J. Thomas Mullins

Lateral branch formation and cellulase production in the water molds.

The formation of lateral branches, in response to induction by metabolites containing amino acids, in the genera Achlya, Saprolegnia and Dictyuchus is accompanied by the production and release of the enzyme cellulase. Treatments which prevent the production and release of cellulase also prevent the formation of lateral branches. Inhibitor studies with cycloheximide indicate that the branching/cellulase response requires protein synthesis.

Parasitism of the Chytrid Dictyomorpha Dioica

SUMMARYThe chytrid, Dictyomorpha dioica, was found capable of infecting 8 identified species of Achlya, 2 unidentified isolates of Achlya, and Thraustotheca clavata. All isolates tested of Saproleg...

A freeze-fracture study of hormone-induced branching in the fungus Achlya.

The induction of the male sexual organ primordia (antheridial hyphae) by the steriod hormone antheridiol in the water mold Achlya ambisexualis Raper requires the production and secretion of the enzyme cellulase. It is postulated that a localized secretion of cellulase produces a limited area of wall hydrolysis that is blown out into a lateral bleb by turgor pressure. Freeze-etch preparations show membrane profiles similar to those seen in other systems where exocytosis is occurring. Such a mechanism would provide the required localized secretion of cellulase. Water stress, imposed by polyethylene glycol, prevents the formation of antheridial hyphae, the secretion of cellulase and the expected membrane profiles. After a period of recovery from water stress antheridial hyphae are formed, cellulase secretion occurs and the expected membrane profiles are restored.

Hyphal tip growth in Achlya bisexualis. II. Distribution of cellulose in elongating and non-elongating regions of the wall

We have approached the problem of hyphal tip growth by comparing the cell wall composition of elongating and non-elongating regions of the hyphae of Achlya bisexualis. To ensure that we could distinguish between elongating and non-elongating hyphae, light microscopic observations were used to determine the rates of elongation under growing and non-growing conditions. When elongation was measured in 10 min intervals it was found to consist of fluctuating periods of fast and slow growth rates, in the form of cycles. Even under our growing conditions, however, a very small number of hyphae in a colony are not elongating. SEM analysis revealed that elongating hyphae have tapered apices, whereas non-elongating hyphae have a rounded apex. The major matrix wall components, 1,3-β-glucans, were localized with an indirect immunogold technique specific for these polymers. This method resulted in their localization to all regions of both elongating and non-elongating hyphae, including the apex.

Cytoplasmic water-soluble β-glucans in Achlya: response to nutrient limitation

Water-soluble /S-glucans are major cyto? plasmic constituents of Achlya bisexualis representing 13% (weight/weight) of mycelial dry weight. This glu- can was fractionated by ion exchange into two types, a neutral form (20%) and a phosphorylated form (80%). The phosphorylated glucan contained 5% (weight/ weight) phosphate when the mycelium was grown in standard medium. A 10-fold reduction in the supply of phosphate reduced the phosphate in the glucan to 1%, and a reduction in nitrogen had a similar effect. When growing mycelium was transferred to medium minus phosphate, total glucan production continued but with a reduced phosphate concentration. During starvation and calcium-induced sporulation, total glu? can declined, the ratio of neutral to phosphorylated glucan remained the same, but phosphate level de- creased by 49%.

Localization of Cytoplasmic Water-Soluble Reserve (1->3)-beta-Glucans in Achlya with Immunostaining

AbstractThe cytoplasmic water-soluble reserve (1→3)-β-glucans of Achlya are of two types: a small neutral and a large phosphorylated. These glucans have been localized using indirect immunolabellin...

Water-Soluble Reserve Polysaccharides from Achlya Are (1->3)-[beta]-Glucans

The cytoplasmic water-soluble p-glucans of Achlya bisexualis are (1?>3)-linked polysaccharides of two types: a small neutral and a large phosphorylat- ed. The carbon-13 nuclear magnetic resonance (NMR) spectrum of neutral glucan is consistent with a (l-^3)-(3-glucan with some (1?>6)-(3-linkages. Meth- ylation analysis suggests a similar structure, but also shows about 6% each of other linkages and other sugars. Acid and enzyme hydrolysis data also support a moderately branched (1??3)-p-glucan probably with single (1?>6)-p-glucopyranosyl segments. The data, however, do not exclude the possibility that the branches are extended (1?>3)-linked residues. The molecular mass of neutral glucan is approximately 9000 with a dp of 50, when compared with algal 1am- inaran by gel filtration chromatography. The carbon- 13 NMR spectrum of phosphoglucan is qualitatively similar to that of neutral glucan. Aqueous hydrofluor- ic acid effectively cleaves phosphate from the phos? phoglucan and produces a polymer of the size of neutral glucan. This material is both qualitatively and quantitatively identical to neutral glucan in its car? bon-13 NMR spectrum. The phosphorus-31 NMR spectrum of the phosphoglucan reveals both mono- and diphosphoester linkages. We propose that the phosphodiester covalent bonds convert the smaller polymer into the very large phosphoglucan.

