Inducible α-glucosidase of Mucor rouxii: Effects of dimorphism on the development of the wall-bound activity

Some properties of the inducible α-glucosidase system of Mucor rouxii were investigated. This enzymatic activity was induced after resuspending glucose-grown cells in a maltose-supplemented medium. The wall-bound activity of α-glucosidase was determined by using intact cells in the enzymatic assay; this activity represented from 80 to 90% of the total activity present in the induced cells. The addition of glucose before, or during, the induction period repressed α-glucosidase synthesis. α-Glucosidase induction was tested under aerobic and anaerobic conditions. It was found that the enzyme synthesis and the appearance of wall-bound activity were not affected by changing the gaseous environment. On the other hand, it was observed that anaerobically grown yeast-like cells were much less efficient than aerobic mycelia to develop wall-bound α-glucosidase activity. This could explain earlier observations about the incapacity of M. rouxii to utilize maltose as a substrate for anaerobic growth. This idea was strengthened by the fact that, if an anaerobic culture was induced to develop under a mycelial morphology by adding to the medium the chemical agent EDTA, these cells also acquired the capacity to grow on maltose and concomitantly possessed wall-bound α-glucosidase activity. The relevance of the structure of the cell wall on the capacity of M. rouxii to metabolize maltose is discussed.     

Changes in the content of cyclic AMP and cyclic GMP during the development of Xenopus laevis.

The levels of the three major DNA-dependent RNA polymerases (enzymes I, II and III) present in the dimorphic fungus Mucor rouxii have been investigated during the transition from yeast-like cells to mycelial growth. Increases in the specific activity of crude extracts were observed at 2 h and at 6 h after induction of mycelium formation by aeration of yeast-like cells. These increases could be attributed to changes in the specific activities of enzymes I and II. Alterations were also found in the relative amounts of enzymes I and II: prior to aeration, 31% of the total polymerase activity of crude extracts was present as enzyme I; after 2 h of aeration, the specific activity of this enzyme doubled and the relative amount increased to 64% of the total activity. After 6 h of aeration, the relative amounts of enzymes I and II were 25 and 65%, respectively, and the specific activity of enzyme II had nearly doubled. The amounts and specific activities of enzyme III did not change significantly during the transition.     

Isolation and Characterization of a Mycelial Cytochrome aa3-Deficient Mutant and the Role of Mitochondria in Dimorphism of Mucor rouxii

Salcedo-Hernandez, R. and Ruiz-Herrera, J. 1993. Isolation and characterization of a mycelial cytochrome aa₃-deficient mutant and the role of mitochondria in dimorphism of Mucor rouxii. Experimental Mycology 17, 142-154. We isolated several mutants of Mucor rouxii unable to utilize triphenyltetrazolium hydrochloride as a final electron acceptor. These mutants did not grow in nonfermentable substrates, and their growth rate was severely reduced in aerobiosis compared to growth of wild type; anaerobic growth was unaffected. Mutation of one selected strain was traced to a decreased level and alteration in the properties of cytochromes aa₃. The fact that the mutant displayed normal mycelial morphology suggests that there is no direct relationship between dimorphism and the operation of the cytochrome respiratory chain. This conclusion was strengthened by the observation that different inhibitors of the latter did not induce yeast-like development. A unitary hypothesis is proposed suggesting that dimorphic Mucorales possess a morphogenetic switch linked to a metabolic O₂ or redox sensor, distinct from the late segments of the respiratory chain. In some species such as Mucor rouxii, the function of the sensor to turn off polarized growth would require a further block brought about by CO₂ or catabolic repression.     

Two alternative pathways for substrate assimilation byMucor circinelloides

Transition of an oleaginous strain ofMucor circinelloides from mycelial to yeast-like form was studied in conditions favoring lipogenesis. In culture media with citric acid as the sole carbon source at concentrations of 7.5 and 10 g/L (C/N ratio of 26 and 35, respectively), the mold accumulated significant quantities of lipid. At a higher citric acid concentration liposynthesis was inhibited and the fermentation mechanism decreased the high substrate concentration in the culture media. Under these conditions yeast-like morphogenesis was observed. In the yeast-like cells, biosynthesis of linoleic and γ-linolenic acid was inhibited. In contrast, no significant changes were observed in the biosynthesis of palmitoleic acid whereas the concentration of oleic acid was increased in the storage lipids of yeast-like cells.     

