Glucose metabolism and dimorphism in Mucor.

Mucor racemosus fermented glucose to ethanol, carbon dioxide, and glycerol. When this fungus was grown anaerobically in either the yeast or mycelial form, the catabolism of glucose was very similar. Yeast cells shifted to aerobic conditions maintained a high flux of glucose carbon through the glycolytic and pentose phosphate pathways. Mycelial cells grown aerobically catabolized glucose in a manner consistent with a respiratory metabolism. Although there was no consistent pattern of glucose metabolism in the mycelial form of Mucor, growth in the yeast form consistently was correlated with a high flux of glucose carbon through the catabolic pathways.     

An immunological study of the induction of polygalacturonases in Botrytis cinerea

Abstract There was no consistent increase with time in the total activity of extracellular polygalacturonase (PG), relative to mycelial dry weight, when Botrytis cinerea was grown on a modified Czapek Dox liquid medium containing galacturonic acid or glucose. However, preparative isoelectric focusing identified a peak of activity between pH 3.7 and 6.3 only when the fungus was grown in the presence of galacturonic acid. This activity was attributed to the induction of exo-PGs (exo-PGI and/or exo-PGII). Antisera raised against one of two endo-PGs (endo-PGI) reacted positively and specifically with endo-PGI and endo-PGII on Western blots, and both of these isozymes were present in Western blots when the fungus was grown in the presence of glucose or galacturonic acid after 48 h in vitro. Plate-trapped antigen-ELISA confirmed that the endo-PGs were constitutively expressed.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of Mucor circinelloides var. lusitanicus INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of gamma-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Influence of carbon and nitrogen sources on glutathione catabolic enzymes inCandida albicans during dimorphism

The effect of carbon sources, glucose and sucrose, and nitrogen sources such as ammonia, glutamate andl-citrulline on the activities of glutathione metabolic enzymes has been studied. Yeast and mycelial cells were used to identify changes in activity levels of glutathione reductase (GSSGR), glutathione transferase (GST), glutathione peroxidase (GPX) and -glutamyl transpeptidase (GGT). Enzyme activities from cells grown in sucrose media were lower than in glucose media regardless of the enzyme tested, morphological form, or the growth interval. In all enzymes except GST, activity was higher in yeast form than in mycelia, regardless of nitrogen source, with lower activity from 24 to 72 h than at 96 h. In citrulline media, yeast form showed the maximum GST, GGT, and GPX activity. In ammonia-amended media, mycelia showed maximum activity in GGT, whereas in glutamate media, mycelia showed the maximum activity in GST. Also, the type of nitrogen source had no effect on GPX activity in the mycelial form. Finally, changing the nitrogen source showed no significant effect on GSSGR activity, either in the yeast or mycelial form.     

Metabolic characteristics and lipid composition of yeastlike cells and mycelium of <Emphasis Type="Italic">Mucor circinelloides</Emphasis> var. <Emphasis Type="Italic">lusitanicus</Emphasis> INMI grown at a high glucose content in the medium

It is shown that the fungus Mucor circinelloides var. lusitanicus INMI grown under aerobic conditions in a medium with a high glucose concentration (20%) is capable of both yeastlike and mycelial growth. In the mycelium, the activity of NAD-dependent isocitrate dehydrogenase was more than twice as high as in yeastlike cells, whereas the isocitrate lyase activity was lower. A number of significant differences were found in the lipid composition of the cells of two different morphological variants. Yeastlike cells contained more polar lipids and free fatty acids and less principal reserve lipids (triacylglycerides) than mycelial cells; the content of γ-linolenic acid and the degree of lipid unsaturation were significantly lower in these cells than in the mycelium. In yeastlike cells, glycolipids composed the bulk of polar lipids; the proportion of phospholipids (primarily phosphatidylserine, phosphatidylcholine, phosphatidylethanolamine, and cardiolipin) was lower. The relationship between cellular metabolism and the lipid composition of fungal cells of different morphotypes grown at high concentrations of glucose, one of the main inducers of dimorphic growth, is discussed.     

