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Changes in both respiratory pathways and mitochondrial structure of Neurospora crassa occurred under conditions of microcycle conidiation. Upon heat-treatment at 46°C, conidia developed a highly cyanide-insensitive, hydroxamate-sensitive respiration associated with morphological alterations in mitochondrial membranes; such changes were time-dependent. When heat-treated conidia were shifted down to 25°C, the alternate, hydroxamate-sensitive respiration decreased significantly, paralleling the recovery of well-cristated mitochondria with an electron-dense matrix in the germ tubes. The decrease in hydroxamate-sensitivity was associated with two periods of increase in cyanide sensitivity corresponding to the events of germination and precocious proconidial budding. [ABSTRACT FROM AUTHOR]

In conidia of Neurospora crassa germinating at 25°C, DNA synthesis measured by incorporation of tritiated adenosine reaches a maximum soon after the outgrowth of the germ tube (6-7h after inoculation). In conidia heat-treated at 46°C (for 15h), a maximum of incorporation of the DNA precursor occurs already 1h after inoculation, then the incorporation progressively declines until the end of the heat-shock. When such conidia are shifted to 25°C, a maximum of DNA synthesis occurs during the development of the presumptive conidiophore as at the outgrowth of normal germ tubes. This wave of DNA synthesis is followed by a second maximum of DNA synthesis, occurring only in the microcyclized cultures, when the premature differentiation of proconidia takes place. Prevention of this second wave of DNA synthesis with hydroxyurea or 5-fluorodeoxyuridine respectively reduces or fully inhibits such induced conidial differentiation. [ABSTRACT FROM AUTHOR]

We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase ( qa-2) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth. [ABSTRACT FROM AUTHOR]

Virus-like particles were observed in thin sections of vegetative hyphae of the Chytridiomycete Allomyces arbuscula strain Burma 1A. These particles were extracted, partially purified by sucrose density gradient ultracentrifugation, and characterized as ribonucleoprotein. No virus-like particles were obtained from extracts of a control strain. [ABSTRACT FROM AUTHOR]

We have purified microtubule-associated proteins from Neurospora crassa on the basis of heat stability and affinity to calmodulin. Two proteins of molecular masses 170 kDa and 190 kDa have been partially purified. A third protein of 145 kDa was purified almost to homogeneity, and we present evidence that this protein is a specific substrate for a Ca2+/calmodulin-dependent protein kinase. The purified 170-, 190-, and 145-kDa proteins induce the assembly of microtubules from purified porcine brain tubulin. We demonstrate that all three proteins are microtubule-associated proteins on the basis of an in vitro microtubule-binding assay. [ABSTRACT FROM AUTHOR]

The enzymatic production of sulfur from l-cysteine was studied in young dormant α-spores of Phomopsis viticola. Cysteine aminotransferase (CAT) and mercaptopyruvate sulfurtransferase (MST) activities could be responsible for the production of endogenous elemental sulfur (S) in α-spores. l-Cysteine was first deaminated, with production of β-mercaptopyruvate, by the CAT. The β-mercaptopyruvate produced is successively desulfurated by the MST with production of sulfur and pyruvate. Deaminase activity was recovered principally in the cytoplasmic fraction, whereas desulfurase activity was recovered mainly in the mitochondrial fraction. l-Cysteine and S sharply affected the respiratory activity, the ATP content, and suppressed germination of α-spores. In contrast, reduced glutathione did not affect these metabolic parameters. Production of S by enzymatic degradation of l-cysteine could be responsible for the inhibitory action of this amino acid. We suggest that CAT and MST, by their capacity to produce sulfur or S, plays a key role in regulation of morphogenetic processes of Phomopsis viticola. [ABSTRACT FROM AUTHOR]

