Mutations in Ophiostoma ulmi induced by N-methyl-N′-nitro-N-nitrosoguanidine

Mutations were induced in Ophiostoma ulmi, the causal agent of Dutch elm disease, by treating yeastlike cells of wild-type strains with the alkylating agent N-methyl-N′-nitro-N-nitrosoguanidine (MNNG). The induced frequency of mutation, assessed by scoring the frequency of benomyl-resistant mutants among the surviving population, was highest in treatments that left from 4 to 10% survivors. The survival rate of the cells was affected by mutagen concentration and length of exposure to MNNG, as well as by cell concentration, cell age, pH, and chemical composition of the incubation buffer. Optimal conditions for routine induction of mutants were obtained by resuspending exponentially growing cultures in phosphate buffer at pH 7.5 (2.0 × 107 cells/mL) and treating the cells with MNNG (20 μg/mL) for 90 min with agitation. The proportion of auxotrophs among the survivors increased at least 200-fold when mutagenesis was followed by nystatin enrichment. Most auxotrophs tested were sexually fertile and carried a recessive mutation at a single nuclear locus. The benomyl-resistant phenotype was dominant. Key words: Ophiostoma ulmi, Dutch elm disease, mutations, N-methyl- N′-nitro-N-nitrosoguanidine.

Speculative cell cycle in yeast-phase growth of Sporothrix schenckii: plasma membrane ultrastructure as revealed by freeze-fracture electron microscopy

The cell cycle in yeast-phase growth of Sporothrix schenckii was investigated by light microscopy and freeze-fracture electron microscopy after a 3- to 7-day cultivation on brain heart infusion agar medium at 37 °C. Mother yeastlike cells were able to bear daughter yeastlike cells. They were also able to produce germ tubes that had the potential to develop into pseudohyhae and hyphae. On the other hand, hyphae or pseudohyphae born from yeastlike cells were able to bear yeastlike cells directly. These results lead us to propose a hypothetical cell cycle for yeast-phase growth involving yeastlike vegetative cells, pseudohyphae, and hyphae.

Freeze-fracture electron microscopic studies of age-related plasma membrane changes in Sporothrix schenckii

Characteristics of the plasma membrane of Sporothrix scheckii cells as revealed by freeze-fracture techniques have been classified into eight types (Y1, Y2a, Y2b, Y3a, Y3b, Y4a, Y4b, and Y5) in yeastlike cells grown under the following two conditions: brain heart infusion agar medium at 27 °C, and brain heart infusion agar medium at 37 °C. Type Y1 cells are yeastlike cells having smooth plasma membranes without any invagination. Typical characteristics of the other types are as follows: type Y2a, smooth plasma membranes with few trenchlike invaginations; type Y2b, wavy plasma membranes with few oval or irregularly formed invaginations; type Y3a, plasma membranes with many randomly distributed trenchlike invaginations; type Y3b, plasma membranes with many cocoonlike or irregularly formed invaginations; type Y4a, plasma membranes with longer trenchlike invaginations; type Y4b, plasma membranes with irregularly formed, enlarged invaginations; and type Y5, smooth or wavy plasma membranes with aggregations of intramembranous particles and with many vacuoles between cell walls and plasma membranes or in the cytoplasm in some cells. By counting the proportion of each type of yeastlike cell under the two conditions and with different cultivation periods, it appears that plasma membrane types change as aging progresses in the following order: type Y1, Y2a, Y3a, Y4a, and Y5 in conidia and type Y1, Y2b, Y3b, Y4b, and Y5 in yeastlike vegetative cells. These observations provide us with an important advantage when studying the effects of antifungal agents on the plasma membrane of Sporothrix scheckii, as it is important to know the natural course of changes in membrane structure during aging.

