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PubMed2000

Fungal degradation of fluorene.

A selection of 30 strains of micromycetes known as good degraders of polychlorinated aromatic compounds, mostly isolated from soil and belonging to various taxonomic groups, have been investigated to degrade fluorene. Toxicity assays, first evaluated on solid media, have shown high growth inhibition at concentrations above 0.001 g l-1 only towards 23% of strains. Degradation of fluorene (0.005 g l-1) was then investigated in liquid synthetic medium for 2 days and evaluated by HPLC. Among the 30 strains tested, 12 could be considered as best degraders because of a rate of degradation at 60% or over. 3 strains of Cunninghamella genus were very efficient (mean of degradation: 96%) but different strains from Ascomycetes. Basidiomycetes and Deuteromycetes were also efficient 11 strains are not yet reported in the literature: Aspergillus terreus, Bjerkandera adusta, Ceriporiopsis subvermispora, Colletotrichum dematium, Cryphonectria parasitica, Cunninghamella blakesleeana, C. echinulata, Drechslera spicifera, Embellisia annulata, Rhizoctonia solani and Sporormiella australis. A metabolic approach with standard compounds (9-fluorenol and 9-fluorenone) indicated the presence of these monooxygenated derivatives for most of the strains.

Biodegradation, Environmental↗
PubMed1999

Fatal Chaetomium cerebritis in a bone marrow transplant patient.

The number of opportunistic infections in the central nervous system (CNS) has been steadily increasing because of a rising number of immunocompromised patients. A rare form of CNS infection can be caused by Chaetomium species, one of the largest genera of saprophytic ascomycetes. The CNS lesions in the present case were caused by Chaetomium atrobrunneum. The main characteristic of almost all Chaetomium species is presence of hairs or setae covering the ascomata. Microbiological studies are the only definitive way to correctly identify this fungal organism. The rapid evolvement of the cerebral infection suggests that the brain tissue provides a favorable environment for growth and proliferation of these fungi. This is the second documented case of a fatal brain abscess caused by Chaetomium atrobrunneum, and the first case report in a bone marrow transplant patient.

Adult↗
PubMed2002

Aging in fungi: role of mitochondria in Podospora anserina.

In experimental gerontology, there is a long tradition in the use of both unicellular and filamentous species of fungi. In the last three decades, biochemical, genetic and molecular approaches have proved very fruitful in elucidating different aspects of ageing. It was shown that various genes and molecular pathways are involved in life span control. The oxygenic energy metabolism plays a central role. During mitochondrial energy transduction, reactive oxygen species (ROS) are generated as by-products. These molecules are able to damage all cellular compounds leading to cellular dysfunctions. Within certain limits, however, cells are able to cope with ROS-related problems. First, ROS scavengers can be induced which are effective in lowering the molecular burden of ROS on cellular functions. Second, if damage occurs, specific repair mechanisms and the general turnover of affected molecules can maintain cellular functions. Finally, if damage of essential components is too severe, cells may induce specific pathways to compensate for the corresponding impairments. A coordinated interaction between different cellular compartments is involved in these processes. In this review I shall concentrate on the ageing in the filamentous ascomycete Podospora anserina. It is clear that both environmental as well as genetic traits are involved in the control of life span and that mitochondrial-nuclear interactions play a paramount role.

Aging↗
PubMed2000

cDNA probes for detection of specific dsRNAs from the fungal pathogen, Monosporascus cannonballus.