Endogenous elemental sulfur (S°) was measured in dormant α‐spores of Phomopsis viticola Sacc. (ATCC 44940) from young (25‐day‐old) and aging (105‐day‐old) cultures grown on malt extract agar medium enriched with [35S]‐MgSO4. Endogenous S° from the mitochondrial fraction, and the lipid and aqueous cytoplasmic fractions of young and aging α‐spores were purified by column chromatography followed by thin-layer chromatography. The purity of mitochondrial pellets were checked by the catalase (EC 1.11.1.6) and acid phosphatase (EC 3.1.3.1) activities. Activities of the mitochondrial enzymes NAD+‐isocitrate dehydrogenase (EC 1.1.1.41) and cytochrome c oxidase (EC 1.9.3.1) were also measured to determine the distribution of the endogenous S° between mitochondria and cytoplasm. In young dormant α‐spores, endogenous S° was mostly found in the cytoplasmic lipid reserves, which were mainly phospholipids. The mitochondrial fraction of these young α‐spores contained ca 10% of the total endogenous S°, whereas in aging α‐spores stored for 105 days the endogenous S° was mainly (ca 90%) localized in the mitochondrial fraction. This accumulation of S° in mitochondria of aging α‐spores was correlated with a sharp decrease in phospholipid reserves, endogenous and exogenous respiratory activities, ATP concentration, uptake of sulfate, and NAD+‐isocitrate dehydrogenase and cytochrome c oxidase activities. These metabolic changes were correlated with an irreversible loss of germination capacity which leads to the natural death of P. viticola α‐spores. During the first min of the breaking of dormancy, the young α‐spores possess a 7.3‐fold capacity to reduce exogenous S° with production of hydrogen sulfide, as compared to the aging α‐spores. In young α‐spores the production of hydrogen sulfide was almost totally inhibited by 40 μM antimycin A (92%), and strongly inhibited by 2mM azide (75%) and by 15 μM 2,4‐dinitrophenol (63%). Our work suggests that endogenous S° plays a key role in the regulation of the dormancy and aging processes of α‐spores of P. viticola. [ABSTRACT FROM AUTHOR]

Mitochondria from young dormant α spores of Phomopsis viticola Sacc. (ATCC 44940) were isolated by grinding and differential centrifugation. They presented a good integrity of their inner and outer membranes as measured by biochemical assays. Electron microscopic analysis revealed an homogenous population. The highest respiratory activities were observed with NADH and ascorbate + tetra‐methyl‐p‐phenylenediamine (TMPD). Malate stimulated the oxidation of pyruvate, citrate or α‐ketoglutarate. The coupling of respiration to oxidative phosphorylation appeared at the time of spore germination. The respiratory activities of mitochondria isolated from young dormant α spores of P. viticola were strongly inhibited by S°. The sensitivity of mitochondrial oxidation of different substrates (NADH, pyruvate + malate, succinate and ascorbate + TMPD) to S° was heterogenous and indicated multiple‐site action. Thus preincubation of mitochondria with 30 μM S° before addition of substrates fully prevented NADH oxidation (>98%), and strongly inhibited oxidation of pyruvate + malate (85%), succinate (60%) and ascorbate + TMPD (74%). S° inhibited more rapidly the oxidation of succinate than that of other substrates. In the presence of dithiothreitol (DTT), S°‐inhibited oxidation of all substrates (except ascorbate + TMPD) could only be transiently and weakly reestablished. The inhibitory action of S° on the oxidation of NADH, pyruvate + malate and succinate was higher than that observed with sulfhydryl group reagents such as mersalyl, Hg‐acetate or p ‐ chloromercuribenzoate. In contrast to S° these SH‐group reagents could not inhibit oxidation of ascorbate + TMPD. S°, by its dual capacity to oxidize the SH‐groups and to self‐reduce, probably at the level of cytochrome c oxidase, could produce a modification of the oxidation state of the respiratory complexes thereby disturbing the electron flux. [ABSTRACT FROM AUTHOR]

