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At least 811 records · Page 45Linked to original sources
PubMed1995

Ultracytochemical evidence of Golgi functions in microvesicles at all phases of cell cycle in Saccharomyces cerevisiae.

The topical question of Golgi compartment identity in the ascomycetous yeast Saccharomyces cerevisiae is illustrated by a multiple ultracytochemical approach. For this eucaryotic single-cell organism the established scheme of secretory transport via a cascade of cisternae housing different functions of Golgi apparatus has been deduced principally of genetic and molecular analyses ex situ and confirms the mammalian secretion scheme. Nevertheless, ultracytochemical in situ localizations of enzyme activities engaged in secretion represented evidence for localization of important steps of secretory glycoprotein maturation in two morphologically distinct populations of transport microvesicles formed from endoplasmic reticulum and Golgi cisternae. Both types of microvesicles function in exocytosis or transport into lysosomal vacuoles and have identical charge. However, their presence differs in interphase and in budding cells of S. cerevisiae. Smooth, larger membrane bound microvesicles are conspicuous at the onset of budding and at construction of scars, while the coated, smaller microvesicles of globular ultrastructure are present constitutively, throughout the cell cycle. Because the established model of the yeast secretory path considers only the part of the budding phase preceding the onset of mitosis, an alternative scheme for the cellular mechanism of glycoprotein secretion in S. cerevisiae that distinguishes interphase and budding yeast, has been established. The lumen of microvesicles contains proteases catalysing maturation of the mating pheromone alpha-factor (yscIV, yscF), vacuolar protease yscY, alkaline phosphohydrolase, polyphosphorylated components of the bud scar and glycoproteins. The in situ approach also reveals a minimum level of alpha-factor precursor processing proteolytic activity at the budding phase of cells, a transient presence of polyphosphorylated compounds in the bud scars and their transport by microvesicles. Ultracytochemical reactions suggest that the nuclear envelope lumen houses certain functions attributed to endoplasmic reticulum and that some steps of outer-chain glycosylation may occur in microvesicles. Microvesicles which contain proteases and polyphosphorylated intermediates also appear in juvenile vacuoles (lysosomes). Ultracytochemical findings show the Golgi compartment of S. cerevisiae to consist not only of discrete endoplasmic cisternae, immunodetected by others as sites of outer chain alpha-1,6-mannosylation and of the Golgi membrane marker proteins Sec7p and Ypt1p, but also of microvesicles moving either to the cell plasma membrane or to vacuoles. The previously hypothesized hierarchy of segregated yeast Golgi cisternae was not revealed by ultracytochemical findings.(ABSTRACT TRUNCATED AT 400 WORDS)

Aminopeptidases↗
PubMed1992

Convergence in ascospore discharge mechanism among pyrenomycete fungi based on 18S ribosomal RNA gene sequence.

Fungi of the class Pyrenomycetes (Ascomycotina) form a morphological series ranging from those that shoot ascospores (sexual spores) forcibly from the ascus (spore sac) to fungi that ooze ascospores or have no obvious mechanism for ascospore release. Did forcible ascospore discharge evolve within these pyrenomycetes, or has it been lost in the group? We determined the sequences of the 18S ribosomal RNA gene from three fungi and used these, along with six sequences from our previous work and three sequences from GenBank, to infer the phylogeny of 12 ascomycetes with various ascospore discharge mechanisms. The 1720 base pairs of sequence data per fungus yielded 361 variable sites, 198 phylogenetically informative sites, and a single most parsimonious tree requiring 562 nucleotide changes. The tree shows that the capacity to shoot ascospores into the air has been lost or, less probably, gained repeatedly and independently. Species lacking forcible ascospore discharge are intercalated among three lineages of species with forcible discharge. In this tree, seven of the nine internal branches appeared in 95% or more of 500 bootstrap replicates. A tree uniting the fungi with forcible ascospore discharge into a monophyletic group required 45 additional steps and fit significantly less well with the data than the most parsimonious tree, based on a maximum likelihood test. Two of the fungi whose sequence we determined, Pseudallescheria boydii and Sporothrix schenckii, are not closely related to one another, even though both are human pathogens and both are from pyrenomycete lineages lacking forcible ascospore discharge. Using the well-resolved, most parsimonious tree, we inferred base substitution patterns in the 18S rRNA. The transition-to-transversion ratio was 1.9. Of all 12 possible substitutions, 29% were from U to C. At sites corresponding to yeast stem positions, A to G transitions were frequent, perhaps compensating for some of the U to C changes, and maintaining secondary structure base pairing (A to G:U to C = 3:4). In loop or bulge positions without secondary structure base pairing, U to C transitions were still frequent, but A to G transitions were rare (A to G:U to C = 1:5).