Stimulation of fermentation and yeast-like morphogenesis in Mucor rouxii by phenethyl alcohol

The germination of fungal spores into hyphae was inhibited by concentrations of phenethyl alcohol (PEA) from 0.05 to 0.3%. Spores of Mucor formed budding spherical cells instead of filaments. These cells were abundant in cultures of Mucor rouxii at 0.22% PEA, provided that the carbon source was a hexose at 2 to 5%. Morphology was filamentous with xylose, maltose, sucrose, or a mixture of amino acids. Removal of PEA resulted in the conversion of yeast-like cells into hyphae. PEA did not inhibit biosynthesis of cytochromes or oxygen uptake, but it stimulated CO₂ and ethyl alcohol production. PEA had no effect on the rate of oxygen uptake, but it inhibited the oxidative-phosphorylation activity of mitochondria. These results suggested that growth inhibition by PEA could result from uncoupling of oxidative phosphorylation and that, in Mucor, yeast-like morphology and fermentation were linked.     

The induction of mycelial development inAureobasidium pullulans (IMI 45533) by yeast extract

Germ tube and subsequent mycelial development from yeast-like and swollen cells ofAureobasidium pullulans (IMI 45533) was induced by yeast extract in defined liquid medium. This morphogenetic transition was dependent on inoculum size; pH effects were not involved and once mycelial development was induced in the cells it continued even in the absence of yeast extract. The progeny of mycelium and future generations were unaffected by yeast extract. Cessation of germination was not due to any obvious medium changes but appeared to be partly due to the production of a germination inhibitor, which could also be produced by control cells grown in the absence of yeast extract.     

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.     

Regulation of ornithine decarboxylase activity in Mucor bacilliformis and Mucor rouxii

Ornithine decarboxylase (ODC) from Mucor bacilliformis and Mucor rouxii was studied. Enzymatic activity was maximal at pH 7.2–7.4 and at 30°C. The K_m was 0.17 mM for the M. bacilliformis enzyme. Putrescine was a competitive inhibitor of ODC with a K_i of 2–3 mM. Enzymatic activity was undetectable in sporangiospores but increased rapidly during the first stages of spore swelling, reaching the highest levels during germ tube or bud emergence, and then decreased. Incubation at 30°C inhibited spore germination in M. bacilliformis and prevented development of ODC activity. More ODC activity was present in mycelial than in yeast cells. Morphological transition of yeast cells into hyphae by an anaerobic-aerobic shift induced a rapid and transient increase in ODC activity. Similar results were obtained when the morphogenetic transformation of M. rouxii was induced by CO₂ elimination in an anaerobic environment. Transfer of mycelial cells to anaerobiosis resulted in a rapid decrease in enzyme activity. Changes in ODC activity were accompanied by a change in the pool of polyamines. The possible role of ODC in growth and cell differentiation in Mucor is discussed.     

Effects of triazoles on fungi: V. Response by a naturally tolerant species,Mucor rouxii

The responses ofMucor rouxii to propiconazole, and in some cases etaconazole, with respect to lipid metabolism were compared with those ofAspergillus ochraceus andRhizopus stolonifer which exhibit higehr sensitivity to this triazole by factors of 50 and 10, respectively. Propiconazole inhibited the C-14 demethylation of lanosterol in each of the species tested, which resulted in a dose-related decrease in ergosterol and increase in C-14 methyl sterols. The principal C-14 methyl sterol that accumulated with inhibitor treatment was 24-methylene dihydrolanosterol. The tolerance ofM. rouxii could not be explained by reduced inhibitor uptake, alteration of the inhibitor binding site, or detoxification through metabolism since C-14 methyl sterols accumulated in mycelium treated with 2.0 μg/ml propiconazole, a concentration 40 times less than that required for 50% growth inhibition and at which no growth inhibition was detected in this species, and one that gave over 50% inhibition ofA. ochraceus. Other alterations in lipid metabolism that accompany treatment with sterol inhibitors in relatively sensitive species, i.e., accumulation of free fatty acids and increase in linoleic acid (C_18:2), were not observed inM. rouxii orR. stolonifer, but they were found inA. ochraceus. The results of this study suggest that the quantitative and perhaps the qualitative nature of the requirement for sterols may be different inM. rouxii, and perhaps other tolerant Mucorales, than in the more sensitive fungi.     