Carbohydrates,invertase activity,growth and dimorphism inSporisorium reilianum

Sporisorium reilianum, the fungus that causes sorghum head smut, was grown with sucrose, lactose, trehalose or raffinose in liquid suspension or on a solid medium. Liquid culture media were analyzed for hydrolysis products of these carbohydrates to determine extracellular enzyme activity of the fungus. Increased amounts of glucose and fructose in the culture medium ofS. reilianum grown with sucrose or raffinose indicated that invertase (-fructofuranosidase, 3.2.1.26) activity was present. No evidence of extracellular galactosidase or trehalase activity was found. Enhanced sporidial colony formation on carbohydrates that can be hydrolyzed to hexoses, and specific forms of mycelial growth on lactose, trehalose or on a carbohydrate-deficient medium might suggest that mycelial growth is a way of foraging for food sources. However, the rapid and profuse mycelial growth on the host cell wall glycoprotein appears to be in response to abundant food supply (probably of a different type). Therefore availability of different kinds of carbon sources in the environment of the growing fungus might determine dimorphism and associated pathogenesis byS. reilianum.Technical Article No: 30699 from the Texas Agricultural Experiment Station.     

Synthesis and regulation of extracellular keratinase in three fungi isolated from the grounds of a gelatin factory,Jabalpur, India

Chrysosporium queenslandicum, Graphium penicilloideus andScopulariopsis brevicaulis were grown on various supplemented basal salts media to compare keratinase induction, activity and repression. All three fungi can utilize keratin as a sole source of carbon, nitrogen and sulfur. Total keratinase activity inC. queenslandicum andS. brevicaulis, was not repressed by supplementation of keratin-containing medium with glucose, ammonium or sulfate. The production of keratinase activity was not derepressible in keratin-free media. Keratin utilization commenced before the detection of significant extracellular keratinase activity which was always associated with mycelial growth.     

Effects of Oxygen and Glucose on Energy Metabolism and Dimorphism of Mucor genevensis Grown in Continuous Culture: Reversibility of Yeast-Mycelium Conversion

Mucor genevensis was grown in both glucose-limited and glucose-excess continuous cultures over a range of dissolved oxygen concentrations (<0.1 to 25 muM) to determine the effects of glucose and the influence of metabolic mode (fermentative versus oxidative) on dimorphic transformations in this organism. The extent of differentiation between yeast and mycelial phases has been correlated with physiological and biochemical parameters of the cultures. Under glucose limitation, oxidative metabolism increased as the dissolved oxygen concentration increased, and this paralleled the increase in the proportion of the mycelial phase in the cultures. Filamentous growth and oxidative metabolism were both inhibited by glucose even though mitochondrial development was only slightly repressed. However, the presence of chloramphenicol in glucose-limited aerobic cultures inhibited mitochondrial respiratory development but did not induce yeast-like growth, indicating that oxidative metabolism is not essential for mycelial development. Once mycelial cultures had been established under aerobic, glucose-limited conditions, subsequent reversal to anaerobic conditions or treatment with chloramphenicol caused only a limited reversal (<35%) to the yeast-like form. Glucose, however, induced a complete reversion to yeast-like form. It is concluded that glucose is the most important single culture factor determining the morphological status of M. genevensis; mitochondrial development and the functional oxidative capacities of the cell appear to be less important factors in the differentiation process.     

Dimerization of 2,6-dimethoxyphenol by Aspergillus flavus: evidence for the reaction occurring close to mycelia

Aspergillus flavus grown on 2,6-dimethoxyphenol as sole carbon source produced tetramethoxy-p-dibenzoquinone by a free radical mechanism. The product was identified by H-nmr and ms. Scanning electron microscopy and light microscopy were used to follow the growth of mycelia and the attachment of crystals to the mycelial surfaces. Formation of dimer was inhibited by the presence of glucose in the medium.     

