Localization of the beta-glucosidases in Neurospora crassa

The beta-glucosidases (EC 3.2.1.21) of Neurospora crassa were studied with respect to their location in conidia and young mycelia. Aryl-beta-glucosidase of conidia was nearly equally divided between extracellular and bound activity. Bound aryl-beta-glucosidase was almost all available to substrate. An induction procedure was used to maximize both beta-glucosidases in 4 to 6-hr cells. Aryl-beta-glucosidase was entirely bound but still mostly (90%) detectable, whereas cellobiase was mostly internal and cryptic. A freeze-thaw cycle or treatment with phenethyl alcohol or deoxycholic acid made the cellobiase detectable without releasing it from the cell. A 10 to 20% increase in cell-bound aryl-beta-glucosidase could be obtained by this treatment. Dilute HCl (0.1 n) destroyed the patent aryl-beta-glucosidase but not the cryptic aryl-beta-glucosidase or the cryptic cellobiase activity in intact cells. This suggested that most aryl-beta-glucosidase activity was exterior to the cell membrane but still within the mural space. The thermal stability of patent aryl-beta-glucosidase and released cellobiase was found to be higher than in corresponding cell-free extracts. Measurements of K(m) suggested a slightly lower affinity for substrate p-nitrophenyl-beta-d-glucopyranoside by the enzymes in intact cells compared to enzymes in extracts.     

Intracellular Localization of Nitrate Reductase in Neurospora crassa

Nitrate reductase was localized in mycelial cells of Neurospora crassa by immunohistochemical labeling with ferritin. The enzyme is found in the cell wall-plasmalemma region and in the tonoplast membranes.     

Effects of Auxin and Gibberellin on Elongation of Young Hyphae in Neurospora crassa

IAA, 2,4-D and GA₃ promoted the elongation of young hyphae inNeurospora crassa at the optimum concentrations of 10^–6,10^–6 and 10^–4 M, respectively. The effects of IAAand GA₃ were additive. (Received June 17, 1983; Accepted December 22, 1983)     

Osmotic Effects on the Polyamine Pathway of Neurospora crassa

Davis, R. H., and Ristow, J. L. 1995. Osmotic effects on the polyamine pathway of Neurospora crassa. Experimental Mycology 19, 314-319. In bacteria, mammals, and certain plants, the induction of the polyamine synthetic enzyme, ornithine decarboxylase (ODC), and the accumulation of its product, putrescine, follows osmotic manipulations of cells. In at least some of these cases, this response is indispensable for survival. We wished to determine whether the polyamine pathway of Neurospora crassa was regulated in response to hyper- or hypoosmotic conditions. Unlike ODC of most other classes of organisms, the N. crassa enzyme and the accumulation of putrescine appears to be relatively indifferent to these conditions, either during sudden transitions or in steady-state. We conclude that other mechanisms of osmotic adjustment or tolerance have evolved in N. crassa and perhaps other fungi that obviate the need for putrescine accumulation.     

End-Product Regulation of the Tryptophan-Nicotinic Acid Pathway in Neurospora crassa

The regulation of the tryptophan-nicotinic acid pathway in Neurospora crassa was examined with mutants (nic-2, nic-3) which require nicotinamide for growth. The accumulation of N-acetylkynurenin and 3-hydroxyanthranilic acid by these mutants served to estimate the level of function of the early reactions in the pathway. In still cultures, maximal accumulation occurred with media containing growth-limiting amounts of nicotinamide; the accumulation of intermediates was almost negligible with nicotinamide in excess. Only nicotinamide and closely related compounds which also supported the growth of these mutants inhibited the accumulation of intermediates. The site of inhibition was assessed to be between tryptophan and kynurenin (or N-acetylkynurenin). The synthesis of N-acetylkynurenin was examined in washed germinated conidia suspended in buffer; the level of N-acetylkynurenin-synthesizing activity was inversely related to the concentration of nicotinamide in the germination medium. The addition of large amounts of nicotinamide to suspensions of germinated conidia did not affect their N-acetylkynurenin-synthesizing activity. Formamidase activity, kynurenin-acetylating activity, and gross tryptophan metabolism in germinated conidia was not influenced by the concentration of nicotinamide in the germination medium. The results obtained indicate that the site of inhibition by nicotinamide is the first step in the pathway, the tryptophan pyrrolase reaction. The data are interpreted as nicotinamide or a product thereof, such as nicotinamide adenine dinucleotide, acting as a repressor of the formation of tryptophan pyrrolase in N. crassa.     

