Effects of Ethylenediaminetetraacetate and Chloramphenicol on Mitochondrial Activity and Morphogenesis in Mucor rouxii

The present study demonstrates the importance of mitochondrial activities in controlling Mucor rouxii morphogenesis. The respiratory capacity of the spores of this facultatively anaerobic, dimorphic fungus becomes repressed if germination and growth take place in the absence of oxygen. The level of activity of mitochondrial enzymes such as cytochrome oxidase and malate dehydrogenase is lower in the anaerobic yeastlike cells than it is in ungerminated spores and in aerobic hyphae, but the reverse is true for glycolytic enzymes such as pyruvate kinase and alcohol dehydrogenase. Following exposure to air, yeastlike cells convert into hyphae after a lag period corresponding to aerobic adaptation. Anaerobic cultures grown in the presence of ethylenediaminetetraacetate (EDTA) at a concentration of 10(-4) M exhibit hyphal morphology. These cells, which are fully adapted to anaerobic fermentation, nevertheless have potentially active mitochondria with the same levels of respiratory enzymes as ungerminated spores. These cells are able to grow immediately after aeration, without an adaptation lag. Evidence is presented which indicates that the morphogenetic effect of EDTA is not the result of elimination of free metals. Additional evidence proving mitochondrial control of morphogenesis in M. rouxii is that chloramphenicol (4 mg/ml) induced the formation of respiratory-deficient, yeastlike cells in aerobic cultures.     

Nutritional Requirements for Growth and Yeastlike Development of Mucor rouxii Under Carbon Dioxide

Unlike five other strains of Mucor rouxii previously studied, certain nutritional factors must be present for rapid growth and completely yeastlike development of M. rouxii (National Regional Research Laboratory 1894) under CO(2); high CO(2) tensions markedly inhibit growth of this strain. Addition of yeast extract, peptone, or enzymatically hydrolyzed casein in substrate amounts to a basal medium (containing acid-hydrolyzed casein) completely relieved CO(2) inhibition of growth and permitted yeastlike development. The "CO(2) growth factor" activity of these supplements proved to be dialyzable and acid labile. These findings, together with the results of gel filtration and amino acid analysis, suggested that CO(2) growth factor activity can be attributed to small peptides.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

THIAMINE AND NICOTINIC ACID: ANAEROBIC GROWTH FACTORS FOR MUCOR ROUXII.

Bartnicki-Garcia, S. (Rutgers, the State University, New Brunswick, N. J.), and Walter J. Nickerson. Thiamine and nicotinic acid: Anaerobic growth factors for Mucor rouxii. J. Bacteriol. 82:142-148. 1961.-Mucor rouxii requires preformed thiamine and nicotinic acid for anaerobic growth. Such requirements are not manifested during aerobic incubation. Aerobically, the fungus was shown to be able to synthesize both vitamins.The yeastlike form and the filamentous form of anaerobically grown M. rouxii exhibit the same vitamin requirements.Thiamine can be substituted by its thiazole moiety. Under certain conditions, nicotinic acid was partly substituted by tryptophan, kynurenine, 3-hydroxykynurenine, and 3-hydroxyanthranilic acid.Anaerobically. the fungus (thiamine requiring) was about ten times more susceptible to pyrithiamine antagonism than the same organism grown aerobically (thiamine independent).     

Effect of hexoses on the levels of pyruvate decarboxylase in Mucor rouxii

Pyruvate decarboxylase activity in the dimorphic fungus Mucor rouxii increased 25- to 35-fold in yeastlike and mycelial cells grown in the presence of glucose as compared to the activity observed in mycelial cultures grown in the absence of glucose.     

