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PubMed2001

The yeast community of sap fluxes of Costa Rican Maclura (Chlorophora) tinctoria and description of two new yeast species, Candida galis and Candida ortonii.

We report on the yeast community associated with sap fluxes of Maclura tinctoria, family Moraceae, in the dry forest of the Area de Conservación Guanacaste, Costa Rica. Eleven samples yielded seven hitherto undescribed ascomycetous yeasts in the genera Candida and Myxozyma. We describe the two most abundant as new species. Candida galis utilizes very few carbon compounds limited to some alcohols and acids. Analysis of rDNA sequences suggests that it occupies a basal position with respect to the Pichia anomala clade, with no obvious sister species. Candida ortonii is also restricted in nutritional breadth, and growth is generally very slow. It is a sister species to Candida nemodendra. The type cultures are: C. galis, strain UWO(PS)00-159.2=CBS 8842; and C. ortonii, strain UWO(PS)00-159.3=CBS 8843.

Candida↗
PubMed2006

Biotechnology, physiology and genetics of the yeast Pichia anomala.

The ascomycetous yeast Pichia anomala is frequently associated with food and feed products, either as a production organism or as a spoilage yeast. It belongs to the nonSaccharomyces wine yeasts and contributes to the wine aroma by the production of volatile compounds. The ability to grow in preserved food and feed environments is due to its capacity to grow under low pH, high osmotic pressure and low oxygen tension. A new application of P. anomala is its use as a biocontrol agent, which is based on the potential to inhibit a variety of moulds in different environments. Although classified as a biosafety class-1 organism, cases of P. anomala infections have been reported in immunocompromised patients. On the other hand, P. anomala killer toxins have a potential as antimicrobial agents. The yeast can use a broad range of nitrogen and phosphor sources, which makes it a potential agent to decrease environmental pollution by organic residues from agriculture. However, present knowledge of the physiological basis of its performance is limited. Recently, the first studies have been published dealing with the global regulation of the metabolism of P. anomala under different conditions of oxygenation.

Animals↗
PubMed2006

The Kluyveromyces lactis repertoire of transcriptional regulators.

We have exploited the recently obtained complete genome sequence of Kluyveromyces lactis to compare the repertoire of transcriptional regulators between K. lactis and Saccharomyces cerevisiae. Looking for similarities with the S. cerevisiae proteins of this functional class, we observed a reduction in gene number, which is not randomly distributed among the different DNA-binding classes, the zinc binuclear cluster class (Zn(II)2Cys6), specific to ascomycetes, being one of the most affected. However, when one examines the number of proteins that, in the K. lactis genome, possess the different DNA-binding signatures, it is not reduced compared to S. cerevisiae. This indicates that transactivator proteins have strongly diverged between the two species and cannot be recognized any more, and/or that each genome has developed a specific set of regulators to adapt the cell to its specific niches. These two aspects are discussed on the basis of available data.

Amino Acid Motifs↗
PubMed2006

Two new species of Kazachstania that form ascospores connected by a belt-like intersporal body: Kazachstania zonata and Kazachstania gamospora.

Four strains of ascomycetous yeasts were isolated from samples collected at two locations in southern Japan. The strains formed two warty ascospores that were joined together by an intersporal body appearing as a belt. Phylogenetic analysis of rRNA gene nucleotide sequences indicated that the strains represented two new and closely related species of the genus Kazachstania. Isolates of one of the species were from Miyazaki Prefecture and those of the other species were from the Iriomote Islands. Genetic separation of the two species was further confirmed by DNA-DNA reassociation, which gave values of 63.3-78.1%, and from interspecific crosses, which gave nonviable ascospores. On the basis of these data, the isolates from Miyazaki Prefecture are described as Kazachstania zonata sp. nov. [type strain NBRC 100504=CBS 10326, mating types NBRC 101821 (+), NBRC 101822 (-)], and the isolates from the Iriomote Islands are described as Kazachstania gamospora sp. nov. [type strain NBRC 11056=CBS 10328, mating types NBRC 101825 (+), NBRC 101826 (-)].

Base Composition↗
PubMed2006

Microeukaryotic diversity in the extreme environments of the Iberian Pyrite Belt: a comparison between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning.

