Calmodulin-like activity in Mucor rouxii

Abstract A modulator factor with properties similar to those of calmodulin was found and partially purified from the soluble fraction of Mucor rouxii . These properties include: heat-stability, stimulation in a Ca²⁺-dependent fashion of cyclic nucleotide phosphodiesterase from bovine brain, and inhibition of this process by trifluoperazine. This calmodulin-like activity was detected in the soluble fraction of both mycelium and yeast-like cells of the fungus.     

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.     

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.     

Production and isolation of chitosan from Mucor rouxii.

A method for the lab-scale production and isolation of chitosan (polyglucosamine) from hyphal walls of Mucor rouxii was developed. Hyphal wall yields were generally 16 to 22% on a dry cell weight basis, of which 35 to 40% was glucosamine. Chitosan was readily extracted from purified, mycelial walls with acetic, formic, and hydrochloric acids; the last named was the most efficient. The yield of chitosan isolated ranged from 4 to 8% of the dry weight of the cell wall material.     

Chitosanases in the autolysis of Mucor rouxii

The mycelium of Mucor rouxii reached a 50% degree of lysis after 50 days incubation, and was then stable with the incubation time. The pH of the medium was 4.3 when autolysis began, rising to pH 7.6 after 6 days of autolysis and remaining there for the duration of the experiment. Maximum degradation of mycelium occurs during the first days of autolysis. Glucosamine is present in the culture liquid during all the autolytic process. Enzymes implicated in the degradation of chitosan and chitin were studied in the culture fluid during autolysis. An exochitosanase activity was detected after a day of autolysis, and its activity increased during 20 days of autolysis and afterwards remained constant until the end of the process. An endochitosanase activity was detected in the culture fluid from the beginning of the autolysis, having its maximum activity after 34 days of incubation. Both activities show an optimum pH of 5.5, but the pH range of activity for endochitosanase was broader than for exochitosanase. Both activities were not inhibited by 0.5 mM glucosamine. Activities of the enzymes B-N-acetylglucosaminidase and chitinase were not found. The chitosan content in the cell walls decreased with the incubation time. In these cell walls the chitin content experienced an increase at the beginning of the autolysis, decreasing afterwards. The enzymatic complex obtained from autolyzed cultures of M. rouxii hydrolyzed 2-day-old cell walls of this fungus. The hydrolysis was 21% after 24 h of incubation, liberating glucose and glucosamine. As a consequence protoplasts from M. rouxii germinated spores were obtained with its own lytic enzymes in adequate osmotic conditions. The involvement of chitosanases in the autolysis of this fungus have been studied.     

Control of dimorphism in Mucor rouxii

Haidle, C. W. (The University of Texas, Austin), and R. Storck. Control of dimorphism in Mucor rouxii. J. Bacteriol. 92:1236-1244. 1966.-Yeastlike cells of Mucor rouxii NRRL 1894 were converted to filaments in a medium containing glucose, mineral salts, casein hydrolysate, nicotinic acid, and thiamine when the gas phase was changed from CO(2)-N(2) or N(2) alone to air. Germ tubes began to appear 3 to 4 hr after exposure to air. Ribonucleic acid (RNA) precursors were incorporated into RNA in a discontinuous fashion during this conversion, but the incorporation was continuous during the anaerobic growth of yeastlike cells and during the aerobic germination of sporangiospores. The incorporation of labeled amino acids during the conversion was exponential. Labeling of ribosomal RNA occurred as shortly as 5 min after replacement of CO(2)-N(2) with air. However, P(32)-labeled RNA isolated 20 min after exposure to air had a guanine plus cytosine (GC) content of 41% (mole%) as compared with the 47% found for labeled and unlabeled RNA isolated at other stages of the life cycle of this organism or later during the conversion. In addition, the overall base composition of this 20-min pulse-labeled RNA resembled that of deoxyribonucleic acid (GC = 39%), suggesting that a significant proportion of this RNA is of the messenger type. Furthermore, the synthesis of cytochrome oxidase was induced upon exposure of yeastlike cells to air. Cyanide, acriflavine, and cycloheximide, which inhibited the action or synthesis of cytochrome oxidase, also inhibited the yeast to filament transition.     

Mode of action of chitin deacetylase from Mucor rouxii on N-acetylchitooligosaccharides.

The mode of action of chitin deacetylase from the fungus Mucor rouxii on N-acetylchitooligosaccharides with a degree of polymerization 1-7 has been elucidated. Identification of the sequence of chitin oligomers following enzymatic deacetylation was verified by the alternative use of two specific exo-glycosidases in conjunction with HPLC. The results were further verified by 1H-NMR spectroscopy. It was observed that the length of the oligomer is important for enzyme action. The enzyme cannot effectively deacetylate chitin oligomers with a degree of polymerization lower than three. Tetra-N-acetylchitotetraose and penta-N-acetylchitopentaose are fully deacetylated by the enzyme, while in the case of tri-N-acetylchitotriose, hexa-N-acetylchitohexaose and hepta-N-acetylchitoheptaose the reducing-end residue always remains intact. Furthermore, the enzyme initially removes an acetyl group from the nonreducing-end residue of all chitin oligomers with a degree of polymerization higher than 2, and further catalyses the hydrolysis of the following acetamido groups in a processive fashion. The results are in agreement with the mode of action that the same enzyme exhibits on partially deacetylated water soluble chitosan polymers.     

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 presence of two heat-stable inhibitors of phosphoprotein phosphatases in the dimorphic fungus, Mucor rouxii

Two heat-stable inhibitors (a and b) of phosphoprotein phosphatases I and II from Mucor rouxii were isolated from mycelium of the fungus. They were partially purified from extracts by heating, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of inhibitors a and b, estimated by gel filtration, are 5,000 and 20,000 respectively. Inhibitor a acts similarly on both enzymes while inhibitor b is relatively more active on enzyme II. Storage of inhibitor b at -20 degrees C for several weeks resulted in a partial conversion to a lower-molecular-weight form with properties similar to those of inhibitor a.     

Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii.

Chitin synthetase was isolated and purified 120-fold from the supernatant fraction (54,500 X g) of broken yeast cells of Mucor rouxii. The purified preparations consisted mainly of chitin synthetase particles (chitosomes) with an average size larger than 7 X 10(6) daltons (by gel filtration) and an average sedimentation coefficient of 105 S. The samples also contained other enzyme complexes (fatty acid synthetase, pyruvate dehydrogenase, and, depending on method, ribosomes). Nearly all of the chitosomal chitin synthetase occurred in a zymogenic form that required proteolytic activation. In most properties, the chitosomal enzyme was similar to crude enzyme (54,000 X g sediment): kinetics, activation by proteases, response to metals, stimulation by N-acetylglucosamine, and inhibition by polyoxin or UDP. One mamor difference was the much greater stability of the chitosomal chitin synthetase zymogen against spontaneous activation and destruction. Product (chitin microfibril) and enzyme (chitin synthetase) remained associated in a complex that was readily separated by centrifugation.     

Trehalose breakdown in germinating spores of Mucor rouxii

Germinating spores of Mucor rouxii rapidly broke down their large (23% of the dry weight) trehalose reserve. More than 50% of this trehalose was broken down to ethanol. About one-third of the trehalose was converted to glycerol, which started to leak out of the spores after some 20 min germination. The synthesis of glycerol was not associated with any major change in glycerol 3-phosphatase activity in the spores. Since its rate of leaking was much smaller and the internal concentration reached was much higher in spores subjected to osmotic stress, glycerol might play a role in the initial water uptake and swelling of the germinating spores.