Stefanie Pöggeler

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

Abstract. 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 α-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 α-receptor Ste2p of S. cerevisiae, respectively. The identification of these homologues suggests the existence of a sexual cycle in A. fumigatus.

Identification of transcriptionally expressed pheromone receptor genes in filamentous ascomycetes

Detection of pheromone genes in filamentous ascomycetes implicated the presence of pheromone receptor genes. Similar to yeasts and basidiomycetes, these might be involved in a G-protein triggered signal transduction pathway during mating. We have identified two pheromone receptor genes, named pre1 and pre2, in the genome of the heterothallic filamentous ascomycete Neurospora crassa and the closely related homothallic Sordaria macrospora. The deduced pre1 gene product is a putative seven-transmembrane protein, which displays a high-level amino acid identity with the a-factor receptor Ste3p of Saccharomyces cerevisiae, and is also homologous to lipopeptide pheromone receptors of basidiomycetes. The deduced pre2 product displays significant sequence similarity with the S. cerevisiae STE2 gene product, the alpha-factor receptor. Pair-wise comparisons between pheromone receptor genes of N. crassa and S. macrospora revealed an extremely low degree of nucleotide conservation in these genes, suggesting that they evolved very rapidly. The two genes are transcriptionally expressed in both N. crassa and S. macrospora. Northern and reverse transcription-polymerase chain reaction analyses indicate that in N. crassa, expression of the receptor genes does not occur in a mating type specific manner. Thus, filamentous ascomycetes appear to posses and express pheromone receptor genes.

Spo76p Is a Conserved Chromosome Morphogenesis Protein that Links the Mitotic and Meiotic Programs

Spo76p is conserved and related to the fungal proteins Pds5p and BIMD and the human AS3 prostate proliferative shutoff-associated protein. Spo76p localizes to mitotic and meiotic chromosomes, except at metaphase(s) and anaphase(s). During meiotic prophase, Spo76p assembles into strong lines in correlation with axial element formation. As inferred from spo76-1 mutant phenotypes, Spo76p is required for sister chromatid cohesiveness, chromosome axis morphogenesis, and chromatin condensation during critical transitions at mitotic prometaphase and meiotic midprophase. Spo76p is also required for meiotic interhomolog recombination, likely at postinitiation stage(s). We propose that a disruptive force coordinately promotes chromosomal axial compaction and destabilization of sister connections and that Spo76p restrains and channels the effects of this force into appropriate morphogenetic mitotic and meiotic outcomes.

Inteins, valuable genetic elements in molecular biology and biotechnology

Inteins are internal protein elements that self-excise from their host protein and catalyze ligation of the flanking sequences (exteins) with a peptide bond. They are found in organisms in all three domains of life, and in viral proteins. Intein excision is a posttranslational process that does not require auxiliary enzymes or cofactors. This self-excision process is called protein splicing, by analogy to the splicing of RNA introns from pre-mRNA. Protein splicing involves only four intramolecular reactions, and a small number of key catalytic residues in the intein and exteins. Protein-splicing can also occur in trans. In this case, the intein is separated into N- and C-terminal domains, which are synthesized as separate components, each joined to an extein. The intein domains reassemble and link the joined exteins into a single functional protein. Understanding the cis- and trans-protein splicing mechanisms led to the development of intein-mediated protein-engineering applications, such as protein purification, ligation, cyclization, and selenoprotein production. This review summarizes the catalytic activities and structures of inteins, and focuses on the advantages of some recent intein applications in molecular biology and biotechnology.

Phylogenetic relationships between mating-type sequences from homothallic and heterothallic ascomycetes

Abstract To gain a deeper insight into the evolution of mating-type genes from filamentous ascomycetes, a comprehensive sequence analysis of PCR-amplified sequences corresponding to A- and a-specific mating-type sequences was undertaken. The study included nine homothallic (compatible) and eight heterothallic (incompatible) members of the genera Neurospora and Sordaria. Distance and parsimony trees based on gene fragments from the mat a-1 and mat A-1 genes were compared with trees derived from partial DNA sequences of the gpd glyceraldehyde-3-phosphate dehydrogenase gene. In contrast to the sequences from the gpd gene, mating-type genes show striking sequence differences, suggesting that these genes evolve very rapidly. Strong inter-relationships were found among homothallic, as well as among heterothallic, members of both genera, indicating that in each genus a change from one reproductive strategy to another might result from one single event.

Fruiting-Body Development in Ascomycetes

Fruiting bodies are multicellular structures that are developed during the sexual life cycle of filamentous ascomycetes and protect the products of meiosis. In this review, we will provide a general overview about the morphology and development of fruiting bodies. This includes an introduction into important model ascomycetes, which have extensively been studied at the molecular level. We will further mention environmental and endogenous factors that affect the development of complex fruiting bodies. Further, we will discuss regulatory networks such as signal transduction pathways, protein degradation mechanisms, as well as transcriptional regulators and chromatin modifiers. This review summarizes our mechanistic understanding of fruiting-body formation in filamentous ascomycetes, which is reminiscent of other complex eukaryotic developmental processes.

