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.     

Evaluation of inoculum performance for enhancing gamma-linolenic acid production from Mucor rouxii

Aims: To facilitate a cost‐effective preparation of spore inoculum with good capacity for gamma‐linolenic acid (GLA) production from Mucor rouxii. Methods and Results: Sporangiospore production, mycelial growth ability and fatty acid composition of M. rouxii were determined. Compared with fungal cultivation on solid semi‐synthetic media, high spore production was achieved from M. rouxii grown on rice grains, particularly polished rice (30·7 g kg^?1 initial substrate). Variations in the fatty acid profiles were found in the spores grown on different types of solid media, whereas the spores obtained at different ages from cultivated polished rice showed a similar fatty acid profile. Using the inocula from different spore‐forming media and culture ages, and low temperature storage, not much change in the vegetative growth of submerged cultures or fatty acid composition of mycelia was observed. Conclusion: The spores generated on polished rice exhibited a high performance for GLA production. Age of spore and timing of spore storage at low temperature did not affect on fatty acid profile of the mycelial cultures. Significance and Impact of the Study: The simple, low cost method of inoculum preparation can be applied for large‐scale production of GLA‐rich oils, which reduce a time constraint and variation in fatty acid composition.     

Developmental and environmental influences in the production of a single NAD-dependent fermentative alcohol dehydrogenase by the zygomycete Mucor rouxii

A soluble NAD-dependent alcohol dehydrogenase (ADH) activity was detected in mycelium and yeast cells of wild-type Mucor rouxii. In the mycelium of cells grown in the absence of oxygen, the enzyme activity was high, whereas in yeast cells, ADH activity was high regardless of the presence or absence of oxygen. The enzyme from aerobically or anaerobically grown mycelium or yeast cells exhibited a similar optimum pH for the oxidation of ethanol to acetaldehyde (∼pH 8.5) and for the reduction of acetaldehyde to ethanol (∼pH 7.5). Zymogram analysis conducted with cell-free extracts of the wild-type and an alcohol-dehydrogenase-deficient mutant strain indicated the existence of a single ADH enzyme that was independent of the developmental stage of dimorphism, the growth atmosphere, or the carbon source in the growth medium. Purified ADH from aerobically grown mycelium was found to be a tetramer consisting of subunits of 43 kDa. The enzyme oxidized primary and secondary alcohols, although much higher activity was displayed with primary alcohols. K _m values obtained for acetaldehyde, ethanol, NADH₂, and NAD⁺ indicated that physiologically the enzyme works mainly in the reduction of acetaldehyde to ethanol. Received: 11 March 1999 / Accepted: 14 July 1999     

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.     

Inhibition of the yeast-mycelial transition and the phorogenesis of Mucorales by diamino butanone

Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.Abbreviations ODC ornithine decarboxylase - DAB 1,4-diamino butanone     

Processing of asparagine-linked saccharides in Mucor rouxii

Asparagine-linked Glc₁Man₉GlcNAc₂, Glc₁Man₈GlcNAc₂ and Glc₁Man₇GlcNAc₂ were detected in mycelial-form cells of the dimorphic fungus Mucor rouxii inculbated with [U-¹⁴C]glucose for 3 min. The oligosaccharides were absent from glycoproteins isolated from cells chased for 15 min with the unlabed monosaccharide. This was due to deglucosylation of the oligosaccharides and not to further addition of mannose residues to them. The half-lives of the glucosylated compounds were much shorter, therefore, in M. rouxii than in other eucaryotic cells. Further processing of N-linked saccharides led to the synthesis of mannan-like glycoproteins, some of whch contained methyl groups in position 3 or the mannose residues. Methylation occurred only at the non-reducing ends and prevented further elongation of the branches.     

The separation and properties of two phosphoprotein phosphatases from the dimorphic fungus Mucor rouxii

Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (M_r 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K_0.5 for MnCl₂ of 2 mm. Enzyme II (M_r 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn²⁺. This enzyme was strongly inhibited by 50 mm NaF or 1 mm ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn²⁺, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn²⁺. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.     