Regulatory role of δ-aminolevulic acid dehydratase in the dimorphic fungus Mucor rouxii

• 1.1. With the aim of finding a possible relationship between the known dimorphism phenomenon existing in the fungus Mucor rouxii and the biosynthesis of respiratory pigments, the activity of aminolevulic acid synthetase (ALA-S) and ALA-dehydratase (ALA-D) was studied in crude extracts and in 15,000 g supernatants of both mycelium and yeast-like cells.
• 2.2. The activity of ALA-S was unusually high (3 nmol ALA/hr/mg protein) compared with that reported for other tissues and did not vary with the fungus morphology.
• 3.3. Instead, ALA-D specific activity was found to be 16.5 nmol PBG/hr/mg protein in mycelium extracts, that is 7-fold greater than that measured in the yeast-like morphology (2.6 nmol PBG/hr/mg protein).
• 4.4. It was of importance to determine the activity levels of ALA-D along with the morphogenic transition from yeast to mycelium. It was observed that the greatest change and enhancing of specific activity occurred 2 hr before the emergence of the germ tubes and was held constant up to the complete development of mycelium.
• 5.5. Both hyphae formation and enhancement of ALA-D activity were diminished when cAMP was added to the culture shifted from the anaerobic atmosphere to air.
• 6.6. These findings and preliminary studies on the characterization of M. rouxii ALA-D indicate that this enzyme plays a regulatory role in porphyrin biosynthesis in this fungus as well as a key function in the characteristic morphogenic transition.

     

Protein composition of purified chitosomes ofMucor rouxii

Different centrifugation conditions were compared for purification of chitosomes (microvesicles containing chitin synthetase). Isopycnic centrifugation of crude chitosome samples from yeast cells ofMucor rouxii on sucrose density gradients, in a vertical rotor, yielded chitosomes of higher purity than before. About 90–96% of the chitin synthetase in purified chitosomes was zymogenic. We estimated that the chitosome population of the yeast form ofM. rouxii comprises a miniscule portion (0.17%) of the total cell protein. The polypeptide composition of purified chitosomes was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining. Prominent polypeptide bands were found at 16, 18, 28, 30, 32, 44, 47, and 55 kDa. The 55-kDa polypeptide was most conspicuous. There were also minor bands at 25, 26, 42, 60, 67, and 80 kDa. Our findings show that highly purified populations of chitosomes consist of microvesicles with a characteristic size range, buoyant density, and a complex polypeptide composition.     

Production in Mice of Ascitic Fluid Containing Antibodies against Plant Lectins

Saccharomycopsis lipolytica developed mycelial cells in media containing both olive oil and bovine milk casein. Olive oil could be replaced by other lipids including triolein, oleic acid, linoleic acid and oleyl alcohol. On the other hand, bovine milk casein could be replaced by a soybean fraction and meat extract, but not by casamino acids or individual common amino acids. The mycelial development was inhibited with a deficiency of magnesium sulfate and ferric chloride or with the addition of cysteine and reduced glutathione.

The mycelial development began after 8 hr from the start of cultivation and the mycelial cell ratio was maximum after 20 hr. Mycelial cells and yeast-form ones were separated from each other on the basis of cellular specific gravity and this method was used to determine the mycelial cell ratio in the present study.     

Change of lipid composition of Zygosaccharomyces rouxii after transfer to high sodium chloride culture medium

The change of the lipid composition was investigated in Zygosaccharomyces rouxii cells transferred from NaCl-free to 2 M NaCl medium. After the transfer thesterol and phospholipid contents increased, and phospholipids with higher oleic acid contents and higher negative charge increased. These changes of membrane lipid may be required for the adaptation of Z. rouxii cells to a high NaCl environment.     

Stability of the Plasma Membrane in Saccharomyces rouxii and Its Relationship to Glucose Tolerance

The stability of spheroplasts from the osmotrophic yeast Saccharomyces rouxii was studied in buffered solutions of mannitol and glucose. The plasma membranes from cells grown in high glucose concentrations were more stable to osmotic lysis than were membranes from cells grown in lower glucose concentrations. Mannitol was a better osmotic stabilizer than glucose, except when the cells were grown in a high glucose concentration. Spheroplasts from a glucose tolerant-deficient mutant were much less stable than the corresponding spheroplasts from the parent strain, especially when suspended in glucose solutions. These results suggest an involvement of the plasma membrane in the glucose-tolerant mechanism of S. rouxii.     

