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PubMed1975

[The enzymatic hydrolysis of cell wall applied to yeasts classification (author's transl)].

The rigid structure of yeast cell wall may be a taxonomic criterion. Using the Helix pomatia juice (with mercaptoethylamine hydrochloride) as a polyenzymatic system, we have always obtained protoplasts from ascomycetous yeasts and never from basidiomycetous yeasts tested. Then, we have applied this enzymatic analysis to the Fungi imperfecti yeasts and, according to this test, separated genus looking like basidiomycetous behaviour: Trichosporon, Pityrosporum, Rhodotorula, Cryptococcus and non fermentative Candida (group II). Oppositely, we have put together the Fungi imperfecti yeasts looking like ascomycetous behaviour: Kloeckera, Trigonopsis, Brettanomyces and fermentative Candida (group I). With the second lytic system made of mercaptoethylamine-HCl and beta-(1-3)-D-glucanase, we have selected ascomycetous yeasts giving protoplasts: Saccharomycoidea with genus Saccharomyces, Kluyveromyces, Pichia, Hansenula, Citeromyces, Debaryomyces; we have joined Saccharomycodes and Hanseniaspora to this second group. The other ascomycetous yeasts studied did not give protoplasts with the definite lytic system made of purified beta-(1-3)-D-glucanase and thiol. These results seem to demonstrate that the structure of cell wall can act as an important taxonomic criterion.

Ascomycota↗
PubMed2006

Cell wall construction in Saccharomyces cerevisiae.

In this review, we discuss new insights in cell wall architecture and cell wall construction in the ascomycetous yeast Saccharomyces cerevisiae. Transcriptional profiling studies combined with biochemical work have provided ample evidence that the cell wall is a highly adaptable organelle. In particular, the protein population that is anchored to the stress-bearing polysaccharides of the cell wall, and forms the interface with the outside world, is highly diverse. This diversity is believed to play an important role in adaptation of the cell to environmental conditions, in growth mode and in survival. Cell wall construction is tightly controlled and strictly coordinated with progression of the cell cycle. This is reflected in the usage of specific cell wall proteins during consecutive phases of the cell cycle and in the recent discovery of a cell wall integrity checkpoint. When the cell is challenged with stress conditions that affect the cell wall, a specific transcriptional response is observed that includes the general stress response, the cell wall integrity pathway and the calcineurin pathway. This salvage mechanism includes increased expression of putative cell wall assemblases and some potential cross-linking cell wall proteins, and crucial changes in cell wall architecture. We discuss some more enzymes involved in cell wall construction and also potential inhibitors of these enzymes. Finally, we use both biochemical and genomic data to infer that the architectural principles used by S. cerevisiae to build its cell wall are also used by many other ascomycetous yeasts and also by some mycelial ascomycetous fungi.

Cell Wall↗
PubMed1986

Orthogonal-field-alternation gel electrophoresis banding patterns of DNA from yeasts.

Chromosomal DNAs from various yeast species were separated by orthogonal-field-alternation gel electrophoresis (OFAGE). To this end we developed a spheroplasting and lysis method to obtain intact DNA from both ascomycetous and basidiomycetous yeasts. The OFAGE banding patterns of 22 ascomycetous and four basidiomycetous yeast strains were compared. The strains represented species from the genera: Brettanomyces, Candida, Cryptococcus, Filobasidiella, Geotrichum, Hansenula, Kluyveromyces, Pachysolen, Pichia, Rhodosporidium, Rhodotorula, Saccharomyces, Saccharomycodes, Saccharomycopsis, Schizosaccharomyces and Zygosaccharomyces. Variations occurred in the number of bands and their positions in the gel, not only among strains of different genera but also among species from the same genus and even between varieties of the same species. The ascomycetous yeasts, with the exception of Saccharomyces cerevisiae, only showed one to five bands of DNA larger than 1000 kilobase pairs (kb) in general none smaller. The patterns of the four basidiomycetous yeasts revealed also a few large DNA bands but in addition one to six bands ranging in size from 500 to 1000 kb, with the exception of a single smaller chromosome in Rhodotorula mucilaginosa. From the OFAGE banding patterns of strains studied here it appears that in Sacch. cerevisiae the partitioning of DNA over chromosomes is unique. But rather than the large number of chromosomes, the presence of four chromosomes with less than 500 kb of DNA is characteristic for Sacch. cerevisiae.

