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At least 721 records · Page 40Linked to original sources
PubMed2000

Genetic transformation and biotechnological application of the yeast Arxula adeninivorans.

The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 degrees C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 degrees C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 degrees C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression.

Biotechnology↗
PubMed1997

Screening for fungi intensively mineralizing 2,4,6-trinitrotoluene.

Within a screening program, 91 fungal strains belonging to 32 genera of different ecological and taxonomic groups (wood- and litter-decaying basidiomycetes, saprophytic micromycetes) were tested for their ability to metabolize and mineralize 2,4,6-trinitrotoluene (TNT). All these strains metabolized TNT rapidly by forming monoaminodinitrotoluenes (AmDNT). Micromycetes produced higher amounts of AmDNT than did wood- and litter-decaying basidiomycetes. A significant mineralization of [14C]TNT was only observed for certain wood- and litter-decaying basidiomycetes. The most active strains, Clitocybula dusenii TMb12 and Stropharia rugosa-annulata DSM11372 mineralized 42% and 36% respectively of the initial added [14C]TNT (100 microM corresponding to 4.75 microCi/l) to 14CO2 within 64 days. Micromycetes (deuteromycetes, ascomycetes, zygomycetes) proved to be unable to mineralize [14C]TNT significantly.

Basidiomycota↗
PubMed2001

Molecular characterization of the actin-encoding gene and the use of its promoter for a dominant selection system in the methylotrophic yeast Hansenula polymorpha.

The actin gene (ACT) from the methylotrophic yeast Hansenula polymorpha was cloned and its structural feature was characterized. In contrast to the actin genes of other ascomycetous yeasts, which have only one large intron, the H. polymorpha ACT gene was found to be split by two introns. The H. polymorpha ACT introns were correctly processed in the heterologous host Saccharomyces cerevisiae despite appreciable differences in the splice site sequences. The promoter region of H. polymorpha ACT displayed two CCAAT motifs and two TATA-like sequences in a configuration similar to that observed in the S. cerevisiae actin promoter. A set of deleted H. polymorpha ACT promoters was exploited to direct expression of the bacterial hygromycin B resistance (hph) gene as a dominant selectable marker in the transformation of H. polymorpha. The resistance level of H. polymorpha transformants to the antibiotic was shown to be dependent on the integration copy number of the hph cassette. The selectivity of the hygromycin B resistance marker for transformants of higher copy number was remarkably increased with the deletion of the upstream TATA-like sequence, but not with the removal of either CCAAT motif, from the H. polymorpha promoter. The dosage-dependent selection system developed in this study should be useful for genetic manipulation of H. polymorpha as an industrial strain to produce recombinant proteins.

Actins↗
PubMed2002

Conservation of XYN11A and XYN11B xylanase genes in Bipolaris sorghicola, Cochliobolus sativus, Cochliobolus heterostrophus, and Cochliobolus spicifer.

Two types of xylanase gene, XYN11A ( XYL1) and XYN11B ( XYL2), were amplified by PCR and partially sequenced in four phytopathogenic species of the ascomycete fungal genus Cochliobolus (anamorph genus Bipolaris). Three of the species, C. heterostrophus ( B. maydis), C. sativus ( B. sorokiniana), and Bipolaris sorghicola (no teleomorph known), are interrelated; the fourth, C. spicifer ( B. spicifera), was found, through analysis of the 5.8S RNA and internal transcribed spacer (ITS) sequences of its ribosomal DNA, to be more distantly related to the other three. Isolates from all four species contain orthologous XYN11A and XYN11B genes, but a set of laboratory strains of C. heterostrophus gave no product corresponding to the XYN11B gene. The patterns of evolution of the two xylanase genes and ribosomal DNA sequences are mutually consistent; the results indicate that the two genes were present in the common ancestor of all Cochliobolus species and are evolving independently of each other.

Ascomycota↗
PubMed2004

Molecular characterization and in planta detection of Sclerotinia sclerotiorum endopolygalacturonase genes.