Mycelial antineoplastic activity of Agaricus blazei

Basidiocarp of Agaricus blazei (=Agaricus brasiliensis; =Agaricus subrufescens) is used as teas or capsules due to its antineoplastic effect but there are few reports of using mycelium for this purpose. The objective of this study was to evaluate the antineoplastic activity on sarcoma 180 cells implanted in mice of two forms of preparation of the mycelium from two A. blazei strains grown in culture medium with different concentrations of isolated soy protein. Mycelia were grown in Pontecorvo medium with different concentrations of isolated soybean protein (ISP). Mycelial hot water extract, moistened mycelial powder, hot water extract of green tea, Ifosfamida^® (ifosfamide drug), and saline solution were administered daily by gavage in mice with sarcoma 180 cells to evaluate antineoplastic activity. It was concluded that antineoplastic activity was the same for both strains, except when used as moistened mycelial powder, which rules out the use of mycelial powder in capsules. Mycelial hot water extract had high antineoplastic activity with lower metabolic demand on the spleen and maintenance of normal blood parameters. Mycelial growth in different ISP concentrations had the same antineoplastic activity. Also the vegetative mycelium was as effective as the basidiocarp for sarcoma 180 tumor inhibition. Green tea was as effective as mycelial hot water extract.     

Filamentous fungal growth assay: correlation between [U-14C] glucose uptake and dry weight determinations

The filamentous fungus Trichophyton mentagrophytes was grown in [U-14C] glucose supplemented nutrient broth. Growth was monitored in 200 microliter microcultures and 5 ml macrocultures by measuring the incorporation of 14C into trichloroacetic acid-insoluble macromolecules and in macrocultures by dry weight determination. Adjustment of the specific activity of the [U-14C] glucose medium supplement from 1080 muCi/mmole to 108 muCi/mmole decreased the rate of isotope uptake and prolonged availability so that growth beyond 36 h could be monitored radiometrically. The dry mycelial weight of the fungus was directly proportional (r = 0.995) to the amount of isotope incorporated. Isotope uptake in microcultures and macrocultures was kinetically identical. These data clearly indicate that the uptake of 14C by T. mentagrophytes can be used to monitor fungal growth accurately in both macrocultures and microcultures.     

Derepressible proteolytic activity in homokaryotic hyphae ofSchizophyllum commune

Proteolytic activity produced bySchizophyllum commune homokaryons was derepressed upon starvation of plate-grown cultures for nitrogen. Decreasing thel-asparagine concentration in the growth medium resulted in slower growth (as total protein), alteration of colonial morphology, and an increase in mycelial and extracellular protease specific activity against hide-powder azure. In comparisons between colonies grown on minimal medium (1 g/literl-Asn), 0.1 ×l-Asn, 0.01 ×l-Asn, and Nol-Asn medium, mycelial proteolytic specific activity increased 2- to 8-fold. Mycelial specific activity also generally increased as the colonies aged. In addition, substantial increases, up to 100-fold, in extracellular protease specific activity occurred under conditions of nitrogen deprivation. This derepression could result in increased mycelial protein turnover and, potentially, proteolysis of extracellular substrates.     

Optimized production of lignolytic manganese peroxidase in immobilized cultures of <Emphasis Type="Italic">Phanerochaete chrysosporium</Emphasis>

Manganese peroxidase (MnP) is a key enzyme involved in the lignolysis of white-rot fungi. The purpose of this study is to investigate the effect of immobilization and culture conditions on MnP production in cultures of Phanerochaete chrysosporium grown on polyurethane foam. Higher concentrations of foam and lower levels of spore inoculums resulted in the formation of scattered mycelial pellets, increased autolysis of chlamydospore-like cells (a reservoir of MnP), and a higher activity of MnP. Even though MnP was a secondary metabolite, the addition of 5 times more glucose and diammonium tartrate, as carbon and nitrogen sources, resulted in a 4 fold increase in the dry cell mass. However, MnP activity decreased under these conditions to less than half, due to the formation of increasingly dense pellets and the inhibited lysis of chlamydospore-like cells.     