Environmental and developmental regulation of carotenogenesis in the dimorphic fungus Mucor rouxii

Aerobic mycelium of wild-type Mucor rouxii accumulated about ten times higher amounts of the carotenoid pigment -carotene when grown continuously in the presence of light than the corresponding cultures grown in the dark. Carotenoid accumulation was dependent on light intensity, with the threshold located at about 10^-2 W.m^-2. Photocarotenogenesis in complex medium was more efficient with glucose as a carbon source. Carotenoid synthesis by M. rouxii mycelium was unaffected by both retinol acetate and retinal, which are stimulators of carotenogenesis in other zygomycetes. Carotenogenesis was significant in aerobic mycelium but was almost undetectable in anaerobic mycelium as well as in aerobic or anaerobic yeast cells. This suggested an involvement of oxygen in carotenoid synthesis by M. rouxii and the existence of developmental regulation of the expression or operation of the pathway.     

Preparation of mycelial and yeast-like spheroplasts fromMucor rouxii by a lytic enzyme preparation fromPenicillium islandicum

Spheroplasts ofMucor rouxii were prepared from mycelial and yeast-like cells by use of aPenicillium islandicum enzymatic complex. This enzyme preparation, which presents high chitosanase and chitinase activities, was produced by growingP. islandicum either on mycelial or yeast-like walls ofM. rouxii. The presence of magnesium sulfate as an osmotic stabilizer was critical to obtain high yields of spheroplasts from mycelial forms. In the case of yeast-like cells, pretreatment with β-mercaptoethanol followed by magnesium sulfate was essential for extensive spheroplast production.     

Production,purification and characterization of an endopolygalacturonase from Mucor rouxii NRRL 1894

An extracellular polygalacturonase (PGase) from Mucor rouxii NRRL 1894 was purified to homogeneity by two chromatographic steps using CM-Sepharose and Superdex 75. The purified enzyme was a monomer with a molecular weight of 43100 Da and a pI of 6. The PGase was optimally active at 35 °C and at pH 4.5. It was stable up to 30 °C and stability of PGase decrease rapidly above 60 °C. The extent of hydrolysis of different pectins was decreased with increasing of degrees of esterification. Except Mn²⁺, all the examined metal cations showed inhibitory effects on the enzyme activity. The apparent K_m and V_max values for hydrolyze of polygalacturonic acid (PGA) were 1.88 mg/ml and 0.045 μmol/ml/min, respectively. The enzyme released a series of oligogalacturonates from polygalacturonic acid indicating that it had an endo-action. Its N-terminal sequence showed homologies with the endopolygalacturonase from the psychrophilic fungus Mucor flavus.     

Electron microscopy of spore germination and cell wall formation in Mucor rouxii

Summary The fine structure of ungerminated and aerobically germinated sporangiospores of Mucor rouxii was compared. The germination process may be divided into two stages: I, spherical growth; II, emergence of a germ tube. In both stages, germination is growth in its strictest sense with overall increases in cell organelles; e.g., the increase in mitochondria is commensurate with the overall increase in protoplasmic mass. Noticeable changes occurring during germination are the disappearance of electron-dense lipoid bodies, formation of a large central vacuole and, most strikingly, formation of a new cell wall. Unlike many other fungi, M. rouxii does not germinate by converting the spore wall into a vegetative wall. Instead, as in other Mucorales, a vegetative wall is formed de novo under the spore wall during germination stage I. This new wall grows out, rupturing the spore wall, to become the germ tube wall. Associated with the apical wall of the germ tube is an apical corpuscle previously described. The vegetative wall exhibits a nonlayered, uniformly microfibrillar appearance in marked distinction to the spore wall which is triple-layered, with two thin electron dense outer layers, and a thick transparent inner stratum. The lack of continuity between the spore and vegetative walls is correlated with marked differences in wall chemistry previously reported. A separate new wall is also formed under the spore wall during anaerobic germination leading to yeast cell formation. On the other hand, in the development of one vegetative cell from another, such as in the formation of hyphae from yeast cells, the cell wall is structurally continuous. This continuity is correlated with a similarity in chemical composition of the cell wall reported earlier.     

