The nuclear DNA-dependent ribonucleic acid polymerases of the dermatophytic fungus Microsporum gypseum.

The DNA-dependent RNA polymerases of the dermatophytic fungus Microsporum gypseum were partially characterized. Nuclear extracts prepared from vegetative mycelia were fractionated by DEAE-Sephadex chromatography into three enzyme species which resembled in most of their characteristics those of other eukaryotic organisms.

Changes in Microsporum gypseum mycelial wall and spore coat glycoproteins during sporulation and spore germination.

Ethylenediamine-soluble glycoproteins were extracted from isolated Microsporum gypseum hyphal walls during sporulation and from spore coats before and after germination. This study was carried out to identify a sporulation-specific cell wall protein that possibly served as a substrate for the alkaline protease which initiated the macroconidial germination of this fungus. Analyses revealed that water-insoluble glycoprotein accounted for 10% of the ungerminated spore coat but only for 4 to 5% of the mycelial wall dry weight. This fraction was modified in its amino acid composition during sporulation, and it decreased in protein content during spore germination. Water-soluble glycoprotein, which accounted for approximately 3 to 3.5% of either the spore coat or mycelial wall dry weight, was of similar amino acid composition from both sources and did not decrease in protein content upon spore germination. The water-insoluble glycoprotein was found to be rich in leucine, aspartic acid, glycine, glutamic acid, and phenylalanine residues. The water-soluble glycoprotein was rich in proline, threonine, glycine, serine, glutamic acid, and alanine.

Phosphate-mediated alteration of the Microsporum gypseum germination protease specificity for substrate: enhanced keratinase activity.

Inorganic phosphate was found to decrease the caseinolytic and ethyl-esterase activities of the Microsporum gypseum germination protease. The germination protease possessed exokeratinase (beta-keratinase) activity immediately after release from the fungal spore. After phosphate treatment of the enzyme, the germination protease also possessed endo-keratinase (alpha-keratinase) activity. Phosphate altered the protease's pH optimum from 9.0 to 7.0 and decreased the molecular weight from 33,000 to 16,000. These values were identical to those found for the keratinase. Alpha- and beta-keratinase activities were stimulated in excess of 200-fold by disulfide reducing agents. Natural and suspected keratin degradation products also enhanced keratinase activity. Cell fractionation and in vitro conversion of the alkaline germination protease into a functional keratinase suggested that the subunits comprising the germination protease and the keratinase were of a common origin.

Physiological and biochemical changes associated with macroconidial germination in Microsporum gypseum.

A study was made of the metabolic processes associated with macroconidial germination in Microsporum gypseum. The optimum conditions for stimulation of endogenous respiration, changes in chemical composition as germination proceeds, and the uptake and synthetic fates of amino acids, glucose, and uracil were investigated. The assimilation and conversion of (14)C-glucose, (14)C-amino acids, and (14)C-uracil into the cell pool and into trichloroacetic acid-precipitable material were studied during the early stages of germination (i.e., prior to germ-tube emergence). The macroconidia were not metabolically inert for any significant period of time after exposure to germination conditions. Rather, the spores rapidly assimilated all metabolites and slowly converted them into macromolecules. Investigations of the effect of inhibitors of nucleic acid and protein synthesis prior to germ-tube emergence and during early germ-tube elongation suggested significant changes in metabolism and cell permeability may be correlated with the emergence of germ tubes. Radioactivity of incorporated glucose was found to be associated largely with the lipid fractions of the macroconidia early in germination.

Initiation of dermatophyte pleomorphic strain sporulation by increased aeration.

