pZHK2, a bi-functional transformation vector, suitable for two step gene targeting

Homologous recombination is a prerequisite for the generation of knock out strains by means of DNA-mediated transformation. In filamentous fungi however, the frequency of ectopic integration events is rather high and the actual efficiency of homologous recombination depends upon the length of homologous DNA flanking the transformation marker. Recently, d´Enfert and coworkers (Chaveroche et al., 2000) presented a two-step technology for the integration of a bi-functional zeocin-pyrG cassette into a target sequence of interest using anEscherichia coli strain expressing the phage lambda Red functions. In the resulting recombinant cosmids, the selection marker is flanked by fungal DNA sequences longer than 1 kb, which can be used to transform appropriate fungal recipient strains. For selection of fungal transformants, those workers used the A. nidulanspyrG gene encoding orotidine-5´-monophosphate decarboxylase, which confers prototrophy in appropriate uridine/uracil auxotrophic recipient strains. Here, we describe the novel bi-functional transformation vector pZHK2, which carries in addition to the zeocin resistance gene the hygromycin B phosphotransferase gene often used as a dominant selectable marker gene in fungal recipient strains. The applicability of the vector is demonstrated by generating a ura3- knock out strain from Sordaria macrospora showing auxotrophy

Application of the nourseothricin acetyltransferase gene (nat1) as dominant marker for the transformation of filamentous fungi

Here, we report the construction of two transformation vectors, pD-NAT1 and pG-NAT1, carrying the nat1 gene encoding the nourseothricin acetyltransferase. The nat1 gene is expressed under the control of the Aspergillus nidulans trpC promoter and thus can be used as a dominant drug-resistance marker for the DNA-mediated transformation of filamentous fungi. The successful application of both vectors was demonstrated by transforming the homothallic ascomycete Sordaria macrospora as well as the β-lactam producerAcremonium chrysogenum. For both fungi and for both vectors, transformation frequencies were between 10 and 40 transformants per 10 µg of plasmid DNA

Superdeep Tests and Experiments at 9.1 km and 4 km

The Continental Deep Drilling Program of Germany (in German: Kontinentales Tiefbohrprogramm der Bundesrepublik Deutschland, abbreviated as KTB) was a scientific drilling project near the town of Windischeschenbach, Bavaria. The KTB Depth Laboratory comprises two 9.1 km and 4 km deep, water-filled boreholes in crystalline basement rocks just 200 meters apart from each other. Available equipment such as cables, winches, geophysical borehole tools as well as workshops and office infrastructure allows for in-situ tests and experiments at different pressure and temperature conditions. The two stable wells are large-diameter steel-cased and have been geophysically monitored in detail since 1996

Genome‐wide identification of target genes of a mating‐type α‐domain transcription factor reveals functions beyond sexual development

Penicillium chrysogenum is the main industrial producer of the β-lactam antibiotic penicillin, the most commonly used drug in the treatment of bacterial infections. Recently, a functional MAT1-1 locus encoding the α-box transcription factor MAT1-1-1 was discovered to control sexual development in P. chrysogenum. As only little was known from any organism about the regulatory functions mediated by MAT1-1-1, we applied chromatin immunoprecipitation combined with next-generation sequencing (ChIP-seq) to gain new insights into the factors that influence MAT1-1-1 functions on a molecular level and its role in genome-wide transcriptional regulatory networks. Most importantly, our data provide evidence for mating-type transcription factor functions that reach far beyond their previously understood role in sexual development. These new roles include regulation of hyphal morphology, asexual development, as well as amino acid, iron, and secondary metabolism. Furthermore, in vitro DNA–protein binding studies and downstream analysis in yeast and P. chrysogenum enabled the identification of a MAT1-1-1 DNA-binding motif, which is highly conserved among euascomycetes. Our studies pave the way to a more general understanding of these master switches for development and metabolism in all fungi, and open up new options for optimization of fungal high production strains.This is the publisher’s final pdf. The published article is copyrighted by the author(s) and published by John Wiley & Sons, Inc. The published article can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291365-295

Sexual reproduction and mating-type-mediated strain development in the penicillin-producing fungus Penicillium chrysogenum