Control of Dimorphism in Mucor by Hexoses: Inhibition of Hyphal Morphogenesis

In anaerobic cultures of Mucor rouxii, morphogenesis was strongly dependent on hexose concentration as well as pCO(2). At low levels of hexose or CO(2), or both, hyphal development occurred; at high levels, the fungus developed as yeast cells. Other dimorphic strains of Mucor responded similarly to hexose and CO(2) but differred in their relative sensitivity to these agents. Glucose was the most effective hexose in eliciting yeast development of M. rouxii; fructose and mannose were next; and galactose was last. The fungus may be grown into shapes covering its entire dimorphic spectrum simply by manipulating the hexose concentration of the medium. Thus, at 0.01% glucose, hyphae were exceedingly long and narrow; at higher sugar concentrations, the hyphae became progressively shorter and wider; finally, at about 8% glucose, almost all cells and their progeny were isodiametric (spherical budding cells). Such yeast development occurred without a manifested requirement for exogenous CO(2). The stimulation of yeast development by hexose is not an artifact due to increased production of metabolic CO(2) (hyphae or yeast cells released metabolic CO(2) at similar rates). Presumably, the effect was caused by some other hexose catabolite which interfered with hyphal morphogenesis (apical growth); deprived of its polarity, the fungus grew into spherical yeastlike shapes. Although 10% glucose inhibited the development of hyphae from germinating spores, it did not prevent the elongation of preformed hyphae. This suggests that hexose inhibits hyphal morphogenesis not by blocking the operation of the enzyme complex responsible for apical growth but by preventing its initiation; such inhibition may be regarded as a repression of hyphal morphogenesis.     

Volatile factor involved in the dimorphism of Mucor racemosus.

Both hyphal and yeastlike development of Mucor racemosus and M. rouxii were demonstrated under 100% N2. Under standardized conditions in yeast extract-peptone-glucose medium, the morphology depended on the N2 flow rate and not on the glucose concentration. The effect was related to the rate of flushing of the atmosphere over the culture medium. The results indicate that a volatile compound produced by Mucor is involved in morphogenesis.     

Oligoglucuronide production in Mucor rouxii: evidence for a role for endohydrolases in hyphal extension.

Culture filtrates of Mucor rouxii contained oligomers of glucuronic acid which were labeled rapidly during pulses with D-[U-14C]glucose. These oligomers were probably derived by enzymatic lysis of acidic polymers in the cell wall. The kinetics of the incorporation of label into oligouronides and cell wall polymers suggested that lysis of the wall was required for active hyphal extension. Experiments with cycloheximide, which inhibited hyphal extension, suggested that wall lysis was also required for the subapical cell wall synthesis which probably occurred under these conditions.     

Phosphofrucktokinase and glucose catabolism of Mucor and Penicillium species.

The primary catabolic pathways in the fungi Penicillium notatum and P. duponti, and Mucor rouxii and M. miehei were examined by measuring the relative rate of 14CO2 production from different carbon atoms of specifically labelled glucose. It was found that these organisms dissimilate glucose predominantly via the Embden--Meyerhof pathway in conjunction with the tricarboxylic acid cycle and to a lesser extent by the pentose phosphate pathway. Phosphofructokinase (EC 2.7.1.11) activity could not be detected initially in Penicillium species because of the interference from mannitol-1-phosphate dehydrogenase (EC 1.1.1.17) and NADH oxidase (EC 1.6.99.3). A combination of differential centrifuging and a heat treatment of Penicillium cell-free extracts in the presence of fructose-6-phosphate removed the interfering enzymes. The kinetic characteristics of phosphofructokinase from P. notatum and M. rouxii are described. The enzyme presents highly cooperative kinetics for fructose-6-phosphate. The kinetics for ATP show no cooperativity and inhibition by excess ATP is observed. The addition of AMP activated the P. notatum enzyme, relieving ATP inhibition; slight inhibition by AMP was observed with the M. rouxii enzyme. In contrast M. rouxii pyruvate kinase (EC 2.7.1.40) is activated 50-fold by fructose-1,6-diphosphate whereas pyruvate kinase from P. notatum and P. duponti were unaffected by fructose-1,6-diphosphate.     

Incorporation of radioactivity from amino acids in the presence of cycloheximide demonstrates gluconeogenic activity in Mucor rouxii.

