Multiple cellular roles of Neurospora crassa plc-1, splA2, and cpe-1 in regulation of cytosolic free calcium, carotenoid accumulation, stress responses, and acquisition of thermotolerance.

Phospholipase C1 (PLC1), secretory phospholipase A2 (sPLA2) and Ca(2+)/H(+) exchanger proteins regulate calcium signaling and homeostasis in eukaryotes. In this study, we investigate functions for phospholipase C1 (plc-1), sPLA2 (splA2) and a Ca(2+)/H(+) exchanger (cpe-1) in the filamentous fungus Neurospora crassa. The Δplc-1, ΔsplA2, and Δcpe-1 mutants exhibited a growth defect on medium supplemented with the divalent ionophore A23187, suggesting that these genes might play a role in regulation of cytosolic free Ca(2+) concentration ([Ca(2+)](c)) in N. crassa. The strains lacking plc-1, splA2, and cpe-1 possessed higher carotenoid content than wild type at 8°C, 22°C, and 30°C, and showed increased ultraviolet (UV)-survival under conditions that induced carotenoid accumulation. Moreover, Δplc-1, ΔsplA2, and Δcpe-1 mutants showed reduced survival rate under hydrogen peroxide-induced oxidative stress and induced thermotolerance after exposure to heat shock temperatures. Thus, this study revealed multiple cellular roles for plc-1, splA2, and cpe-1 genes in regulation of [Ca(2+)](c), carotenoid accumulation, survival under stress conditions, and acquisition of thermotolerance induced by heat shock.

Isolation, characterization and application of a cellulose-degrading strain Neurospora crassa S1 from oil palm empty fruit bunch.

BACKGROUND: Oil palm empty fruit bunch (EFB) is a lignocellulosic waste produced in palm oil industry. EFB mainly consists of cellulose, hemicellulose (mainly xylan) and lignin and has a great potential to be reused. Converting EFB to fermentable sugars and value-added chemicals is a much better choice than treating EFB as waste. RESULTS: A cellulase-producing strain growing on oil palm empty fruit bunch (EFB) was isolated and identified as Neurospora crassa S1, which is able to produce cellulases using EFB as the sole carbon source. The strain started to secret cellulases into the medium after 24 h of cultivation at 30°C and reached its maximal cellulase activity at 240 h. Mass spectroscopy (MS) analysis showed that more than 50 proteins were secreted into the medium when EFB was used as the sole carbon source. Among them, 7 proteins were identified as putative enzymes associated with cellulose degradation. The whole cell culture of Neurospora crassa S1 was used to hydrolyze acid-treated EFB, giving a total sugar yield of 83.2%, which is comparable with that (82.0%) using a well-known cellulase producer Trichoderma reesei RUT-C30 (ATCC56765). CONCLUSION: Neurospora crassa S1 is a commercially promising native cellulase producer for EFB hydrolysis especially when the sugars obtained are to be fermented to products that require use of non-genetically engineered strains.

The antifungal activity of the Penicillium chrysogenum protein PAF disrupts calcium homeostasis in Neurospora crassa.

The antifungal protein PAF from Penicillium chrysogenum exhibits growth-inhibitory activity against a broad range of filamentous fungi. Evidence from this study suggests that disruption of Ca(2+) signaling/homeostasis plays an important role in the mechanistic basis of PAF as a growth inhibitor. Supplementation of the growth medium with high Ca(2+) concentrations counteracted PAF toxicity toward PAF-sensitive molds. By using a transgenic Neurospora crassa strain expressing codon-optimized aequorin, PAF was found to cause a significant increase in the resting level of cytosolic free Ca(2+) ([Ca(2+)](c)). The Ca(2+) signatures in response to stimulation by mechanical perturbation or hypo-osmotic shock were significantly changed in the presence of PAF. BAPTA [bis-(aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid], a Ca(2+) selective chelator, ameliorated the PAF toxicity in growth inhibition assays and counteracted PAF induced perturbation of Ca(2+) homeostasis. These results indicate that extracellular Ca(2+) was the major source of these PAF-induced effects. The L-type Ca(2+) channel blocker diltiazem disrupted Ca(2+) homeostasis in a similar manner to PAF. Diltiazem in combination with PAF acted additively in enhancing growth inhibition and accentuating the change in Ca(2+) signatures in response to external stimuli. Notably, both PAF and diltiazem increased the [Ca(2+)](c) resting level. However, experiments with an aequorin-expressing Deltacch-1 deletion strain of N. crassa indicated that the L-type Ca(2+) channel CCH-1 was not responsible for the observed PAF-induced elevation of the [Ca(2+)](c) resting level. This study is the first demonstration of the perturbation of fungal Ca(2+) homeostasis by an antifungal protein from a filamentous ascomycete and provides important new insights into the mode of action of PAF.