Monosporascus cannonballus is an ascomycete fungus that is the causative agent of Monosporascus root rot/vine decline, a serious disease of muskmelon and watermelon. Double-stranded RNA (dsRNA) was identified in approximately 60% of M. cannonballus isolates recovered from infected muskmelon plants in 1993. After repeated laboratory transfer on culture media, the majority of the isolates harboring dsRNAs developed degenerate culture phenotypes and showed reduced virulence (hypovirulence) to muskmelon. Initially, dsRNA purification and cDNA synthesis were attempted in three M. cannonballus isolates harboring dsRNAs. However, numerous difficulties were encountered due to the stable, double-stranded nature of the dsRNAs and contamination of the preparations by fungal rRNA. Several purification and cDNA protocols were evaluated and eventually modified into methods that were ultimately highly effective for cloning dsRNAs from M. cannonballus. The cDNAs derived from purified dsRNA preparations were cloned into a pUC119 plasmid vector and amplified in Escherichia coli. Nine cDNA clones were identified that are specific for medium-sized (ca. 3 kbp) dsRNAs associated with M. cannonballus isolate Ca91-17(96+). The methods used to make the cDNA clones of the dsRNAs in M. cannonballus may be useful for those working on fungal dsRNAs. In addition, these cDNAs may be useful for identifying dsRNAs associated with the hypovirulence phenotype.

Ascomycota↗
PubMed2001

Expression of Pleurotus eryngii aryl-alcohol oxidase in Aspergillus nidulans: purification and characterization of the recombinant enzyme.

Aryl-alcohol oxidase (AAO) is an extracellular flavoenzyme involved in lignin biodegradation by some white-rot fungi. The enzyme catalyzes the extracellular oxidation of aromatic alcohols to the corresponding aldehydes. The electron acceptor is molecular oxygen yielding H(2)O(2) as the product. Herein we describe, for the first time, the expression of AAO from Pleurotus eryngii in the ascomycete Aspergillus nidulans. The activity of the recombinant enzyme in A. nidulans cultures is much higher than found in the extracellular fluid of P. eryngii. The recombinant enzyme showed the same molecular mass, pI and catalytic properties as that of the mature protein secreted by P. eryngii. The enzymic properties are also similar to those reported from other Pleurotus and Bjerkandera species.

Alcohol Oxidoreductases↗
PubMed2000

Analysis of the dynamics of fungal communities in soil via fungal-specific PCR of soil DNA followed by denaturing gradient gel electrophoresis.

A molecular method for profiling of fungal communities in soil was applied in experiments in soil microcosms, with two objectives, (1) to assess the persistence of two selected fungal species in soil, and (2) to analyze the response of the natural fungal community to a spill of sulphurous petrol in the same soil. To achieve the aims, two soil DNA extraction methods, one originally designed for the direct extraction of bacterial community DNA and the other one aimed to obtain fungal DNA, were tested for their efficiency in recovering DNA of fungal origin from soil. Both methods allowed for the efficient extraction of DNA from introduced Trichoderma harzianum spores as well as Arthrobotrys oligospora mycelial fragments, at comparable rates. Several PCR amplification systems based on primers specific for fungal 18S ribosomal RNA genes were tested to design strategies for the assessment of fungal communities in soil. The PCR systems produced amplicons of expected size with DNA of most fungi studied, which included members of the Ascomycetes, Basidiomycetes, Zygomycetes and Chytridiomycetes. On the other hand, the 18S rRNA genes of Oomycetes (including key plant pathogens) were poorly amplified. Plant (Solanum tuberosum), nematode (Meloidogyne sp.) and bacterial DNA was not amplified. For studies of soil fungal communities, a nested PCR approach was selected, in which the first PCR provided the required specificity for fungi, whereas the second (nested) PCR served to produce amplicons separable on denaturing gradient gels. Denaturing gradient gel electrophoresis (DGGE) allowed the resolution of mixtures of PCR products of several different fungi, as well as products resulting from mixed-template amplifications, into distinct banding patterns. The persistence of fungal species in soil was assessed using T. harzianum spores and A. oligospora hyphal fragments added to silt loam soil microcosms. Using PCR-DGGE, these fungi were detectable for about 14 days and 2 months, respectively. Both singly-inoculated soils and soils that had received mixed inoculants revealed, next to bands resulting from indigenous fungi, the expected bands in the DGGE profiles. The A. oligospora specific amplicon, by virtue of its unique migration in the denaturing gradient, was well detectable, whereas the T. harzianum specific product comigrated with products from indigenous fungi. PCR-DGGE analysis of DNA obtained from the silt loam soil treated with dibenzothiophene-containing petrol showed the progressive selection of specific fungal bands over time, whereas this selection was not observed in untreated soil microcosms. Cloning of individual molecules from the selected bands and analysis of their sequences revealed a complex of targets which clustered with the 18S rDNA sequences of the closely-related species Nectria haematococca, N. ochroleuca and Fusarium solani. Fungal isolates obtained from the treated soil on PDA plates were identified as Trichoderma sp., whereas those on Comada agar fell into the Cylindrocarpon group (anamorph of Nectria spp).

