Experimental Mycology最新文献

In a medium containing cellulose as the carbon source, the rapid growth of Sporotrichum thermophile, the secretion of cellulases and the utilization of cellulose were well-correlated events. The production of β-glucosidase in culture medium lagged behind cellulases, coinciding with the time of extensive autolysis of mycelia. By contrast, neither apparent autolysis nor secretion of β-glucosidase occurred when S. thermophile was grown in medium containing cellobiose; the enzyme activity remained associated with mycelia. The release of β-glucosidase in cellulose-grown cultures was correlated with the activity of the lytic enzyme in the cell wall. Immunocytochemical localization and biochemical characterization showed that a β-glucosidase released in the cellulose medium was the same as that which remained associated with mycelia grown on cellobiose. The results indicated that the release of β-glucosidase in the cellulose culture is incidental to the activity of the lytic enzymes which are strongly induced by cellulose. The observations minimize a functional role of the culture fluid P-glucosidase in cellulolysis by the fungus. Rather, the available information suggests that the cellulases and β-glucosidases associated with the hyphal cell wall may play a role in cellulolysis by the fungus.

Morphological and molecular aspects of the dimorphic pathogenic fungus Paracoccidioides brasiliensis, strain Pb 01, were investigated in vitro. Morphological alterations during temperature shifts were preceded by significant changes in protein synthesis during the yeast → mycelium (36°C → 26°C) transition. The reverse process, mycelium → yeast differentiation (26°C → 36°C) is characterized by alterations in the synthesis of few proteins. We have also reported the occurrence of some stage-specific proteins in both yeast and mycelium forms; the classical heat shock protein hsp70 is detected only in yeast cells. The dimorphic characteristics of P. brasiliensis make it an attractive model for gene expression and cellular differentiation studies.

We have previously shown that in the ascomycete Saccobolus platensis the presence of cyclic AMP can overcome the requirement for light to produce apothecia. With the aim to gain insight into the phototransduction mechanism the existence of G protein-like molecules was investigated. By using the GTP analog [³⁵S]GTP-γS we detected the existence of GTP-binding proteins; the dissociation constant for the binding of the nucleotide was calculated (K_d = 52 nM); the binding was effectively competed by GTP and GDPPS and to a lesser extent by ATP. We also demonstrated by SDS-PAGE the existence of proteins of 48, 41, 26, and 15 kDa that are substrates of the cholera toxin-dependent ADP-ribosylation reaction. Proteins resolved by SDS-PAGE were identified by Western blotting with antisera specific for vertebrate G protein subunits; the antibody anti-α_common revealed two main bands at 48 and 41 kDa; the AS/7 antibody (anti-α_t, −α₁, and −a_i2) led to the identification of one band at 41 kDa; the antibody anti-β subunit revealed a polipeptide band at around 37 kDa. The antibody anti-α_common also abolished the binding of [³⁵S]GTP-γS to crude membranes. The results presented in this paper provide evidence for the existence in S. platensis of proteins that proved to have a behavior similar to that of mammalian G proteins.

Sutherland, J. B., Freeman, J. P., Williams, A. J., and Cerniglia, C. E. 1994. N-oxidation of quinoline and isoquinoline by Cunninghamella elegans. Experimental Mycology 18: 271-274. Cultures of Cunninghamella elegans were grown for 7 days in liquid Sabouraud medium containing 1.9mM quinoline or isoquinoline. The spent culture media were extracted with ethyl acetate; metabolites were purified by high-performance liquid chromatography (HPLC) and thin-layer chromatography. The major metabolite produced from each compound was identified by the HPLC elution time, ultraviolet/visible absorption spectrum, and mass spectrum. Under similar conditions, approximately 65% of the added quinoline and 3% of the added isoquinoline were metabolized to quinoline N-oxide and isoquinoline N -oxide, respectively.