Functional characterization of tzn1 and tzn2-zinc transporter genes in Neurospora crassa.

Previous work from our laboratory involved the description of the Neurospora metal transportome, which included seven hypothetical zinc transporters belonging to the ZIP family. The aim of the present study was to make a comparative functional evaluation of two hypothetical zinc transporters named tzn1 (NCU07621.3) and tzn2 (NCU11414.3). Phenotypic analysis of tzn1 and tzn2 mutants and a double mutant (tzn1tzn2) revealed that the deletion of tzn1 causes aconidiation and a greater defect in growth than the single deletion of tzn2. Supplementation with zinc restores growth but not conidiation in tzn1 and tzn1tzn2. TZN1 complemented a zinc-uptake-deficient Saccharomyces cerevisiae mutant (zrt1zrt2) in zinc-deficient conditions, while tzn2 restored growth upon supplementation with zinc (0.05 mM). Furthermore, the Deltatzn1 mutant was found to have severely reduced zinc content indicating that tzn1 functions as a key regulator of intracellular zinc levels in Neurospora crassa. Zinc uptake studies indicate tzn1 is a specific transporter of zinc, while tzn2 transports both zinc and cadmium. Quantitative RT-PCR showed up-regulation of tzn1 (128-fold) under zinc-depleted conditions and down-regulation (>1,000-fold) in zinc-replete conditions. The present study indicates that the zinc transport proteins encoded by tzn1 and tzn2 are members of the zinc uptake system regulated by zinc status in N. crassa.

Developmental expression of two forms of arginase in Neurospora crassa.

N. crassa has two forms of arginase. The physiological role of multiple arginases is not understood. The two forms were shown to be differentially expressed from a single locus (aga) and both proteins are localized to the cytoplasm. The 36-kDa protein was expressed in minimal and arginine supplemented medium, whereas the 41-kDa form was detected only in the presence of arginine. In this study we examined developmental expression of the two arginase transcripts and proteins in conidia and during conidial germination. Two novel observations are revealed, storage of both arginase proteins in conidia and temporal expression of aga transcripts during early germination. To better understand the role of arginase in conidia and the nature of the temporal expression, we examined the effects of related metabolites, arginine, ornithine, proline, glutamate and glutamine on protein storage and temporal expression. These metabolites were used as supplements or sole nitrogen sources. Storage of arginase protein was detected in all conidial samples examined except when glutamate was used as the nitrogen source. The aga temporal RNA expression early in germination was abolished when arginine related metabolites were used as nitrogen sources. The exception to this result is observed with glutamate where temporal expression was seen when glutamate was the sole nitrogen source and abolished with glutamate supplementation. The temporal expression result supports a unique role for arginase in glutamate accumulation early in germination whereas the protein storage result supports the existence of a novel pathway utilizing arginase for glutamate synthesis in asexual spore development.

Time series analysis demonstrates the absence of pulsatile hyphal growth.

Hyphal tip growth has been previously reported as pulsatile, defined as regularly alternating fast and slow rates of extension. The growth of pollen tubes, and hyphae of Neurospora crassa and Saprolegnia ferax were analysed using high spatial and temporal resolution. By using long (100-500 s) records of growth rate, sampled every second, it was possible to apply rigorous statistical analysis of the time series. As previously demonstrated, pollen tubes can show pulsatile growth, detectable with this system. In contrast, hyphal growth rates do not show any evidence of pulsatile growth; instead, growth rates appear to fluctuate randomly. It is concluded that pulsatile growth is not a common feature of hyphal tip growth.

Characterization and expression of the Neurospora crassa nmt-1 gene.