Ca2+-Induced Sporulation in Achlya bisexualis: Reserve 1,3-beta-glucans Provide Both Carbon and Phosphorus

Reserve water-soluble 1,3-3-glucans represent some 13% (wt/wt) of the mycelial dry weight of Achlya, and consist of a small neutral form and a very large phosphorylated one. The phosphoglucan appears to be formed via aggregation of neutral molecules by phosphodiester linkages. Asexual sporulation in Achlya bisexualis, induced by 0.5 mM Ca2+, was accompanied by a 43% reduction in total glucan after 4 h, and the ratio of neutral glucan to phosphoglucan changed between 4 and 12 h. Phosphate content of the phosphoglucan declined by 50%, but the molecular size remained large and the ratio of monoand diester linkages changed only slightly. Appropriate enzyme activities of {3-glucanase and phosphoesterases capable of releasing both glucose and phosphate were found, and are described.

Association of UDPG transferase activity with cell walls of Achlya ambisexualis

Fungal cell wall growth has been postulated to involve the cooperative action of both wall-hydrolyzing and wall-synthesizing enzymes (Park and Robinson, 1966; Gooday and Trinci, 1980). Investigations of cell wall morphogenesis in Achlya have dealt primarily with hydrolytic events, particularly those mediated by endo-l,4-D-glucanases (Thomas and Mullins, 1967, 1969; Hill and Mullins, 1979, 1980). Much less attention has been devoted to wall-synthesizing enzymes in Achlya and other cellulosic fungi because the activity of glucan-synthesizing enzymes is difficult to demonstrate in vitro, and the low level of activity makes characterization of the products uncertain. In a previous investigation, uridinediphosphoglucose (UDPG) transferase (EC 2.1.4.12) was demonstrated at low activity in association with subcellular particles that also exhibited cellulase activity (Hill and Mullins, 1980). Since these particles were similar to apical vesicles this association suggests that UDPG transferase in Achlya may be involved in cell wall synthesis. In the present paper, activity of UDPG transferase is demonstrated in cell walls of Achlya, and the solubility properties of the in vitro reaction products are described. Achlya ambisexualis Raper strain E87 was cultured for 48 h as described previously (Mullins, 1973). UDPG tranferase activity was assayed by a modification of the techniques of Ray et al. (1969) and Shore and Maclachlan (1975). The reaction mixture contained (mM): UDPG, 0.24; cellobiose, 5; MgCl2, 11; dithiothreitol (DTT), 1.7; pH 5.8 sodium phosphate buffer, 67; and incubation was 20 min at 23 C. The UDPG was glucose-uL-14C (250 utCi/Aumole, New England Nuclear) and as used gave 7.3 x 10-8 moles with 119 nCi of radioactivity. Products were recovered by centrifugation of 70% ethanol-insoluble material after addition of 30 mg of powdered Whatman cellulose as a carrier. To determine the distribution of transferase activity between wall and protoplasm fractions, mycelia were homogenized by ultrasonication at 5-10 C in a buffered homogenizing solution containing 20% w/w sucrose, 10 mM DTT, and 20 mM tris HCl buffer, pH 7.6. Hyphal fragments were sedimented by centrifugation, then resuspended, and the entire process repeated three times. The supernatant fractions were pooled and the final sediment, when examined by phase contrast microscopy, consisted of wall fragments without apparent cytoplasmic contamination. UDPG transferase activity was assayed in each fraction and protein was estimated with the Branford (1976) Bio-Rad assay. Results are displayed in TABLE I. Although less transferase activity was found in the wall fraction than 851

Preparation of Coenocytes for Freeze-Etching

Successful freeze-etching of a coenocyte has been accomplished with glutaraldehyde stabilization followed by infiltration with cryoprotectant. Hyphae of the coenocytic water mold Achlya were stabilized with 5% glutaraldehyde in phosphate buffer. Gradual infiltration by dropwise addition of the cryoprotectant (25% glycerol, 10% ethylene glycol, distilled water, v/v) is accomplished over a period of 8-10 hr on a shaker. Subsequent freeze-etching is carried out by standard procedures.