from cultures ofSerratia marcescens, could hydrolyse the cell wall completely apart from being capable of hydrolysing preparations of chitin and chitosan. The free and N-acetyl glucosamines, released by chitinase hydrolysis, were determined by a modified Morgan-Elson reaction carried out in the presence and absence of acetic anhydride. The method is capable of estimating chitin and chitosan contents in as little as 100 μg of cell wall material.     

B-Vitamins in root exudates of cotton

Summary Cotton field soils fromFusarium wilt-free zone (KPT) and a wilt-sick zone (PLD), and root exudates of diploid and amphidiploid strains of cotton grown in these two soils were analyzed for B-vitamins by microbiological assay technique employing X-ray mutants ofNeurospora crassa andN. sitophila.PLD soil was found to contain choline, pyridoxine,p-aminobenzoic acid, traces of biotin, and inositol. KPT soil was found to contain pyridoxine, choline, thiamine,p-aminobenzoic acid, and traces of biotin and inositol.Thiamine, biotin, pyridoxine, andp-aminobenzoic acid were found in higher amounts in the exudates of diploid cotton strains, especially K 2 and 6186.9 in KPT soil. In addition to the above, choline was also found in the exudates of diploid strains grown in PLD soil.Amphidiploid strains showed comparatively lower amounts of these vitamins in the root exudates with a few exceptions.In the inoculated series, inositol was absent in most cases and the contents of other vitamins were reduced to varying extents compared to the respective healthy controls.Part of Doctoral thesis, University of Madras.     

In vivo absorption spectra ofNeurospora crassa white collar photomutants

White collar (wc) mutants ofNeurospora crassa, which lack several responses to blue light, did not have altered absorption spectra when compared to a control strain. We conclude either that thewc mutants do not lack photoreceptor pigments or alternatively that the photoreceptors are present at levels too low to detect. The results show that carotenoids can be genetically reduced to a level at which they should not interfere with spectrophotometric attempts to detect the blue light receptors.     

Isolation and mapping of fluoropyrimidine-resistant mutants of Neurospora crassa

Summary Growth characteristics of wild type Neurospora crassa on 5-fluorouracil, 5-fluorouridine and 5-fluorodeoxyuridine and methods for selecting mutants resistant to these pyrimidine analogues are described. The mutants are mapped and characterized for resistance to the analogues. Some mutants are found to be allelic with the previously described ud-1 (fdu-1) ad uc-4 genes.Supported by S.R.C. grant GR/A/64655F. Buxton was supported during the period of this work by an S.R.C. Research Studentship     

Comparative analysis of respiratory activity in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and its photoreceptor complex mutants

Cell respiratory activity of protoplasts obtained from the wild type of Neurospora crassa and photoreceptor complex WCC—white collar 1 (wc-1) and white collar 2 (wc-2)—mutants of Neurospora crassa strains was investigated. Respiration inhibition by KCN in the presence of 25 mM succinate was similar in all strains and did not exceed 83–85% against control. The significant induction of KCN-resistant respiratory pathway occurred under 1% glucose oxidation in wc-1 and wc-2 mutants if compared with the wild type strains. The inhibitors of the main (cytochrome) pathway of electron transfer in mitochondria—1 mM KCN and antimycin A (4 μg/ml)—blocked the respiration rate of the protoplasts from N. crassa wild type by 75%, while the cell respiration of wc-1 and wc-2 strains was suppressed by approximately 50%. The specific inhibitor of alternative oxidase—10 mM salicylhydroxamic acid (SHAM)—in combination with the blockers of mitochondrial electron transfer chain caused the total suppression of respiratory activity of protoplasts in all studied strains. It is supposed that an increase of KCN-resistance in WCC mutants under glucose oxidation is connected with alternative oxidase activation as the result of failure in reception and signal transduction of active oxygen species.     

Calcineurin Subunit B Is Required for Normal Vegetative Growth inNeurospora crassa

A series ofNeurospora crassamutants affected in the ability to regulate entry into conidiation (an asexual developmental program) were isolated by using an insertional mutagenesis procedure followed by a screening protocol. One of the mutants isolated by this approach consisted entirely of cells with an abnormal morphology. The mutant produces chains of swollen septated cells. The developmentally regulatedccg-1gene is constitutively expressed in these cells, suggesting that they have entered the conidial developmental program. The insertionally disrupted genecnb-1was isolated by plasmid rescue and found to encode calcineurin B, the regulatory subunit of the Ca²⁺and calmodulin-dependent protein phosphatase calcineurin. The data demonstrate that calcineurin B is required for normal vegetative growth inN. crassaand suggest that thecnb-1mutant is unable to repress entry into the asexual developmental program. The results suggest that Ca²⁺may play an important role in regulating fungal morphology.     

Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of <Emphasis Type="Italic">Neurospora crassa</Emphasis> and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups, in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-(wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction of stress factor signals that promotes reactive oxygen species (ROS) formation in the WCC mutants.     

Circadian period lengths of lipid synthesis mutants (cel,chol-1) of Neurospora show defective temperature,but intact pH-compensation

The influence of extracellular pH on the circadian sporulation rhythm of Neurospora crassa has been investigated for the mutants chol-1 and cel. Both mutants have a defect in the lipid synthesis pathway and require either choline or palmitate, respectively, as supplements for normal growth. The chol-1 and cel mutants also show an impaired temperature-compensation when growing on minimal medium. We investigated the possible correlation between loss of temperature- and pH-compensation in cel and chol-1 similar to the correlation found earlier for the frq⁷ mutant. Our results show that the cel and the chol-1 mutants, although defective in temperature-compensation have an intact pH-compensation of their circadian rhythms. At present, the products of the frq-locus are the only components of the clock that affect the sporulation rhythm of Neurospora both through pH- and temperature-compensation.     