Ascomycota↗
PubMed2005

Conservation of cysteine residues in fungal histidine acid phytases.

Amino acid sequence analysis of fungal histidine acid phosphatases displaying phytase activity has revealed a conserved eight-cysteine motif. These conserved amino acids are not directly associated with catalytic function; rather they appear to be essential in the formation of disulfide bridges. Their role is seen as being similar to another eight-cysteine motif recently reported in the amino acid sequence of nearly 500 plant polypeptides. An additional disulfide bridge formed by two cysteines at the N-terminus of all the filamentous ascomycete phytases was also observed. Disulfide bridges are known to increase both stability and heat tolerance in proteins. It is therefore plausible that this extra disulfide bridge contributes to the higher stability found in phytase from some Aspergillus species. To engineer an enhanced phytase for the feed industry, it is imperative that the role of disulfide bridges be taken into cognizance and possibly be increased in number to further elevate stability in this enzyme.

6-Phytase↗
PubMed2006

MRSP1, encoding a novel Trichoderma secreted protein, is negatively regulated by MAPK.

A novel gene, MRSP1 (MAP kinase repressed secreted protein 1) is strongly overexpressed in the tmkA MAPK mutant of the biocontrol fungus Trichoderma virens. Transcriptional regulation of MRSP1 is determined by presence or absence of TmkA, rather than by light or sporulation, making it a molecular marker for the unusual, negative, regulation by TmkA. The predicted protein is 15.9 kDa, has a secretory signal, and the four-cysteine pattern, C-X29-CP(G)C-X31-C, may define a new cysteine-rich motif. This is a novel protein with functions not known from any other organism. Conservation in ascomycete, basidiomycete, and Dictyostelium homologs, as well as tight MAPK regulation, might indicate important cellular functions.

Cells, Cultured↗
PubMed2005

Electrochemical investigation of cellobiose dehydrogenase from new fungal sources on Au electrodes.

Following previous electrochemical investigations of cellobiose dehydrogenase (CDH), the present investigation reports on the initial screening of the electrochemistry of three new CDHs, two from the white rot basidiomycetes Trametes villosa and Phanerochaete sordida and one from the soft rot ascomycete Myriococcum thermophilum, for their ability to directly exchange electrons with 10 different alkanethiol-modified Au electrodes. Direct electron transfer (DET) between the enzymes and some of the modified Au electrodes was shown, both, in the presence and in the absence of cellobiose. However, the length and the head functionality of the alkanethiols drastically influenced the efficiency of the DET reaction and also influenced the effect of pH on the biocatalytic/redox currents, suggesting the importance of structural/sequence differences between these CDH enzymes. In this respect, the white rot CDHs exhibit excellent biocatalytic and redox currents, whereas for the soft rot CDH the DET communication is much less efficient. Cyclic voltammograms indicate that the heme domain of the CDHs is the part of the enzymes that most readily exchanges electrons with the electrode. However, for P. sordida CDH on 11-mercaptoundecanol or dithiopropionic acid-modified Au electrodes, a second voltammetric wave was noticed suggesting that for some orientations of the enzyme, DET communication with the FAD cofactor can also be obtained.

Biosensing Techniques↗
PubMed2006

Rate of iodine volatilization and accumulation by filamentous fungi through laboratory cultures.

Five strains of basidiomycetes (Lentinula edodes, Coprinus phlyctidosporus, Hebeloma vinosophyllum, Pleurotus ostreatus and Agaricus bisporus), one strain of ascomycete (Hormoconis resinae) and six strains of imperfect fungi (Penicillium chrysogenum, Penicillium roquefortii, Cladosporium cladosporioides, Alternaria alternata, Aspergillus niger and Aspergillus oryzae) were cultured in a liquid medium containing a radioactive iodine tracer ((125)I), and were tested for their abilities to volatilize or accumulate iodine. Of the fungal strains tested, 11 strains volatilized a considerable amount of iodine, with L. edodes showing the highest volatilization rate of 3.4%. The volatile organic iodine species emitted from imperfect fungi cultures was identified as methyl iodide (CH(3)I). In contrast, six fungal strains in 12 strains accumulated a considerable amount of iodine from the medium with concentration factors of more than 1.0. Among these, Alt. alternata and Cl. cladosporioides accumulated more than 40% of the iodine in their hyphae, and showed high concentration factors of 22 and 18, respectively. These results suggest that filamentous fungi have a potential to influence the mobility and speciation of iodine by volatilization and accumulation. Considering their great biomass in soils, filamentous fungi may contribute to the global circulation of stable iodine and also the long-lived radioiodine, (129)I (half-life: 1.6 x 10(7)years), released from nuclear facilities into the environment.