The Neurospora crassa homologue of the yeast no message in thiamine ( nmt-1) gene was characterized. The deduced 342-amino-acid gene product has more than 60% identity with other fungal homologues and 42% similarity to a putative bacterial permease. In addition to three introns disrupting the coding sequence, a differentially spliced intron in the 5' untranslated region was also detected. Unlike other fungi, the N. crassa nmt-1 gene is repressed only 6- to 8-fold by exogenous thiamine concentrations above 0.5 microM; and a high basal level of nmt-1 mRNA persists even at 5 microM thiamine. Immuno-blotting with purified antibodies detected two variants of NMT-1 which differ in size and charge. The more abundant 39-kDa form is more strongly repressed by thiamine than the 37-kDa protein. NMT-1 abundance modulates slowly in response to changes in the concentration of exogenous thiamine, suggesting that N. crassa maintains thiamine reserves in excess of immediate needs. Disruption of the nmt-1 gene demonstrated that it is essential for growth in the absence of exogenous thiamine. NMT-1-deficient strains had a growth rate and colony density which was about 70% of the wild type, despite supplementation with a wide range of exogenous thiamine. These results suggest that the nmt-1 gene plays some other role in addition to thiamine biosynthesis.

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.

Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells.

The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.

31P-NMR-studies on intracellular pH and metabolite concentrations in relation to the circadian rhythm, temperature and nutrition in Neurospora crassa.

Perfused mycelia of Neurospora crassa were analyzed in vivo with 31P-NMR. Both the cytoplasmatic and the vacuolar pH and the concentrations of phosphate metabolites were followed up to 30 h under constant conditions. No circadian changes were detected. However, slight changes in the nutrition or oxygen supply induced distinct changes in the intracellular pH and in the concentrations of metabolites. An increase of temperature from 21 to 43 degrees C lowered the intracellular pH and the metabolite concentrations. Changes in the perfusion rate affect the temperature responses, probably due to different availability of carbon sources and exhaustion of acid catabolites.

Circadian rhythms in Neurospora crassa: membrane composition of a mutant defective in temperature compensation.

The cel mutant of Neurospora, partially blocked in fatty acid synthesis and lacking temperature compensation of its circadian rhythm below 22 degrees C, had a phospholipid fatty acid composition in liquid shaker culture distinctly different from that of a cel+ control strain. During growth, cel+ exhibited a reproducible increase in its linoleic acid level from about 32 to a plateau at 63 mol%, and a corresponding decrease in its linolenic acid level from about 40 to a plateau at 10 mol%. The level of palmitic acid was constant at 19 mol%. In the cel strain, the linoleic acid level was constant at 54 mol% while the palmitic acid level increased from about 12 to about 23 mol%. Supplementation with palmitic or linoleic acids altered the patterns of fatty acid composition of cel, but did not affect the pattern of cel+. Altered fatty acid composition cosegregated with the cel marker. The mitochondrial phospholipids of cel in liquid culture also had abnormal fatty acid composition, as did the whole mycelial phospholipids on solid medium. These results are consistent with the involvement of membrane homeostasis in the temperature compensation of circadian rhythms.

Regulation of amino acid synthetic enzymes in Neurospora crassa in the presence of high concentrations of amino acids.

Ornithine carbamoyl transferase and leucine aminotransferase of Neurospora crassa represent two of many amino acid synthetic enzymes which are regulated through cross-pathway (or general) amino acid control. In the wild-type strain both enzymes display derepressed activities if the growth medium is supplemented with high (mM range) concentrations of L-amino acids derived from branched pathways, i.e. the aspartate, pyruvate, glycerophosphate and aromatic families of amino acids. A cpc-1 mutant strain, impaired in cross-pathway regulation i.e. lacking the ability to derepress, shows delayed growth under such conditions. In the presence of glycine, homoserine and isoleucine various cpc-1 isolates do not grow at all. Derepression of the wild-type enzymes and the retarded growth of the mutant strain can be reversed if certain amino acids are present in the medium in addition to the inhibitory amino acids.

Preparation of functional group analogs of unsaturated fatty acids and their effects on the circadian rhythm of a fatty-acid-deficient mutant of Neurospora crassa.