A normally asporogenous pleomorphic strain of Microsporum gypseum was induced to sporulate by controlled aeration and dehydration. Aeration of the pleomorphic strain under optimal cultivation conditions caused the initiation of a sporulation cycle with equivalent growth parameters and percentage intracellular water loss as the wild-type strain. Initiation of sporulation was not due to alteration of the medium's nutrient concentration or consistency, concentration of fungal growth by-products, or removal of volatile "staling factors." Macroconidia formed by the pleomorphic colonies were of characteristic wildtype morphology, but germinated to form typical pleomorphic colonies, indicating that the induced sporulation was strictly phenotypic and reversible. Other asporogenous pleomorphic strains from different dermatophyte genera also were induced to form macroconidia by aeration, suggesting a similarity in sporulation induction in Microsporum sp., Epidermophyton floccosum, and Trichophyton violaceum. Initiation of sporulation by aeration further suggested that the pleomorphic mutation was one which affected the sensitivity of the pleomorphic aerial hyphae to natural sporulation inducers (i.e., decreased humidity) and did not represent a loss in the ability to form fertile macroconidia.

Isolation and characterization of Microsporum gypseum lysosomes: role of lysosomes in macroconidia germination.

Three types of lysosomes containing either acid protease, alkaline protease, or phosphodiesterase were isolated from a Microsporum gypseum macroconidial homogenate on Ficoll gradients. The acid protease was contained in an assimilative lysosome since its activity was affected by the complexity of the exogenous nitrogen source. Ultracentrifugation and electron microscopy revealed that the alkaline protease-containing vesicles were associated with the spore coat material prior to macroconidial germination. During macroconidial germination, zones of spore coat hydrolysis were seen surrounding these vesicles. Other larger vesicles, believed to contain the phosphodiesterase, were also observed in the spore coat during macroconidial germination.

Regulation and self-inhibition of Microsporum gypseum Macroconidia germination.

Germination of Microsporum gypseum macroconidia was accompanied by the release of alkaline protease, calcium ions, and inorganic phosphate into the germination fluid. The rate of germination was greatest during the first 2 hr, decreasing thereafter. This decrease in rate was accompanied by a decrease in protease activity, which was caused by an interaction of the enzyme with the inorganic phosphate released from the spores and accumulated in the germination medium after 2 hr. Germination of high spore densities was regulated by the ratio of released phosphate to protease protein, resulting in a constant percentage of germination at both high and low spore densities. A germination-defective mutant strain failed to germinate normally and released excessively high concentrations of phosphate into the germination medium during the initial 2 hr of incubation. Addition of calcium ions to germination mutant macroconidia stabilized spore morphology, prevented protease inactivation, and allowed normal germ-tube outgrowth. The germination of macroconidia appears to be regulated by the release of phosphate ions, which then inhibit the alkaline protease.

Absence of histones from the chromosomal proteins of fungi.

Interphase chromosomes were isolated in good yield from four species of fungi. In no case does the chromatin contain histones such as are characteristic of the chromosomes of other eukaryotic organisms.

Isolation and preliminary characterization of developmental mutants from Microsporum gypseum.

Developmental mutants affected in either sporulation or spore germination have been isolated from Microsporum gypseum with the aid of nitrosoguanidine or as spontaneously occurring mutants. The time course levels of several proteins temporally associated with conidial development have been assayed in the wild-type and mutant strains. The spore germination characteristics of two of the mutants are described. The relationship of alkaline protease accumulation to tyrosinase accumulation and spore germination is discussed.

Relationship of nuclear segregation and macroconidial germination in Microsporum gypseum.

Microsporum gypseum macroconidia germinated at 37 C possessed from one to eight nuclei per germinated spore compartment. The distribution of nuclei per spore compartment was the result of a random packaging of nuclei from the available nuclear population. Partial inhibition of germination by incubation at 25 C or at 37 C in the presence of 10(-4)m phenyl methyl sulfonyl-fluoride resulted in an enrichment of germinated spores containing high numbers of nuclei per compartment. The selection for higher nuclear numbers was statistically significant. Compartments possessing high numbers of nuclei appeared to be precommitted to spore germination since they were not sensitive to germination inhibition. The effect of incubation temperature variation on spore germination is discussed with respect to the organism's natural environment.