Hemagglutinins of some Aspergilli

Hemagglutinins for human red blood cells have been found in hot-water soluble mycelial extracts of a strain of Aspergillus flavus and two mutant strains of A. parasiticus. The agglutinin from one strain of A. parasiticus was specific for blood group A cells while the other two agglutinins were non-specific. With the A. flavus strain, the greatest hemagglutination activity (HA) was found at 10 days for the mycelial extract, and at 12 days for culture fluid preparations. More agglutinin was produced by fungi grown on sucrose than on d-glucose as carbon source. Solubilities in ammonium sulfate solutions and protein and carbohydrate analyses show that the agglutinins from the mycelial extract and culture fluid preparation are different. The mycelial agglutinin was inhibited by a number of different sugars, many of which possess common stereochemical features.     

Specificity of DNA Methylation Changes During Fungal Dimorphism and Its Relationship to Polyamines

We utilized our modification of the amplified fragment length polymorphism technique for the determination of changes occurring in the DNA methylation patterns during the dimorphic transition of the fungi Mucor rouxii, Yarrowia lipolytica, and Ustilago maydis. To determine the specificity of differential methylation in regards to dimorphism, we obtained the yeast-like form of the three fungi under conditions that induced mycelial growth, by addition of 1,4-diaminobutanone (DAB), an inhibitor of ornithine decarboxylase in the case of M. rouxii and Y. lipolytica. In an odc null mutant of U. maydis, repression of the dimorphic transition was brought about by limitation in the amounts of exogenous putrescine. Yeasts from the three fungi thus obtained conserved a significant number of the differential DNA fragments with the methylation pattern displayed by normal yeasts, indicating their true correlation with dimorphism. Our results also confirm a role of polyamines in differential DNA methylation and fungal dimorphic transition.     

Further studies on cyclic adenosine 3':5'-monophosphate protein kinase from dimorphic fungus Mucor rouxii

In this paper, cyclic adenosine-3′:5′-monophosphate-dependent protein kinase from yeast-like cells of Mucor rouxii is characterized. A scheme of partial purification is described together with K_m for ATP (15 μm), histone (0.2 mg/ml), half-maximal activation constant for cyclic AMP (30 nm), and dissociation constant for the binding of cyclic AMP (40 nm). This enzyme is similar to type II protein kinases in two main aspects: the elution position in DEAE-cellulose chromatography and the readiness of its reassociation. But it has a singular characteristic: it does not dissociate completely with cyclic AMP alone (even at concentrations as high as 0.3 mm) unless histone or NaCl is present. NaCl displays several roles: helps dissociation, prevents inactivation of the catalytic subunit, inhibits enzyme activity, and does not prevent reassociation as occurs with type II protein kinases. Once the holoenzyme is dissociated, cyclic AMP is essential to maintain the enzyme in the dissociated state.     

Studies on cyclic adenosine 3' ,5'-monophosphate levels, Adenylate cyclase and phosphodiesterase activities in the dimorphic fungus Mucor rouxii.

The germination of spores of Mucor rouxii into hyphae was inhibited by 2 mm dibutyryl cyclic adenosine 3′,5′-monophosphate or 7 mm cyclic adenosine 3′,5′-monophosphate; under these conditions spores developed into budding spherical cells instead of filaments, provided that glucose was present in the culture medium. Removal of the cyclic nucleotides resulted in the conversion of yeast cells into hyphae. Dibutyryl cyclic adenosine 3′,5′-monophosphate (2 mm) also inhibited the transformation of yeast to mycelia after exposure of yeast culture to air.Since in all living systems so far studied adenylate cyclase and cyclic adenosine 3′,5′-monophosphate phosphodiesterase are involved in maintaining the intracellular cyclic adenosine monophosphate level, the activity of both enzymes and the intracellular concentration of cyclic adenosine monophosphate were investigated in yeast and mycelium extracts. Cyclic adenosine monophosphate phosphodiesterase and adenylate cyclase activities could be demonstrated in extracts of M. rouxii. The specific activity of adenylate cyclase did not vary appreciably with the fungus morphology. On the contrary, cyclic adenosine monophosphate phosphodiesterase activity was four- to sixfold higher in mycelial extracts than in yeast extracts and reflected quite accurately the observed changes in intracellular cyclic adenosine monophosphate levels; these were three to four times higher in yeast cells than in mycelium.