Base Composition↗
PubMed1999

Detection of siderophore production from several fungi and bacteria by a modification of chrome azurol S (CAS) agar plate assay.

A well-known and widely used method for detection of siderophore production by microorganisms in solid medium is the universal chrome azurol S (CAS)-agar plate assay. However, the high toxicity of CAS-blue agar medium caused by the presence of a detergent impedes its utilization with many varieties of fungi and Gram-positive bacteria. To solve this problem, a modification of the CAS-agar plate assay was made by incorporating the CAS-blue dye in a medium with no contact with the microorganisms tested. Half of each plate used in our experiments was filled with the most appropriate culture medium for each type of microorganism and the other half with CAS-blue agar. This modification allowed us to study several strains of fungi (basidiomycetes, deuteromycetes, ascomycetes and zygomycetes) and bacteria (Gram positive and negative), some of them appearing for the first time in the literature. All the microorganisms grew properly and reacted in different manners to the CAS assay. Some strains of wood-decaying basidiomycetes (mainly white-rot fungi) and Aspergillus species produced the fastest color-change reactions in the CAS-blue agar. This modified method could facilitate optimization of culture conditions, since both CAS-blue agar and growth medium were prepared and added in the Petri plate separately.

Agar↗
PubMed2002

Intra-species chromosome-length polymorphism in Geotrichum candidum revealed by pulsed field gel electrophoresis.

Geotrichum candidum is an ascomycetous anamorph yeast-like fungus found in various habitats. It is a component of the natural flora of milk and is used as a maturing agent for both soft and hard cheeses. This microorganism displays phenotypic variability and may act as an opportunist pathogen, causing geotrichosis. Cytological analysis of G. candidum strain ATCC 204307 showed this strain to have eight chromosomes. We prepared chromosomal DNA from 13 strains of G. candidum differing in habitat and morphotype. We used pulsed field gel electrophoresis (PFGE) in two sets of conditions to determine the size of the chromosomal DNA molecules. The strains investigated had five to eight chromosomes, 0.6 to 4.5 Mb in size. We estimated genome size in these 13 strains to be between 11 and 19 Mb. Pulsed-field gel electrophoresis profiles showed a high degree of polymorphism, indicating considerable variability between strains. Genome size and the presence of large chromosomes appeared to be correlated with morphotype. Strains with a mold-like or intermediate morphotype tended to have larger genomes than strains with a yeast-like morphotype did.

DNA, Bacterial↗
PubMed1998

Nonradioactive in situ hybridization for detection of hydrophobin mRNA in the phytopathogenic fungus Claviceps purpurea during infection of rye.

Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts.

Claviceps↗
PubMed1999

Comparative analysis of Ca2+ and H+ flux magnitude and location along growing hyphae of Saprolegnia ferax and Neurospora crassa.