Optimization of some culture parameters and properties of β-glucosidase and β-xylosidase from Penicillium funiculosum NRRL – 13033

Penicillium funiculosum NRRL 13033 produced β-glucosidase and β-xylosidase activities when grown on wheat straw. The addition of some inducers (individually or in combination) to the fermentation medium were tested for the production of both enzymes. The relation of mycelial bound enzyme to cell free enzyme was studied during incubation period of fermentation. The optimum activity of β-glucosidase and β-xylosidase were found to be in the pH 4.5 using phosphate-citrate buffer at 50°C for 60 min and at 55°C for 40 min respectively. β-Glucosidase lost about 40% of its original activity by heating to 65°C for 60 min, while, β-xylosidase activity was found to be nearly stable with the same treatment. Both enzyme activities were greatly inhibited when 1.0% (w/v) of xylose and glucose were added to the assay mixture.     

Glycolysis in Ustilago maydis

The kinetic parameters of the 10 glycolytic enzymes and glycolytic fluxes were determined for the first time in Ustilago maydis. Enzyme activities in yeast grown in minimal medium and harvested in the stationary stage were twofold higher than those from yeast grown in rich medium. In contrast, in yeast harvested in the exponential stage, the enzyme activities were higher in cells grown in rich medium. Phosphofructokinase activity was the lowest in the four culture conditions analyzed, suggesting that this enzyme is a flux-controlling step in U. maydis glycolysis. The V(max) and K(m) values of hexokinase and pyruvate kinase were similar under all conditions. The results revealed that U. maydis aldolase belongs to the class II type of metalo-aldolases. 3-Phosphoglycerate mutase (PGAM) activity was 2,3-bisphosphoglycerate cofactor independent, which contrasted with the cofactor dependency predicted by the amino acid sequence alignment analysis. Pyruvate was secreted by U. maydis yeast in the presence and absence of external glucose. The glycolytic enzyme activities in the U. maydis mycelial form were similar to those found in yeast, except for one order of magnitude higher phosphofructokinase and PGAM activities, thus suggesting differences in the glycolysis regulatory mechanisms between the two cellular forms.     

The effect of alkanes on the physiology and metabolism of the entomopathogenic fungus,Isaria fumosorosea

The influence of different alkanes on spore morphology, glyoxlate pathway enzyme activities, total lipid contents and fatty acid composition of Isaria fumosorosea were investigated under laboratory conditions. Fungal spores grown on different alkanes showed higher germination and mycelial growth when compared to control. A strong induction of glyoxlate cycle enzymes in cell-free extracts was observed for cells grown on different alkanes when compared to glucose and control. Higher activities of glyoxlate cycle enzymes were observed for cells grown on alkanes when compared to other treatments. Even numbered fatty acids accounted for the majority of fatty acid production with a significant increase in relative amounts of linoleic acid and palmatic acid observed for conidia grown on alkanes. These results indicate that addition of alkanes to culture media can be a tool to pre-induce metabolic adaptations that can facilitate successful infection of insect host by entomopathogenic fungi.     

Growth of Trichoderma harzianum and Myceliophthora thermophila in palm oil sludge

Uncentrifuged palm oil mill sludge (POS) diluted to about 50% (v/v in tap water) supports good mycelial growth of Myceliophthora thermophila and Trichoderma harzianum. Both of the selected fungi are non-toxic to mice. After 24 h M. thermophila grown in batch culture in POS yielded 28.6 g/l of mycelial biomass with biological oxygen demand (BOD) and chemical oxidation demand (COD) reductions of 72% and 74% respectively. T. harzianum yielded 24.4 g/l of mycelial biomass with BOD and COD reductions of 67% and 68% respectively. The crude protein of the mycelial biomass of M. thermophila and T. harzianum was twice that of untreated POS. T. harzianum showed amylolytic activity while M. thermophila was cellulolytic and lipolytic.     

