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PubMed2000

Molecular characterization of actin genes from homobasidiomycetes: two different actin genes from Schizophyllum commune and Suillus bovinus.

The actin-encoding genes Scact1 and Scact2 of the homobasidiomycete Schizophyllum commune are the first actin genes isolated from higher filamentous fungi. Their isolation shows that homobasidiomycetes have two actin encoding genes instead of one typical to yeasts and filamentous ascomycetes. This result was further confirmed by cloning two actin encoding genes, Sbact1 and Sbact2, from another homobasidiomycete Suillus bovinus. The comparison of the genomic and cDNA sequences of the actin genes showed that Scact1 and Scact2 genes of S. commune contain seven introns, five of which are at the same position in the two genes while S. bovinus actin genes contain nine similarly positioned introns. In the four genes, five intron positions are shared, which indicates a close relationship between the actin encoding genes from S. commune and S. bovinus. Northern hybridization and analysis of two-dimensional immunoblots showed a difference in the expression levels between the two actin genes in each fungus. No actin protein could be detected from S. commune Scact2. The deduced amino acid sequence of the Scact2 gene also differs considerably from any other known actin protein. These data suggest that the Scact2 gene either has a special as yet unidentified function in S. commune life cycle or is a transcribed but no longer translated pseudogene. Scact2 gene has a putative microORF (short open reading frame) and Scact1 gene an intron in the 5'-untranslated region, which could reduce the translational efficiency and increase the transcriptional efficiency of the Scact2 and Scact1 genes, respectively. During mating in S. commune or at formation of ectomycorrhiza in S. bovinus, the expression of actin genes was similar to that in vegetative hyphae. This result suggests that the reorganization of actin cytoskeleton in response to extra- and intracellular signals in higher filamentous fungi could be directly regulated by members of signalling pathways well characterized in yeast and mammalian cells.

Actins↗
PubMed2002

Mitochondrial functions and aging.

In the filamentous ascomycete Podospora anserina mitochondria play a major role in lifespan control. Since the function of these organelles depends on a large number of individual components it is no surprise that a complex network of interacting branches of individual molecular pathways is involved in this process. Recently, the nuclear encoded transcription factor GRISEA was found to significantly affect mitochondrial functions. GRISEA is involved in the control of cellular copper homeostasis. Most importantly, the high affinity uptake of copper from the environment is controlled by this transcription factor. Once copper has entered the cell, it becomes distributed to different compartments and different target molecules. This process depends on a group of proteins, termed copper chaperones. PaCOX17, a homologue of the yeast copper chaperone yCOX17, appears to be involved in copper delivery to mitochondria. Most importantly, the metal is crucial for the assembly and the function of complex IV of the respiratory chain. However, although P. anserina is an obligate aerobe and therefore depends on mitochondrial energy transduction, impairments in the copper delivery pathway are not lethal. This is due to the induction of a molecular back-up system able to compensate for deficiencies in complex IV. The system utilizes an alternative oxidase (PaAOX) which uses iron instead of copper as a cofactor. The alternative respiratory pathway is characterized by a decreased ATP generation but, most significantly, also a decrease in the production of reactive oxygen species. Consequently, molecular damage is reduced which contributes to an increased lifespan of this type of mutant. In addition, modifications in the availability of cellular copper have other relevant consequences. Most significantly, the characteristic age-related rearrangements occurring in the mitochondrial DNA of wild-type strains of P. anserina were found to be dependent on the availability of copper.

Ascomycota↗
PubMed1998

Killer activity of Tilletiopsis albescens Gokhale: taxonomic and phylogenetic implication.

Killer activity expressed at pH values ranging from 3.5 to 8.0 was found in the Tilletiopsis albescens VKM-2822. Its killer phenotype was cureless. The toxin excreted with a molecular mass above 10 kDa is fungicidal, thermolabile, sensitive to proteinase K and was specified as a mycocin. The latter does not act against ascomycetous, sporidiobolaceous and tremellaceous yeasts. In contrast to all other Rhodotorula species, Rh. bacarum, Rh. hinnulea and Rh. phylloplana are sensitive to the mycocin studied. Also, its killing pattern includes the species of the genera Exobasidium, Farysia, Protomyces, Pseudozyma, Sporisorium, Taphrina and Ustilago. The host range of T. albescens mycocin is discussed from taxonomic and phylogenetic viewpoints.