Calcium and proton ion fluxes were mapped at the growing apices of two hyphal organisms, the oomycete Saprolegnia ferax and the ascomycete Neurospora crassa and pseudohyphal Saccharomyces cerevisiae using self-referencing ion-selective probes. S. ferax exhibited well-defined transport zones absent in N. crassa. Ca2+ fluxes were located within 8 microm of the growing hyphal tip; the net Ca2+ flux was either inward (75% of all experiments) or outward. The inward component of the net flux was inhibited by Gd3+, known to inhibit Ca2+ permeable stretch-activated channels. Because the Ca2+ flux is located at the region of maximal hyphal expansion, exocytosis may contribute to Ca2+ efflux, in addition to the stretch-activated channel mediated influx. Maximal inward H+ flux was observed 10-30 microm behind the hyphal tip where peak mitochondria densities taper off at the onset of a vacuolation zone, presumably due to highly localized H+ cotransporter activity. By contrast, N. crassa exhibited no net Ca2+ flux and a consistently inward H+ flux (93% of all experiments) that was homogeneously distributed up to 60 microm behind the hyphal apex. Both hyphal organisms have similar tip morphology and growth rates, and are reported to have tip-high cytosolic Ca2+ gradients associated with growth. Only S. ferax exhibited tip-localized Ca2+ fluxes and a well defined H+ influx zone just behind the tip. Differences in ecological habitats and cytology--S. ferax is an aquatic organism that grows as a migrating plug of cytoplasm while N. crassa is normally terrestrial with a cytoplasm-rich mycelium and highly active cytoplasmic streaming behind the growing margin--may account for the differences in the 'architecture' of ion transport occurring during the process of tip growth. Net Ca2+ efflux and H+ influx of growing S. cerevisiae pseudohyphae were also measured but localization was not possible due to small cell size.

Calcium↗
PubMed1999

The contribution of cell wall proteins to the organization of the yeast cell wall.

Our knowledge of the yeast cell wall has increased rapidly in the past few years, allowing for the first time a description of its structure in molecular terms. Two types of cell wall proteins (CWPs) have been identified that are covalently linked to beta-glucan, namely GPI-CWPs and Pir-CWPs. Both define a characteristic supramolecular complex or structural unit. The GPI building block has the core structure GPI-CWP-->beta1,6-glucan-->beta1,3-glucan, which may become extended with one or more chitin chains. The Pir building block is less well characterized, but preliminary evidence points to the structure, Pir-CWP-->beta1,3-glucan, which probably also may become extended with one or more chitin chains. The molecular architecture of the cell wall is not fixed. The cell can make considerable adjustments to the composition and structure of its wall, for example, during the cell cycle or in response to environmental conditions such as nutrient and oxygen availability, temperature, and pH. When the cell wall is defective, dramatic changes can occur in its molecular architecture, pointing to the existence of cell wall repair mechanisms that compensate for cell damage. Finally, evidence is emerging that at least to a considerable extent the cell wall of Saccharomyces cerevisiae is representative for the cell wall of the Ascomycetes.

Cell Cycle↗
PubMed2000

Characterization of fungal 17beta-hydroxysteroid dehydrogenases.

To promote understanding of the evolution of the steroid hormone signalling and hydroxysteroid dehydrogenases (HSDs), comparative characterization of fungal 17beta-HSDs was performed. Constitutive 17beta-HSD activity was determined in cytosols of the fungi: Cochliobolus lunatus, Pleospora herbarum, Fusarium lini, Trichoderma viride, Mucor spinosus, Rhizopus nigricans and Pleurotus ostreatus. The reaction equilibrium in all species except P. ostreatus was shifted towards reduction. The preferential coenzyme for reduction of androstenedione was NADPH, while for oxidation of testosterone, NAD4 was preferred. The highest enzyme activities were found in the Ascomycete C. lunatus (152.4 nmol mg(-1) h(-1)) and in the Basidiomycete P. ostreatus (69.1 nmol mg(-1) h(-1)). No similarities on the protein and mRNA level between fungal 17beta-HSDs and the purified enzyme from C. lunatus were observed. To investigate the nature of these enzymes, 17beta-HSD was purified from P. ostreatus using ammonium sulphate precipitation, hydrophobic interaction chromatography, and affinity chromatography. The purified enzyme has an apparent molecular mass of approximately 35 kDa and is probably a dimer as determined by gel filtration. Chemical modifications exposed Lys, His and Tyr as important for enzyme activity. Additionally, no similarities of C. lunatus and P. ostreatus enzymes were found to bacterial 3alpha,20beta-HSD from Streptomyces hydrogenans, 3beta,17beta-HSD from Comamonas testosteroni and mammalian 17beta-HSD types 1 and 4. The results thus suggest that there are most probably different enzymes responsible for 17beta-HSD activity in filamentous fungi.