Sclerotinia sclerotiorum, a plant pathogenic ascomycete, secretes multiple pectinolytic enzymes that facilitate penetration, colonization, and maceration of the plant tissues. Molecular analysis has previously revealed that the pectinolytic system of the fungus is organized as a multigene family, among which a subfamily of three members encoding for neutral endopolygalacturonase (endoPG) isoforms has been characterized. Here we describe the isolation and characterization of three additional endoPG-encoding genes ( pg5, pg6, and pg7) that belong to distinct phylogenetic groups. Pairwise sequence comparison between the known endoPGs from S. sclerotiorum revealed 43% to 97% identity, and the genomic organization of the pectinolytic system showed a great similarity to that of the related necrotroph Botrytis cinerea. During plant pathogenesis, a sequential expression of the endoPG-encoding genes was shown.

Ascomycota↗
PubMed2004

The Metarhizium anisopliae trp1 gene: cloning and regulatory analysis.

The trp1 gene from the entomopathogenic fungus Metarhizium anisopliae, cloned by heterologous hybridization with the plasmid carrying the trpC gene from Aspergillus nidulans, was sequence characterized. The predicted translation product has the conserved catalytic domains of glutamine amidotransferase (G domain), indoleglycerolphosphate synthase (C domain), and phosphoribosyl anthranilate isomerase (F domain) organized as NH2-G-C-F-COOH. The ORF is interrupted by a single intron of 60 nt that is position conserved in relation to trp genes from Ascomycetes and length conserved in relation to Basidiomycetes species. RT-PCR analysis suggests constitutive expression of trp1 gene in M. anisopliae.

Amino Acid Sequence↗
PubMed2001

Molecular characterization of the host-adapted pathogen Verticillium longisporum on the basis of a group-I intron found in the nuclear SSU-rRNA gene.

Verticillium wilt of oilseed rape is caused by the host-adapted pathogen Verticillium longisporum comb. nov. With one set of nuclear SSU-rRNA gene primers, a PCR amplification product of ca. 2.5 kb was generated from all isolates of V. longisporum tested (36 from Europe, Japan, and USA), with the exception of two recombinant isolates. On the contrary, all the other phytopathogenic and nonphytopathogenic species of Verticillium tested (18 species, 46 isolates), with the exception of one isolate of V. lecanii and two of Cordyceps sp., generated a product of ca. 1.65 kb. Sequence analysis of the SSU-rRNA gene of two typical isolates of V. longisporum (wild radish, Japan, and oilseed rape, Germany) revealed that this dimorphism was due to the presence of an identical 839-bp intron located in a highly conserved insertion position (nt 1165 of Saccharomyces cerevisiae). The intron sequence was classified as group-I intron on the basis of conserved sequence and secondary structural elements. Primers designed from the 839-bp intron sequence amplified only the V. longisporum. Phylogenetic analysis based on SSU-rDNA sequences showed that V. longisporum was closely related to the genera of other filamentous Ascomycetes with fruiting bodies.

Base Sequence↗
PubMed1996

Identification of a group I intron in the nuclear small subunit rDNA of Pseudohalonectria lignicola.

A 865-bp insertion was detected within the nuclear small subunit (SSU) rDNA in two isolates (intron+) of the freshwater ascomycete Pseudohalonectria lignicola by the polymerase chain reaction (PCR). The intron sequences from the two isolates were identical to each other, and the exon sequences from the two intron+ isolates were identical to those in the intron- isolates in the PCR-amplified region of rDNA. Reverse transcription PCR (RT-PCR) indicated that the intron was absent in the mature rRNA. The intron sequence has all the characteristics of a group I intron, including four conserved sequence elements (P, Q, R, and S), the presence of a U at the 5' splice site of the exon, a G at the 3' splice site of the intron, a putative internal guiding sequence, and the sequence fit a secondary structure model for group I introns. Like most introns found in nuclear rDNA, this intron was located at a highly conserved region and was devoid of long open reading frames.