In Allomyces arbuscuIa Butl., strain Bali, the ratio of protein to dry weight remained constant in exponentially growing but decreased in differentiating cultures. The adenylate pools (ATP, ADP, AMP) and energy charge which integrates them, increased during zoospore germination and stabilized around 0.9 during differentiation. The level of ATP increased early during the induction of zoosporangia for up to 1 h and then declined. The ADP and AMP remained low for most of the time except for a transient increase in ADP (first 30 min induction). The energy charge was low in spores. The rate of turnover of proteins, during growth and differentiation was more or less similar for up to 1.5 h after transfer. Subsequently very little turnover of proteins occurred in the growing plants. In differentiating plants, the rate of degradation was maintained and by the end of the 4 h experimental period 30% of the vegetative proteins were degraded. The intracellular ammonium showed a peak between 30 to 60 min of induction and was higher in the differentiating mycelia than in actively growing plants, while the glutamate pool remained around 1 μmol (mg protein)-1 in both types of plants. The physiological role of these protein degradation products is discussed. [ABSTRACT FROM AUTHOR]

Cytochrome oxidase (EC 1.9.3.1) activity of conidia of wild-type Neurospora crassa was detected cytochemically at the ultrastructural level by 3,′-diaminobenzidine (DAB) reaction during heat treatment at 46 °C and after shift down to 25 °C. At 46 °C the decrease in the enzyme activity was shown to be time-dependent (0-10 hours) in the progressively overswelling conidia. Gradual recovery of the DAB reaction occurred in mitochondria of conidia outgrowing conidiogenous germ tubes (microcycle conidiation) when shifted down to 25 °C. Inactivation of cytochrome oxidase was efficiently prevented during heat treatment in the presence of Tween 20. Such conidia no longer overswelled but still required shift down to 25 °C to produce germ tubes with delayed conidiogenous ability. [ABSTRACT FROM AUTHOR]

An escape reaction of the hyphae from virus-containing strain Bali 1 of A. arbuscula was detected when intra- and interspecific Allomyces cultures were opposed on solid media. Hyphae of this virus-containing strain proliferate into witch-broom structures only detectable on solid media in which bunches of rhizoid-like structures remain more superficial than those of the control cultures, suggesting that metabolism of the Bali 1 strain is altered into a more aerobic type. [ABSTRACT FROM AUTHOR]

Pregerminative polarization of the cytoplasmic content of a spore following its awakening by water imbibition is ultrastructurally featured by a 2-step sequential process: (i) general partition into a vacuole-enriched pole endowed with increased turgor pressure opposite to an energized pole of clustered mitochondria; such mitochondrial positioning would predetermine the bulging site of the germ tube by optimizing the chance event of (ii) physical contact between that plasma membrane site with the membrane of a few of the most peripherically located mitochondria of the cluster. In such a membrane interaction, the plasma membrane would presumably be electrically depolarized by mitochondrial protons vectorially extruded towards its negatively charged inside face acting as proton sink. That primary spatio-physical event would set off a train of events, the first being a local acidification of the thereby gelified cytosol excluding mitochondria while attracting vesicles into this prospective hyphal tip. It is further suggested that the primary axiation of the germinative conidium can be controlled by slight changes in the peripheral O tension guiding the mitochondrial cluster towards the elected membrane site of germ tube outgrowth. [ABSTRACT FROM AUTHOR]

Thus, from the gene to the character, and back from the typical character (standard conidium) to the gene, we have traced a few of the biosynthetic pathways which must obligatorily be triggered (switched on or off) to effect the transition from the relatively undifferentiated vegetative hyphae through the fertile, conidiogenous hyphae to the differentiated cells, the conidia. There is good promise that further, deeper studies will finally reveal Neurospora to have one of the most highly integrated control systems for eucaryotic sporogenesis when considered as a model of cell differentiation. [ABSTRACT FROM AUTHOR]