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

Abstract. 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.

Cell Differentiation during Sexual Development of the Fungus Sordaria macrospora Requires ATP Citrate Lyase Activity

ABSTRACT During sexual development, mycelial cells from most filamentous fungi differentiate into typical fruiting bodies. Here, we describe the isolation and characterization of the Sordaria macrosporadevelopmental mutant per5, which exhibits a sterile phenotype with defects in fruiting body maturation. Cytological investigations revealed that the mutant strain forms only ascus precursors without any mature spores. Using an indexed cosmid library, we were able to complement the mutant to fertility by DNA-mediated transformation. A single cosmid clone, carrying a 3.5-kb region able to complement the mutant phenotype, has been identified. Sequencing of the 3.5-kb region revealed an open reading frame of 2.1 kb interrupted by a 66-bp intron. The predicted polypeptide (674 amino acids) shows significant homology to eukaryotic ATP citrate lyases (ACLs), with 62 to 65% amino acid identity, and the gene was named acl1. The molecular mass of the S. macrospora ACL1 polypeptide is 73 kDa, as was verified by Western blot analysis with a hemagglutinin (HA) epitope-tagged ACL1 polypeptide. Immunological in situ detection of the HA-tagged polypeptide demonstrated that ACL is located within the cytosol. Sequencing of the mutant acl1 gene revealed a 1-nucleotide transition within the coding region, resulting in an amino acid substitution within the predicted polypeptide. Further evidence that ACL1 is essential for fruiting body maturation comes from experiments in which truncated and mutated versions of theacl1 gene were used for transformation. None of these copies was able to reconstitute the fertile phenotype in transformed per5 recipient strains. ACLs are usually involved in the formation of cytosolic acetyl coenzyme A (acetyl-CoA), which is used for the biosynthesis of fatty acids and sterols. Protein extracts from the mutant strain showed a drastic reduction in enzymatic activity compared to values obtained from the wild-type strain. Investigation of the time course of ACL expression suggests that ACL is specifically induced at the beginning of the sexual cycle and produces acetyl-CoA, which most probably is a prerequisite for fruiting body formation during later stages of sexual development. We discuss the contribution of ACL activity to the life cycle of S. macrospora.

Carbonic anhydrases in fungi

Carbonic anhydrases (CAs) are metalloenzymes that catalyse the interconversion of carbon dioxide and bicarbonate with high efficiency. This reaction is fundamental to biological processes such as respiration, photosynthesis, pH homeostasis, CO(2) transport and electrolyte secretion. CAs are distributed among all three domains of life, and are currently divided into five evolutionarily unrelated classes (alpha, beta, gamma, delta and zeta). Fungal CAs have only recently been identified and characterized in detail. While Saccharomyces cerevisiae and Candida albicans each have only one beta-CA, multiple copies of beta-CA- and alpha-CA-encoding genes are found in other fungi. Recent work demonstrates that CAs play an important role in the CO(2)-sensing system of fungal pathogens and in the regulation of sexual development. This review focuses on CA functions in S. cerevisiae, the fungal pathogens C. albicans and Cryptococcus neoformans, and the filamentous ascomycete Sordaria macrospora.

Functional Characterization of MAT1-1-Specific Mating-Type Genes in the Homothallic Ascomycete Sordaria macrospora Provides New Insights into Essential and Nonessential Sexual Regulators†

ABSTRACT Mating-type genes in fungi encode regulators of mating and sexual development. Heterothallic ascomycete species require different sets of mating-type genes to control nonself-recognition and mating of compatible partners of different mating types. Homothallic (self-fertile) species also carry mating-type genes in their genome that are essential for sexual development. To analyze the molecular basis of homothallism and the role of mating-type genes during fruiting-body development, we deleted each of the three genes, SmtA-1 (MAT1-1-1), SmtA-2 (MAT1-1-2), and SmtA-3 (MAT1-1-3), contained in the MAT1-1 part of the mating-type locus of the homothallic ascomycete species Sordaria macrospora. Phenotypic analysis of deletion mutants revealed that the PPF domain protein-encoding gene SmtA-2 is essential for sexual reproduction, whereas the α domain protein-encoding genes SmtA-1 and SmtA-3 play no role in fruiting-body development. By means of cross-species microarray analysis using Neurospora crassa oligonucleotide microarrays hybridized with S. macrospora targets and quantitative real-time PCR, we identified genes expressed under the control of SmtA-1 and SmtA-2. Both genes are involved in the regulation of gene expression, including that of pheromone genes.