Ascomycota↗
PubMed1996

Purification and Characterization of an Extracellular beta-Glucosidase from the Wood-Grown Fungus Xylaria regalis

Xylaria regalis, a wood-grown ascomycete isolated in Taiwan, produces beta-glucosidase (EC 3.2.1.21) extracellularly. The beta-glucosidase was purified to homogeneity by ammonium sulfate precipitation, ion-exchange, and gel filtration chromatography. The molecular mass of the purified enzyme was estimated to be 85 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. With p-nitrophenyl beta-D-glucopyranoside (PNPG) as the substrate at pH 5.0 and 50°C, the Km was 1.72 mM and Vmax was 326 &mgr;mol/min/mg. Optimal activity with PNPG as the substrate was at pH 5.0 and 50°C. The enzyme was stable at pH 5.0 at temperatures up to 50°C. The purified beta-glucosidase was active against PNPG, cellobiose, sophorose, and gentiobiose, but did not hydrolyze lactose, sucrose, Avicel, and o-nitrophenyl beta-D-galactopyranoside. The activity of beta-glucosidase was stimulated by Ca2+, Mg2+, Mn2+, Cd2+ and beta-mercaptoethanol, and inhibited by Ag+, Hg2+, SDS, and p-chloromercuribenzoate (PCMB).

Journal Article↗
PubMed2002

Nht2, a copia LTR retrotransposon from a conditionally dispensable chromosome in Nectria haematococca.

In the plant pathogenic ascomycete Nectria haematococca mating population (MP) VI, the conditionally dispensable chromosomes are unstable during sexual reproduction. During mapping of such a chromosome, three dispersed repeats were identified. Nht2, one of these repeated elements, is a long terminal repeat (LTR) retrotransposon that is 5.9 kb in length. Its deduced amino acid sequence is homologous to the four enzymatic domains characteristic of copia retrotransposons, but it contains multiple stop codons and probably is no longer able to transpose autonomously. Nht2's LTRs differ at ten positions and the characteristics of these differences resemble the changes induced by repeat-induced point mutation (RIP) in Neurospora crassa. The likelihood that Nectria haematococca MP VI has a RIP-like process, however, is reduced by the fact that a multi-copy transposon cloned from the same ascospore isolate as Nht2 encodes an intact open reading frame. Nht2 is broadly distributed among isolates collected from a variety of host plants. A limited survey of three field isolates suggests that Nht2 is on only one or a few chromosomes in every genome. Nht2's degeneracy and its widespread distribution within the species both suggest that it is an ancient element within N. haematococca MP VI.

Amino Acid Sequence↗
PubMed2002

Nuclear small subunit rRNA group I intron variation among Beauveria spp provide tools for strain identification and evidence of horizontal transfer.

An optional group I intron was characterized at a single insertion point in nuclear small subunit rRNA (nuSSU rRNA) genes of the imperfect entomopathogenic fungi, Beauveria bassiana and B. brongniartii. Insertion points were conserved among nuSSU rRNA genes from 35 Beauveria isolates. PCR-RFLP and DNA sequencing identified 12 group I intron variants and were applied to the identification of strains isolated from insect hosts. Alignment of 383-404-nt subgroup IB3 group I introns indicated that four insertion/deletion (indel) mutations were the main basis of fragment length variation. Phylogeny reconstruction using parsimony and neighbor-joining methods suggested six lineages may be present among nuSSU rRNA group I intron sequences from Beauveria and related ascomycete fungi. Terminal node placement of Beauveria introns conflicted with previously published phylogenies constructed from gene sequences, suggesting horizontal transfer of group I introns. PCR-RFLP among introns provided a means for the differentiation of Beauveria isolates.

Base Sequence↗
PubMed2002

Genomic evidence for mating abilities in the asexual pathogen Aspergillus fumigatus.