Enzyme activities have been measured and compared at several stages of the development of Neurospora crassa i.e. from free conidia (inoculum) to conidiated mycelia grown on sucrose versus acetate (poor versus highly conidiogenous) media. Glucose-6-phosphate dehydrogenase (G-6-PDH) and NADP nucleotidase (NADPase) show inverse activity-time curves, both on sucrose and acetate media. G-6-PDH has its higher activity at the preconidiating stage while NADPase progressively increases to reach its maximal value in the mature conidia. Malate dehydrogenase (MDH) has a higher activity in extracts from acetate compared to sucrose cultures; in both conditions, maximal MDH activity corresponds with the initiation of conidiation. Malate synthetase (MS) has a delayed activity which is much higher in acetate extracts. Succinate dehydrogenase (SDH) is more active in sucrose than acetate extract with a sharp activity peak just preceding conidiation proper. Isozymes of G-6-PDH and MDH, as well as total soluble proteins from extracts of sucrose versus acetate cultures have been compared after their separation on polyacrylamide columns. [ABSTRACT FROM AUTHOR]

Copyright of Archiv für Mikrobiologie is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

β-carotene has been identified as the major carotenoid pigment in two Ascomycetous fungi, the parasitic Epichloë typhina (with restriction to the sexual stroma and additional presence of traces of α-carotene) and the ascocarp of Helotium citrinum. In the Basidiomycete Calocera viscosa, cryptoxanthin appears as the second major carotenoid to β-carotene. Neurosporene has been found as the major carotenoid in the dull yellow Cantharellus infundibuliformis. This partially saturated polyene is accompanied by traces only of lycopene whereas a reverse neurosporene/lycopen ratio is apparently present in the related, yellow Cantharellus lutescens. No carotenoids but quinone-like reacting pigments were detected in the grey Cantharellus cinereus and the orange red Guepinia helvelloides. [ABSTRACT FROM AUTHOR]

Copyright of Protoplasma is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The gametes of Allomyces javanicus exhibit a nuclear cap essentially made of ribonucleoprotein with sulphydryl groups. When dipped in phosphate buffered solutions of boric acid M/20-M/400, the male and female gametangia of Allomyces liberate giant male and female gametes. Such giant gametes exhibit either a single regular nuclear cap (with 2 or 3 nuclei) or a massive and irregular basophilic formation (with 4-10 nuclei). Boric acid may interfere with ribonucleic acid, essential constituent of the nuclear cap, during the cleavage of the gamete-units in the gametangia. [ABSTRACT FROM AUTHOR]

Morphogenesis of carotene-containing male gametangia of the gametophytic generation of Allomyces macrogynus and A. arbusculus is selectively disturbed by heavy, subtoxic doses of diphenylamine (1/60,000 DPA) which fully inhibit carotenogenesis. DPA-effects on growth, carotenogenesis, and conidial differentiation have been studied in Neurospora crassa. The rate of growth on Fries agar medium (measured in growth tubes) is reduced ⅓ with DPA 1/80,000 and ¾ with DPA 1/40,000. In Fries liquid medium, growth is less or not affected by similar doses of DPA. In these conditions, 1/40,000 DPA strongly inhibits biosynthesis of carotenoids but requires the additional presence of Fe+2 for effective activity al the concentration 1/80,000. Conidial differentiation, normally accompanied by a prominent increase of the acidic carotenoids and the spirilloxanthin, is suppressed by 1/40,000 DPA or 1/80,000 DPA + Fe which have an especially strong inhibitory effect on these "conidial oxycarotenoids". A marked accumulation of phytoene has been defected in such sterile mycelia of Neurospora. Residual DPA bas not been detected with the nitric acid coloration test in the phytoene fraction of wild type extracts. Moreover, phytoene and DPA are absent in the insaponifiable extract of au albino mutant of Neurospora crassa also grown on a DPA medium. These facts and the relation DPA-Fe point lo a bio-oxidative destruction of DPA which might well act as an antioxidant progressively destroyed in preventing the oxidation of the precursors of the caroteuoids. [ABSTRACT FROM AUTHOR]