Functional group analogs of oleic, linoleic and linolenic acids were prepared by coverting their double bonds to dibromo, cyclopropyl, epoxy, methoxy, and, in the case of oleic acid, hydroxy groups. These compounds were supplemented to the bd csp cel strain of the mold Neurospora crassa. The cel mutation confers a partial requirement for saturated fatty acids and, also, perturbs the circadian rhythm of spore formation. For example, the period of bd csp cel's rhythm is dramatically lengthened upon supplementation by natural cis-unsaturated fatty acids. Of the analogs tested, only the monoepoxy, monomethoxy, dibromo, and hexabromo stearic acids gave significant period lengthening. Other analogs, which should have comparable abilities to disrupt lipid bilayer packing, gave no rhythm effect. Further, the inactive di- and tri-methoxystearic acid analogs were incorporated to a greater extent than the active mono-methoxystearic acid. The results do not, therefore, support a direct alteration in membrane "fluidity' as the mode of action of the period-lengthening fatty acids.

Unsaturated fatty acid isomers: effects on the circadian rhythm of a fatty-acid-deficient Neurospora crassa mutant.

The fatty acids oleic, linoleic, and linolenic, each of which has a cis double bond at the delta 9 position, are known to lengthen the circadian period of conidiation (spore formation) of strains of Neurospora crassa carrying the cel mutation. cel confers a partial fatty acid requirement on the organism and has been used to promote incorporation of exogenous fatty acids. To test whether a physical effect imparted by the cis double bonds, such as increased membrane fluidity, is critical for the perturbation of the rhythm, various isomers of these fatty acids were supplemented to the bd csp cel strain. Positional isomers of oleic acid, such as petroselinic (delta 6) and vaccenic (delta 11) acids, and longer-chain isomers, such as eicosenoic (delta 11) and erucic (delta 13) acids, did not lengthen the rhythm. The shorter-chain palmitoleic (delta 9) acid did not give a consistent lengthening of the rhythm; it may be elongated to vaccenic acid. In contrast, gamma-linolenic acid (delta 6,9,12) dramatically lengthened the period. Linoelaidic acid (the trans,trans isomer of linoleic acid) lengthened the period at 22 degrees C, but elaidic acid (the trans isomer of oleic acid) did not. Elaidic acid was shown to exert a lengthening effect, but only at lower temperatures. The data do not support a direct physical action as the source of the fatty acids' "chronobiotic" ability.

Cell-biology of ageing. III. Malondialdehyde as an index of free radical reactions in the early senescent mutants of Neurospora crassa and study of the effect of free radical scavengers on malondialdehyde contents.

Malondialdehyde - a product of lipid peroxidation due to free radical reaction was estimated in the culture filtrates of early senescent mutants of Neurospora crassa and the effects of vitamin E, vitamin C and Sodium selenite (free radical scavengers) in malondialdehyde contents were studied. From the results obtained, it could not be established that increased free radical reaction was the sole factor for the early senescence of all the mutants; and the free radical scavengers had very little effect on the malondialdehyde content of the selected early senescent mutants.

Inositol-limited growth, repair, and translocation in an inositol-requiring mutant of Neurospora crassa.

The biochemical consequences of inositol limitation in an inositol auxotroph of Neurospora crassa have been examined as a means of disclosing the cellular role of inositol. The cellular levels of inositol in the inl mutant were proportional to the concentration of inositol in the growth medium whereas inositol phosphate levels remained relatively constant at about 0.1 mumol/g (dry weight). After 72 h of growth, about 57-fold more protein per milligram (dry weight) was released by the mutant grown on limiting inositol than by the inositol-supplemented control. When the inositol-limited growth medium was osmotically buffered with 1% NaCl, 3% NaCl, or 6% sorbitol, there was about 33, 74, or 54%, respectively, less protein released by the mutant. These results are consistent with cell lysis occurring in the mutant grown on limiting inositol because of a structurally weakened cell wall and membrane deterioration. When sufficient inositol for normal mycelial growth was supplied to an inositol-deficient mycelium, there was within 2 h a rapid incorporation of inositol to 85% of control levels. This incorporation occurred without significant growth by any area of the mycelium. About 10 to 15% of the total cell inositol was translocated forward from the older mycelial areas to the growing tips; only 2 to 5% of the total cell inositol was translocated backward toward the older mycelial areas. Possible mechanisms of translocation are discussed.

Circadian rhythms in Neurospora crassa: effects of saturated fatty acids.