Distribution of sterols in fungi. II. Brassicasterol in Tuber and Terfezia species

Sterols from ascocarps of Tuber (truffle) and Terfezia species were identified by gas–liquid chromatography, gas chromatography – mass spectrometry, and 1H nuclear magnetic resonance, and the structure of brassicasterol from the yeastlike cells of Taphrina deformans confirmed by 1H nuclear magnetic resonance. Sterols of the fruiting bodies ranged from 1.2 to 2.3 μg/mg dry weight. Ergosterol was the principal sterol of Tuber species which also contained 28–44% brassicasterol depending on the species and source of the sample. Terfezia sp., on the other hand, contained about 98% brassicasterol with only small amounts of ergosterol. Brassicasterol is common to several families of the subdivision Ascomycotina, and to our knowledge has not been reported for a nonascomycetous species.

Defect of In Vitro Digestive Ability of Polymorphonuclear Leukocytes in Paracoccidioidomycosis

Selected functions of polymorphonuclear leukocytes were studied in patients with paracoccidioidomycosis (South American blastomycosis), in healthy control individuals, and in patients with diseases unrelated to paracoccidioidomycosis. Patients with paracoccidioidomycosis were also evaluated by standard immunological techniques. Phagocytosis and digestion of Paracoccidioides brasiliensis yeastlike cells in vitro was estimated by an original method. It was based on the appearance of phagocytosed P. brasiliensis in preparations stained by a modification of the Papanicolaou method and examined with phase-contrast optics. Interpretation of such findings was confirmed by electron microscopy. Two strains of P. brasiliensis were used. Strain 8506 was freshly isolated from a patient. Strain Pb9 was known to be nonpathogenic and to have a peculiar cell wall composition. Yeastlike cells of the Pb9 strain were digested significantly better than those of strain 8506. A higher number of leukocytes per fungus cells led to a higher proportion of digested P. brasiliensis. Leukocytes from patients with paracoccidioidomycosis phagocytosed the fungus in a normal way, but had a significant lower ability to digest it in vitro. When individual cases were analyzed, there was an excellent correlation between clinical evolution and digestive ability of polymorphonuclear leukocytes. There was good correlation between both of these and immunological parameters. Leukocytes from all groups behaved comparably in tests of general leukocyte function and in their abilities to kill and digest Candida albicans. Our results indicate that, as a group, polymorphonuclear leukocytes from patients with paracoccidioidomycosis had a significant, rather specific, defect in their in vitro digestive capacity against phagocytosed P. brasiliensis. There was also an inverse correlation between strain pathogenicity and its susceptibility to in vitro digestion by polymorphonuclear leukocytes. Our findings are concordant with and relevant to clinical reality.

Structure and chemical composition of yeast chlamydospores of Aureobasidium pullulans

Cellular form in Aureobasidium pullulons can be partially controlled by nitrogen nutrition. Ammonium nitrogen supports a mixture of filamentous and yeastlike growth, whereas only a few filaments develop on nitrate nitrogen. On nitrate 97% of the cell material consists of a mixture of yeastlike cells and chlamydospores. Chlamydospores are produced on both nitrogen sources; however, with ammonium nitrogen chlamydospores occur in an intercalar position, whereas nitrate nitrogen supports development of chlamydospores as separate structures containing one, two, or occasionally three cells. This mode of production allows separation of yeast chlamydospores from other cell types and subsequent isolation of their cell walls. Yeast chlamydospores and filaments have an electron dense, melanin-rich, granular, outer cell-wall layer which yeastlike cells lack. This granular material is also found in cross walls of filaments and chlamydospores. Glucose is the main component of chlamydospore walls and accounts for 36% of the dry weight. Yeastlike cell walls contain only 13% glucose, but more mannose, galactose, and bound lipid. Most of the glucan portion of chlamydospore walls is insoluble in dilute alkali; methylation analysis indicates that this material contains linear chains of (1 → 3) and (1 → 6) linked glucose. About one residue in five forms a branch point having both (1 → 3) and (1 → 6) linkages.