The filamentous fungus Aspergillus fumigatus is one of the causes of invasive lung disease in immunocompromised individuals. It is classified as asexual because no direct observation of mating or meiosis has been reported. Sequencing of the complete genome by an international collaboration, including the Wellcome Trust Sanger Institute (UK) and The Institute for Genomic Research (TIGR, USA), has made most of the genomic sequence information from A. fumigatus publicly available. By searching the incomplete genome sequence of A. fumigatus, I have identified the coding capacity for a set of proteins that could be involved in mating and the pheromone response pathway. These include one putative mating-type gene, one gene encoding a pheromone and two pheromone-receptor genes. The mating-type gene encodes a high-mobility group domain protein exhibiting significant similarity with mating-type proteins from sexually reproducing filamentous ascomycetes. The pheromone gene is predicted to encode a precursor pheromone that is processed by a KEX2-like protease to yield a pheromone that is structurally similar to the alpha-factor of the yeast Saccharomyces cerevisiae. In addition, the deduced gene products of the receptor genes are putative seven-transmembrane proteins, which display a high-level amino acid identity with the a-factor receptor Ste3p and the alpha-receptor Ste2p of S. cerevisiae, respectively. The identification of these homologues suggests the existence of a sexual cycle in A. fumigatus.

Amino Acid Sequence↗
PubMed2002

A Blumeria graminisf.sp. hordei BAC library--contig building and microsynteny studies.

A bacterial artificial chromosome (BAC) library of Blumeria graminis f.sp. hordei, containing 12,000 clones with an average insert size of 41 kb, was constructed. The library represents about three genome equivalents and BAC-end sequencing showed a high content of repetitive sequences, making contig-building difficult. To identify overlapping clones, several strategies were used: colony hybridisation, PCR screening, fingerprinting techniques and the use of single-copy expressed sequence tags. The latter proved to be the most efficient method for identification of overlapping clones. Two contigs, at or close to avirulence loci, were constructed. Single nucleotide polymorphism (SNP) markers were developed from BAC-end sequences to link the contigs to the genetic maps. Two other BAC contigs were used to study microsynteny between B. graminis and two other ascomycetes, Neurospora crassa and Aspergillus fumigatus. The library provides an invaluable tool for the isolation of avirulence genes from B. graminis and for the study of gene synteny between this fungus and other fungi.

Ascomycota↗
PubMed2003

Versatile EGFP reporter plasmids for cellular localization of recombinant gene products in filamentous fungi.

The recent development of variants of the green fluorescent protein (GFP) with altered codon composition facilitated the efficient expression of this reporter protein in a number of fungal species. In this report, we describe the construction and application of a series of plasmids, which support the expression of an enhanced gfp (egfp) gene in filamentous fungi and assist the study of diverse developmental processes. Included were a promoterless egfp vector for monitoring the expression of cloned promoters/enhancers in fungal cells and vectors for creating translation fusions to the N-terminus of EGFP. The vectors were further modified by introducing a variant hygromycin B phosphotransferase (hph) gene, lacking the commonly found NcoI site. Instead, this site, which contained an ATG start codon, was placed in front of the egfp gene and thus was made suitable for the cloning of translational fusions. The applicability of these vectors is demonstrated by analyzing transcription regulation and protein localization and secretion in two ascomycetes, Acremonium chrysogenum and Sordaria macrospora. In the latter, the heterologous egfp gene is stably inherited during meiotic divisions, as can easily be seen from fluorescent ascospores.

Acremonium↗
PubMed2003

Sequence diversity of mating-type genes in Phaeosphaeria avenaria.

Phaeosphaeria avenaria, one of the causal agents of stagonospora leaf blotch diseases in cereals, is composed of two subspecies, P. avenaria f. sp. triticea (Pat) and P. avenaria f. sp. avenaria (Paa). The Pat subspecies was grouped into Pat1-Pat3, based on restriction fragment length polymorphism (RFLP) and ribosomal DNA (rDNA) internal transcribed spacer (ITS) sequences in previous studies. Mating-type genes and their potential use in phylogeny and molecular classification were studied by DNA hybridization and PCR amplification. The majority of Pat1 isolates reported to be homothallic and producing sexual reproduction structures on cultural media had only the MAT1-1 gene. Minor sequence variations were found in the conserved region of MAT1-1 gene in Pat1 isolates. However, both mating-type genes, MAT1-1 and MAT1-2, were identified in P. avenaria isolates represented by ATCC12277 from oats (Paa) and the Pat2 isolates from foxtail barley ( Hordeum jubatum L.). Cluster analyses based on mating-type gene conserved regions revealed that cereal Phaeosphaeria is not phylogenetically closely related to other ascomycetes, including Mycosphaerella graminicola (anamorph Septoria tritici). The sequence diversity of mating-type genes in Pat and Paa supports our previous phylogenetic relationship and molecular classification based on RFLP fingerprinting and rDNA ITS sequences.