Fungi↗
PubMed2005

Influence of temperature and nutritional requirements for mycelial growth of Eutypa lata, a vineyard pathogenic fungus.

Eutypa dieback (dying arm disease, eutypiosis) is a very devastating disease in many grape-producing areas around the world. This vascular disease is induced by the ascomycete Eutypa lata Pers. Fr. Tul & C. Tul. invading the trunk by pruning wounds. The environmental factors and the nutritional requirements regulating fungus development are yet poorly known. This work shows that the isolated strain of E. lata was able to grow in a large temperature range (2-30 degrees C). However, a higher temperature (35 degrees C) presented inhibitory effects on mycelial growth. E. lata was able to use various osidic molecules (C5, C6, C12, C18, C24, and starch); showing thus a large adaptation to the carbon source supplied. As nitrogen source, it used salts and numerous natural amino acids. A significant result was obtained with cysteine presenting obvious antifungal properties. This effect can further be used with the aim of setting up a curative treatment of the disease.

Ascomycota↗
PubMed2005

The Aspergillus nidulans phytochrome FphA represses sexual development in red light.

Phytochrome photoreceptors sense red and far-red light through photointerconversion between two stable conformations, a process mediated by a linear tetrapyrrole chromophore. Originally, phytochromes were thought to be confined to photosynthetic organisms including cyanobacteria, but they have been recently discovered in heterotrophic bacteria and fungi, where little is known about their functions. It was shown previously in the ascomycetous fungus Aspergillus nidulans that asexual sporulation is stimulated and sexual development repressed by red light. The effect was reminiscent of a phytochrome response, and indeed phytochrome-like proteins were detected in several fungal genomes. All fungal homologs are more similar to bacterial than plant phytochromes and have multifunctional domains where the phytochrome region and histidine kinase domain are combined in a single protein with a C-terminal response-regulator domain. Here, we show that the A. nidulans phytochrome FphA binds a biliverdin chromophore, acts as a red-light sensor, and represses sexual development under red-light conditions. FphA-GFP is cytoplasmic and excluded from the nuclei, suggesting that red-light photoperception occurs in the cytoplasm. This is the first phytochrome experimentally characterized outside the plant and bacterial kingdoms and the second type of fungal protein identified that functions in photoperception.

Amino Acid Sequence↗
PubMed2005

Fungal adenylyl cyclase integrates CO2 sensing with cAMP signaling and virulence.

The ascomycete Candida albicans is the most common fungal pathogen in immunocompromised patients . Its ability to change morphology, from yeast to filamentous forms, in response to host environmental cues is important for virulence . Filamentation is mediated by second messengers such as cyclic adenosine 3',5'-monophosphate (cAMP) synthesized by adenylyl cyclase . The distantly related basidiomycete Cryptococcus neoformans is an encapsulated yeast that predominantly infects the central nervous system in immunocompromised patients . Similar to the morphological change in C. albicans, capsule biosynthesis in C. neoformans, a major virulence attribute, is also dependent upon adenylyl cyclase activity . Here we demonstrate that physiological concentrations of CO2/HCO3- induce filamentation in C. albicans by direct stimulation of cyclase activity. Furthermore, we show that CO2/HCO3- equilibration by carbonic anhydrase is essential for pathogenesis of C. albicans in niches where the available CO2 is limited. We also demonstrate that adenylyl cyclase from C. neoformans is sensitive to physiological concentrations of CO2/HCO3-. These data demonstrate that the link between cAMP signaling and CO2/HCO3- sensing is conserved in fungi and reveal CO2 sensing to be an important mediator of fungal pathogenesis. Novel therapeutic agents could target this pathway at several levels to control fungal infections.

Adenylyl Cyclases↗
PubMed2006

Evolution of filamentous plant pathogens: gene exchange across eukaryotic kingdoms.