The Iberian Pyrite Belt extends from Portugal to Spain and is one of the most important pyrite regions in the world. Its aquatic reservoirs display extreme conditions characterized by low pH and high concentrations of heavy metals. In this study, the diversity of microeukaryotes was analysed at the abandoned mines of São Domingos (Portugal) and at Rio Tinto (Spain). DNA was extracted from water samples and a set of eukaryotic universal primers directed to the small subunit rRNA genes (rDNA) was used. The amplicons were analysed by molecular cloning and temperature gradient gel electrophoresis (TGGE). In addition, a fungi-specific primer set was also used in TGGE experiments. The fungi-specific primers contributed to a substantial increase in the number of fungal taxa found due, probably, to the relative low density of fungal structures. Several microorganisms, belonging (or closely related) to the ascomycetous yeast Pichia acaciae, the basidiomycetous yeasts Cryptococcus humicola and Cystofilobasidium bisporidii, the green algae Chlamydomonas noctigama and Chlorella protothecoides var. acidicola and some uncultured microeukaryotes were present at both localities, which suggests that specific microorganisms are adapted to the peculiar conditions of the Iberian Pyrite Belt extreme environments. However, in spite of the similarities, a higher algal richness was observed at S. Domingos, whereas for R. Tinto the richness of fungi was more prominent.

DNA, Ribosomal↗
PubMed2006

Growth kinetics of microorganisms isolated from Alaskan soil and permafrost in solid media frozen down to -35 degrees C.

We developed a procedure to culture microorganisms below freezing point on solid media (cellulose powder or plastic film) with ethanol as the sole carbon source without using artificial antifreezes. Enrichment from soil and permafrost obtained on such frozen solid media contained mainly fungi, and further purification resulted in isolation of basidiomycetous yeasts of the genera Mrakia and Leucosporidium as well as ascomycetous fungi of the genus Geomyces. Contrary to solid frozen media, the enrichment of liquid nutrient solutions at 0 degrees C or supercooled solutions stabilized by glycerol at -1 to -5 degrees C led to the isolation of bacteria representing the genera Polaromonas, Pseudomonas and Arthrobacter. The growth of fungi on ethanol-microcrystalline cellulose media at -8 degrees C was exponential with generation times of 4.6-34 days, while bacteria displayed a linear or progressively declining curvilinear dynamic. At -17 to -0 degrees C the growth of isolates and entire soil community on 14C-ethanol was continuous and characterized by yields of 0.27-0.52 g cell C (g of C-substrate)(-1), similar to growth above the freezing point. The 'state of maintenance,' implying measurable catabolic activity of non-growing cells, was not confirmed. Below -18 to -35 degrees C, the isolated organisms were able to grow only transiently for 3 weeks after cooling with measurable respiratory and biosynthetic (14CO2 uptake) activity. Then metabolic activity declined to zero, and microorganisms entered a state of reversible dormancy.

Alaska↗
PubMed1994

Microcin production by the yeast Cryptococcus humicola.

Cryptococcus humicola strains secrete killer toxins inhibitory (at pH values ranging from 3 to 5.5) to many ascomycetous and basidiomycetous yeast-like fungi. RNA or DNA plasmids were not detected in the killers. The amino acid-containing toxins were of low M(r), soluble in methanol, resistant to proteolysis, thermostable, cellophane-diffusible and were specified as microcins. These findings show that the killer phenomenon in yeasts such as bacteriocinogeny may be due to excretion of two types of killer toxins: mycocins and microcins.

Anti-Bacterial Agents↗
PubMed1994

Catabolism of 4-hydroxybenzoate in Candida parapsilosis proceeds through initial oxidative decarboxylation by a FAD-dependent 4-hydroxybenzoate 1-hydroxylase.