Mucor rouxii cells induced for gluconeogenesis incorporated radioactivity from [14C]glutamic acid into trichloroacetic acid-precipitable material in the presence of 200 micrograms of cycloheximide per ml. This metabolic capacity was repressed by hexoses and required amino acids for induction. These results suggest that the incorporation of amino acids in the presence of cycloheximide represents gluconeogenic activity with associated polysaccharide synthesis.     

Paracoccidioides brasiliensis: Conversion of Yeastlike Forms into Mycelia in Submerged Culture

Details of the sequential morphological changes occurring during yeastlike to mycelial-form conversion of the dimorphic pathogen Paracoccidioides brasiliensis are described and illustrated by photomicrographs. Conversion of yeastlike to hyphal morphology was initiated by changing the temperature of incubation from 37 to 23 C. Production by the parent yeastlike cells of elongated buds developing into hyphae started to be conspicuous after 24 hr of incubation at 23 C. After 120 hr of incubation, growth was almost exclusively filamentous. Direct transformation of parent yeastlike cells into hyphae was not observed. Dry weight increased continuously during the conversion process in spite of the gradual disappearance of the parent yeastlike cells. Concurrent studies showed that changes in ribonucleic acid and deoxyribonucleic acid content per unit dry weight are about the same whether the yeastlike cells are undergoing conversion at 23 C or growing normally at 37 C, and that deoxyribonucleic acid synthesis is apparently required for bud formation in both cases.     

Role of exogenous hemin in the synthesis of hemoproteins and nonheme proteins during glucose repression in Saccharomyces cerevisiae

Exogenous addition of hemin to glucose-repressed cells of Saccharomyces cerevisiae stimulates the incorporation of amino acid into cytoplasmic proteins twofold. There was no significant change in the synthesis of total cytoplasmic RNA whereas a 40% increase in the synthesis of poly(A)-containing RNA was observed upon hemin treatment. Cell-free translation of cytoplasmic mRNAs and immunoprecipitation analysis of the translated products with antibodies against subunit V of cytochrome oxidase and the alpha and beta subunits of F1-ATPase reveals that there is an eightfold enrichment of the mRNA for subunit V of cytochrome oxidase upon hemin treatment. The effect of hemin on the alpha and beta subunits of F1-ATPase is only marginal, suggesting a differential role for heme in the synthesis of hemoproteins and nonheme proteins during glucose repression.     

Composition of lipids from yeast-like and mycelial cells of the fungus Mucor hiemalis, grown in the presence of 4-chloroaniline

The fungus Mucor hiemalis, which is commonly thought to be monomorphic, produced two types of cells, yeastlike and mycelial, during growth in a medium containing 4-chloroaniline. Among the polar lipids of yeastlike cells, diphosphatidylglycerol was dominant, while phosphatidylcholine and phosphatidylethanolamine were present in minor amounts. Conversely, mycelial cells mainly contained phosphatidylcholine and phosphatidylethanolamine, whereas the content of diphosphatidylglycerol was low. The neutral lipids of yeastlike cells were dominated by diacylglycerides, sterols, and fatty acids. The content of triacylglycerides and sterol esters was low. Yeastlike cells contained higher amounts of saturated fatty acids and lower amounts of unsaturated fatty acids than the mycelium. The content of stearic acid in the fatty acids of the mycelium grown in the presence of 4-chloroaniline was as high as 25.3-29.9%.     

Genetic modification of essential fatty acids biosynthesis in Hansenula polymorpha

The Delta(6)-desaturase gene isoform II involved in the formation of gamma-linolenic acid (GLA) was identified from Mucor rouxii. To study the possibility of alteration of the synthetic pathway of essential fatty acids in the methylotrophic yeast, Hansenula polymorpha, the cloned gene of M. rouxii under the control of the methanol oxidase (MOX) promoter of H. polymorpha, was used for genetic modification of this yeast. Changes in flux through the n-3 and n-6 pathways in the transgenic yeast were observed. The proportion of GLA varied dramatically depending on the growth temperature and media composition. This can be explained by the effects of either substrate availability or enzymatic activity. In addition to the potential application for manipulating the fatty acid profile, this study provides an attractive model system of H. polymorpha for investigating the deviation of fatty acid metabolism in eukaryotes.     