Ascomycota↗
PubMed1998

Evolutionary relationships among putative RNA-dependent RNA polymerases encoded by a mitochondrial virus-like RNA in the Dutch elm disease fungus, Ophiostoma novo-ulmi, by other viruses and virus-like RNAs and by the Arabidopsis mitochondrial genome.

The nucleotide sequence (2617 nucleotides) of virus-like double-stranded (ds) RNA 3a in a diseased isolate, Log1/3-8d2 (Ld), of the ascomycete fungus Ophiostoma novo-ulmi has been determined. One strand of the dsRNA contains an open reading frame (ORF) with the potential to encode a protein of 718 amino acids, and the complementary strand contains two smaller ORFs with the potential to encode proteins of 178 and 182 amino acids, respectively. The large ORF contains 12 UGA codons which code for tryptophan in ascomycete mitochondria and has a codon bias typical of mitochondrial genes, consistent with the localization of Ld dsRNAs within the mitochondria. The amino acid sequence contains motifs characteristic of RNA-dependent RNA polymerases (RdRps). This putative RdRp was shown to be related to putative RdRps of mitochondrial dsRNAs of another ascomycete and a basidiomycete fungus and also to a putative RdRp encoded by the mitochondrial genome of Arabidopsis thaliana. In multiple sequence alignments, the fungal mitochondrial dsRNA-encoded RdRp-like proteins formed a cluster, ancestrally related to the RdRps of the yeast 20S and 23S RNA replicons and of the positive-stranded RNA bacteriophages of the Leviviridae family, but distinct from RdRps of other families and genera of fungal RNA viruses and related plant and animal RNA viruses. Northern blot analysis with RNA 3a strand-specific probes indicated that nucleic acid extracts of Ld contain more single-stranded (positive-stranded) RNA than dsRNA, consistent with an evolutionary relationship between RNA 3a and positive-stranded RNA phages.

Amino Acid Sequence↗
PubMed1995

Characterization of the "promoter region" of the enolase-encoding gene enol from the anaerobic fungus Neocallimastix frontalis: sequence and promoter analysis.

The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5' to the major transcriptional start point. The base composition of the enolase upstream sequence revealed a very A + T-rich profile (13.5% G + C) leading to many putative hairpin structures. The functional organization of the N. frontalis enolase promoter was investigated by heterologous transient-expression assays. DNA fragments obtained by the sequential removal of sequences upstream of the translation start codon were fused to the Escherichia coli lacZ gene and the resulting plasmids were used to transform the ascomycetes Aspergillus nidulans and Penicillium roqueforti and the oomycete Saprolegnia monoica. Transient expression of the lacZ reporter gene was observed in regenerating proteoplasts of S. monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region. In contrast no beta-galactosidase activity was detected in ascomycete protoplasts. DNA hybridization analysis revealed the integration of vector DNA in the genomic DNA of S. monoica and the presence of free copies of the transformation plasmid which could be rescued in E. coli. Our results indicate that the transcriptional machinery of the anaerobic chytrid N. frontalis may differ significantly from that of ascomycetes but that enough conservation exists within the lower fungi to allow a transient-driven expression of a reporter gene in an oomycete fungus.

Base Sequence↗
PubMed1990

Yeasts and fungi occurring in ensiled whole-crop maize and other ensiled vegetable crops.

The yeast flora of whole-crop maize ensiled for two weeks was predominated by Candida holmii, C. lambica, C. milleri, Hansenula anomala and Saccharomyces dairensis. Inoculation with other yeast species reported in the literature to prevail in maize or wheat silages did not alter the yeast flora. At 25 or 30 degrees C the ascomycetous fermentative species found at 20 degrees C were accompanied with ascomycetous non-fermentative fungi, i.c. Exophiala jeanselmei and Verticillium psalliotae, by the non-fermentative imperfect basidiomycetous yeast Rhodotorula mucilaginosa and by the weakly fermentative imperfect ascomycetous yeast Trichosporon adeninovorans. The yeast flora of other vegetable crops, ensiled at 20 degrees C for two weeks, was predominated by the same species that prevailed in ensiled maize, provided the crop did not contain mustard oils or menthol. If these compounds occurred in the crops, the yeast flora was predominated by nonfermentative species like Candida famata, Stephanoascus ciferrii, Rhodotorula minuta, Rh. rubra and Trichosporon cutaneum.