To assess their effects on the conidiation rhythm in Neurospora, 14 saturated fatty acids from 6 to 24 carbons long were used to supplement the bd csp and bd csp cel strains. Both strains express a circadian spore-forming rhythm when grown on solid media; the cel mutation confers a partial fatty acid requirement. Fatty acid supplements from 8 to 13 carbons long lengthened the free-running period of bd csp cel compared with the control value of 21 h; the maximal effect (33 h) was obtained with nonanoic acid (9:0) at a concentration of 5 x 10(-4) M. In contrast, the period of bd csp remained unchanged under all experimental conditions. The short-chain fatty acids (<14 carbons) reduced the rate of advance of the growth front in both strains, compared with unsupplemented controls. However, this inhibition did not appear to be responsible for the lengthened periods in bd csp cel. Nor was direct incorporation of the short-chain (period-lengthening) fatty acids into mycelial total lipids responsible, since such incorporation was not observed. In fact, extensive metabolic conversion of these supplements by both strains was indicated by the disappearance of short-chain fatty acids from the agar media coupled with their absence in mycelial lipids, and by the liberation of (14)CO(2) from cultures supplemented with [1-(14)C]lauric acid (12:0).

Conidiation of Neurospora crassa induced by treatment with natrium fluoride in submerged culture.

A transient treatment of pregerminated conidia of Neurospora crassa with NaF induced young, submerged cultures to prematurely differentiate conidia. The inductive treatment decreased the rate of respiration (with lower RQ), reduced the relative concentration of nucleoside triphosphates, and inhibited leucine incorporation into protein and adenosine incorporation into RNA.

[Studies on the conidial differentiation of "Neurospora crassa" VI.--Functional anomalies of the conditional aconidial mutant "amycelial" (author's transl)]. / Recherches sur la différenciation conidienne de Neurospora crassa VI.--Anomalies fonctionnelles du mutant aconidien conditionnel "amycelial"

Studies using the morphological mutant "amycelial"--a conditional, aconidial strain of N. crassa--have revealed certain consistent functional and biochemical differences from wild type strains showing normal morphology and conidia formation. Thus it was found that the mutant strain showed an abnormal utilization of protein reserves at a time when there is extensive degradation during conidiogenesis in wild type. In addition, the mutant exhibited abnormally low oxygen consumption and free amino acid pools depressed nearly 3/4 with respect to wild type. All of these findings are consistent with earlier work linking conidiogenesis to aerobic oxidative processes and suggest that catabolic reactions particularly regarding protein breakdown may be important to conidiogenesis from the standpoint of the synthesis of new conidial proteins and for the supply of endogenous energy reserves during the process. The aconidial character of "amycelial" could be partially alleviated on solid acetate medium supplemented with amino acids, especially tryptophan or histidine. Conidiation was also induced by cyclic-AMP, even on sucrose medium. A polymer strongly antigenic in rabbits was found to be released into growth medium by the mutant but not by wild type cultures, providing a possible clue to the immediate cause of the abnormal morphology seen in "amycelial". This substance appears to be a glycopeptide containing a few hexoses and amino acids associated with polyphosphate.

Effects of ribonuclease A on amino acid transport in Neurospora crassa.

Incubation of Neurospora crassa conidia with ribonuclease (RNase) A reduces transport of L-phenylalanine by those cells. Under similar conditions, oxidized RNase A, RNase T1, and RNase T2 do not have this effect. Incubation of conidia with active RNase covalently attached to polyacrylamide beads reduces L-phenylalanine transport. This indicates that the site of enzymatic action is at the cell surface. At the lower concentration of enzyme used in this study, incubation with RNase A reduces transport of L-phenylalanine by the general (G) amino acid permease. Increasing the enzyme concentration results in reduction of transport by the neutral aromatic (N)-specific permease. The increased transport activity that accompanies onset of conidial germination is also sensitive to incubation with RNase A. Application of the enzyme to actively transporting cells does not release amino acid transported prior to enzyme addition. Cells cultured on media supplemented with [2-14C] uridine release isotopic activity after RNase A incubation. Analogous treatments with Pronase, RNase T1, RNase T2, or deoxyribonuclease I do not release isotope activity. Pronase treatment does reduce L-phenylalanine transport. Incubation of conidia with RNase A also inhibits germination of those conidia.