Michel Flipphi

Comparative genome sequence analysis underscores mycoparasitism as the ancestral life style of Trichoderma

BackgroundMycoparasitism, a lifestyle where one fungus is parasitic on another fungus, has special relevance when the prey is a plant pathogen, providing a strategy for biological control of pests for plant protection. Probably, the most studied biocontrol agents are species of the genus Hypocrea/Trichoderma.ResultsHere we report an analysis of the genome sequences of the two biocontrol species Trichoderma atroviride (teleomorph Hypocrea atroviridis) and Trichoderma virens (formerly Gliocladium virens, teleomorph Hypocrea virens), and a comparison with Trichoderma reesei (teleomorph Hypocrea jecorina). These three Trichoderma species display a remarkable conservation of gene order (78 to 96%), and a lack of active mobile elements probably due to repeat-induced point mutation. Several gene families are expanded in the two mycoparasitic species relative to T. reesei or other ascomycetes, and are overrepresented in non-syntenic genome regions. A phylogenetic analysis shows that T. reesei and T. virens are derived relative to T. atroviride. The mycoparasitism-specific genes thus arose in a common Trichoderma ancestor but were subsequently lost in T. reesei.ConclusionsThe data offer a better understanding of mycoparasitism, and thus enforce the development of improved biocontrol strains for efficient and environmentally friendly protection of plants.

Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation.

This article reviews our knowledge of the ethanol utilization pathway (alc system) in the hyphal fungus Aspergillus nidulans. We discuss the progress made over the past decade in elucidating the two regulatory circuits controlling ethanol catabolism at the level of transcription, specific induction, and carbon catabolite repression, and show how their interplay modulates the utilization of nutrient carbon sources. The mechanisms featuring in this regulation are presented and their modes of action are discussed: First, AlcR, the transcriptional activator, which demonstrates quite remarkable structural features and an original mode of action; second, the physiological inducer acetaldehyde, whose intracellular accumulation induces the alc genes and thereby a catabolic flux while avoiding intoxification; third, CreA, the transcriptional repressor mediating carbon catabolite repression in A. nidulans, which acts in different ways on the various alc genes; Fourth, the promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA) and the regulatory alcR gene, which exhibit exceptional strength compared to other genes of the respective classes. alc gene expression depends on the number and localization of regulatory cis-acting elements and on the particular interaction between the two regulator proteins, AlcR and CreA, binding to them. All these characteristics make the ethanol regulon a suitable system for induced expression of heterologous protein in filamentous fungi.

The 2008 update of the Aspergillus nidulans genome annotation: A community effort

The identification and annotation of protein-coding genes is one of the primary goals of whole-genome sequencing projects, and the accuracy of predicting the primary protein products of gene expression is vital to the interpretation of the available data and the design of downstream functional applications. Nevertheless, the comprehensive annotation of eukaryotic genomes remains a considerable challenge. Many genomes submitted to public databases, including those of major model organisms, contain significant numbers of wrong and incomplete gene predictions. We present a community-based reannotation of the Aspergillus nidulans genome with the primary goal of increasing the number and quality of protein functional assignments through the careful review of experts in the field of fungal biology.

Cloning and characterization of the abfB gene coding for the major α-l-arabinofuranosidase (ABF B) of Aspergillus niger

Based on amino-acid sequence data from Aspergillus niger α-l-arabinofuranosidase B (ABF B), and cyanogen bromide fragments derived thereof, deoxyoligonucleotide mixtures were designed to the employed as primers in a polymerase chain reaction (PCR) on A. niger genomic DNA. This resulted in amplification of three related PCR products. The abfB gene encoding ABF B was isolated from a genomic library using such an amplification product as a probe. A 5.1-kb BamHI fragment was subcloned to result in plasmid pIM991. Upon introduction by co-transformation into both A. niger and A. nidulans uridine auxotrophic strains, pIM991 was shown to contain the functional gene since prototrophic transformants overproduced ABF B upon growth on the inducing carbon source sugar beet pulp. A plate assay was developed enabling quick selection of ABF B-overproducing transformants. The sequence of a 4122-bp long BamHI/SstI fragment was determined. The abfB gene does not contain introns and codes for a protein of 499 amino acids. The mature ABF B, 481 amino acids in length, has a deduced molecular weight of 50.7 kDa. A. niger abfB is the first eukaryotic gene encoding an ABF to be characterized.