Penicillium chrysogenum is a filamentous fungus of major medical and historical importance, being the original and present-day industrial source of the antibiotic penicillin. The species has been considered asexual for more than 100 y, and despite concerted efforts, it has not been possible to induce sexual reproduction, which has prevented sexual crosses being used for strain improvement. However, using knowledge of mating-type (MAT) gene organization, we now describe conditions under which a sexual cycle can be induced leading to production of meiotic ascospores. Evidence of recombination was obtained using both molecular and phenotypic markers. The identified heterothallic sexual cycle was used for strain development purposes, generating offspring with novel combinations of traits relevant to penicillin production. Furthermore, the MAT1-1–1 mating-type gene, known primarily for a role in governing sexual identity, was also found to control transcription of a wide range of genes with biotechnological relevance including those regulating penicillin production, hyphal morphology, and conidial formation. These discoveries of a sexual cycle and MAT gene function are likely to be of broad relevance for manipulation of other asexual fungi of economic importance

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

A nucleosome assembly protein-like polypeptide binds to chloroplast group II intron RNA in Chlamydomonas reinhardtii

In the unicellular green alga Chlamydomonas reinhardtii, the chloroplast-encoded tscA RNA is part of a tripartite group IIB intron, which is involved in trans-splicing of precursor mRNAs. We have used the yeast three-hybrid system to identify chloroplast group II intron RNA-binding proteins, capable of interacting with the tscA RNA. Of 14 candidate cDNAs, 13 encode identical polypeptides with significant homology to members of the nuclear nucleosome assembly protein (NAP) family. The RNA-binding property of the identified polypeptide was demonstrated by electrophoretic mobility shift assays using different domains of the tripartite group II intron as well as further chloroplast transcripts. Because of its binding to chloroplast RNA it was designated as NAP-like (cNAPL). In silico analysis revealed that the derived polypeptide carries a 46 amino acid chloroplast leader peptide, in contrast to nuclear NAPs. The chloroplast localization of cNAPL was demonstrated by laser scanning confocal fluorescence microscopy using different chimeric cGFP fusion proteins. Phylogenetic analysis shows that no homologues of cNAPL and its related nuclear counterparts are present in prokaryotic genomes. These data indicate that the chloroplast protein described here is a novel member of the NAP family and most probably has not been acquired from a prokaryotic endosymbiont

De novo Assembly of a 40 Mb Eukaryotic Genome from Short Sequence Reads: Sordaria macrospora, a Model Organism for Fungal Morphogenesis

Filamentous fungi are of great importance in ecology, agriculture, medicine, and biotechnology. Thus, it is not surprising that genomes for more than 100 filamentous fungi have been sequenced, most of them by Sanger sequencing. While next-generation sequencing techniques have revolutionized genome resequencing, e.g. for strain comparisons, genetic mapping, or transcriptome and ChIP analyses, de novo assembly of eukaryotic genomes still presents significant hurdles, because of their large size and stretches of repetitive sequences. Filamentous fungi contain few repetitive regions in their 30–90 Mb genomes and thus are suitable candidates to test de novo genome assembly from short sequence reads. Here, we present a high-quality draft sequence of the Sordaria macrospora genome that was obtained by a combination of Illumina/Solexa and Roche/454 sequencing. Paired-end Solexa sequencing of genomic DNA to 85-fold coverage and an additional 10-fold coverage by single-end 454 sequencing resulted in ∼4 Gb of DNA sequence. Reads were assembled to a 40 Mb draft version (N50 of 117 kb) with the Velvet assembler. Comparative analysis with Neurospora genomes increased the N50 to 498 kb. The S. macrospora genome contains even fewer repeat regions than its closest sequenced relative, Neurospora crassa. Comparison with genomes of other fungi showed that S. macrospora, a model organism for morphogenesis and meiosis, harbors duplications of several genes involved in self/nonself-recognition. Furthermore, S. macrospora contains more polyketide biosynthesis genes than N. crassa. Phylogenetic analyses suggest that some of these genes may have been acquired by horizontal gene transfer from a distantly related ascomycete group. Our study shows that, for typical filamentous fungi, de novo assembly of genomes from short sequence reads alone is feasible, that a mixture of Solexa and 454 sequencing substantially improves the assembly, and that the resulting data can be used for comparative studies to address basic questions of fungal biology