Isolation and characterization of a laccase gene fromPodospora anserina

The genome of the filamentous ascomycetePodospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene ofNeurospora crassa. One of these regions contains a gene (lac2) encoding a protein that displays 62% identity with theN. crassa laccase. In shaken cultures,lac2 mRNA is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. Addition of aromatic xenobiotics (guaiacol, hydroquinone, benzoquinone) to the medium during the growth phase results in a rapid, drastic and temporary increase in the abundance oflac2 mRNA. The promoter region oflac2 contains two sequences which display complete homology with the eukaryotic Xenobiotic Responsive Element and two sequences homologous to the eukaryotic Antioxidant Responsive Element. The identity and function of the laccase encoded bylac2 are discussed.     

Superoxide Dismutase and Catalase Activities in Carotenoid-Synthesizing Fungi Blakeslea trispora and Neurospora crassa Fungi in Oxidative Stress

The addition of menadione into the medium during cultivation ofNeurospora crassa in the dark activated its constitutive superoxide dismutase. Exposure to light not only activated superoxide dismutase and catalase, but also increased the content of neurosporaxanthin. Superoxide dismutase activity in the mixed (+/–) cultures of Blakeslea trispora synthesizing -carotene in the dark was much lower than that inNeurospora crassa. The superoxide dismutase activity and catalase activity further decreased in oxidative stress with a parallel increase in the content of -carotene. Our results indicate that neurosporaxanthin possesses photoprotective properties in Neurospora crassa. In Blakeslea trispora (+/–) fungi, -carotene acts as a major antioxidant during inactivation of enzymes that detoxify reactive oxygen species.     

The effect of copper on histidine biosynthesis inNeurospora crassa

The inhibition of growth of a wild strain ofNeurospora crassa by Cu²⁺ is counteracted by histidine, histidine methyl ester, histidinol and Mn²⁺. In the presence of Cu²⁺, the total free amino acid content decreased by 30%. The decreased free amino acid pools of arginine, histidine and tyrosine were restored on the addition of Mn²⁺. Histidinol phosphate phosphatase showed a decrease in activity in the presence of Cu²⁺. This inhibition was reversed on the addition of excess Mn²⁺. The data suggest that copper toxicity in the mould is due to suppression of histidine biosynthesis.     

Lipids associated with microconidial differentiation in a mutant ofNeurospora crassa

Thepeach-fluffy-cot mutant ofNeurospora crassa produces neither macroconidia nor ascospores but does differentiate microconidia after a defined length of time. Changes in the composition of sterols, sterol esters, triglycerides, free fatty acids, and phospholipids were followed during vegetative growth and differentiation of microconidia. The changes in free sterols before and during microconidial differentiation indicate a change in lipid metabolism associated with differentiation. Free sterols and sterol esters accumulated in the developing microconidia, but decreased rapidly during microconidial maturation. The fatty acid components remained relatively unchanged except for a significant increase in linoleic acid. The linoleic acid change might be associated with the development of microconidia or it might simply be a reflection of the NADP-deficiency common in many morphological mutants ofN. crassa.     

The effect of thiourea on ureide metabolism in Neurospora crassa

The wild-type strain of Neurospora crassa Em 5297a can utilize allantoin as a sole nitrogen source. The pathway of allantoin utilization is via its conversion into allantoic acid and urea, followed by the breakdown of urea to ammonia. This is shown by the inability of the urease-less mutant, N. crassa 1229, to grow on allantoin as a sole nitrogen source and by the formation of allantoate and urea by pre-formed mycelia of this mutant. In the wild strain (Em 5297a) thiourea is tenfold more toxic on an allantoin medium than on an inorganic nitrogen medium; allantoin as well as urea counteract thiourea toxicity in the allantoin nitrogen medium. This selective toxicity of thiourea for the mould utilizing allantoin nitrogen does not, however, result in an impairment of allantoin uptake, allantoinase activity or the formation of urea from allantoin. The only process affected by thiourea is the synthesis of urease; urea antagonizes this effect of thiourea in N. crassa.     

Induced expression of the Aspergillus nidulans QUTE gene introduced by transformation into Neurospora crassa

Summary Theqa-2 gene ofNeurospora crassa encodes catabolic dehydroquinase which catabolizes dehydroquinic acid to dehydroshikimic acid. TheQUTE gene ofAspergillus nidulans corresponds to theqa-2 gene ofN. crassa. The plasmid pEH1 containing theQUTE gene fromA. nidulans was used to transform aqa-2 ^– strain ofN. crassa. In Southern blot analyses, DNAs isolated from these transformants hybridized specifically to theQUTE gene probe. Northern blot analyses indicated thatQUTE mRNA was produced in the transformants. The functional integrity of theQUTE gene inN. crassa was indicated by transformants which had regained the ability to grow on quinic acid as sole carbon source. Enzyme assays indicated that the specific activities of catabolic dehydroquinase induced by quinic acid in the transformants ranged from 4% to 32% of that induced in wild-typeN. crassa. The evidence that theQUTE structural gene ofA. nidulans is inducible when introduced into theN. crassa genome implies that theN. crassa qa activator protein can recognize, at least to a limited extent, DNA binding sequences 5 to theQUTE gene.