Association and dissociation of Neurospora crassa cytoplasmic ribosomes.

Dissociation and association factors of ribosomal particles were detected in extracts from Neurospora crassa at different stages of growth. The dissociation factor was easily released into the S100 supernatant fraction, whereas the association factor remained bound to the ribosomes.     

Conidiation in Neurospora crassa

Summary Conidiation in Neurospora crassa has been studied in vivo by time-lapse microphotography and shown to be most generally (in aerial, dry conditions) a budding-fission process. Such a two-phase process is characterized by an initial basifugal budding of proconidial elements which are then secondarily separated as maturing conidia by interconidial septa. Dry macroconidia of Neurospora are thus blasto-arthrospores, i.e. blastospores basifugally budded on conidiophores and secondarily disarticulated from the proconidial chain as arthrosporal elements. Inception and median splitting of the interconidial septum have been electron microphotographed.In the vegetative hyphae, ethanol dehydrogenase has been cytochemically detected by oxidative assay and demonstrates a dense, uniform distribution of activity except at the hyphal tips. In the conidiating hyphae, the ethanol dehydro-genase becomes less dense in distribution, especially in the budding apices. Cytochrome oxidase activity, localized in the mitochondria, is confined in the subapical zone of vegetative hyphae while at the initiation of conidiation it becomes dispersed throughout the proconidial buds.     

Effect of Auxin and Gibberellin on Conidial Germination in Neurospora crassa II. "Conidial Density Effect" and Auxin

Conidial germination in liquid shake culture of Neurospora crassawas affected by the number of conidia in the medium (conidialdensity effect) with the optimum density being around 2?10⁶conidia/ml. The conidial density effect at 2?10⁴ and 2?10⁵ conidia/mlwas eliminated by the addition of auxin, IAA or 2,4-D with theoptimum concentrations of IAA and 2,4-D being around 10^–6M. IAA and 2,4-D had no effect on the effect at 2?10⁷ conidia/ml.An active substance(s) for conidial germination which was relatedto the conidial density effect was detected in the cell-freefiltrate of the germination medium, and was found to be non-dialyzableand thermolabile. At the early stage of germination, the concentrationof active substance(s) in the medium increased in proportionto the conidial density and reached a supraoptimum amount forgermination at 2?10⁷ conidia/ml. IAA (10^–6M) enhancedthe concentration. Endogenous auxin concentrations in filtratesof the germination media containing 2?10⁵, 2?10⁶ and 2?10⁷ conidia/mlwere 5.8?10^–12, 4.6?10^–11 and 1.7?10^–10M IAAequivalent, respectively. The conclusion reached was that auxinmay control conidial germination with mediation by the activesubstance(s). (Received June 8, 1982; Accepted September 27, 1982)     

Postreplication repair in Neurospora crassa

Summary Changes in the molecular weight of nascent DNA made after ultraviolet (UV) irradiation have been studied in the excision-defective Neurospora mutant uvs-2 using isotopic pulse labeling, alkaline gradient centrifugation and alkaline filter elution. Both the size of nascent DNA and the rate of incorporation of label into DNA was reduced by UV light in a dose dependent manner. However, this DNA repair mutant did recover the ability to synthesize control-like high molecular weight DNA 3 hours after UV treatment, although the rate of DNA synthesis remained depressed after the temporary block to elongation (or ligation) had been overcome. Photoreactivation partially eliminated the depression of DNA synthesis rate and UV light killing of cells, providing strong evidence that the effects on DNA synthesis and killing were caused by pyrimidine cyclobutane dimers. The caffeine inhibition repair studies performed were difficult to quantitate but did suggest either partial inhibition of a single repair pathway or alternate postreplication DNA repair pathways in Neurospora. No enhancement in killing was detected after UV irradiation when cells were grown on caffeine containing plates.     