The first two steps in the catabolism of 4-hydroxybenzoate by the ascomycetous yeast Candida parapsilosis CBS604 were investigated. In contrast to the well-known bacterial pathways and to what was previously assumed, metabolism of 4-hydroxybenzoate in C. parapsilosis proceeds through initial oxidative decarboxylation to give 1,4-dihydroxybenzene. This reaction is catalyzed by a NAD(P)H and FAD-dependent 4-hydroxybenzoate 1-hydroxylase. Further metabolism of 1,4-dihydroxybenzene to the ring-fission substrate 1,2,4-trihydroxybenzene is catalyzed by a NADPH-specific FAD-dependent aromatic hydroxylase acting on phenolic compounds. 19F-NMR experiments with cell extracts and 2-fluoro-4-hydroxybenzoate as the model compound confirm this metabolic pathway and exclude the alternative pathway proceeding through initial 3-hydroxylation followed by oxidative decarboxylation in the second step.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1996

Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum.

DNA fragments homologous to chitin synthase were amplified from the genomic DNA of Penicillium chrysogenum by PCR. Cloning and sequencing of the PCR-amplified fragments led to the identification of four different genes, designated PcCHS1, PcCHS2, PcCHS3, and PcCHS4. By comparison of the deduced amino acid sequences, PcCHS1 was identified as a gene for class I chitin synthase, PcCHS2 and PcCHS3 were for class II, and PcCHS4 was for class III. Among these only PcCHS4 includes an intervening sequence of 56 bp. The analysis of the deduced amino acid sequences revealed a close evolutionary relationship between Penicillium and ascomycetous fungi.

Amino Acid Sequence↗
PubMed1997

Mycocinogeny in the genus Bullera: taxonomic specificity of sensitivity to the mycocin produced by Bullera sinensis.

A strain of Bullera sinensis that secretes a fungicidal thermolabile and protease-sensitive toxin was found. Its killer phenotype was cureless. Ascomycetous, ustilaginaceous yeasts and the members of the Sporidiales are insensitive to the B. sinensis killer toxin. The mycocin acts against tremellaceous yeasts only, except for Cystofilobasidium, Fellomyces and Trichosporon spp. The genera Mrakia, Bullera, Cryptococcus and Udeniomyces are heterogeneous in sensitivity pattern. The different responses of some anamorphic species to this mycocin are often consistent with the origin of the strains and the differences in sexual, chemotaxonomic and physiological characteristics between them.

Drug Resistance, Microbial↗
PubMed1998

Phylogeny of nematode-trapping fungi based on 18S rDNA sequences.

The small subunit (SSU) ribosomal DNA (18S rDNA) from 15 species of nematode-trapping fungi and closely related non-parasitic species were sequenced. Phylogenetic analysis indicated that species within the genera of Arthrobotrys, Dactylaria, Dactylella, Monacrosporium and Duddingtonia formed a monophyletic and isolated clade among an unresolved cluster of apothecial ascomycetes. The phylogenetic patterns within this clade were not concordant with the morphology of the conidia nor the conidiophores, but rather with that of the infection structures. The results from the different methods of tree reconstruction supported three lineages; the species having constricting rings, the non-parasitic species and the species having various adhesive structures (nets, hyphae, knobs and non-constricting rings) to infect nematodes.

Animals↗
PubMed1998

PKC1, encoding a protein kinase C, and FAT1, encoding a fatty acid transporter protein, are neighbors in Cochliobolus heterostrophus.

A protein kinase C gene (PKC1) and adjacent DNA of the filamentous ascomycete Cochliobolus heterostrophus was cloned and sequenced. The deduced amino acid sequence of PKC1 shows high homology to PKCs of other filamentous fungi and all define a new subgroup of PKCs. All attempts to disrupt PKC1 failed, suggesting, but not proving, that disruption of PKC1 function is lethal. About 1 kb 3' of PKC1 is FAT1 encoding a putative bifunctional fatty acid transporter/very-long-chain acyl-CoA synthetase.

Amino Acid Sequence↗
PubMed2000

Basidiomycetous fungus Flammulina velutipes harbors two linear mitochondrial plasmids encoding DNA and RNA polymerases.

Basidiomycetous fungus Flammulina velutipes R15 strain had two linear plasmids in its mitochondria designated pFV1 and pFV2. They were double-stranded DNAs, whose sizes were 8.3 and 8.9 kb, respectively. Sequencing analysis of 7364 bases of the pFV1 and 6861 bases of the pFV2 revealed that the both plasmids had one set of two open reading frames (ORFs) each of that encoded putative DNA and RNA polymerases similar to those of mitochondrial plasmids in other filamentous fungi. In phylogenetic analysis of deduced amino acid sequences of the ORFs and counterparts of other filamentous fungi, the pFV2 was expectedly clustered with plasmids of basidiomycetous fungi. whereas the pFV1 with kalilo plasmid of ascomycetous fungus Neurospora intermedia.