Ascomycota↗
PubMed1999

Occurrence of neutral and alkaline cellulases among alkali-tolerant micromycetes

600 different natural strains of alkali-tolerant micromycetes belonging to 4 classes, 38 genera and 119 species were tested on their ability to produce neutral and alkaline cellulases. Frequency of occurrence of neutral and alkaline cellulases producers among the studied strains was estimated. It is concluded that 72% of all tested strains produce cellulases at neutral and alkaline pH values. But the largest number of neutral and alkaline cellulases producers belong to the following classes which are tolerant to extreme environmental factors: namely Hyphomycetes (genera Acremonium, Alternaria, Cylindrocarpon, Fuzarium, Penicillium, Verticillium, Myrothecium, Ulocladium, Gliocladium), Ascomycetes (genus Chaetomium) and Coelomycetes (genera Phoma, Microsphaeropsis, Aposphaeria).

Journal Article↗
PubMedDate not supplied

Determination of the stability of protein pools from the cell wall of fungi.

Stability of the protein populations present in the cell wall of three ascomycetous fungi Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica was investigated. Cell wall proteins were either labeled with biotin or radiolabeled with amino acids, and chased for a period of time representing several generations. Proteins linked by non-covalent or covalent bonds were separated and their turnover was analyzed. No significant turnover took place during the chase period, and in fact radioactive proteins were accumulated in the wall during the period possibly by transfer through the secretory pathway. This transfer did not involve de novo protein synthesis; it was inhibited by azide, and by incubation of a sec1 mutant of S. cerevisiae at the non-permissive temperature. It is concluded that proteins bound to the cell wall are stable and that there is no precursor-product relationship among those linked by non-covalent bonds and the covalently bound ones.

Ascomycota↗
PubMed1991

Temporal and spatial controls of Aspergillus development.

The mechanisms regulating elaboration of the multicellular asexual reproductive apparatus, the conidiophore, of the filamentous ascomycete Aspergillus nidulans provide a model for the control of fungal development. Recent advances have been made on three fronts. First, new physical and chemical signals have been discovered that control commitment of cells to the conidiation pathway. Second, positively acting developmental regulatory genes have been cloned and characterized. In addition, evidence has been obtained for the existence of negatively acting regulatory loci. Finally, an anonymous developmentally regulated gene has been used to make a directed mutation that has revealed the morphogenetic function of the gene.

Aspergillus nidulans↗
PubMedDate not supplied

Pds5 cooperates with cohesin in maintaining sister chromatid cohesion.

BACKGROUND: Sister chromatid cohesion depends on a complex called cohesin, which contains at least four subunits: Smc1, Smc3, Scc1 and Scc3. Cohesion is established during DNA replication, is partially dismantled in many, but not all, organisms during prophase, and is finally destroyed at the metaphase-to-anaphase transition. A quite separate protein called Spo76 is required for sister chromatid cohesion during meiosis in the ascomycete Sordaria. Spo76-like proteins are highly conserved amongst eukaryotes and a homologue in Aspergillus nidulans, called BimD, is required for the completion of mitosis. The isolation of the cohesin subunit Smc3 as a suppressor of BimD mutations suggests that Spo76/BimD might function in the same process as cohesin. RESULTS: We show here that the yeast homologue of Spo76, called Pds5, is essential for establishing sister chromatid cohesion and maintaining it during metaphase. We also show that Pds5 co-localizes with cohesin on chromosomes, that the chromosomal association of Pds5 and cohesin is interdependent, that Scc1 recruits Pds5 to chromosomes in G1 and that its cleavage causes dissociation of Pds5 from chromosomes at the metaphase-to-anaphase transition. CONCLUSIONS: Our data show that Pds5 functions as part of the same process as cohesin. Sequence similarities and secondary structure predictions indicate that Pds5 consists of tandemly repeated HEAT repeats, and might therefore function as a protein-protein interaction scaffold, possibly in the cohesin-DNA complex assembly.

Animals↗
PubMed2001

The hydrophobin EAS is largely unstructured in solution and functions by forming amyloid-like structures.