Lipid Composition of the Yeastlike and Mycelial Mucor hiemalis Cells Grown in the Presence of 4-Chloroaniline

The fungus Mucor hiemalis F-1156, which is commonly thought to be monomorphic, produced two types of cells, yeastlike and mycelial, during growth in a medium containing 4-chloroaniline. Among the polar lipids of yeastlike cells, diphosphatidylglycerol was dominant, while phosphatidylcholine and phosphatidylethanolamine were present in minor amounts. Conversely, mycelial cells mainly contained phosphatidylcholine and phosphatidylethanolamine, whereas the content of diphosphatidylglycerol was low. The neutral lipids of yeastlike cells were dominated by diacylglycerides, sterols, and fatty acids. The content of triacylglycerides and sterol esters was low. Yeastlike cells contained higher amounts of saturated fatty acids and lower amounts of unsaturated fatty acids than the mycelium. The content of stearic acid in the fatty acids of the mycelium grown in the presence of 4-chloroaniline was as high as 25.3–29.9%.     

Respiratory-deficient,yeastlike mutant of Mucor

Stable mutants of Mucor bacilliformis having lost the ability to grow filamentously and to sporulate occur spontaneously with a frequency of about one in every 3000 colonies. On solid and in liquid medium these mutants have a typical yeastlike morphology and reproduce by budding. The detailed study of one of these mutants shows that the inability to form filaments and spores is accompanied by the loss of cytochrome oxidase activity. This mutant is unable to take up oxygen but has a high level of alcoholic fermentation, which appears to be the major if not the sole source of energy.This investigation was supported by Public Health Service Grant AI 07804 05.     

Development of Respiration and Mitochondria in Mucor genevensis After Anaerobic Growth: Absence of Glucose Repression

Respiration and mitochondria in Mucor genevensis, a facultatively anaerobic dimorphic mold, have been studied in aerobically and anaerobically grown cells and in anaerobically grown cells adapting to aerobic conditions. Respiration in hyphae continues at a high level during aerobic growth but drops rapidly on exhaustion of glucose. In anaerobically grown yeastlike cells, containing no recognizable aerobic cytochromes, a small cyanide-insensitive respiration occurs. Mitochondria with well defined cristae are visible in negative contrast after KMnO(4) fixation of stringently anaerobic cells containing low amounts of fatty acid of which 10% or less are unsaturated. On aeration of anaerobically grown cells, respiratory capacity and cytochromes develop rapidly, even in the presence of 10% glucose, indicating that glucose does not repress development of respiration. However, mycelium formation by adapting yeastlike cells is repressed by high glucose concentration. In adapting cells, apparent changes in mitochondrial ultrastructure appear to be more related to changes in fixation properties of cells than to changes in the structure of mitochondria.     

Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system

Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.     

Ethanol-induced changes in expression profiles of cell growth, fatty acid and desaturase genes of Mucor rouxii

We investigated the response of Mucor rouxii to ethanolic stress conditions. A differential response was found that was dependent on growth phase and ethanol concentration. 5% (v/v) ethanol showed an inhibitory effect on the mycelial growth of all stages. However, the ethanol sensitivity was specifically observed in active growing phases (12 and 21 h-grown cultures), in which the biomass and ratio of unsaturated/saturated fatty acids (UFA/SFA) decreased greatly after ethanol exposure compared to non-ethanol adding culture. With respect to different ethanol concentrations, M. rouxii was tolerant to low ethanol concentrations (about 1-3%, v/v) such that there was not much change in biomass and UFA/SFA ratio, in contrast to the 5% ethanol-added culture. We also showed the molecular basis of this response mechanism, demonstrating that expression of Delta(9)-, Delta(12)- and Delta(6)-desaturase genes, responsible for fatty acid desaturation in M. rouxii, were coordinately down-regulated upon exposure to ethanol stress.