Biodiversity and evolution of primary carbon metabolism in Aspergillus nidulans and other Aspergillus spp.

Some of the Aspergilli are reputed for their versatile and efficient catabolism of soluble carbon sources and related metabolites as well as raw polymeric materials. Here, we present a detailed investigation of the genomic and evolutionary basis for this versatility, using seven Aspergillus and one Neosartorya genome sequences. We manually annotated about 155 genes per genome covering glycolysis, the pentose phosphate cycle, alternative routes of D-glucose metabolism, catabolism of D-galactose and pentoses, and the TCA cycle, as well as the utilization of acetate and ethanol, propionate metabolism, and gluconeogenesis.The annotation reveals that the Aspergilli have re-enforced several areas of their primary metabolism(notably glycolysis, TCA cycle, ethanol utilization, and pentose and polyol metabolism) by gene duplications,horizontal gene transfer or gene clustering. Results from the phylogenetic analysis of several enzymes encoded by duplicated genes also suggests that some gene products may have acquired new(physiological) functions, that render primary carbon metabolism of the Aspergilli more complex than previously thought.

Arabinase gene expression in Aspergillus niger: indications for coordinated regulation.

Aspergillus niger secretes three glycosylated glycosyl hydrolases which are involved in degradation of the plant cell wall polysaccharide L-arabinan: alpha-L-arabinofuranosidases (ABF) A and B, and endo-1,5-alpha-L-arabinase (ABN) A. The nucleotide sequence of the previously cloned gene encoding ABF A (abfA) from A. niger was determined. The coding region contains seven introns. Mature ABF A comprises 603 amino acids with a molecular mass of 65.4 kDa as deduced from the nucleotide sequence. The secreted enzyme is N-glycosylated. The primary structures of the three A. niger arabinases characterized lack similarity. Regulation of arabinase expression upon induction by sugar beet pulp and by L-arabitol was studied as a function of time. This was done in wild-type A. niger as well as in transformants carrying multiple copies of either one of the ABF-encoding genes. Each arabinase gene responded differently upon a mycelial transfer to L-arabitol-containing medium. Extra copies of abfA or abfB led to a decreased expression level of ABN A, though the repression elicited by abfB is stronger and more persistent than that effected by abfA. Multiple copies of both abf genes influence expression of the other ABF similarly, but to a far less pronounced degree than they affect ABN A synthesis. Four putative promoter elements, shared by all three arabinase genes, could be involved in coordination of L-arabinan degradation by A. niger.

Induction of extracellular arabinases on monomeric substrates in Aspergillus niger

The induction of extracellular arabinases by pentose sugars and polyols generated by the metabolic pathway of l-arabinose and d-xylose catabolism in Aspergillus niger was investigated. Induction occurred with l-arabinose and l-arabitol but not with d-xylose or xylitol. l-arabitol in particular was found to be a good inducer for α-l-arabinofuranosidase and endo-arabinase activities. Western blotting analysis showed both α-l-arabinofuranosidase A and B to be present. No induction was observed using d-arabitol. Unlike the wild type A. niger N402 strain, the A. niger xylulose kinase negative mutant N572 also showed induction of α-l-arabinofuranosidases A and B and endo-arabinase activity on d-xylose and xylitol. This is due to metabolic conversion of these compounds leading to the accumulation of both xylitol and l-arabitol in this mutant, the latter of which then acts as inducer. The induction of the two α-l-arabinofuranosidases and endo-arabinase is under the control of two regulatory systems namely pathway specific induction and carbon catabolite repression. Under derepressing conditions in the wild type only α-l-arabinofuranosidase B could be detected by Western blotting analysis. This indicates that α-l-arabinofuranosidase B is of importance in the initiation of specific induction of the various arabinose activities in A. niger grown on arabinose containing structural polysaccharides.