BACKGROUND: Fungal hydrophobin proteins have the remarkable ability to self-assemble into polymeric, amphipathic monolayers on the surface of aerial structures such as spores and fruiting bodies. These monolayers are extremely resistant to degradation and as such offer the possibility of a range of biotechnological applications involving the reversal of surface polarity. The molecular details underlying the formation of these monolayers, however, have been elusive. We have studied EAS, the hydrophobin from the ascomycete Neurospora crassa, in an effort to understand the structural aspects of hydrophobin polymerization. RESULTS: We have purified both wild-type and uniformly 15N-labeled EAS from N. crassa conidia, and used a range of physical methods including multidimensional NMR spectroscopy to provide the first high resolution structural information on a member of the hydrophobin family. We have found that EAS is monomeric but mostly unstructured in solution, except for a small region of antiparallel beta sheet that is probably stabilized by four intramolecular disulfide bonds. Polymerised EAS appears to contain substantially higher amounts of beta sheet structure, and shares many properties with amyloid fibers, including a characteristic gold-green birefringence under polarized light in the presence of the dye Congo Red. CONCLUSIONS: EAS joins an increasing number of proteins that undergo a disorder-->order transition in carrying out their normal function. This report is one of the few examples where an amyloid-like state represents the wild-type functional form. Thus the mechanism of amyloid formation, now thought to be a general property of polypeptide chains, has actually been applied in nature to form these remarkable structures.

Amino Acid Sequence↗
PubMed2002

Endopolygalacturonase is encoded by a multigene family in the basidiomycete Chondrostereum purpureum.

The basidiomycete Chondrostereum purpureum produces several plant cell wall-degrading enzymes, including endopolygalacturonase (endoPG). Degenerate oligonucleotide primers were designed according to conserved regions of endoPG genes from various fungi, plants, and bacteria and used to amplify members of this gene family from C. purpureum. Four different amplification products showed significant similarity to known endoPGs and were used as hybridization probes to screen a library of genomic DNA sequences and to retrieve five full-length endoPG genes (epgA, epgB1, epgB2, epgC, and epgD). The identities between the deduced polypeptides for epgA, epgB1, epgC, and epgD ranged from 61.8 to 80.0%, while the deduced polypeptides for epgB1 and epgB2 shared 97.1% identity. Phylogenetic analysis suggested that the duplication of existing endoPG genes occurred after the divergence of the ascomycetes and basidiomycetes. C. purpureum is the first basidiomycete fungus for which the endoPG gene family has been described.

Agaricales↗
PubMed2002

An IQGAP-related protein, encoded by AgCYK1, is required for septation in the filamentous fungus Ashbya gossypii.

In filamentous ascomycetes hyphae are compartmentalized by septation in which the cytoplasm of the compartments are interconnected via septal pores. Thus, septation in filamentous fungi is different from cytokinesis in yeast like fungi. We have identified an Ashbya gossypii orthologue of the Saccharomyces cerevisiae CYK1 gene which belongs to the IQGAP-protein family. In contrast to S. cerevisiae disruption of AgCYK1 yields viable mutant strains that exhibit wildtype-like polarized hyphal growth rates. In the Agcyk1 mutant cortical actin patches localize to growing hyphal tips like wildtype, however, mutant hyphae are totally devoid of actin rings at presumptive septal sites. Septation in wildtype results in the formation of chitin rings. Agcyk1 mutant hyphae are aseptate and do not accumulate chitin in their cell walls. Agcyk1 mutant strains are completely asporogenous indicating that septation is essential for the formation of sporangia in A. gossypii. AgCyk1p-GFP localizes to sites of future septation as a ring prior to chitin depositioning. Furthermore, decrease in Cyk1p-ring diameter was found to be a prerequisite for the accumulation of chitin and septum formation.

Actins↗
PubMed2002

The polyketide synthase gene pks4 from Gibberella fujikuroi encodes a key enzyme in the biosynthesis of the red pigment bikaverin.

The ascomycete Gibberella fujikuroi mating population C (MP-C) is well known for the production of gibberellins, but also produces many other secondary metabolites, including the red polyketide pigment bikaverin. Here, we used a differential display method to clone a polyketide synthase gene pks4 responsible for the first step of bikaverin biosynthesis. Sequence analysis indicated that pks4 encoded a 2009-amino acid polypeptide consisting of four functional domains: beta-ketoacyl synthase (KS), acyltransferase (AT), acyl carrier (ACP), and thioesterase (TE). Disruption of pks4 resulted in the loss of both pks4 transcripts and bikaverin biosynthesis in G. fujikuroi cultures. Expression of pks4 is strongly repressed by high amounts of ammonium and basic pH. Unexpectedly, pks4 was overexpressed in mutants of the regulatory gene, areA, which is responsible for the activation of nitrogen assimilation genes. Three additional polyketide synthase genes have been cloned from G. fujikuroi MP-C by heterologous hybridization. The presence of these four PKS genes demonstrates the diversity of polyketide biosynthetic pathways in this fungus.

