Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis.

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sits increased during the first 2 days in culture to a plateau that was maintained for the next 2-3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.

Recharacterization of fungal dinucleoside polyphosphate (HS3).

Three polyphosphorylated dinucleosides given the pseudonyms of HS3, HS2, and HS1 that were erroneously described as diguanosine polyphosphates (LéJohn, H. B., Cameron, L. E., McNaughton, D. R. & Klassen, G. R. (1975) Biochem, Biophys, Res, Commun. 66, 460-467) have been repurified and partially recharacterized. They have proved to be extremely complex molecules; chemical (HCl and KOH hydrolysis), physical (ultraviolet-light spectral analysis and ion-exchange chromatography), and enzymic (nucleotide pyrophosphatase and bacterial alkaline phosphatase hydrolysis) studies showed that (i) all three HS compounds are uracil rich and (ii) only HS3 contains a purine nucleoside and glutamate. The partial structure of HS3 was deciphered as a moiety of ADP--sugar X--glutamate (the mode of attachment of glutamate is obscure) that is covalently linked to another moiety composed of UDP, mannitol, and four phosphates. Sugar X had chromatographic characteristics of ribitol, but the chromatographic isolate also contained a ninhydrin-sensitive entity presumed to be an amino group. Sugar X, THEREFore, may be an amino sugar polyol. Only the general chemical compositions of HS2 and HS1 were determined. Each contained two uridines and HS2 had 10 phosphates whereas HS1 had 12.

Glucose transport in Achlya: characterization and possible regulatory aspects.

The freshwater fungus Achlya transported D-(+)glucose (glucose) and 2-deoxy-D-glucose (deoxyglucose) by an energy-related system. Their transport4 was inhibited by uncouplers of metabolic energy such as 2,4-dinitrophenol, cyanide, azide, and carbonylcyanide-p-chlorophenylhydrazone. Besides inhibiting each other, glucose and deoxyglucose transport was inhibited by D-(+)galactose, D-(+)mannose, and D-(+)xylose. Many other sugars tested failed to inhibit glucose transport implying a certain degree of specificity. Glucose transport was pH (optimum at 6.5) and temperature (optimum at 30-40 degrees C) dependent. Glucose transport was also inhibited by citrate, N6-substituted adenines (cytokinins), and iodine. None of these agents penetrated the cell membrane within the brief (1-3-min) period in which glucose transport was measured. In every case, transport was inhibited within 10 s (the shortest time in which measurements could be made). When cells were osmotically shocked to release a cell-wall membrane phosphorylated proteoglycan (PPG), they became incapable of transporting glucose for several hours until new PPG material was reisolable from the membrane by osmotic-shock treatment. The osmotically shocked cells could not transport glucose or deoxyglucose. No glucose-binding protein was detected in the shock fluid. Practically all of the glucose transported within 1-2 min was recovered as glucose-6-phosphate. No other phosphorylated sugar was detected suggesting that glucose may be phosphorylated in transport. Related studies have shown that citrate removed calcium bound by PPG; N6-substituted adenines were bound by PPG while three polyphosphorylated dinucleosides, HS3, HS2, and HS1, were displaced from it. Iodine formed stable complexes with the HS compounds. All of these agents inhibited glucose transport without entering the cell. It is therefore possible that HS compounds, calcium and PPG may be involved in maintaining the cell membrane in proper form for glucose transport.

Glycogen and other soluble glucans from chytridiomycete and oomycete species.

Dry weight, protein, lipid, and glycogen were determined at various times during cultivation of the Chytridiomycetes, Rhizophydium sphaerotheca and Monoblepharella elongata. M. elongata had relatively stable levels of glycogen, but, in R. sphaerotheca, glycogen levels showed significant changes, particularly in older cultures in which a depletion of glycogen was accompanied by a marked thickening of the cell walls. Glycogen was a significant cellular constituent in both chytridiomycete species. In R. sphaerotheca and M. elongata, respectively, glycogen accounted for as much as 6% and 8.1% of the dry weight. In purified glycogens of both species, only alpha-1,4- and alpha-1,6-linked glucosyl residues were detected and the absorbance spectra of I2-complexes were similar to those of other well characterized glycogens. Purified Rhizophydium glycogen had a beta-amylolysis limit of 43%, and a CL of approximately 12. For the Monoblepharella polysaccharide, the respective values were 45% and 11. In extracts of the Oomycetes, Pythium debaryanum, Mindeniella spinospora, and Apodachlya sp., only beta-1,3- and beta-1,6-linked glucosyl residues were detected. These glucans were not iodophilic nor were they sensitive to alpha-amylase and beta-amylase. The properties of the oomycete polysaccharides suggested that they were similar to the mycolaminarans of Phytophthora spp. Although both investigated chytridiomycete species produced glycogen with typical properties, glycogen was apparently absent in the investigated Oomycetes.

Fractionation of Saprolegnia diclina (Oomycetes) satelite DNAs by AgNO3/Cs2SO4 density gradient centrifugation.

Saprolegnia diclina DNA has been fractionated using preparative AgNO3/Cs2SO4 and CsCl density gradients. In addition to the previously identified major satellite DNA, there are two minor DNA components banding at 1.682 and 1.701 g - cm(-3) in CsCl. Purified major satellite DNA bands at 1.707 g - cm(-3) giving a base composition of 48% G + C in good agreement with 47% G + C calculated from its Tm value. The nuclear DNA base composition is 58% G + C by both methods. The base composition of the major satellite DNA suggests that it may represent ribosomal DNA cistrons.

Quantitative microanalysis of cell walls of Saprolegnia diclina Humphrey and Tremella mesenterica Fries.

Cell walls of the fungi Saprolegnia declina Humphrey and Tremella mesenterica Fries were analyzed quantitatively. Particular attention was paid to the hydrolysis and analysis of neutral sugars, amino sugars and amino acids. These components, together with total lipids, total uronic acids and the ashed residue, accounted for more than 90% by weight of the original dry cell wall preparation. There were substantial losses of amino acids during hydrolysis; however, analytical recovery approached 100% when total protein was calculated from the total nitrogen analysis. The analytical procedures were reproducible (+/- 3% for amino acids and amino sugars, and +/- 5-10% for other components) when applied to individual cell wall preparations. However, even under carefully standardized conditions, different cell wall preparations from the same species showed variable composition. Glucose was the predominant neutral sugar in the cell wall polymers of both species. The amino acid compositions were remarkable in that neither species contained detectable levels of cyst(e)ine. Hydroxyproline was detected in both species. The report from Tremella mesenterica is the first for this amino acid from the cell wall of a Basidiomycete.

Polyphosphate granules in encysted zoospores of Rozella allomycis.

Methods of ultracytochemistry and of X-ray energy dispersive analysis have been used to demonstrate that the "gamma-like" granules in encysted zoospores of the chytrid Rozella allomycis contain polyphosphate. The possibility that cysts contain two classes of polyphosphate granules which differ in structure, in function, and in origin is discussed.

Composition of cellulin, the unique chitin-glucan granules of the fungus, Apodachlya sp.

Cellulin granules, the polysaccharide inclusions found uniquely in oomycetous fungi of the order Leptomitales, were isolated from Apodachyla sp. The granules were prepared free of cell wall and cytoplasmic contaminants. Biochemical analyses and X-ray diffraction studies demonstrated that the granules were composed of 60% chitin and 39% glucan consisting of beta-1,3- and beta-1,6-linked glucose units. A protein content of only 0.1% was attributed to an insignificant amount of cytoplasmic contamination. Isolated granules and those in situ showed no apparent differences in their microscopic form.