Cloning of the Aspergillus niger gene encoding α-l-arabinofuranosidase A

Using l-arabitol as an inducer, simple induction conditions were established that resulted in high-level expression of α-l-arabinofuranosidase A by an Aspergillus nigerd-xylulose kinase mutant strain. These conditions were adapted to construct a cDNA expression library from which an α-l-arabinofuranosidase A cDNA clone was isolated using specific antiserum. The corresponding gene encoding α-l-arabinpfuranosidase A (abfA) was isolated from a genomic library and cloned into a high copy plasmid vector. By co-transformation of uridine auxotrophic mutants lacking orotidine-5-phosphate decarboxylase activity, the afbA gene was introduced both in A. niger and A. nidulans, using the A. niger pyrA gene as selection marker. The identity of the abfA gene was confirmed by overexpression of the gene product by A. niger and A. nidulans transformants, upon growth using sugar beet pulp as the carbon source.

Identification of the mstE Gene Encoding a Glucose-inducible, Low Affinity Glucose Transporter in Aspergillus nidulans

The mstE gene encoding a low affinity glucose transporter active during the germination of Aspergillus nidulans conidia on glucose medium has been identified. mstE expression also occurs in hyphae, is induced in the presence of other repressing carbon sources besides glucose, and is dependent on the function of the transcriptional repressor CreA. The expression of MstE and its subcellular distribution have been studied using a MstE-sGFP fusion protein. Concordant with data on mstE expression, MstE-sGFP is synthesized in the presence of repressing carbon sources, and fluorescence at the periphery of conidia and hyphae is consistent with MstE location in the plasma membrane. Deletion of mstE has no morphological phenotype but results in the absence of low affinity glucose uptake kinetics, the latter being substituted by a high affinity system.

Contributions of the Peroxisome and β-Oxidation Cycle to Biotin Synthesis in Fungi

Background: Pimeloyl-CoA is an intermediate of the biotin pathway, but it is unknown how it is synthesized in fungi. Results: Aspergillus nidulans mutants in β-oxidation are biotin-deficient, and the enzyme utilizing pimeloyl-CoA is targeted to the peroxisome. Conclusion: Pimeloyl-CoA is synthesized in the peroxisome via the β-oxidation cycle in filamentous fungi. Significance: This is the first implication of peroxisomal β-oxidation in biotin synthesis. The first step in the synthesis of the bicyclic rings of d-biotin is mediated by 8-amino-7-oxononanoate (AON) synthase, which catalyzes the decarboxylative condensation of l-alanine and pimelate thioester. We found that the Aspergillus nidulans AON synthase, encoded by the bioF gene, is a peroxisomal enzyme with a type 1 peroxisomal targeting sequence (PTS1). Localization of AON to the peroxisome was essential for biotin synthesis because expression of a cytosolic AON variant or deletion of pexE, encoding the PTS1 receptor, rendered A. nidulans a biotin auxotroph. AON synthases with PTS1 are found throughout the fungal kingdom, in ascomycetes, basidiomycetes, and members of basal fungal lineages but not in representatives of the Saccharomyces species complex, including Saccharomyces cerevisiae. A. nidulans mutants defective in the peroxisomal acyl-CoA oxidase AoxA or the multifunctional protein FoxA showed a strong decrease in colonial growth rate in biotin-deficient medium, whereas partial growth recovery occurred with pimelic acid supplementation. These results indicate that pimeloyl-CoA is the in vivo substrate of AON synthase and that it is generated in the peroxisome via the β-oxidation cycle in A. nidulans and probably in a broad range of fungi. However, the β-oxidation cycle is not essential for biotin synthesis in S. cerevisiae or Escherichia coli. These results suggest that alternative pathways for synthesis of the pimelate intermediate exist in bacteria and eukaryotes and that Saccharomyces species use a pathway different from that used by the majority of fungi.