Amino Acid Sequence↗
PubMed2003

Congruent evidence from alpha-tubulin and beta-tubulin gene phylogenies for a zygomycete origin of microsporidia.

The origin of microsporidia and the evolutionary relationships among the major lineages of fungi have been examined by molecular phylogeny using alpha-tubulin and beta-tubulin. Chytrids, basidiomycetes, ascomycetes, and microsporidia were all recovered with high support, and the zygomycetes were consistently paraphyletic. The microsporidia were found to branch within zygomycetes, and showed relationships with members of the Entomophthorales and Zoopagales. This provides support for the microsporidia having evolved from within the fungi, however, the tubulin genes are difficult to interpret unambiguously since fungal and microsporidian tubulins are very divergent. Rapid evolutionary rates a characteristic of practically all microsporidian genes studied, so determining their evolutionary history will never be completely free of such difficulties. While the tubulin phylogenies do not provide a decisive conclusion, they do further narrow the probable origin of microsporidia to a zygomycete-like ancestor.

Animals↗
PubMed2003

Carbohydrate and amino acid metabolism in Tuber borchii mycelium during glucose utilization: a (13)C NMR study.

The metabolism of [1-13C]glucose in the vegetative mycelium of the ectomycorrhizal ascomycete Tuber borchii was studied in order to characterize the biochemical pathways for the assimilation of glucose and amino acid biosynthesis. The pathways were characterized using nuclear magnetic resonance spectroscopy in conjunction with [1-13C]glucose labeling. The enzymes of mannitol cycle and ammonium assimilation were also evaluated. The majority of the 13C label was incorporated into mannitol and this polyol was formed via a direct route from absorbed glucose. Amino acid biosynthesis was also an important sink of assimilated carbon and 13C was mainly incorporated into alanine and glutamate. From this intramolecular 13C enrichment, it is concluded that pyruvate, arising from [1-13C]glucose catabolism, was used by alanine aminotransferase, pyruvate dehydrogenase and pyruvate carboxylase before entering the Krebs cycle. The transfer of 13C-labeled mycelium on [12C]glucose showed that mannitol, alanine, and glutamate carbon were used to synthesize glutamine and arginine that likely play a storage role.

Amino Acids↗
PubMed2003

ESTs from the basidiomycete Schizophyllum commune grown on nitrogen-replete and nitrogen-limited media.

Lambda phage cDNA libraries were constructed using mRNAs from the basidiomycete Schizophyllum commune grown on media with high or low nitrogen concentrations. A total of 440 clones were sequenced, representing 373 distinct transcripts. Of these, 166 showed significant similarity to annotated genes in GenBank. Those that could be tentatively identified using BLAST searches were classified by function using the Gene Ontology (GO) database. Genes with products involved in cell-cycle processes were more frequent in the nitrogen-limited libraries, while genes with products involved in protein biosynthesis were more frequent in the nitrogen-replete library. Overall, clones showed much greater similarity to the one publicly available basidiomycete genome, Phanerochaete chrysosporium, than to any of the ascomycete genomes.

Bacteriophage lambda↗
PubMed2003

Fungus-specific microsatellite primers of lichens: application for the assessment of genetic variation on different spatial scales in Lobaria pulmonaria.

We isolated 12 microsatellite loci for the epiphytic lichen-forming ascomycete Lobaria pulmonaria and studied their patterns of variation within and among populations from Canada and Switzerland. Even though several microsatellites exhibited high levels of variability at different spatial scales, we did not find any evidence for intrathalline variation. Most of the genetic variation was attributed to differences among individuals within populations. High genetic variation was also detected among L. pulmonaria samples taken from individual trees, suggesting that either multiple colonization events had occurred or that local recombination is frequent. The geographically structured distribution of alleles from several microsatellites indicated that L. pulmonaria from Canada and Switzerland represent two distinct evolutionary lineages. The potential to identify multiple alleles, and their transferability to closely related species, make microsatellites an ideal tool to study dispersal, population differentiation, and microevolution in lichens.