Filamentous Growth of Mucor rouxii Under Nitrogen

The form of growth of Mucor rouxii (National Regional Research Laboratory 1894) under nitrogen is dependent on inoculum size. With a large inoculum (10⁶ spores inoculated per ml), the morphology consists mainly of swollen spores with some filaments and yeastlike cells. At lower inoculum levels growth is filamentous. The morphology of this strain on incubation under nitrogen and the dependence of the form of growth on inoculum size are similar to those found previously for other strains of M. rouxii.     

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.     

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.     

Isolation, characterization and expression analysis of the ornithine decarboxylase gene (ODC1) of the entomopathogenic fungus, Metarhizium anisopliae

The gene ODC1, which codes for the ornithine decarboxylase enzyme, was isolated from the entomopathogenic fungus, Metarhizium anisopliae. The deduced amino acid sequence predicted a protein of 447 amino acids with a molecular weight of 49.3 kDa that contained the canonical motifs of ornithine decarboxylases. The ODC1 cDNA sequence was expressed in Escherichia coli cells; radiometric enzyme assays showed that the purified recombinant protein had ornithine decarboxylase activity. The optimum pH of the purified Odc1 protein was 8.0-8.5, and the optimum reaction temperature was 37 °C. The apparent K_m for ornithine at a pyridoxal phosphate concentration of 20 mM was 22 μM. The competitive inhibitor of ODC activity, 1,4-diamino-2-butanone (DAB), at 0.25 mM inhibited 95% of ODC activity. The ODC1 mRNA showed an increase at the beginning of appressorium formation in vitro. During the M. anisopliae invasion process into Plutella xylostella larvae, the ODC1 mRNA showed a discrete increase within the germinating spore and during appressorium formation. The second expression peak was higher and prolonged during the invasion and death of the insect. The ODC1 gene complements the polyamine auxotrophy of Yarrowia lipolytica odc null mutant.     

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.     

Phosphodiesterase activity and cyclic AMP content during early germination ofMucor rouxii spores

Incubation ofMucor rouxii spores in complex medium under aerobic conditions resulted in a very rapid transient increase in the level of soluble, low-K_m cAMP phosphodiesterase. Maximum activity was reached after 2–4 min. Simultaneously, the cAMP content increased quickly, reproducing the profile exhibited by phosphodiesterase. Activation of the enzymein vitro by a cAMP-dependent phosphorylation process showed that its stimulation was minimal at those times when its activity was highest. The correlation between cellular cAMP content, phosphodiesterase activity, and sensitivity of the enzyme to activation by phosphorylation suggests that thein vivo regulation of phosphodiesterase activity by cAMP-dependent phosphorylation may be the mechanism for shutoff of the cAMP signal elicited by glucose.     

Sialoglycoproteins in Morphological Distinct Stages of Mucor polymorphosporus and their Influence on Phagocytosis by Human Blood Phagocytes

The possible role of sialic acids in host cells–fungi interaction and their association with glycoproteins were evaluated using a clinical isolate of the dimorphic fungus Mucor polymorphosporus. Lectin-binding assays with spores and yeast cells denoted the presence of surface sialoglycoconjugates containing 2,3- and 2,6-linked sialylglycosyl groups. Western blotting with peroxidase-labeled Limulus polyphemus agglutinin revealed the occurrence of different sialoglycoprotein types in both cell lysates and cell wall protein extracts of mycelia, spores, and yeasts of M. polymorphosporus. Sialic acids contributed to the surface negative charge of spores and yeast forms as evaluated by adherence to a cationic substrate. Sialidase-treated spores were less resistant to phagocytosis by human neutrophils and monocytes from healthy individuals than control (untreated) fungal suspensions. The results suggest that sialic acids are terminal units of various glycoproteins of M. polymorphosporus, contributing to negative charge of yeasts and spore cells and protecting infectious propagules from destruction by host cells.     

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.