Animal Feed↗
PubMed2006

Genetic diversity of naturally established ectomycorrhizal fungi on Norway spruce seedlings under nursery conditions.

We have assessed ectomycorrhizal fungi colonizing Norway spruce (Picea abies L.) seedlings in nine forest nurseries using restriction fragment length polymorphism (RFLP) and sequencing analyses of the internal transcribed spacers (ITS1-5.8S-ITS2) amplicons. Restriction analysis of the amplified DNA fragments with HinfI, MboI, and TaqI enzymes allowed the definition of 17 RFLP genotypes; five of them could be unambiguously assigned to Thelephora terrestris, Hebeloma longicaudum, H. crustuliniforme, Tricharina ochroleuca, and Cenococcum geophilum species by comparison with the sporocarp RFLP-pattern database. The remaining genotypes have been sequenced and compared with sequences deposited in the GenBank database. The phylogenetic analysis of resulting sequences and their identified matches indicated that isolated genotypes have formed seven clades. The ascomycetes were predominant: we have determined eight species--Wilcoxina mikolae, Phialophora finlandia, Tuber sp., Cenococcum geophilum, Tricharina ochroleuca, Pulvinula constellatio, and two unidentified ascomycetes--whereas the basidiomycetes were less common (four species denoted: Amphinema byssoides, Hebeloma crustuliniforme, H. longicaudum, and Thelephora terrestris). Wilcoxina mikolae and Phialophora finlandia were the most frequent fungi. Analysis of variance revealed that ascomycetes abundance was higher in nurseries that used organic fertilizer.

Analysis of Variance↗
PubMed1997

3-Hydroxy-3-methylglutaryl-CoA reductase gene of Gibberella fujikuroi: isolation and characterization.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMGR) is the first specific enzyme of the isoprenoid pathway, which leads to several classes of primary and secondary metabolites such as sterols, quinones, carotenoids and gibberellins. The structural gene of HMG-CoA reductase was isolated from the ascomycetous fungus Gibberella fujikuroi. Additionally, the most conserved region of this gene was also isolated from another plant pathogenic fungus, Sphaceloma manihoticola. Both ascomycetous fungi use the plant hormone gibberellin to induce an elongation of infected host plants, and in the case of S. manihoticola of plant tumors. Sequence analysis revealed a high degree of similarity between the deduced amino-acid sequences in the C-terminal catalytic domains of all known HMG-CoA reductases, but the highest degree was found between the sequences of both analysed ascomycetes. In contrast to Saccharomyces cerevisiae, Ustilago maydis and plants, G. fujikuroi and S. manihoticola possess only a single copy of this gene, although the product of HMGR (mevalonate) is the precursor for essential sterol and quinone biosynthesis and secondary metabolites such as gibberellins. RNA-blot and hybridization experiments showed that gene expression is not influenced by either glucose or ammonium excess.

Amino Acid Sequence↗
PubMed1997

Disparate sequence characteristics of the Erysiphe graminis f.sp. hordei glyceraldehyde-3-phosphate dehydrogenase gene.