Agaricales↗
PubMed2001

Morphological and molecular characterisation of Pulvinula constellatio ectomycorrhizae.

The ascomycete Pulvinula constellatio was found to form competing ectomycorrhizal relationships with plants that had been inoculated with Tuber spp. and other edible ectomycorrhizal fungi. In order to be able to distinguish P. constellatio mycorrhizae from those of the Tuber spp., we determined the morphological and bio-molecular characteristics of P. constellatio. The complete sequence of the ITS regions was determined, in order to select specific primers. The ITS region was also studied using restriction fragment length polymorphism analyses with several restriction enzymes that allowed an unambiguous identification of the species.

Ascomycota↗
PubMed2001

Chloride channel antagonists perturb growth and morphology of Neurospora crassa.

The chloride channel antagonists anthracene-9-carboxylic acid, ethacrynic acid and niflumic acid were found to be fungistatic and morphogenic when tested against the ascomycete Neurospora crassa. Potency increased with decreasing pH, suggesting that the protonated forms of the compounds were active. Niflumic acid produced the most pronounced growth aberrations which may reflect an ability to acidify the cytoplasm and block the plasma membrane anion channel of N. crassa.

Agar↗
PubMed2002

Carnitine biosynthesis in Neurospora crassa: identification of a cDNA coding for epsilon-N-trimethyllysine hydroxylase and its functional expression in Saccharomyces cerevisiae.

The biosynthesis of L-carnitine in eukaryotic organisms was first elucidated in the ascomycete Neurospora crassa. The first step of the pathway is catalysed by epsilon-N-trimethyllysine hydroxylase (TMLH), which converts epsilon-N-trimethyllysine into beta-hydroxy-N-epsilon-trimethyllysine in a reaction dependent on alpha-ketoglutarate, Fe2+ and oxygen. Here we report on the cloning of the N. crassa TMLH cDNA and its functional expression in Saccharomyces cerevisiae. The TMLH cDNA contains an open reading frame of 1413 base pairs encoding a predicted polypeptide of 471 amino acids. The Michaelis-Menten constants of the heterologously expressed enzyme were determined for epsilon-N-trimethyllysine, alpha-ketoglutarate, Fe2+ and correspond to 0.33 mM, 133 microM and 46 microM, respectively.

Amino Acid Sequence↗
PubMed2002

A molecular approach for early monitoring of decay basidiomycetes in wood chips.

A DNA-based method for the early detection and identification of decay basidiomycetes on wood chips is described. From 26 species of wood-decaying basidiomycetes and 20 species of wood-inhabiting ascomycetes, DNA fragments containing the internal transcribed spacers, 5.8S and partial 28S rDNA were amplified by PCR using the ITS1-F-NL2 rDNA primer pair and analysed through sequencing and restriction digestion. A decay basidiomycete-specific restriction fragment length polymorphism (RFLP) pattern was generated by DraI. Using this PCR-RFLP method the detection of decay fungi is possible 4 days after the inoculation of the wood chips with either a single culture or mixed species.

Basidiomycota↗
PubMed2002

Characterization of PG2, an early endoPG produced by Sclerotinia sclerotiorum, expressed in yeast.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, contains a neutral endopolygalacturonase (endoPG) subfamily of genes that was previously isolated. We report here that pg2, a member of this subfamily, is early and strongly expressed during the first steps of pathogenesis of sunflower cotyledons. The corresponding protein, PG2, was produced in the heterologous Kluyveromyces lactis system and purified. Characterization of the recombinant enzyme revealed a narrow pH activity curve with an optimal pH of 4.5. Hydrolysis of polygalacturonic acid by PG2 resulted in the accumulation of oligomers ranging from 2- to 9-mer. This degradation profile indicates a random attack on the polymer and demonstrates an endo-mode of action. These results provide evidence that pg2 contributes to the infection process during the early phase of host colonization.

Ascomycota↗