A preliminary study of the applicability of sweet potatoes for mycological fat production

Summary For mycological fat production,Aspergillus nidulans andPenicillium lilacinum were grown on media made up of crushed and boiled sweet potatoes. Mycelial mats containing a fair amount of fat were obtained, namely, 15 % in case ofAspergillus and 24 % in case ofPenicillium. Media containing an external supply of glucose gave rise to increase in percentage fat in mycelium, but the amount of mycelium and consequently the total fat content decreased.Addition of sodium nitrate gave heavier mycelial mats but the total fat content could not be raised.Addition of potassium phosphate or magnesium sulphate or both to the sweet potato medium gave rise to heavier mycelial mats but with lower fat content.     

Carbohydrate Storage in the Entomopathogenic Fungus Beauveria bassiana

The entomopathogenic fungus Beauveria bassiana was grown in 1% (wt/vol) gelatin-liquid media singly supplemented with a monosaccharide (glucose or fructose), a disaccharide (maltose or trehalose), a polyol (glycerol, mannitol, or sorbitol), or the amino sugar N-acetyl-d-glucosamine. The relative contributions of the carbohydrate, protein, and water contents in the fungal biomass were determined. Carbohydrates composed 18 to 42% of the mycelial dry weight, and this value was lowest in unsupplemented medium and highest in medium supplemented with glucose, glycerol, or trehalose. Biomass production was highest in liquid cultures supplemented with trehalose. When liquid cultures were grown in medium supplemented with 0 to 1% (wt/vol) glucose, trehalose, or N-acetyl-d-glucosamine, there was an increase in the biomass production and the contribution of carbohydrate to mycelial dry weight. Regardless of the glucose concentration in the culture, water content of the mycelia remained about 77.5% (wt/wt). Mycelial storage carbohydrates were determined by capillary gas chromatography. In gelatin-liquid medium supplemented with 1% (wt/vol) glucose, B. bassiana stored glycogen (12.0%, wt/dry wt) and the polyols mannitol (2.2%), erythritol (1.6%), glycerol (0.4%), and arabitol (0.1%). Without glucose, B. bassiana stored glycogen (5.4%), mannitol (0.8%), glycerol (0.6%), and erythritol (0.6%) but not arabitol. To our knowledge, this is the first report of carbohydrate storage in an entomopathogenic fungus, and the results are discussed in relation to other fungi and the potential implications to commercial formulation and insect-fungus interactions.     

Production of Fungal Mycelial Protein in Submerged Culture of Soybean Whey

Various soybean whey media were tested as substrate for seven species of fungi in submerged culture. Very little mycelial growth was obtained with Morchella hybrida, Collybia velutipes, Cantharellus cibarius, and Xylaria polymorpha. Agaricus campestris failed to grow. Tricholoma nudum and Boletus indecisus showed the greatest rate of growth and production of mycelial protein and the best utilization of soybean whey solids, with much shorter incubation times compared with those of the other species. T. nudum developed as spheres having diameters of about 5 to 8 mm, instead of the usual slurry or yeastlike form, in the presence of added ammonium acetate. B. indecisus always developed as spheres. Mycelial yields and production of protein by T. nudum greatly decreased with the addition of more than 1% glucose to soybean whey, whereas with B. indecisus the yield of protein almost doubled when up to 3% glucose was added. The effect of minerals on mycelial growth was determined. With soybean whey concentrated to 50%, the rate of mycelial growth of T. nudum was nearly doubled, but protein content of mycelia was greatly reduced. Mycelial growth and yield of protein of B. indecisus grown in concentrated whey were increased greatly. About 4 to 6 g of mycelial protein per liter can be obtained from fermentation in soybean whey, depending upon the medium used. Utilization of soybean whey by fungal fermentation may have economic value in whey disposal and in the production of products of high protein content.