The Erysiphe graminis f.sp. hordei (Egh) glyceraldehyde-3-phosphate dehydrogenase (gpd) gene was isolated and characterized. It contains typical promoter elements and has three introns, one of which is positioned in the 5' untranslated region of the gene. The deduced amino-acid sequence has 87% similarity to gpd genes from other Ascomycete fungi. This is at the same level as previously estimated among these fungi. Comparison at the DNA level reveal similarities of only around 70%, which is 10% lower than previously reported. In an evolutionary tree based on the sequences from 18 fungal gpd genes, Egh falls into the group of Ascomycetes located at a basal position. The regulatory region of the Egh gpd gene has no homology to corresponding sequences in other filamentous Ascomycetes. Codon usage was determined for the four characterized Egh genes (tub2, Egh7, Egh16 and gpd) and found to be similar for all four genes. The results of the codon-usage analysis suggest that Egh is more flexible than other fungi in the choice of nucleotides at the wobble position. Codon-usage preferences in Egh and barley genes indicate a level of difference which may be exploited to discriminate between fungal and plant genes in sequence mixtures. The Egh gpd promoter appears to be superior to that of the Egh beta-tubulin gene (tub2) for driving the E. coli beta-glucuronidase (GUS) gene in transformation experiments.

Ascomycota↗
PubMed2000

Comparative analysis of the mating-type loci from Neurospora crassa and Sordaria macrospora: identification of novel transcribed ORFs.

The mating-type locus controls mating and sexual development in filamentous ascomycetes. In the heterothallic ascomycete Neurospora crassa, the genes that confer mating behavior comprise dissimilar DNA sequences (idiomorphs) in the mat a and mat A mating partners. In the homothallic fungus Sordaria macrospora, sequences corresponding to both idiomorphs are located contiguously in the mating-type locus, which contains one chimeric gene, Smt A-3, that includes sequences which are similar to sequences found at the mat A and mat a mating-type idiomorphs in N. crassa. In this study, we describe the comparative transcriptional analysis of the chimeric mating-type region of S. macrospora and the corresponding region of the N. crassa mat a idiomorph. By means of RT-PCR experiments, we identified novel intervening sequences in the mating-type loci of both ascomycetes and, hence, concluded that an additional ORF, encoding a putative polypeptide of 79 amino acids, is present in the N. crassa mat a idiomorph. Furthermore, our analysis revealed co-transcription of the novel gene with the mat a-1 gene in N. crassa. The same mode of transcription was found in the corresponding mating-type region of S. macrospora, where the chimeric Smt A-3 gene is co-transcribed with the mat a-specific Smt a-1 gene. Analysis of a Smt A-3 cDNA revealed optional splicing of two introns. We believe that this is the first report of co-transcription of protein-encoding nuclear genes in filamentous fungi. Possible functions of the novel ORFs in regulating mating-type gene expression are discussed.

Alternative Splicing↗
PubMed2006

Yeasts in an industrial malting ecosystem.

The malting ecosystem consists of two components: the germinating cereal grains and the complex microbial community. Yeasts and yeast-like fungi are an important part of this ecosystem, but the composition and the effects of this microbial group have been largely unknown. In this study we surveyed the development of yeasts and yeast-like fungi in four industrial scale malting processes. A total of 136 malting process samples were collected and examined for the presence of yeasts growing at 15, 25 and 37 degrees C. More than 700 colonies were isolated and characterized. The isolates were discriminated by PCR-fingerprinting with microsatellite primer (M13). Yeasts representing different fingerprint types were identified by sequence analysis of the D1/D2 domain of the 26S rRNA gene. Furthermore, identified yeasts were screened for the production of alpha-amylase, beta-glucanase, cellulase and xylanase. A numerous and diverse yeast community consisting of both ascomycetous (25) and basidiomycetous (18) species was detected in the various stages of the malting process. The most frequently isolated ascomycetous yeasts belonged to the genera Candida, Clavispora, Galactomyces, Hanseniaspora, Issatchenkia, Pichia, Saccharomyces and Williopsis and the basidiomycetous yeasts to Bulleromyces, Filobasidium, Cryptococcus, Rhodotorula, Sporobolomyces and Trichosporon. In addition, two ascomycetous yeast-like fungi (black yeasts) belonging to the genera Aureobasidium and Exophiala were commonly detected. Yeasts and yeast-like fungi produced extracellular hydrolytic enzymes with a potentially positive contribution to the malt enzyme spectrum. Knowledge of the microbial diversity provides a basis for microflora management and understanding of the role of microbes in the cereal germination process.

Ascomycota↗
PubMed2005

Fungal diversity from western redcedar fences and their resistance to beta-thujaplicin.