17-Hydroxysteroid Dehydrogenases↗
PubMed1996

Cancer is a hybrid produced by a relationship between a plant bacterium and a mammalian cell: an original concept.

Facts presented in previously published articles (1-7) support the conclusion that the malignant cell, although of animal origin, metabolizes and respires anaerobically. Facts also support the conclusion that plant bacterial conidia (spores) derived, under duress, from one of the several groups belonging to the Ascomycete family or from the Staphylococcus aureus coagulase positive micro-organism are present intracellularly. It is this group of conidia, when formed, that survives as a primitive oval, spheroidal type of a unicell. They are identical in all respects to the various carbohydrate, protein, and fat molecules. Although these conidia have lost their original outside cell wall and all their enzymes and metabolites, they survive none the less as bacteria by retaining within their cytoplasm a genetically viable anion acceptor complex (oxidant), and their asexual procreative unit. It is this oxidant factor that survives within a sac or cell, as long as it is free of the atmospheric environment. Ultimately, with an ensuing circulating but compatible flow of blood by the host, there develops an annealing process whereby the genes of each species unite to form a plant animal intracellular hybridization. This somatic association accounts for the origin of the metabolic and respiratory anaerobiosis and also for the subsequent growth and pathophysiology that occurs in those living human beings suffering from the various malignant diseases.

Anaerobiosis↗
PubMed1998

'Ascospore O', Oxidant, host assistance: the pathway to carcinogenesis.

Sufficient evidence exists to conclude that a cycle of sequential physiological events is the pathway contributing to the transformation of the mammalian cell to its malignant phase. The cycle of events involves a reticulum or network of cells. These consist either of the reticuloendothelial cells, the squamous, or the epithelial group. A second factor requires the presence, intracellularly, of conidia derived from one of the various groups of the anaerobic micro-organisms known as Ascomycetes. These conidia as a separate entity, totally free of any part of their parental origin, constitute the invasive agent which, however, can or may lie quiescent at the onset. They retain genetically an electronegative viable chemical complex (Oxidant) and also an asexual procreative unit. This Oxidant can alternately accept the negative charged ions (anions) while releasing oxygen. The subsequent ensuing circulation and compatible flow of blood by the host provides the source for the electrolytes as well as the metabolites, enzymes, etc. It is this continued flow of blood that promotes the next stage of the conidia's lifecycle and reactivates their anaerobic respiration, metabolism, and reproduction of new asexual conidia. This is the cycle of physiological events that ultimately contributes to the transformation of an animal (human) cell to its malignant phase. It also accounts for the pathological consequences as clinically present in malignantly ill patients.

Animals↗
PubMed1978

The influence of water-logging stage upon the paddy soil mycoflora in the Ukraine.