Ascomycota↗
PubMed2004

Agrobacterium tumefaciens-mediated transformation of the antitumor clavaric acid-producing basidiomycete Hypholoma sublateritium.

The basidiomycete Hypholoma sublateritium produces clavaric acid, an antitumor isoprenoid compound. Arthrospores of this fungus were transformed by Agrobacterium tumefaciens-mediated conjugation. Five plasmids carrying different regulatory sequences to drive expression of the hph (hygromycin phosphotransferase) gene were tested. The promoter used was critically important in order to express heterologous genes in H. sublateritium. Constructions carrying the Agaricus bisporus glyceraldehyde-3-phosphate dehydrogenase promoter (P gpd) showed a good transformation efficiency, whereas constructions with the gpd promoter from ascomycetes were ineffective. Transformant clones showed a random integration pattern of plasmid DNA. Most transformants showed a single integrated copy of the transforming plasmid, but about 1.5% showed double or multiple integrations. All the analyzed transformants were mitotically stable and maintained the integrated exogenous DNA in the absence of antibiotic. The green fluorescent protein gene was expressed from the A. bisporus gpd promoter, as shown by RT-PCR studies, but no significant fluorescence was observed. Transformation of H. sublateritium opens the way for the genetic manipulation of clavaric acid biosynthesis in this fungus.

Agrobacterium tumefaciens↗
PubMed2005

Characterization of the Lentinula edodes exg2 gene encoding a lentinan-degrading exo-beta-1,3-glucanase.

Lentinan, an antitumor substance purified from Lentinula edodes, is degraded during post-harvest preservation as a result of increased glucanase activity. We isolated an exo-beta-1,3-glucanase encoding gene, exg2, from L. edodes which is a homologue of an exo-glucanase-encoding gene conserved in ascomycetous fungi. The exg2 gene was cloned as an approximately 2.4-kbp cDNA, and as a genomic sequence of 3.9-kbp. The product of the exg2 gene is predicted to contain 759 amino acids with a molecular weight of 79 kDa and a pI value of 4.6. The putative N-terminus of EXG2 is identical to the N-terminal sequences of lentinan-degrading enzymes, GNase I and II, and a custom-made anti-EXG2 peptide anti-serum cross-reacted with purified GNase I and II. Transcription and translation of exg2 was low in the gills of mature fruiting bodies, but increased after harvesting. We conclude that the exg2 gene is a lentinan-degrading enzyme-encoding-gene in L. edodes.

Amino Acid Sequence↗
PubMed2005

Genome-wide analysis of differentially expressed genes from Penicillium chrysogenum grown with a repressing or a non-repressing carbon source.

Penicillium chrysogenum is an economically important ascomycete used as industrial producer of penicillin. However, with the exception of penicillin biosynthesis genes, little attention has been paid to the genetics of other aspects of the metabolism of this fungus. In this article we describe the first attempt of systematic analysis of expressed genes in P. chrysogenum, using a suppression subtractive hybridization approach to clone and identify sequences of genes differentially expressed in media with glucose or lactose as carbon source (penicillin-repressing or non-repressing conditions). A total of 167 clones were analysed, 95 from the glucose condition and 72 from the lactose condition. Genes differentially expressed in the glucose condition encode mainly proteins involved in the mitochondrial electron transport chain and primary metabolism. Genes expressed differentially in lactose-containing medium include genes for secondary metabolism (pcbC, isopenicillin N synthase), different hydrolases and a gene encoding a putative hexose transporter or sensor. The results provided information on how the metabolism of this fungus adapts to different carbon sources. The expression patterns of some of the genes support the hypothesis that glucose induces higher rates of respiration in P. chrysogenum while repressing secondary metabolism.

Amino Acid Sequence↗