The work reported here investigated the fungal community inhabiting western redcedar fence material with a focus on species colonizing wood below the surface, of which little is known. From seven pieces of fence material, twenty-three different fungal species were isolated and characterized using both traditional morphology and molecular identification methods. The species identified included thirteen ascomycetous and ten basidiomycetous fungi. Isolates were tested for their resistance to beta-thujaplicin - one of the principle fungicidal agents of western redcedar heartwood extractives. Generally, ascomycetous fungi exhibited greater resistance to beta-thujaplicin than basidiomycetous fungi. Interestingly, three ascomycetous and two basidiomycetous species frequently isolated had high tolerance to this compound. These species could be candidate 'pioneer' species that invade and detoxify western redcedar extractives, paving the way for colonization by decay fungi.

Antifungal Agents↗
PubMed2006

Polysaccharides and phenolic compounds as substrate for yeasts isolated from rotten wood and description of Cryptococcus fagi sp.nov.

Pieces of rotten wood collected in the forest were screened for the presence of yeasts. In spring time 3 tree species were sampled, followed by 9 species in summer. Yeast strains were identified by traditional methods. Identifications were confirmed by sequencing of ribosomal DNA in case of doubt. In total 14 yeast species of ascomycetous affiliation and 6 anamorphic basidiomycetous yeasts were isolated and identified. Most species were represented by only one strain, but Candida bertae by two and Trichosporon porosum by six strains, all from different wood samples. Three strains represented novel species, one of which is described as Cryptococcus fagi Middelhoven et Scorzetti. The type strain is CBS 9964 (JCM 13614). All strains were tested for growth on several polysaccharides as sole carbon source. Only some of these polymers supported growth of ascomycetous yeasts. Basidiomycetous yeasts assimilated soluble starch, pullulan, dextran, xylan, polygalacturonate, galactomannan and tannic acid or at least some of these. Cryptococcus podzolicus and T. porosum were the most active in this respect. None of the isolated strains grew on carboxymethyl cellulose, colloidal chitin, arabinogalactan and gum xanthan. Phenolic compounds were assimilated by several strains, belonging to the Trichosporonales and the Microbotryum and Stephanoascus/Blastobotrys clades, but not by members of the Tremellales (Cryptococcus musci excepted) and the Debaryomyces/Lodderomyces clade. Most of the ascomycetes assimilated n-hexadecane.

Alkanes↗
PubMed1992

Detecting morphological convergence in true fungi, using 18S rRNA gene sequence data.

For the true fungi, phylogenetic relationships inferred from 18S ribosomal DNA sequence data agree with morphology when (1) the fungi exhibit diagnostic morphological characters, (2) the sequence-based phylogenetic groups are statistically supported, and (3) the ribosomal DNA evolves at roughly the same rate in the lineages being compared. 18S ribosomal RNA gene sequence data and biochemical data provide a congruent definition of true fungi. Sequence data support the traditional fungal subdivisions Ascomycotina and Basidiomycotina. In conflict with morphology, some zygomycetes group with chytrid water molds rather than with other terrestrial fungi, possibly owing to unequal rates of nucleotide substitutions among zygomycete lineages. Within the ascomycetes, the taxonomic consequence of simple or reduced morphology has been a proliferation of mutually incongruent classification systems. Sequence data provide plausible resolution of relationships for some cases where reduced morphology has created confusion. For example, phylogenetic trees from rDNA indicate that those morphologically simple ascomycetes classified as yeasts are polyphyletic and that forcible spore discharge was lost convergently from three lineages of ascomycetes producing flask-like fruiting bodies.

DNA, Fungal↗
PubMed2002

Mate recognition in fungi.

The ascomycete and basidiomycete fungi have contributed much to our understanding of eukaryotic cell biology. The study of mate recognition, in particular, has provided detailed understanding of cell signalling pathways and cell-specific gene transcription. Sexual dimorphism has little relevance to mating in these organisms, indeed specialised cells for mating are found only in filamentous ascomycetes and even here, a single individual produces both male and female structures. None the less, most species have genetic barriers to prevent selfing. The genes that determine self-incompatibility divide populations into different mating types, and only individuals with different mating types can engage in sexual reproduction. Ascomycetes have just two mating types, but basidiomycetes may have several thousands. Despite apparent differences in the biology and numbers of mating types in these fungi, it is becoming increasingly apparent that many components of their mating pathways are highly conserved.