Macroconidial germination in Microsporum gypseum.

Biochemical events which occur during macroconidial germination have been studied in the dermatophyte Microsporum gypseum. The specific activity levels of various metabolic enzymes have been assayed during germination time periods. The accumulated levels of several of these enzymes, as a function of exogenous carbohydrate source, have been investigated. M. gypseum was found to possess a constitutive glyoxalate shunt, a constitutive glucokinase, a fructose phosphoenolpyruvate transferase, and a mannitol phosphoenolpyruvate transferase. The integration of endogenous reserve utilization during germination is discussed. The purification and properties of an alkaline phosphatase and its possible relationship to sporulation and spore germination also are described.

Biochemical changes during fungal sporulation and spore germination. I. Phenyl methyl sulfonyl fluoride inhibition of macroconidial germination in Microsporum gypseum.

Macroconidia of Microsporum gypseum release free amino acids into the medium during germination. A single alkaline protease is also found in the germination supernatant fraction. The purified protease is capable of hydrolyzing isolated spore coats in vitro. Phenyl methyl sulfonyl fluoride (PMSF) is an effective inhibitor of the protease. Incorporation of PMSF at 10(-4)m into the germination system inhibits spore germination and the release of free amino nitrogen. Addition of PMSF after germ tube emergence is completed has no effect on subsequent outgrowth. The addition of exogenous purified protease to quiescent spores results in more than a 2.5-fold increase in germinated spores. It is concluded that spore coat proteolysis is an essential event in the germination of dermatophyte macroconidia. A model system to explain macroconidial germination response to inhibition, temperature shift, and addition of protease is presented.

Heat-induced macroconidia germination in Microsporum gypseum.

A method for obtaining purified ungerminated macroconidia is described, and a technique for obtaining 85 to 90% germination of macroconidia under normal nutritional conditions is presented.

Oxidative metabolism of dermatophytes.

A method for preparing young, actively respiring dermatophyte mycelia was obtained through the use of concentrated spore inocula and short growth periods in static culture. These hyphal elements were uniform in appearance, and vacuoles were absent. Concentrated mycelial suspensions were obtained which could be transferred easily and accurately. Glucose stimulated oxygen uptake in young mycelia which had been grown in a medium with low carbohydrate content. The level of endogenous respiration was affected by exogenous glucose only when this substrate stimulated oxygen uptake by less than 14%. Low nicotinamide adenine dinucleotide phosphate (NADP) dehydrogenase activity was noted in microconidia which have a low endogenous Q(o(2) ) value, whereas the activity of this enzyme was greater in macroconidia and mycelia which possess higher endogenous Q(o(2) ) values. Microsporum gypseum oxidizes 50% of exogenous glucose and assimilates the remainder. A large percentage of this substrate was assimilated into nitrogenous substances.

Biochemical Alterations of Dermatophytes during Growth.

Alterations in the biochemical constituents of mycelia were studied during the growth, development, and starvation of Microsporum quinckeanum. On the basis of dry weight, growth of this dermatophyte could be divided into four phases: lag, log, stationary, and death. The percentage of total nitrogen, inorganic phosphorus, ribonucleic acid (RNA), and protein increased rapidly during the lag phase. The percentage of protein remained constant after the initial increase; however, inorganic phosphate and RNA decreased in older mycelia. Acid-soluble materials in the cells increased in concentration as the organism aged. Chitin was present in the spores at a much higher concentration than in the mycelia. The percentage of this compound decreased rapidly until the end of the lag phase. An increase and subsequent decrease in per cent chitin occurred during the log phase. Inorganic phosphorus in the mycelia increased from the value in the spore stage to a maximum in the early log phase, and then decreased rapidly during the remainder of the growth cycle. Compounds involved in protein synthesis increased rapidly during the lag phase of growth. Changes in chemical composition of the mold during starvation indicate that carbohydrate does not form the principal endogenous reserve of M. quinckeanum, whereas lipids may represent the primary reserve material.