Filamentous fungi and oomycetes are eukaryotic microorganisms that grow by producing networks of thread-like hyphae, which secrete enzymes to break down complex nutrients, such as wood and plant material, and recover the resulting simple sugars and amino acids by osmotrophy. These organisms are extremely similar in both appearance and lifestyle and include some of the most economically important plant pathogens . However, the morphological similarity of fungi and oomycetes is misleading because they represent some of the most distantly related eukaryote evolutionary groupings, and their shared osmotrophic growth habit is interpreted as being the result of convergent evolution . The fungi branch with the animals, whereas the oomycetes branch with photosynthetic algae as part of the Chromalveolata . In this report, we provide strong phylogenetic evidence that multiple horizontal gene transfers (HGT) have occurred from filamentous ascomycete fungi to the distantly related oomycetes. We also present evidence that a subset of the associated gene families was initially the product of prokaryote-to-fungi HGT. The predicted functions of the gene products associated with fungi-to-oomycete HGT suggest that this process has played a significant role in the evolution of the osmotrophic, filamentous lifestyle on two separate branches of the eukaryote tree.

Algal Proteins↗
PubMed2006

Safety evaluation of pigment containing Aspergillus carbonarius biomass in albino rats.

Aspergillus carbonarius, an ascomycetes fungus, is known to produce pectinase in solid-state fermentation. A mutant strain of A. carbonarius UV-10046 selected for temperature tolerance over produced polygalacturonase and during growth accumulated an yellow pigment in its biomass. Since the colored fungus suggested its application for food use, the freeze-dried biomass was evaluated to assess its safety in experimental animals. Acute and sub-acute toxicity studies were conducted on both sexes of albino rats. Feeding acute doses of A. carbonarius freeze-dried biomass at 0.5-5.0g/kg body weight to adult rats did not show any symptoms of toxicity or mortality of the rats. Similarly, dietary feeding of A. carbonarius at 0.25-2.0% level (w/w) for 14 weeks did not produce any significant changes in food intake or gain in body weight of the experimental rats compared to control rats. There were no significant differences in the relative weight of vital organs, hematological parameters, macroscopic and microscopic changes in vital organs and serum enzyme levels between the experimental and control groups. The results clearly showed that acute and sub-acute oral feeding of freeze-dried whole cells of A. carbonarius mutant for 14 weeks did not produce any toxic effects in male and female rats.

Administration, Oral↗
PubMed2006

The transition metal chelator nicotianamine is synthesized by filamentous fungi.

Nicotianamine is an important metal ligand in plants. Surprisingly, recent genome sequencing revealed that ascomycetes encode proteins with similarity to plant nicotianamine synthases (NAS). By expression in a Zn2+-hypersensitive fission yeast mutant we show for a protein from Neurospora crassa that it indeed possesses NAS activity. Using electrospray-ionization-quadrupole-time-of-flight mass spectrometry we prove the formation of nicotianamine in N. crassa. Transcript level is strongly upregulated under Zn deficiency as shown by real-time PCR. These findings demonstrate that nicotianamine is more widespread in nature than anticipated and provide further evidence for a function of nicotianamine as a cytosolic chelator of Zn2+ ions.

Alkyl and Aryl Transferases↗
PubMed2004

Oxygen flux magnitude and location along growing hyphae of Neurospora crassa.

Oxygen fluxes were mapped at the growing apices and along mycelial hyphal segments of the ascomycete Neurospora crassa. High spatial resolution was obtained using micro-oxygen probes (2-3 microm tip diameters) and the self-referencing technique to maximize the sensitivity of oxygen flux measurements. As expected, oxygen influx was inhibited by cyanide, although oxygen influx (and hyphal growth) resumed with the induction of an alternate oxidase activity. Along hyphal segments, variations in oxygen influx were not correlated with location, near or far from septa, and varied over time along the same hyphal segment. Growing hyphae had a region of maximal oxygen influx greater than 10 microm behind the hyphal tip, the oxygen influx was correlated with hyphal growth rate. The region of maximal oxygen influx did not correspond with mitochondrial density, which is maximal (about 30% of hyphal volume) 5-10 microm behind the tip. Therefore, tip-localized mitochondria do not contribute to the respiratory requirements of the growing hypha. The tip-localized mitochondria may function in clearing calcium from the cytoplasm, although a decline in chlortetracycline fluorescence after cyanide inhibition could also be due to ATP-depletion due to inhibition of actively respiring mitochondria. Alternatively, the growing tip may be the site of mitochondrial biogenesis.

Calcium↗
PubMed2004

Glutamic protease distribution is limited to filamentous fungi.