The soil fungal flora of paddy soils from the southern districts of the Ukraine was investigated. From 287 soil samples 170 species were isolated. They belonged to the following genera: Oomycetes 1.2%, Zygomycetes 8.8%, Ascomycetes 5.8%, Deuteromycetes 84.2%. Certain fungi were found to be dominant forms, among them Mortierella alpina Peyron., Penicillium funiculosum Thom, P. rugulosum Thom, P. chrysogenum Thom, Acremonium strictum W. Gams, Acr. breve W. Gams, Alternaria alternata (Fr.) Riessler, Cladosporium brevi-compactum Pidopl. et Deniak, Gliocladium roseum (Link) Thom, Harposporium sp., Fusarium oxysporum Schlect. var. orthoceras (App. et Wr.) Bilai, Trichoderma lignorum (Tode) Harz etc. The total number of fungi decreased in the paddy fieldsoils. It was found low frequency of Penicillium, Aspergillus, and high frequency of Mucorales.

Ecology↗
PubMed1979

Distribution of extracellular proteolytic activities among various yeast.

A general screening test for extracellular proteolytic activities was carried out on 24 cultures belonging to 14 genera of yeasts. The screening was performed using gelatine and casein as sole nitrogen sources under two sets of pH conditions. Potent proteolytic yeast cultures belonging to both Ascomycetes and Deuteromycetes were detected, particularly certain members of the genera Endomycopsis, Metschnikowia, Debaromyces, Rhodotorula, and Candida spec. Most detected proteolytic cultures were active on both casein and gelatine substrates, possibly suggesting one enzyme system in each case with wide substrate specificity. Physiological studies revealed that maximum proteolysis occurred after 3--4 days of aerobic incubation and that some strains were able to hydrolyze egg albumin.

Caseins↗
PubMed2004

Evidence for 1,8-dihydroxynaphthalene melanin in three halophilic black yeasts grown under saline and non-saline conditions.

The ascomycetous black yeasts Hortaea werneckii, Phaeotheca triangularis, and Trimmatostroma salinum are halophilic fungi that inhabit hypersaline water of solar salterns. They are characterized by slow, meristematic growth and very thick, darkly pigmented cell walls. The dark pigment, generally thought to be melanin, is consistently present in their cell walls when they grow under saline and non-saline conditions. We used the inhibitor tricyclazole to test the fungi in this study for the presence of 1,8-dihydroxynaphthalene (DHN)-melanin biosynthesis, since fungal melanins reportedly are derived either from DHN, tyrosine via 3,4-dihydroxyphenylalanine, gamma-glutaminyl-3,4-dihydroxybenzene, or catechol. Tricyclazole-treated cultures of the fungi were reddish-brown in color and contained typical intermediates of the DHN-melanin pathway, as demonstrated by high-performance liquid chromatography. This investigation showed that the three fungi synthesized DHN-melanin under saline and non-saline growth conditions.

Chromatography, High Pressure Liquid↗
PubMed2000

Cloning, characterization and chromosomal location of three genes encoding host-cell-wall-degrading enzymes in Leptosphaeria maculans, a fungal pathogen of Brassica spp.

The ascomycete, Leptosphaeria maculans, causes blackleg disease of oilseed Brassica spp. such as canola (Brassica napus). We have cloned a gene encoding endopolygalacturonase, pg1, and two genes encoding cellulases, cel1 and cel2, in L. maculans. These genes are not clustered in the genome, as they are located on different chromosomes. The deduced amino acid sequences of all three genes predict an N-terminal signal sequence, as is common for secreted fungal enzymes that degrade plant cell walls. The endopolygalacturonase encoded by pg1 shows the highest similarity (54% amino acid identity) to endopolygalacturonase 4 from Botrytis cinerea. Both cel1 and cel2 appear to encode cellobiohydrolase, and neither gene encodes a recognizable cellulose-binding domain or linker region. Transcription of pg1 is induced in cultures containing 1% polygalacturonic acid or pectin, and cel1 is induced in 1% cellulose or carboxymethylcellulose, as shown by Northern analysis. Glucose represses the induction of cel1 caused by cellulose and carboxymethylcellulose, but does affect transcription of pg1. Transcription of cel2 (but not cel1 or pg1) is detectable during infection of B. napus and B. juncea cotyledons and leaves using reverse transcription-PCR.

Amino Acid Sequence↗