Differences in the carbohydrate composition between the yeastlike and mycelial cells of Mucor hiemalis

The use of sporangiospores from a 20-day culture for inoculation enabled us to obtain yeastlike cells of strain Mucor hiemalis F-1431 (for which capacity for dimorphic growth was not previously studied) by cultivation in liquid medium under aerobic conditions. The carbohydrate composition of the fat-free biomass of the mycelial and yeastlike forms grown in the same culture under aerobic conditions was studied. In the fat-free biomass consisting of yeastlike cells, as compared to the mycelium, the contents of chitin and glucose decreased from 25 and 30.9% of dry biomass to 14 and 17.5% of dry biomass, respectively. We failed to detect any changes in the contents of fucose, galactose, mannose, and uronic acids among the heteropolysaccharide monomers of fungal cells of different morphotypes, which is probably due to the aerobic cultivation conditions. It was suggested that the composition of heteropolysaccharide monomers does not play such a significant role in the formation of yeastlike cells in the culture grown under aerobic conditions as the content of chitin, the main supporting biopolymer.     

Respiratory-competent conditional developmental mutant of Mucor racemosus

A conditional developmental mutant of Mucor racemosus which is capable of oxidative energy metabolism is described. Unlike the wild-type strain the mutant was highly fermentative and exhibited the yeast morphology when grown aerobically in glucose-containing media. The high fermentative activity and yeast morphology under these conditions correlated well with maximal expression of glycolytic enzymes and with expression of some polypeptides characteristic of anaerobic growth. Aerobic growth of the mutant on amino acids as the sole carbon source resulted in growth in the mycelial morphology. The mutant was fully capable of oxidative metabolism as judged by its ability to grow on amino acids, respiratory capacity, and complement of tricarboxylic acid cycle enzymes. The results support the hypothesis that oxygen controls both the expression of glycolytic enzymes and the expression of proteins involved in morphogenesis. Moreover, they suggest that there are common regulatory elements in the control of these two classes of gene products. Abnormally high levels of aconitase and isocitrate dehydrogenase in the mutant are consistent with the proposal that pool sizes of citrate may act as a regulator of genes responsive to environmental oxygen concentration.     

Respiratory-deficient Mutants in Saccharomyces lactis

Six independent ultraviolet-induced respiratory-deficient mutants (petites) of Saccharomyces lactis were isolated and characterized. Two possessed a normal cytochrome spectrum, another displayed an increased level of all the cytochromes, and three suffered from a partial or complete loss of one or more of the cytochromes a, b, c, and c₁. All of the mutants were segregational petites; none was vegetative. Determination of linkage relationships between mutants was restricted because matings between mutants, homozygous or heterozygous, for loci affecting cytochrome content were blocked at various stages in the mating-sporulating sequence. At least three of the petites were genetically nonidentical. Three of the mutations appeared to occupy loci within the same linkage group; two of the three mutations that mapped within this region were cytochrome-deficient. Growth at high or low temperatures, under increased osmotic pressure or in media supplemented with various fatty acids or sterols, did not relieve the physiological defects in these mutants. Reasons for the differences in survival of segregational and vegetative petites within this species are examined.     

A Mucor pusillus mutant defective in asparagine-linked glycosylation.

A Mucor pusillus mutant defective in asparagine-linked glycosylation was found in our stock cultures. This mutant, designated 1116, secreted aspartic proteinase (MPP) in a less-glycosylated form than that secreted by the wild-type strain. Analysis of enzyme susceptibility, lectin binding, and carbohydrate composition indicated that this mutant secreted three glycoforms of MPPs, one of which contained no carbohydrate; the other two had truncated asparagine-linked oligosaccharide chains such as Man0-1GlcNAc2. Further analysis using oligosaccharide processing inhibitors, such as castanospermine, 1-deoxynojirimycin and N-methyldeoxynojirimycin, suggested that MPPs in the mutant were glycosylated through a transfer of the truncated lipid-linked oligosaccharides, Man0-1GlcNAc2, to the MPP protein but not through an aberrant processing. In addition, genetic studies with forced primary heterokaryons indicated that the mutation in strain 1116 was recessive.     