Alleles↗
PubMed2003

The Snf1 kinase of the filamentous fungus Hypocrea jecorina phosphorylates regulation-relevant serine residues in the yeast carbon catabolite repressor Mig1 but not in the filamentous fungal counterpart Cre1.

In Saccharomyces cerevisiae, the SNF1 gene product phosphorylates the carbon catabolite repressor protein Mig1 under conditions when glucose is limiting, thereby relieving the fungus from catabolite repression. We have investigated whether the corresponding counterpart of filamentous fungi-the Cre1 protein-is also phosphorylated by Snf1. To this end, snf1, an ortholog of SNF1, was isolated from the ascomycete Hypocrea jecorina. The gene encodes a protein with high similarity to Snf1 kinases from other eukaryotes in its N-terminal catalytic domain, but little similarity in the C-terminal half of the protein, albeit some short aa-areas were detected, however, which are conserved in filamentous fungi and in yeast. Expression of snf1 is independent of the carbon source. An overexpressed catalytic domain of H. jecorina Snf1 readily phosphorylated yeast Mig1, but not a Mig1 mutant form, in which all four identified Snf1 phosphorylation sites (Phi XRXXSXXX Phi) had been mutated. The enzyme did neither phosphorylate H. jecorina Cre1 nor histone H3, another substrate of Snf1 kinase in yeast. H. jecorina Snf1 also phosphorylated peptides comprising the strict Snf1 consensus, but notably did not phosphorylate peptides containing the regulatory serine residue in Cre1 (=Ser(241) in H. jecorina Cre1 and Ser(266) in Sclerotinia sclerotiorum CRE1). The use of cell-free extracts of H. jecorina as protein source for Snf1 showed phosphorylation of an unknown 36 kDa protein, which was present only in extracts from glucose-grown mycelia. We conclude that the Snf1 kinase from H. jecorina is not involved in the phosphorylation of Cre1.

Amino Acid Sequence↗
PubMed2006

Pandora's mycological box: molecular sequences vs. morphology in understanding fungal relationships and biodiversity.

Fundamental reappraisals of diverse traditional ideas in mycology have become necessary as a result of molecular insights. These different insights are discussed in relation to: the positions of microsporidia, slime moulds and oomycetes; the basal position of lichen fungi in the evolution of ascomycetes forming fruit bodies; remodelling of orders and families; changed generic concepts; the issue of whether permitting a dual nomenclature for the different states of pleomorphic fungi should be continued; and the recognition of additional cryptic species within a "species". The molecular data has necessitated a reassessment of the systematic importance of many types of characters. Also, the techniques open exciting horizons and undreamed of abilities through being able to identify non-sporing fungi in ecological samples and plant material, and revealing unexpected levels of diversity in hitherto little-explored habitats. Major advances in understanding how fungi operate through total genomic approaches can be anticipated as more are completely sequenced. The Pandora's box of molecular surprises is to be seen as one of blessings and not one of miseries and evils.

Agaricales↗
PubMed2000

Intracellular accommodation of microbes by plants: a common developmental program for symbiosis and disease?

Plant cells engage in mutualistic and parasitic endosymbioses with a wide variety of microorganisms, ranging from Gram-negative (Rhizobium, Nostoc) and Gram-positive bacteria (Frankia), to oomycetes (Phytophthora), Chytridiomycetes, Zygomycetes (arbuscular mycorrhizal fungi) and true fungi (Erysiphe, ascomycete; Puccinia, basidiomycete). Endosymbiosis is characterised by the 'symbiosome', a compartment within host cells in which the symbiotic microorganism is either partially or completely enclosed by a host-derived membrane. The analysis of plant mutants indicates that the genetic requirements for the interaction with rhizobia and arbuscular mycorrhiza fungi are partially overlapping. The extent to which plants use similar or identical developmental programs for the intracellular accommodation of different microorganisms is, however, not clear. For example, plant cells actively weaken their cell wall to facilitate bacterial colonisation, whereas penetration by fungal symbionts appears not to be assisted in this manner. Moreover, different transport requirements are imposed on the symbiotic interface of different interactions indicating that additional system-specific components are likely to exist.

Bacterial Physiological Phenomena↗