Braun, E. J., and Howard, R. J. 1994. Adhesion of Cochliobolus heterostrophus conidia and germlings to leaves and artificial surfaces. Experimental Mycology 18, 211-220. We have examined the nonspecific attachment of Cochliobolus heterostrophus germlings to a variety of surfaces (glass, cellophane, Mylar, polystyrene, Teflon, maize leaves) in an effort to more fully characterize this important stage of pathogenesis. Washing experiments showed that conidia began adhering to glass just prior to germ tube emergence, about 20 min after hydration and inoculation. By 50-60 min after inoculation, over 90% of the germinating conidia resisted washing and remained firmly attached. Similar results were obtained with the other surfaces. Both sodium azide and cycloheximide prevented attachment, indicating that metabolic activity was required for adhesion. Light microscopy and cryo scanning electron microscopy were used to document a temporal and spatial relationship between attachment, appearance of extracellular matrix materials, and germ tube emergence. Attachment of conidia to the substratum was correlated with the appearance of extracellular material exuded from the tips of conidia just prior to germination. The two-layered sheath of matrix materials associated with germ tubes also surrounded appressoria and appeared to aid in attachment of these structures to leaves and artificial surfaces. We conclude that extracellular matrix is produced and/or secreted within 20 min of hydration and serves in the nonspecific attachment of germlings to the substrate.

Redman, R. S., and Rodriguez, R. J. 1994. Factors affecting the efficient transformation of Colletotrichum species. Experimental Mycology, 18, 230-246. Twelve isolates representing four species of Colletotrichum were transformed either by enhanced protoplast, restriction enzyme-mediated integration (REMI), or electroporation-mediated protocols. The enhanced protoplast transformation protocol resulted in 100- and 50-fold increases in the transformation efficiencies of Colletotrichum lindemuthianum and C. magna , respectively. REMI transformation involved the use of Hin dIII and vector DNA linearized with HindIII to increase the number of integration events and potential gene disruptions in the fungal genome. Combining the enhanced protoplast and the REMI protocols resulted in a 22-fold increase in the number of hygromycin/nystatin-resistant mutants in C. lindemuthianum . Electroporation-mediated transformation was performed on mycelial fragments and spores of four Colletotrichum species, resulting in efficiencies of up to 1000 transformants/μg DNA. The pHA1.3 vector which confers hygromycin resistance contains telomeric sequences from Fusarium oxysporum , transforms by autonomous replication and genomic integration, and was essential for elevated transformation efficiencies of 100 to 10,000 transformants/μg DNA. Modifications of pHA1.3 occurred during bacterial amplification and post fungal transformation resulting in plasmids capable of significantly elevated transformation efficiencies in C. lindemuthianum.

Banuett, F., and Herskowitz, I. 1994. Morphological transitions in the life cycle of Ustilago maydis and their genetic control by the a and b loci. Experimental Mycology 18: 247-266. Two forms characterize the life cycle of Ustilago maydis: a haploid yeast-like form and a filamentous dikaryotic form. Dimorphism and other aspects of the life cycle (including tumor induction) are governed by two mating type loci, a and b . Here we report characterization of two different morphological transitions in the life cycle of U. maydis. First, we describe an assay for conjugation tube formation in which cellular response is rapid and occurs synchronously and uniformly in the population. Using this assay, we demonstrate that different alleles of the a locus (but not the b locus) are necessary for conjugation tube formation. We also show that the b locus determines the type of filament formed after cell fusion: different b alleles lead to formation of true filaments, whereas identical b alleles result in production of pseudofilaments. Second, we analyze the role of a and b in postfusion events leading to filament formation in diploid strains. We show that diploid strains heterozygous for both a and b are capable of a dimorphic transition from yeast-like to filamentous growth when shifted from rich medium to low-nitrogen medium. This transition has two components: the first is dependent on the a locus and generates structures similar to conjugation tubes; the second is dependent on the b locus and produces true hyphal structures. We surmise that similar events take place in formation of the dikaryotic filament.