Cyanide-Resistant Respiration in Neurospora crassa

Cell respiration in wild type and poky was studied as part of a long-term investigation of cyanide-resistant respiration in Neurospora. Respiration in wild type proceeds via a cytochrome chain which is similar to that of higher organisms; it is sensitive to antimycin A or cyanide. Poky, on the other hand, respires by means of two alternative oxidase systems. One of these is analogous to the wild-type cytochrome chain in that it can be inhibited by antimycin A or cyanide; this system accounts for as much as 15% of the respiration of poky f(-) and 34% of the respiration of poky f(+). The second oxidase system is unaffected by antimycin A or cyanide at concentrations which inhibit the cytochrome chain maximally. It can, however, be specifically inhibited by salicyl hydroxamic acid. The cyanide-resistant oxidase is not exclusive to poky, but is also present in small quantities in wild type grown under ordinary circumstances. These quantities may be greatly increased (as much as 20-fold) by growing wild type in the presence of antimycin A, cyanide, or chloramphenicol.     

Protease secretion in Neurospora crassa

Secreted and constitutive intracellular proteases of $\text{Neurospora crassa}$ differ with regard to inhibitor sensitivity, substrate specificity, isoelectric points and other properties. Upon the induction of protease secretion the enzymes released from the mycelium are formed $\text{de novo}$ as demonstrated by density labelling with D₂O. Vesicles which contain the constitutive intracellular proteases are, therefore, not involved in the secretion of proteases.     

Rubidium transport in Neurospora crassa

Rb⁺ transport in low-K⁺ cells of Neurospora crassa is biphasic, transport at millimolar Rb⁺ being added to a transport process which saturates in the micromolar range. Both processes exhibit Michaelis-Menten kinetics, but in the micromolar phase the kinetic parameters depend on the K⁺ content of the cell (the lower the K⁺ content the lower the K_m and the higher the V_max). Normal-K⁺ cells, suspended in a buffer with millimolar K⁺, do not present Rb⁺ transport in the micromolar range. Millimolar transport in these cells presents kinetics which depend on the K⁺ in buffer (the higher the K⁺ the higher the K_m), although the K⁺ content of the cells is constant. Na⁺ inhibits competitively Rb⁺ transport in low-K⁺ and normal-K⁺ cells, but, even when the differences between the Rb⁺K_m values are more than three orders of magnitude, the apparent dissociation constant for Na⁺ is the same, and millimolar, in both cases.     

Biochemical and Histochemical Localization of Invertase in Neurospora crassa During Conidial Germination and Hyphal Growth

The intracellular localization of Neurospora invertase, an enzyme partially secreted and partially retained by Neurospora at the cell periphery, was investigated. A cell wall fraction was isolated, to which 24% of the cell-bound invertase was firmly attached. A sensitive osmiophilic stain for invertase was developed and used in conjunction with the technique of indirect immunofluorescence to follow the pattern of invertase localization during the development of Neurospora from the germination of conidia to the mature hypha. These studies revealed that: (i) conidial invertase was uniformly distributed along the cell periphery; (ii) growing hyphal tips of germinating conidia showed pronounced invertase activity as the rest of the conidial cell wall lost its peripheral activity; (iii) hyphae in early log-phase growth had strong enzyme activity associated with the cell wall, and in late log phase the activity became associated with the plasma membrane and points where new hyphal branches were being formed; and (iv) hyphae in early stationary phase had strong fluorescence at incipient branching points, in "dots" close to the plasma membrane, and in the cytoplasm.     

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-¹⁴C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [¹⁴C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [¹⁴C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [¹⁴C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [¹⁴C]geranylgeranyl pyrophosphate from [1-¹⁴C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.