Impact of morphogenetic effectors on the growth pattern and the lipid composition of the mycelium and the yeastlike cells of the fungus Mucor hiemalis

We investigated the growth and the cell lipid composition of the mycelium and of the yeast-like form of Mucor hiemalis VKMF-1431 obtained under aerobic conditions by treatment with the morphogenetic agents itraconazole, exogenous triacylglycerols (TAGs), and trehalose. The sporangiospores of a 20-day culture were inoculated on the medium with glucose. Under these conditions, the fungus produced both mycelium and yeast-like cells. It was established that, upon the germination of old (20-day) sporangiospores, the fungus predominantly used the mycelium development strategy in the presence of trehalose and TAGs. It was characterized by a low ratio between the two bulk membrane lipids (PEA/PC) and increased levels of PC and polyunsaturated fatty acids (FA). Compared to the mycelium, the yeast cell morphotype obtained on the medium with glucose was distinguished by an elevated PEA/PC ratio, lowered TAG, free sterol (FS) and esterified sterol (ES) levels, a decreased ES/FS ratio that correlated with the reserve sterol pool size, and a lowered content of unsaturated fatty acids (the linoleic and the ??-linolenic acid). These peculiarities of the lipid composition of yeastlike cells correlated with the intensity of yeastlike growth. Light and electron microscopy revealed differences between the above cell morphotypes. With itraconazole, yeast-like cells were characterized by the destruction of the endoplasmic reticulum membranes and formation of a large number of vacuoles. The suggestion was confirmed that the state/age of inoculum sporangiospores exerts an influence on the capacity for dimorphism in mucorous fungi such as M. hiemalis. The data obtained testify to an involvement of lipids in the process of adaptation to environmental factors and to their regulatory role in morphogenetic processes associated with the formation of alternative morphotypes of the mucorous fungus.     

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.     

Secretion of Mucor rennin, a fungal aspartic protease of Mucor pusillus, by recombinant yeast cells

Summary The aspartic protease gene of a zygomycete fungus Mucor pusillus was expressed in Saccharomyces cerevisiae under the control of the yeast GAL7 promoter. A putative preproenzyme with an NH₂-terminal extension of 66 amino acids directed by the gene was processed in yeast cells and the mature enzyme, whose NH₂-terminus was identical to that of the Mucor enzyme, was efficiently secreted into the medium at a concentration exceeding 150 mg/l. The enzyme secreted from the recombinant yeast was more glycosylated than the native Mucor enzyme but its enzymatic properties were almost identical with those of the native enzyme, which has been used as a milk coagulant in cheese manufacture.     

Slow Growth Induces Heat-Shock Resistance in Normal and Respiratory-deficient Yeast

Yeast cells respond to a variety of environmental stresses, including heat shock and growth limitation. There is considerable overlap in these responses both from the point of view of gene expression patterns and cross-protection for survival. We performed experiments in which cells growing at different steady-state growth rates in chemostats were subjected to a short heat pulse. Gene expression patterns allowed us to partition genes whose expression responds to heat shock into subsets of genes that also respond to slow growth rate and those that do not. We found also that the degree of induction and repression of genes that respond to stress is generally weaker in respiratory deficient mutants, suggesting a role for increased respiratory activity in the apparent stress response to slow growth. Consistent with our gene expression results in wild-type cells, we found that cells growing more slowly are cross-protected for heat shock, i.e., better able to survive a lethal heat challenge. Surprisingly, however, we found no difference in cross-protection between respiratory-deficient and wild-type cells, suggesting induction of heat resistance at low growth rates is independent of respiratory activity, even though many of the changes in gene expression are not.     

Lipid composition of yeastlike cells of the fungusPullularia pullulans

AlthoughPullularia pullulans is a polymorphic fungus, cultures have been obtained consisting exclusively of yeastlike cells. These cells can be considered as “medium lipid content” yeasts (5.7%). Thirty percent of the total lipids are phosphoglycerides, the most abundant of which are phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine. The bulk of the nonpolar lipids is made up of unsaponifiable matter, sterols, and hydrocarbons. Eighteen fatty acid species have been detected, but the C₁₆ and C₁₈ species are by far the most abundant. The major unsaturated species is oleic acid. Linolenic acid is found in significant amounts only in triglycerides and esterified sterols. Fatty acid moieties associated with phosphatidylcholine and phosphatidylethanolamine are more unsaturated than those associated with phosphatidylserine or cardiolipin. Considerable proportions of the phosphoglycerides exist in the form of plasmalogen, which is unusual in yeasts.     

Cloning and sequencing of a gene for Mucor rennin, an aspartate protease from Mucor pusillus.

The aspartate protease of Mucor pusillus (Mucor pusillus rennin; MPR) is a milk-clotting enzyme used in the cheese industry. The partial amino acid sequence of MPR was determined and oligonucleotide probes were synthesized for cloning of the MPR gene. A clone giving positive hybridization with the probes was selected from the cosmid library. Sequencing of the cloned DNA revealed an open reading frame of 1281 bp without introns which encodes 361 amino acids for the expected MPR with an NH2-terminal extension of 66 amino acids. MPR seems to be synthesized as a prepro enzyme.     

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.