Glutamic proteases are a distinct, and recently re-classified, group of peptidases that are thought to be found only in fungi. We have identified and analysed the distribution of over 20 putative glutamic proteases from all fungal species whose genomes have been sequenced so far. Although absent from the Saccharomycetales class, glutamic proteases appear to be present in all other ascomycetes species examined. A large number of coding regions for glutamic proteases were also found clustered together in the Phanerochaete chrysosporium genome, despite apparently being absent from three other species of Basidiomycota.

Fungi↗
PubMed2005

Yeast diversity in hypersaline habitats.

Thus far it has been considered that hypersaline natural brines which are subjected to extreme solar heating, do not contain non-melanized yeast populations. Nevertheless we have isolated yeasts in eight different salterns worldwide, as well as from the Dead Sea, Enriquillo Lake (Dominican Republic) and the Great Salt Lake (Utah). Among the isolates obtained from hypersaline waters, Pichia guilliermondii, Debaryomyces hansenii, Yarrowia lipolytica and Candida parapsilosis are known contaminants of low water activity food, whereas Rhodosporidium sphaerocarpum, R. babjevae, Rhodotorula laryngis, Trichosporon mucoides, and a new species resembling C. glabrata were not known for their halotolerance and were identified for the first time in hypersaline habitats. Moreover, the ascomycetous yeast Metschnikowia bicuspidata, known to be a parasite of the brine shrimp, was isolated as a free-living form from the Great Salt Lake brine. In water rich in magnesium chloride (bitterns) from the La Trinitat salterns (Spain), two new species provisionally named C. atmosphaerica - like and P. philogaea - like were discovered.

Biodiversity↗
PubMed2005

Agrobacterium tumefaciens-mediated transformation of Leptosphaeria spp. and Oculimacula spp. with the reef coral gene DsRed and the jellyfish gene gfp.

Four filamentous ascomycetes, Leptosphaeria maculans, L. biglobosa, Oculimacula yallundae and O. acuformis, were transformed via Agrobacterium tumefaciens-mediated transformation with the genes encoding DsRed and GFP. Using vectors pCAMDsRed and pCAMBgfp, either germinated conidia of Leptosphaeria spp. and O. yallundae or physically fragmented cultures of Oculimacula spp. were transformed. In vitro, the expression of the two reporter proteins in mycelium of both Oculimacula and both Leptosphaeria species was sufficient to distinguish each species in co-inoculated cultures. In planta, transformants of L. maculans or L. biglobosa expressing DsRed or GFP could be observed together in leaves of Brassica napus. Either reporter protein could be used to view the colonization of leaf petioles by both Leptosphaeria spp. and growth in the xylem vessels could be clearly observed. With the generation of these transformants, further studies on interactions between pathogen species involved in disease complexes on various host species and between opposite mating types of the same species are now possible.

Agrobacterium tumefaciens↗
PubMed2005

Tuber borchii mycelial protoplasts isolation, characterization and functional delivery of liposome content, a new step towards truffles biotechnology.

The filametous ascomycete Tuber borchii is a plant-symbiotic ectomycorrhizal microrganism with an high value due to the production of hypogeous fruitbodies (truffles). The present work was undertaken to develop a procedure for the release of T. borchii viable protoplasts from Tuber mycelium, isolate ATTC 96540; several factors which affect the isolation, morphology and viability were examined and developed in order to improve applications of T. borchii protoplasts in morphological, biochemical and genetic investigations (protoplast fusion or transformation). Functional delivery of liposome content into T. borchii protoplasts has also been examined with a cytotoxic ribosome inactivator as saporin. T. borchii protoplasts incubation/fusion with saporin containing liposomes were made to demonstrate the absence of cell wall of 16 days cultured protoplasts.

Ascomycota↗
PubMed2004

Polyphasic identification of yeasts isolated from bark of cork oak during the manufacturing process of cork stoppers.

A two-step protocol was used for the identification of 52 yeasts isolated from bark of cork oak at initial stages of the manufacturing process of cork stoppers. The first step in the identification was the separation of the isolates into groups by their physiological properties and RFLPs of the ITS-5.8S rRNA gene. The second step was the sequencing of the D1/D2 domains of the 26S rRNA gene of selected isolates representing the different groups. The results revealed a predominance of basidiomycetous yeasts (11 species), while only two species represented the ascomycetous yeasts. Among the basidiomycetous yeasts, members representing the species Rhodosporidium kratochvilovae and Rhodotorula nothofagi, that have been previously isolated from plant material, were the most abundant. Yeasts pertaining to the species Debaryomyces hansenii var. fabryii, Rhodotorula mucilaginosa and Trichosporon mucoides were isolated in small numbers.

Base Sequence↗