Fungi↗
PubMed2003

Shifting fungal reproductive mode by manipulation of mating type genes: obligatory heterothallism of Gibberella zeae.

Fungi capable of sexual reproduction use heterothallic (self-sterile) or homothallic (self-fertile) mating strategies. In most ascomycetes, a single mating type locus, MAT, with two alternative forms (MAT1-1 and MAT1-2) called idiomorphs, controls mating ability. In heterothallic ascomycetes, these alternative idiomorphs reside in different nuclei. In contrast, most homothallic ascomycetes carry both MAT1-1 and MAT1-2 in a single nucleus, usually closely linked. An example of the latter is Gibberella zeae, a species that is capable of both selfing and outcrossing. G. zeae is a devastating cereal pathogen of ubiquitous geographical distribution, and also a producer of mycotoxins that threaten human and animal health. We asked whether G. zeae could be made strictly heterothallic by manipulation of MAT. Targeted gene replacement was used to differentially delete MAT1-1 or MAT1-2 from a wild-type haploid MAT1-1; MAT1-2 strain, resulting in MAT1-1; mat1-2, mat1-1; MAT1-2 strains that were self-sterile, yet able to cross to wild-type testers and, more importantly, to each other. These results indicated that differential deletion of MAT idiomorphs eliminates selfing ability of G. zeae, but the ability to outcross is retained. They also indicated that both MAT idiomorphs are required for self-fertility. To our knowledge, this is the first report of complete conversion of fungal reproductive strategy from homothallic to heterothallic by targeted manipulation of MAT. Practically, this approach opens the door to simple and efficient procedures for obtaining sexual recombinants of G. zeae that will be useful for genetic analyses of pathogenicity and other traits, such as the ability to produce mycotoxins.

Crosses, Genetic↗
PubMed2004

Differential evolution of the Saccharomyces cerevisiae DUP240 paralogs and implication of recombination in phylogeny.

Multigene families are observed in all genomes sequenced so far and are the reflection of key evolutionary mechanisms. The DUP240 family, identified in Saccharomyces cerevisiae strain S288C, is composed of 10 paralogs: seven are organized as two tandem repeats and three are solo ORFs. To investigate the evolution of the three solo paralogs, YAR023c, YCR007c and YHL044w, we performed a comparative analysis between 15 S.cerevisiae strains. These three ORFs are present in all strains and the conservation of synteny indicates that they are not frequently involved in chromosomal reshaping, in contrast to the DUP240 ORFs organized in tandem repeats. Our analysis of nucleotide and amino acid variations indicates that YAR023c and YHL044w fix mutations more easily than YCR007c, although they all belong to the same multigene family. This comparative analysis was also conducted with five arbitrarily chosen Ascomycetes-specific genes and five arbitrarily chosen common genes (genes that have a homolog in at least one non-Ascomycetes organism). Ascomycetes-specific genes appear to be diverging faster than common genes in the S.cerevisiae species, a situation that was previously described between different yeast species. Our results point to the strong contribution, during DNA sequence evolution, of allelic recombination besides nucleotide substitution.

Base Sequence↗
PubMed1993

Phylogenetic relationships among Taphrina, Saitoella, and other higher fungi.

To determine the phylogenetic placement of the major groups of higher fungi, we sequenced the DNA sequences from the small-subunit ribosomal RNA (18S rRNA) coding regions from Taphrina wiesneri (synonym: T. cerasi) and Saitoella complicata and compared them to 18S rRNA sequences from the oomycetes, chytridiomycetes, zygomycetes, ascomycetes, and basidiomycetes. Here we demonstrate that the ascomycetes have at least two major evolutionary lineages. Taphrina wiesneri and Saitoella complicata form a monophyletic branch that diverged prior to the separation of other ascomycetes. The same treatment could be accorded to Schizosaccharomyces pombe.

Ascomycota↗